Growth Factors, Part A (Growth Factors & Cytokines in Health & Disease) (Growth Factors & Cytokines in Health & Disease)

GROWTH FACTORS AND CYTOKINES IN HEALTH AND DISEASE A Multi-Volume Treatise Volume IA • 1996 GROWTH FACTORS

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GROWTH FACTORS AND CYTOKINES IN HEALTH AND DISEASE A Multi-Volume Treatise GROWTH FACTORS Editors: DEREK LEROITH Diabetes Branch NIDDK National Institutes of Health' Bethesda, Maryland CAROLYN BONDY Developmental Endocrinology Branch NICHD National Institutes of Health Bethesda, Maryland VOLUME 1A • 1996

(yu)

Greenwich, Connecticut

JAI PRESS INC.

London, England

Copyright © 1996 byJAI PRESS INC 55 Old Post Road, No. 2 Greenwich, Connecticut 06836 JAI PRESS LTD. 38 Tavistock Street Covent Garden London WC2E 7PB England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher. ISBN: 0-7623-0091-4 Manufactured in the United States of America

CONTENTS LIST OF CONTRIBUTORS

vii

PREFACE Derek LeRoith and Carolyn Bondy

xi

INSULIN-LIKE GROWTH FACTORS Derek LeRoith and Carolyn Bondy GROWTH HORMONE Gerhard Baumann GROWTH HORMONE RECEPTOR Lisa S. Smit and Christin Carter-Su

1

27

43

EPIDERMAL GROWTH FACTOR: CELLULAR AND MOLECULAR FUNCTION Douglas K. Tadaki and Salil K. Niyogi

85

PLATELET-DERIVED GROWTH FACTOR Carl-Henrik Heldin, Arne Ostman, and Bengt Westermark

123

FIBROBLAST GROWTH FACTORS Ann Logan and Andrew Baird

147

PDGF AND FGF RECEPTORS IN HEALTH AND DISEASE Wendy j. FantI, Kevin C. Peters and Lewis T. Williams

179

THE NERVE GROWTH FACTOR FAMILY Mari Oshima, Yoke Hirata, and Cordon Guroff

229

NGF RECEPTORS Mariano Barbacid

259

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LIST OF CONTRIBUTORS

Andrew

Baird

Department of Cell Biology The Scripps Research Institute La Jolla, California

Mariano

Barbacid

Department of Molecular Biology Bristol-Myers Squibb Pharmaceutical Research Institute Princeton, New Jersey

Craig H. Bassing

Department of Pharmacology Duke University Medical Center Durham, North Carolina

Gerhard Baumann

Center for Endocrinology, Metabolism and Molecular Medicine Department of Medicine Northwestern University Medical School Chicago, Illinois

Carolyn Bondy

Developmental Endocrinology Branch NIHCD National Institutes of Health Bethesda, Maryland

Cristin Carter-Su

Department of Physiology University of Michigan Medical School Ann Arbor, Michigan

Paolo M.

Department of Biochemical Sciences

Comoglio

University of Torino School of Medicine Torino, Italy

Wei Cui

Department of Medical Genetics University of Glasgow Duncan Guthrie Institute Yorkhill Hospitals Glasgow, United Kingdom

VII

LIST OF CONTRIBUTORS Michael B. Datto

Department of Pharmacology Duke University Medical Center Durham, North Carolina

Wendy J. FantI

Chiron Corporation Emeryville, California

Denis

Cospodarowicz

Laboratory of Cellular Chemistry Chiron Corporation Emeryville, California

Gordon

Guroff

Section on Growth Factors National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland

Carl-Henrik

Heldin

Ludv^ig Institute for Cancer Research Biomedical Center Uppsala, Sweden

Yoko Hirata

Section on Growth Factors National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland

Robert T. Jensen

David C. Lee

Digestive Diseases Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland Lineberger Comprehensive Cancer Center and Department of Microbiology and Immunology School of Medicine University of North Carolina Chapel Hill, North Carolina

Se-Jun Lee

Department of Molecular Biology and Genetics Johns Hopkins University School of Medicine Baltimore, Maryland

List of Contributors Ann Logan

Molecular Biology Research Group The Wolfson Research Laboratories Queen Elizabeth Medical Centre Edgbaston, Birmingham, United Kingdom

Derek LeRoith

Diabetes Branch NIDDK National Institutes of Health Bethesda, Maryland

Alexandra C. McPherron

Department of Molecular Biology and Genetics Johns Hopkins University School of Medicine Baltimore, Maryland

Sheldon

Milstien

Laboratory of Neurochemistry National Institute of Mental Health National Institutes of Health Bethesda, Maryland

Terry W. Moody

Biomarkers and Prevention Research Branch National Cancer Institute Rockville, Maryland

Salil K. Niyogi

Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee

Mari

Oshima

Arne Ostman

Section on Growth Factors National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland Ludwig Institute for Cancer Research Biomedical Center Uppsala, Sweden

Kevin G. Peters

Duke University Medical Center Durham, North Carolina

Lisa S. Smit

Department of Physiology University of Michigan Medical School Ann Arbor, Michigan

LIST OF CONTRIBUTORS Sarah Spiegel

Department of Biochemistry and Molecular Biology Georgetown University Medical Center Washington, D.C.

Douglas K. Tadaki

Naval Medical Research Institute Immune Cell Biology Program Bethesda, Maryland

Xiao-Fan Wang

Department of Pharmacology Duke University Medical Center Durham, North Carolina

Bengt Westermark

Department of Pathology University Hospital Uppsala, Sweden

Lewis T. Williams

Chiron Corporation Emeryville, California

PREFACE

Advances in molecular technology in recent years have catalyzed an explosive growth of information about intercellular peptide messengers and their receptors. For example, ten years ago the only neurotrophin characterized at the molecular level was nerve growth factor (NGF) and the only recognized neurotrophin receptor was the p75 NGF receptor. At present, the number of described neurotrophic peptides approaches 30 and the number of receptors is increasing apace. Just six years ago, the characterized interleukins numbered about three while now there are at least 16. Because many of these new peptide ligands and receptors were identified by "reverse genetic" techniques the understanding of their biological roles lags behind the knowledge of their molecular structures. Over the past few years, however, a new era of functional studies has begun because recombinant proteins have become available for clinical studies. In addition, animal models have been and are being developed using recombinant DNA techniques. Both the clinical studies and studies of transgenic and target deleted mice will allow for further physiologic elucidation of the biological roles of these messenger peptides and their receptors. This series on Growth Factors and Cytokines is divided into three main sections: Growth Factors (Volume I), Cytokines (Volume II) and Systems (Volume III). Although volumes I and II are separate the distinction between "growth factors" and "cytokines" is probably more historical or pragmatic than indicative of differences in function. The term "growth factors" refers to a wide variety of locally or systemically produced proteins with pleiotropic actions on tissue growth and

XI

xii

PREFACE

differentiation. The term "cytokines" describes a group of proteins identified primarily within the immune and hematopoietic systems, although it is likely that such a narrow view of cytokines will not survive for long. For example it appears that some interleukins and interleukin receptors are expressed by neuroepithelial cells in vivo suggesting that these interleukins may have intrinsic roles within the nervous system. Furthermore, tumor necrosis factor (TNF) has been identified as a potential adipose tissue regulatory factor which is both produced and acts locally. The third volume entitled Systems deals more directly with the role of these factors in both normal physiology and the disease processes resulting from the deficiency or excess of growth factors/cytokines and their receptors. The first volume deals with peptide growth factors and their receptors. Here too there is an arbitrary division of ligands and their receptors. In some instances (e.g., insulin-like growth factors) the proteins and their corresponding receptors are discussed in the same chapter, whereas in other cases, for example, NGF and platelet-derived growth factor they are discussed separately. While we have attempted to be as comprehensive and inclusive as possible, there will always be some regrettable omissions. At the publishing date we recognize that a few growth factors and cytokines have not been included in this review. These new discoveries will for certain be reviewed in similar pages in the future. Derek Le Roith Carolyn Bondy

INSULIN-LIKE GROWTH FACTORS

Derek LeRoith and Carolyn Bondy

Abstract I. Introduction II. Molecular and Cellular Aspects A. IGFs B. Receptors C. IGF Binding Proteins III. Physiological and Clinical Aspects A. Embryonic Growth and Development B. Postnatal Growth C. IGF-I and Intermediary MetaboHsm D. Clinical Uses of IGF-I E. IGF-II F. IGFs and Neoplasia IV. Conclusion References

1 2 2 3 5 9 11 11 12 13 15 17 18 18 19

ABSTRACT The insulin-like growth factor family of peptides, binding proteins and receptors is involved in normal growth and development. Later they are important in the differentiated function of a number of tissues. Aberrations in this growth factor system are associated with different diseases, rangingfromshort stature and diabetes to malignancy. Growth Factors and Cytokines in Health and Disease Volume lA, pages 1—26. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0091-4 1

DEREK LEROITH and CAROLYN BONDY

With the advent of recombinant DNA technology, sufficient quantities of the ligands (and binding proteins) have become available for clinical testing in the therapy of certain diseases. These exciting new possibilities need to be assessed carefully for side-effects.

I. INTRODUCTION The insulin-like growth factors (IGF-I and IGF-II) regulate growth and development of multiple tissues during embryonic and fetal stages (reviewed in Daughaday and Rotwein, 1989; Werner et al., 1994). During postnatal stages they continue to affect growth and maintain the differentiated function in these numerous tissues and in specific cell types. While the liver produces large amounts of both IGFs, many extrahepatic tissues synthesize and secrete these factors as well (Lowe et al., 1987; Hoyt et al., 1988). Circulating IGFs are of hepatic origin and act in a classical endocrine mode, whereas extrahepatic IGFs act locally in a paracrine or autocrine mode. The biological actions of the IGFs are mediated primarily by the type I IGF receptor (IGF-I receptor) which is ubiquitously expressed (LeRoith et al., 1995). The actions of the IGFs are also affected by a family of IGF-specific binding proteins (IGFBPs) found in circulation and in extracellular fluids; these proteins may enhance or inhibit the actions of the IGFs primarily by affecting their availability to cell surface receptors (Baxter and Martin, 1989; Rechler, 1993; Jones andClemmons, 1995). In this review we will initially discuss the basic molecular and cellular aspects of the IGFs, their binding proteins and receptors, and use examples from normal physiology and pathology to highlight their importance. Then we summarize the available data on the clinical studies of recombinant human IGF-I (rhIGF-I) and, to a lesser extent, IGF-II which have recently become available for clinical research.

II. MOLECULAR AND CELLULAR ASPECTS The IGFs are structurally similar demonstrating -65% amino acid similarity with each other and -50% with insulin (Blundell et al., 1983; Daughaday and Rotwein, 1989; Sussenbach, 1989; Rechler and Nissley, 1990) (Figure 1). Circulating insulin consists of an A- and B-chain, because the connecting(C) peptide is proteolytically cleaved out during processing of the prohormone. Mature, circulating IGF-I and IGF-II retain the smaller C-peptide and have a D-extension to the A-chain. The E-peptide in the prohormone is cleaved off during processing (see below, Figure 2). A. IGFs

The human IGF-I gene, on the long arm of chromosome 12 (Tricoli et al., 1984), spans more than 90 kb of chromosomal DNA and contains at least six exons. Exons

Insulin-Like Growth Factors

B30

A21 INSULIN

IGF (I)

PROINSULIN

IGF (II)

Figure 1. Predicted tertiary structures of the insulin-iii<e growth factor (IGF) family of peptides.

1 and 2 encode distinct, mutually exclusive 5'-untranslated regions (UTRs) as well as distinct N-termini of the signal peptide (Figure 2) (Rotwein et al., 1986; Shimatsu and Rotwein. 1987a). Exons 3 and 4 encode the mature peptide sequence, whereas the E-peptide coding sequences are contained in exons 4, 5 and 6. Transcriptional and posttranscriptional events are extremely complex. For example, the exon 1 promoter lacks core promoter elements, such as TATA and CCAAT boxes, and transcription of this exon is, therefore, initiated from at least four sites dispersed over a ^350 bp region. Transcription from exon 2, which contains TATAand CCAAT-like motifs, is initiated over a smaller region (Jensen et al., 1991). Exon 1-containing transcripts are expressed ubiquitously, and transcription is regulated by multiple factors, generally, specific to each particular tissue. Exon 2-

DEREK LEROITH and CAROLYN BONDY

•GF-1 rmM

Pi

'Gi^-^ J 1

wym^^mv^^

n n 2 3

Pz

in

P3 P4

n nn I 4 5 6 7 8 9

Figure 2. Structure of mammalian IGF-I and IGF-II genes. Exons are numbered. Known promoter sites in the IGF-II gene are labeled P1-P4.

containing transcripts, on the other hand, are particularly abundant in the liver and are generally more responsive to growth hormone (GH). Thus, during development, exon 2-containing transcripts appear after exon 1-containing transcripts in the liver, but increase markedly at the onset of GH-dependent linear growth (Jensen et al., 1991; Kim et al., 1991; Kikuchi et al, 1992). At the posttranscriptional level there appears to be regulation of mRNA splicing, in certain species of IGF-I mRNA (Shimatsu and Rotwein, 1987b), of a 186 bp region of exon 1 that potentially influences translatability of that transcript. Furthermore, two alternative E-peptides may be transcribed depending on exon 5 or exon 6 usage; GH seems to favor exon 5 retention. At the level of mRNA stability, the longer -'7.5 kb transcripts, derived from distal polyadenylation site usage in the long exon 6, are more unstable than the shorter -'1 kb mRNAs derived by more proximal polyadenylation site usage (Lowe et al., 1988; Lund et al., 1989; Heppler et al., 1990; Steenbergh et al., 1991). IGF-U

The IGF-II gene spans ^30 kb of chromosomal DNAon the distal end of the short arm of human chromosome 11 (Tricoli et al., 1984), immediately 3' to the insulin gene. Like IGF-I, the IGF-II gene is complex consisting of nine exons. The mature peptide is encoded by exons 7,8 and 9. Transcription is controlled by four different promoters (PI - P4) (Dull et al., 1984) (Figure 2). The promoters are activated in a tissue- and development-specific manner; promoter PI is activated in adult liver, whereas promoters P2, P3 and P4 are active in most fetal tissues and adult nonhepatic tissues (dePagter-Holthuizen et al., 1987; dePagter-Holthuizen, 1988; Holthuizen et al, 1990). PI is a TATA-less, GC-rich promoter with heterogeneous transcription initiation. Liver-specific expression of PI is regulated by the CAAT/enhancer binding protein (C/EBP) and the liver-enriched activator protein

Insulin-Like Growth Factors

5

(LAP) (Sussenbach et al, 1994). Promoters P3 and P4 contain a TATA box and P3 also has a CCAAT box. P3 and P4 exhibit transcription from specific sites and are more highly regulated than PI. Human IGF-II promoter P3 is expressed in many fetal and non-hepatic adult tissues and is regulated by the krox 20/egr2 and krox 24/egrl transcription factors (Sussenbach et al., 1994). Multiple IGF-II mRNA transcripts are produced as a function of specific promoter usage and different lengths of 3' UTRs resulting from use of multiple polyadenylation sites. Transcripts from P2 and P4 have shorter 5'-UTRs and are preferentially translated (Irminger et al., 1987; Rechler, 1991). In human tissues, the IGF-II gene demonstrates promoter-specific genomic imprinting (Vu and Hoffman, 1994). When promoter P1 is used, as with adult liver, both maternal and paternal alleles are transcribed, but in the case of promoters P2—P4 usage, only the paternal allele is expressed. Because promoters P2—P4 are clustered in a '-5 kb DNA region, whereas PI is ~20 kb upstream (Figure 2), this suggests that the imprinting signals lie between promoters PI and P2 (Vu and Hoffman, 1994). In murine species, the equivalent of promoter PI is absent and the remaining three promoters are all imprinted except for the choroid plexus and leptomeninges where biallelic expression occurs (Pedone et al., 1994; DeChiara et al., 1991a; DeChiara et al., 1991b). Interestingly, in the mouse and rat, postnatal IGF-II expression decreases dramatically leading to undetectable circulating IGF-II levels (except in the CNS), whereas in humans, where the PI promoter is active in liver, IGF-II expression persists throughout life. Because the PI promoter is biallelically expressed, it has been postulated that this may allow more abundant synthesis of the IGF-II peptide which is released into the circulation. Loss of genomic imprinting has been implicated as a reason for the high level of expression of IGF-II in many cancers (Steenman et al, 1994; Wemer and LeRoith, 1995), where the IGF-II peptide may be important as an autocrine growth factor enhancing tumor growth (Zhan et al., 1994). B. Receptors

The biological effects of the IGFs are mediated by a family of specific membraneassociated glycoprotein receptors including the insulin IGF-I and IGF-II receptors and the insulin receptor-related-receptor (IRR) (Zhang and Roth, 1992; Reinhardt et al., 1993; Kovacina and Roth, 1995; LeRoith et al., 1995). The insulin IRR, and IGF-I receptors are closely related tyrosine kinase receptors, whereas the IGF-II receptor is identical to the cation-independent mannose-6-phosphate (M-6-P) receptor (Figure 3). Because the biological effects of the IGFs on growth, development, and differentiated functions are primarily via the IGF-I receptor, we will concentrate on its structural and functional characteristics. Because the IRR has limited tissue distribution and does not bind the IGFs, it does not apparently affect IGF action.

Figure3. Comparison of IGF-I, insulin, insulin receptor-relatedreceptor, insulin-IGF-l hybrid, and IGF-II/M-6-P receptors. The biological actions of insulin and the IGFs are initiated by their interaction with specific cell surface receptors. Insulin and IGF-I receptors are tetrameric proteins composed of two extracellular a subunits and two transmembrane subunits. The insulin receptor-related receptor shows limited tissue expression. Naturally occurring hybrid receptors have been described in which an insulin a / p hemireceptor is linked to an IGF-I a/j3 hemireceptor. The IGF-II/M-6-P receptor i s a single chain polypeptide comprising 15 repeat sequences and a short cytoplasmic domain.

Insulin-Like Growth Factors

7

IGF-! Receptor The human IGF-I receptor spans more than 100 kb of chromosomal DNA at the distal end of the long arm of chromosome 15 and consists of 21 exons (Abbott et al., 1992). The gene encodes contiguous a and p subunits which are cleaved during processing and the mature functional receptor is a heterotetrameric glycoprotein in an (aP)2 configuration where the subunits are joined by disulfide bridges. The a subunits are entirely extracellular and bind the ligands, primarily, in the region of the cysteine-rich domain. The P subunits are anchored in the membrane and contain a cytoplasmic tyrosine kinase domain which has 84% homology with the equivalent region in the insulin receptor and IRR (Ebina et al., 1985; Ullrich et al., 1985; Ullrich etal, 1986). The IGF-I receptor is widely expressed at high levels during embryogenesis, suggesting an important role in tissue development (see below). Extensive studies on characterizing the promoter region have revealed a number of features involved in regulating IGF-I receptor gene expression (Werner et al., 1989; Werner et al., 1990; Cooke et al., 1991; Mamula and Goldfme, 1992; Werner et al., 1992). Both the unusually long (-^1 kb) 5' UTR and the proximal -^500 bp 5' flanking region are very GC-rich with multiple Spl binding sites and no TATA or CCAAT boxes. However, transcription initiation begins at a single site surrounded by an "initiator" sequence. Spl binding to the promoter region may regulate transcription initiation in the absence of TATA and CCAAT boxes (Smale and Baltimore, 1989). Ligand binding to the IGF-I receptor initiates receptor autophosphorylation which involves the phosphorylation of a cluster of three tyrosines (1131,1135 and 1136) in the kinase domain (Gronberg et al., 1993; Kato et al., 1993; Kato et al., 1994). As with the insulin receptor, autophosphorylation activates the tyrosine kinase activity of the receptor, leading to tyrosine phosphorylation of cellular substrates. The substrate that has been best characterized to date is the insulin substrate-1 (IRS-1) (Sun et al., 1991; Myers and White, 1993) (Figure 4). IRS-1 contains multiple tyrosine residues in a Tyr-Met-X-Met (YMXM) or related motifs. Phosphorylation of tyrosines in these motifs mediate binding of other substrates, such as the 85-kDa subunit of phosphatidyl-inositol 3' (PI3') kinase and Grb2 (Myers et al., 1992; Yamamoto et al., 1992; Baltenspergan et al., 1993; Giorgetti et al., 1993; Myers et al., 1993; Skolnik et al., 1993). IGF-I stimulates PI3' kinase activity and MAP kinase activity, the latter being the result of activation of the ras/raf kinase pathway initiated by Grb2 binding to IRS-1. Other known pathways involved in IGF-I receptor activation include protein tyrosine phosphatase-IB (Kenner, 1993) and SHC and crk (Beitner-Johnson and LeRoith, 1995). IGF'II/M-6'P Receptor The IGF-II/M-6-P receptor is a bifunctional receptor with a large extracellular region containing 15 contiguous repeats and a very short cytoplasmic tail (Lobel et al., 1988; MacDonald et al., 1988; Morgan et al., 1987). Unlike the insulin and

DEREK LEROITH and CAROLYN BONDY

MAP Kinases (ERKs)

Transcription Factors

Gene Expression

Figure 4. Schematic representation of intracellular signaling pathways of the IGF-I receptor. Upon binding IGF-i, the IGF-I receptor undergoes autophosphorylation at multiple tyrosine residues. The intrinsic kinase activity of the receptor also phosphorylates IRS-1 at multiple tyrosine residues. Various SH-domain-containing proteins, including PI3-kinase, Grb2, Syp, Nek and crk associate with specific phosphotyrosine-containing motifs within IRS-1, as shown. Activation of IGF-I receptors also results in tyrosine phosphorylation of She, which then complexes with Grb2. Grb2 is tightly associated with the mammalian guanine nucleotide exchange factor Sos, which activates Ras. IGF-I can apparently activate Ras via both the IRS-1-Grb2-Sos or the Shc-Grb2-Sos pathways. This leads to the activation of a cascade of protein kineses including Raf-1 and one or more related kineses, MAP kinase kineses (or MEKs), the MAP kineses, and S6 kinase. These protein kineses, in turn, activate various other elements, including nuclear transcription factors. IGF-I receptors, the tail does not contain any tyrosine kinase activity. The major function of this receptor is to target recently synthesized lysosomal enzymes from the tmnS'Golgi network to lysosomes and to internalize lysosomal enzymes that have escaped the cell. It also internalizes surface-bound IGF-II, targeting it to the lysosomal compartment for degradation. Certain studies have suggested that, in a limited number of cells, the IGF-II/M-6-P receptor may mediate Ca^"^ uptake, cell growth, and generation of inositol phosphate (IP3) and diacylglycerol (Hari et al., 1987; Matsmaga et al., 1988; Rogers and Hammerman, 1988). These effects were mediated by a G-protein (Gi2) -related mechanism (Nishimoto et al., 1987; Nishimoto et al., 1989; Okamoto et al., 1990). However, a recent study has failed to

Insulin-Like Growth Factors

9

reproduce these results, and the general conclusion is that the IGF-n/M-6-P receptor does not contain any signaling function (Komer et al., 1995). The role of the IGFs and their receptors in embryonic and postnatal growth is strongly supported by studies in which targeted disruption of various components has been obtained in mice by homologous recombination (DeChiara et al., 1991b; Baker et al., 1993; Liu et al., 1993; Powell-Braxton et al., 1993). Most mice homozygous for a null mutation of the IGF-I gene die perinatally, though a small number survive to adulthood. These mice demonstrate a 40% reduction in body weight at birth and a 70% reduction at eight weeks of age. They are infertile and have delayed ossification, underdeveloped muscles, and poorly organized lungs, suggesting an important role for IGF-I in tissue differentiation and development. On the other hand, targeted disruption of the IGF-II gene results in mice that weigh 60% of their normal littermate weight but survive, grow postnatally, albeit at a lower weight, and are fertile. Thus, IGF-II may be important for determining overall body size, but not essential for organogenesis. The IGF-I receptor plays a very important role in organ development and probably mediates the effects of both IGF-I and IGF-II. Mice lacking the IGF-I receptor gene are severely growth retarded by birth, weighing only 45% of controls and die due to impaired muscular development leading to respiratory failure. Many other organs are also severely retarded in their development. Both the naturally occurring Tme mutation and the IGF-II/M-6-P receptor-lacking mice show the same phenotype, that is, increased body size and polydactylic and embryonic/neonatal lethality. Because this phenotype is rescued by breeding mice lacking both the IGF-II/M-6-P receptor and IGF-II, it suggests that the phenotype of the Tme mutation is due to excess IGF-II acting through the IGF-I receptor, which is normally expressed in these mice. The lack of the IGF-II/M-6-P receptor in the Tme mutants probably results in reduced clearance of IGF-II resulting in overstimulation of the IGF-I receptor, again, strongly supporting a role for the IGF-I receptor in signal transduction and for the IGF-II/M-6-P receptor in IGF-II internalization and degradation (Lau et al., 1994; Wang et al., 1994). C. IGF Binding Proteins

In addition to the IGFs and their receptors, the IGFBPs play a critical role in IGF action. These are a family of six proteins, IGFBP-1 to 6, that specifically bind the IGFs with high affinity and modulate their bioavailability and biological actions (Table 1) (Baxter and Martin, 1989; Rechler, 1993; Jones and Clemmons, 1995). Molecular The IGFBPs are a family of closely related proteins, with N- and C-termini that are highly conserved at the amino acid level, including 18 cysteine residues, 12 in the NH2-terminal region and six in the COOH-terminal region. Most of the cysteines are disulfide bonded, giving rise to the tertiary structure (Wood et al., 1988; Rechler, 1993).

10

DEREK LEROITH and CAROLYN BONDY Table 1, Insulin-Like Growth Factor Binding Proteins

IGFBPs IGFBP-1 IGFBP-2 IGFBPS IGFBP-4 IGFBPS IGFBPS

Protein RGD Mass (kDa) Glycosylation Phosphorylation Sequence Proteolysis 25 31.3 28.7 25.9 28.5 22.8

N-linked N-linked 0-linked 0-linked

+

+

+ 7

+ +

-

+ + + + ?

Relative Binding Affinity IGF-I = IGF-II IGF-II > IGF-I IGF-I = IGF-II IGF-I = IGF-II IGF-I = IGF-II IGF-II»IGF-I

The IGFBPs bind the IGFs with high affinity (-10 to ~11 M), often greater than the affinity demonstrated by the IGF-I receptor. IGFBP-2 and 6 have greater affinity for IGF-II than IGF-I. The IGFBPs are ubiquitously expressed and are found in most body fluids including plasma, cerebrospinal fluid, amniotic fluid, lymph, milk, etc. In some, IGFBP-3 and 4, N-glycosylation adds to the heterogeneity seen, whereas IGFBP-6 is 0-linked glycosylated. In plasma, the maj or portion of the IGFs circulate bound to a '^ 150-kDa molecular weight ternary complex consisting of the ligand (IGF-I or IGF-II), IGFBP-3 and an acid-labile subunit (ALS) (Baxter and Martin, 1989; Leong et al., 1992). Because ALS usually circulates in excess, IGFBP-3 may play a more significant role in determining the total concentration of circulating IGF. The IGFs are also found, to a lesser degree, in a 40-50 molecular weight complex that includes IGFBP-2,4 or 6 and, to the least extent, IGFBP-1. Very little free IGF is measurable because of the short half-life of the free hormone. While the 150-kDa molecular weight complex controls the bioavailability of IGFs, the smaller molecular weight complexes may be important in transporting IGFs out of circulation to the target tissue (Baretal., 1990). Modulation of IGF Action Circulating IGFs are bound to IGFBPs which prolong their half-lives and deliver them to their target tissues. At the cellular level, IGFBPs control IGF action either by restricting access to cell surface receptors and, thereby, inhibiting growth and related anabolic frmctions or by augmenting this interaction and thereby potentiating the cellular response (Elgin et al., 1987; Bar et al., 1989; Blum et al., 1989). Most of the studies have demonstrated that, with the exception of IGFBP-4 which seems to be uniformly inhibitory, all of the IGFBPs have the capacity to potentiate and inhibit IGFs biological responses. The affinity of released IGFBPs (i.e., IGFBP-3 and 5) for IGFs is significantly higher than cell-surface associated forms of IGFBP. Therefore, if the majority of the IGFBPs are in the interstitial fluids or culture medium, they will sequestrate

Insu I in-Like Growth Factors

11

IGFs and inhibit their interaction with the receptor. On the other hand, when cell-surface bound, the IGFBPs have reduced affinity for IGFs, and the IGFs are then able to interact with their receptors. How this effect results in potentiated function is not clear. Some studies suggest that cell-surface-associated IGFBP-3 inhibits the rate of receptor internalization, thereby, prolonging its activity at the cell surface. Another study using IGF analogues suggested that potentiation required binding of the IGF to both IGFBP-1 and the IGF-I receptor (Conover and Powell, 1991; Camacho-Hubner et al., 1992). The mechanism(s) of interaction of the IGFBPs with the cell surface membrane is also not clear. Some IGFBPs contain Arg-Gly-Asp (RGD) sequences that allow interaction with integrin receptors; others such as IGFBP-3, 4, and 5 are glycosylated (Table 1) (Jones et al., 1993; Arai et al., 1994a; Arai et al, 1994b). Whether these features are important for the functions described above is yet to be clearly defined. IGFBP-1 and 3 contain multiple serine residues that are phosphorylated intracellularly. Phosphorylated IGFBP-1 inhibits IGF action on certain tissues, whereas the dephosphorylated form has manyfold lower affinity for IGF-I (Frost and Tseng, 1991). Finally, multiple serine and metallothionein proteases have been isolated that proteolytically cleave the IGFBPs and aid in the release of the IGFs; they, too, may be significant in modulating IGF biological activity at the tissue level (Fowlkes and Freemark, 1992; Nam et al., 1994)

III. PHYSIOLOGICAL AND CLINICAL ASPECTS There is a very large literature on the effects of IGF-I and II in cell culture systems demonstrating pleiotropic actions largely dependent upon the cell type, culture conditions, and assay system employed. The IGF-I receptor, which is thought to mediate most, if not all, of the biological effects of IGF-I and II, is very widely expressed in vivo and in cell lines, and, thus, IGF actions have been demonstrated in a very wide array of cell types. These in vitro studies have been comprehensively reviewed (Lowe, 1991; Jones and Clemmons, 1995). This survey will focus on in vivo or clinical studies, which have been proliferating recently with the advent of targeted deletion of IGF and IGF receptor genes in mice and the availability of recombinant IGF-I for humans. A. Embryonic Growth and Development IGF-I and II have important effects in murine embryonic development as demonstrated by the dwarfism of mice bom with targeted deletion of either of these genes or the IGF-I receptor (Baker et al., 1993; Liu et al., 1993). Deletion of the active IGF-II allele results in proportionate dwarfism which is apparent from midgestation (Baker et al., 1993), that is after placentation but not before, suggesting that the dwarfism may result from limited placental development. These mice grow and reproduce normally, although they maintain their diminutive size

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DEREK LEROITH and CAROLYN BONDY

throughout life. IGF-I deletants are also bom small but their growth retardation appears late in gestation and is not associated with reduction in placental size. In contrast to the IGF-II null mice, most of the IGF-I null mice die in the first days or weeks after birth (Liu et al., 1993; Powell-Braxton et al., 1994). The cause of death in these mice is not clear. The few that survive demonstrate extremely retarded postnatal growth, so that, by two months of age, they are approximately 30% the size of wild-type littermates and both sexes are infertile. Combined IGF-I/IGF-II deletants and IGF-I receptor null mice are bom very small and uniformly die within hours of birth, apparently due to hypoplasia of respiratory muscles (Liu et al., 1993; Powell—Braxton et al., 1994). IGF-I and II are present in human gestation (Han et al., 1987; Hill, 1990) and, presumably, have important roles in fetal growth and development, although as yet there are few fetal syndromes attributed to IGF-I or -II deficiency. Dismption of the distal portion of chromosome 15q, which includes the locus of the IGF-I receptor gene, is associated with severe fetal and postnatal growth retardation (Roback et al., 1991), and a child with intrauterine growth retardation was reported to have a 50% decrease in IGF-I binding to cultured fibroblasts (Bierich et al., 1984). Overproduction of IGF-II during gestation has been implicated in fetal overgrowth in the Beckwith—Wiedeman syndrome (Weksberg et al, 1994). B. Postnatal Growth

The most well-recognized clinical role of IGF-I is the promotion of statural growth. Growth hormone (GH) secreted by the anterior pituitary acts on the liver to stimulate hepatocyte synthesis and secretion of IGF-I into circulation. Circulating IGF-I levels normally reflect GH activity, peaking around the time of the pubertal growth spurt and gradually declining during the aging process (Juul et al., 1994). Deficient GH secretion or action results in abnormally low circulating IGF-I levels and short stature, known as pituitary dwarfism. In addition to short stature, affected individuals demonstrate poor muscular development, suggesting that IGF-I promotes muscle and long bone growth. GH treatment results in normalization of the growth process in most cases. However, some individuals are resistant to GH due to genetic defects in the GH receptor or to antibodies to the hormone, which may develop after GH treatment in patients with absent endogenous GH (Laron, 1993). These GH-resistant patients grow in stature in response to treatment with recombinant human IGF-I (Laron et al., 1992; Walker et al., 1992), supporting the view that GH's growth-promoting effects are largely mediated by IGF-I (the somatomedin hypothesis). Several more years will be necessary to determine if fully normal growth pattems are produced in these children by IGF-I in the absence of GH action. IGF-I appears to enhance linear growth by acting directly on growth plate chondrocytes during the time of their proliferation and hypertrophy. An increased number of cell divisions and an increased dimension of individual chondrocytes result in progressive linear expansion of the cartilage template upon which bone growth depends. Elevation of IGF-I levels due to GH-producing tumors during

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childhood results in gigantism, as epiphyseal chondrocytes undergo increased cycles of division or increased hypertrophy, or perhaps both, augmenting long bone growth. Elevation of IGF-I levels due to GH-producing tumors later in life causes overgrowth of articular cartilage and soft tissues, but further growth in stature is not seen due to the terminal differentiation of growth plate chondrocytes and fusion of the epiphyses which occur in late puberty. These patients also have increased bone mineral density, while GH and IGF-I-deficient adults often have reduced bone density (Halse et al., 1981; Bouillon, 1991). These observations, together with the fact that IGF-I promotes osteoblast activity in vitro (reviewed in Delaney et al., 1994), suggest that IGF-I may have significant anabolic effects on bone mineralization in adults. Clinically, it has been difficult to isolate the effects of IGF-I on bone from those of other important effectors of bone mineral homeostasis, such as sex steroids, thyroid, and glucocorticoid hormones, which also may be disturbed in patients with pituitary disease. However, a recent study employing a primate model of menopausal bone loss showed that GH treatment prevented osteoporosis induced by suppression of ovarian flmction in rhesus monkeys (Mann et al, 1992). Thus, although IGF-I's promotion of linear growth is its most clinically obvious effect, this anabolic hormone may also have an important role in maintaining skeletal mass in adults. This review addresses primarily the endocrine role(s) and regulation of circulating IGFs. IGFs and IGFBPs are also synthesized locally for presumed autocrine or paracrine functions in many different tissues. Significant interspecies differences are apparent in local patterns of IGF system expression. The many studies directed at elucidating specific local IGF system functions are beyond the scope of this review. C. IGF-I and Intermediary Metabolism

IGF-I has significant insulin-like metabolic effects, including the stimulation of glucose and amino acid uptake, protein and glycogen synthesis (Guler et al., 1987; Boulware et al., 1992). However, compared to insulin, IGF-I promotes substrate uptake preferentially in lean tissue (Cascieri et al., 1986; Werner et al., 1989) and promotes protein as opposed to fat synthesis (Bolinder et al., 1987; Sinha et al., 1989). Although its anabolic effects are most notable during childhood and adolescence when IGF-I secretion and growth are maximal, this hormone has continuing effects in the adult, with a strong correlation found between decreasing IGF-I levels and loss of musculoskeletal mass during aging (Juul et al., 1994). There is complex interplay between GH, IGF-I, and insulin in regulating fuel homeostasis and specific tissue anabolism (Figure 5). Portal insulin appears necessary for GH to maximally stimulate IGF-I synthesis, because, when portal insulin is reduced or absent, as in starvation, hyperalimentation, and insulin-dependent diabetes mellitus (IDDM), hepatic IGF-I output in response to GH is reduced. This may be due to a downregulation of hepatic GH receptor expression reflected by a reduction in circulating GH binding protein levels (HoU et al., 1993). Insulin priming of hepatic IGF-I synthesis is also deficient in patients with severe genetic

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DEREK LEROITH and CAROLYN BONDY -->► anti-insulin effects - > direct anabolic effects?

IGF-II

/

>- insulin-like effects ~ > anabolic effects

Figures. Interrelationships between IGF system, growth hormone (GH), and insulin. GH and insulin both stimulate hepatic IGF-I production and IGF-I feeds back to suppress both GH and insulin release. IGF-binding protein-3 (BP3) and the associated acid-labile subunit (ALS) levels are positively regulated by G H , and IGF-binding protein-1 (BP1) levels are negatively regulated by insulin. Regulation of IGF-II is unclear at present.

insulin receptor defects, as in leprechaunism and the Rabson—Mendenhall syndrome. These patients demonstrate growth retardation and extremely low levels of circulating IGF-I and resistance to GH. Decreased portal insulin (or hepatic insulin receptor deficiency) impairs IGF-I secretion and alters the serum IGF-binding protein profile, resulting in accelerated clearance of IGF-I from the circulation. IGFBP-1 is synthesized by hepatocytes and is negatively regulated by insulin (Sukkari et al., 1988; Conover et al., 1992), so that IGFBP-1 production is increased, when portal insulin is low, and IGF-I bound to IGFBP-1 is rapidly cleared from circulation. In contrast, components of the high molecular weight IGF-binding complex, which normally stabilizes IGF-I in the serum, are reduced in GH deficiency or resistance. In addition to stimulating IGF-I production, GH has direct metabolic effects counter to those of insulin, for example, the promotion of gluconeogenesis and lipolysis. Finally, IGF-I participates in a classical endocrine negative feedback loop, suppressing GH production and insulin secretion, even under euglycemic conditions (Leahy et al., 1990; Phillips et al., 1991; Rennert et al., 1993; Bach et al., 1994). IGF-I also suppresses glucagon (Rennert et al., 1993). Thus, IGF-I, insulin and GH each have independent, tissue-specific effects on fuel utilization and each participates in the regulation of the other hormones, making for a complex integrated system of fuel homeostasis.

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D. Clinical Uses of IGF-I GH Resistance-Laron Syndrome and Insulin-Dependent Diabetes Mellitus (IDDM) As noted above, the first clinical use of IGF-I was in GH-resistant short children. Other clinical applications have recently been summarized (Bondy et al., 1994). IGF-I has been investigated in the treatment of conditions associated with relative or functional GH resistance, such as IDDM. Low portal insulin levels in IDDM result in diminished IGF-I production in response to GH (Lanes et al., 1985) and elevation of GH levels due to decreased negative feedback by IGF-I. Elevated GH levels, particularly during adolescence, exacerbate hyperglycemia and increase insulin requirements (Press et al., 1984). Intensive peripheral insulin therapy may succeed in normalizing IGF-I and GH levels, but at the expense of significant risk for hypoglycemia and excessive adiposity. Recent clinical studies have demonstrated that addition of IGF-I to insulin treatment in adolescents with IDDM reduces GH levels and insulin requirements, at least over the short term (Cheetham et al., 1993; Bach et al., 1994). Based on the considerations discussed above, there is reason to believe that reduced systemic IGF-I levels, particularly during adolescence, may also have primary adverse effects on musculoskeletal development, independent of the secondary derangements of counterregulatory hormones GH and glucagon. Further studies are required to determine if normalization of IGF-I levels by exogenous IGF-I in addition to insulin treatment may improve metabolic/anabolic balance in IDDM over the long term. Catabolic States IGF-I treatment is also being used to improve lean tissue anabolism in patients with a variety of catabolic conditions, as recently reviewed (Clemmons and Underwood, 1994). There seems to be a clear rationale for this approach to hyperalimentation, where patients may have reduced IGF-I levels secondary to reduction in portal insulin and could better utilize substrate in lean tissues with normalization of IGF-I levels. If available substrate is inadequate however, the shutdown in insulin and IGF-I production is probably an adaptive mechanism. Indeed, the use of IGF-I in fasting patients incurs a significant risk of hypoglycemia, likely due to IGF-I induced suppression of counterregulatory hormones GH and glucagon, in addition to IGF-I stimulated glucose utilization. IGF-I may not be helpful where there is a primary catabolic insult affecting substrate utilization by muscle, as, for example, in glucocorticoid-induced protein wasting (Mauras et al., 1992). Furthermore, treatment with IGF-I in situations where endogenous IGF-I levels are not low may be self-limited because significant elevation of circulating IGF-I and suppression of GH alters the IGFBP balance resulting in accelerated clearance of the costly recombinant hormone (Lieberman et al., 1993). Recent work has

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DEREK LEROITH and CAROLYN BONDY

shown that treating fasting volunteers with a combination of both GH and IGF-I may overcome some of these difficulties (Kupfer et al., 1993). In summary, it seems likely that if catabolic patients are receiving adequate nutrients but endogenous IGF-I levels are suppressed, supplementation with exogenous IGF-I and GH may be beneficial. Insulin-Resistance Another area in which IGF-I may have significant therapeutic potential is hyperglycemic disorders characterized by insulin resistance. Over the short-term, recombinant IGF-I reduces blood glucose and triglycerides in insulin-resistant obese patients with noninsulin-dependent diabetes mellitus (NIDDM; Zenobi et al., 1992). These salutary effects have been attributed to an IGF-I-induced reduction in GH and insulin secretion, improving insulin sensitivity, in addition to direct, insulin-like metabolic effects of IGF-I. A more recent study (Moses, 1996) has shown a significant reduction in glycosylated hemoglobin, a marked increase in insulin sensitivity (measured by frequently sampled intravenous glucose tolerance test) and a 50% decrease in circulating insulin levels with a 2% reduction in body fat after 4 to 6 weeks of IGF-I in obese NIDDM patients treated with 100 |ag/kg subcutaneously twice daily. Several studies have reported that recombinant IGF-I treatment improves the hyperglycemia of nonobese patients with extreme insulin resistance (Quin et al., 1990; Schoenle, 1991; Usala et al., 1992; Kuzuya, 1992; Morrow et al., 1993). Not all insulin-resistant patients improve with IGF-I treatment, however (Skarulis, 1994), and many patients cannot tolerate IGF-I treatment at the doses necessary to achieve hypoglycemic effects (Guler, 1994). Common side effects include edema, nerve compression syndromes, parotid gland swelling, and tenderness and joint pain. Treatment side effects are prominent in NIDDM patients probably because this group does not have low endogenous IGF-I levels and supranormal levels resulting from exogenous IGF-I treatment may rapidly produce acromegaloid effects in this older patient population. System-Specific Anabolic Effects As mentioned above, there is an association between declining GH/IGF-I levels in aging and loss of muscle and bone mineral mass. Other important hormonal factors, such as gonadal and adrenal steroids, are doubtless involved in age-related diminution in lean tissue mass. Thus far, no clear independent correlation between IGF-I levels and osteoporosis has been documented (reviewed in Delaney et al., 1994). It may be, however, that local osteoblast IGF-I production is diminished in aging. Studies are underway to evaluate potential therapeutic effects of systemic IGF-I treatment in clinical osteoporosis. GH and IGF-I have also been implicated in stimulating a variety of immune system functions, although GH and IGF-Ideficient patients do not demonstrate clinically significant immunodeficiency (reviewed in Gelato, 1993). Interestingly, a number of young, short-stature patients

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treated with IGF-I have demonstrated hypertrophy of lymphatic tissues, including tonsils, adenoid, and spleen. A number of clinical studies are investigating the efficacy of GH and/or IGF-I in improving immune system parameters as well as in counteracting protein catabolism in AIDS patients. A variety of evidence suggests that IGF-I may have a role in therapy of neurological diseases. One group has found that intrathecal IGF-I treatment reduces histological damage in the rat brain following hypoxic-ischemic injury (Gluckman, 1993). IGF-I levels are reduced in peripheral nerves and spinal cord of streptozotocin-diabetic rats and systemic IGF treatment improves sensory innervation in this model of diabetes (Ishii, 1995; Ishii and Lupien, 1995). Peripheral IGF-I treatment has been shown to reduce demyelination and significantly improve neurological function in rats with experimental autoimmune encephalomyelits (Yao et al., 1995). Recent clinical studies reported by Cephalon at the 1995 Endocrine Society meeting demonstrated beneficial effects of subcutaneous IGF-I treatment on neuromuscular function in patients with amyotrophic lateral sclerosis (ALS). Long term studies will be necessary to confirm and extend the preliminary data as well as determine the safety of IGF-I treatment in neurological patients. A number of clinical studies are investigating the efficacy of GH and/or IGF-I in improving immune system parameters as well as in counter-acting protein catabolism in AIDS patients. An initial study of IGF-I treatment in humans with chronic renal failure showed improvement in some functional parameters, although most patients could not tolerate IGF-I side effects (O'Shea et al., 1993). Another potential therapeutic use for IGF-I is in wound healing, although no clinical studies are available as yet. It may be predicted that chronic, systemic use of exogenous IGF-I in conditions where endogenous IGF-I levels are essentially normal will encounter significant difficulties. Treatment with exogenous IGF-I to the point of substantially elevating circulating levels will lead to suppressing GH, glucagon, and insulin secretion and alterations in circulating IGFBP levels, which, taken together, are expected to have unintended effects. For example, suppression of GH leads to significant reduction in levels of the major IGF-binding protein complex which stabilizes IGF-I in the serum and, consequently, accelerates clearance of IGF-I. Furthermore, if treatment is sufficient to produce sustained elevation of circulating IGF-I levels despite accelerated clearance, acromegaloid tissue changes are highly likely. To avoid these unwanted effects, mechanisms must be developed to deliver IGF-I selectively to target tissues so as to avoid the ramifications of elevating systemic levels. E. IGF-II

Very little is known about the physiological role or mechanisms of regulating IGF-II in humans. In rodents, from which most information on IGF-II has been obtained, high levels of IGF-II are expressed by the placenta (Zhou and Bondy, 1992), embryonic liver and mesenchymal tissues, and a variety of other tissues later in development (Lund et al., 1986; Stylianopolou, 1988; Bondy et al., 1990). Expression is dramatically suppressed postnatally, however, except in adventitial

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DEREK LEROITH and CAROLYN BONDY

structures of the CNS (choroid plexus, meninges, vasculature) and blood vessels generally. Deletion of the active paternal murine IGF-II allele results in proportionate dwarfism which affects the placenta in parallel with the embryo (Baker et al., 1993), leading to the view that the dwarfism may result from limited placental development (Zhou and Bondy, 1992). It may be predicted, by analogy with the rodent, that IGF-II has a role in human placental development. But, aside from the fact that IGF-II mRNA is present in the human trophoblast/placenta, little information is now available. The regulation of IGF-II expression in humans is quite different from the rodent, in that circulating and tissue IGF-II levels remain high throughout life and are considerably more abundant than IGF-I. High IGF-II mRNA and peptide levels are present in the liver. Presumably, this is the source of most of the circulating IGF-II, although it is possible that IGF-II synthesized by the vascular endothelium also contributes directly to the circulating pool. Unlike IGF-I, IGF-II levels are not primarily regulated by GH; furthermore, they may be negatively regulated by insulin, because two studies have reported a substantial increase in circulating IGF-II levels in patients with IDDM (Hall et al., 1989; Bach et al., 1994). At this time, there is very little understanding of IGF-II's normal physiological role, nor any clear association of this factor with disease states. F. IGFs and Neoplasia

The presence of IGF's and their potential roles in neoplasia have recently been summarized (LeRoith et al., 1995). Though there is no direct evidence that the IGFs, their binding proteins and receptors induce neoplasia there is a growing body of experimental studies that suggest that this family of growth factors may play a significant role in tumor growth and metastases (Baserga, 1995; LeRoith et al., 1995). Transgenic mice overexpressing IGF-II develop a wide array of neoplasias (Rogler et al., 1994), whereas the effect of the Simian virus-40 large T-antigen on tumor formation is enhanced in transgenic mice with upregulated levels of IGF-II (Christofori et al., 1994). In many tumors, where IGF-II is overexpressed and affects tumor cell proliferation, the effect is blocked by aIR3, an antibody directed against the IGF-I receptor (Gansler et al., 1989). The role of the IGF-I receptor in tumor growth is further supported by experiments using antisense strategies toward the IGF-I receptor that resulted in decreased levels of endogenous IGF-I receptor mRNA and protein and inhibited tumor growth (Kappel et al., 1994; Shapiro et al., 1994). Thus, the IGF system has strong growth-promoting function and may enhance progression once the transforming characteristics have been initiated by the oncogenic process.

IV. CONCLUSION As outlined in this review, the IGF system is essential for normal growth and development and maintaining the integrity of adult tissues. Current investigations are pursuing the regulation of various components of the system mechanisms of

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action at the target tissue. The advent of rhIGF-I will allow investigating possible therapeutic uses in various disease states. REFERENCES Abbott, A. M., Bueno, R., Pedrini, M. T., Murray, J. M., & Smith, R. J. (1992). Insulin-like growth factor I receptor gene structure. J. Biol. Chem. 267, 10759-10763. Aral. T., Aria, A., Busby, Jr., W. J., & Clemmons, D. R. (1994a). Glycosaminoglycans inhibit degradation of insulin-like growth factor-binding protein-5. Endocrinology 135, 2358-2363. Arai, T., Parker, A., Busby, Jr., W. H., & Clemmons, D. R. (1994b). Heparin, heparin sulfate, and dermatan sulfate regulate formation of the insulin-like growth factor I and insulin-like growth factor-binding protein complexes. J. Biol. Chem. 269, 20366-20393. Bach, M. A., Chin, E., & Bondy, C. A. (1994). The effects of recombinant insulin-like growth factor I (IGF-I) on growth hormone, IGF-II, IGF binding protein and blood glucose levels in normal and diabetic adolescents. J. C. E. M. 79, 1040-1046. Baker, J., Liu. J.-R, Robertson, E. J., & Efstratiadis, A. (1993). Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75, 73-82. Baltensperger, K., Kozma, L. M., Chemiak, A. D., Klarlund, J. K., Chawla, A., Banerjee, U., & Czech, M. P. (1993). Binding of the ras activator son of sevenless to insulin receptor substrate-1 signaling complexes. Science 260, 1950-1952. Bar, R. S., Booth, B. A., Bowes, M., & Drake, B. L. (1989). Insulin-like growth factor binding proteins from cultured endothelial cells: Purification, characterization, and intrinsic biologic activities. Endocrinology 125, 1910-1920. Bar. R. S., Bowes, M., Clemmons. D. R., Busby, W. H., Sandra, A., Drake, B. L., & Booth, B. A. (1990). Insulin differentially alters transcapillary movement of intravascular IGFBP-1, IGFBP-2 and endothelial cell IGF binding proteins in rat heart. Endocrinology 127, 497-499. Baserga, R. (1995). The insulin-like growth factor I receptor: A key to tumor growth? Cancer Res. 55, 249-252. Baxter, R. C, & Martin, J. L. (1989). Binding proteins for insulin-like growth factors: Structure, regulation, and function. Prog. Growth Fact. Res. 1, 49-69. Baxter, R. C, & Martin, J. L. (1989). Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: Determination by reconstitution and affinity labeling. Proc. Natl. Acad. Sci. USA 86, 6898-6902. Beitner—Johnson, D., & LeRoith, D. (1995). Insulin-like growth factor I stimulates tyrosine phosphorylation of endogenous c-Crk. J. Biol. Chem, 270, 5187-5190. Bierich, F. R., Moeller, H., Ranke, M. B., & Rosenfeld, R. G. (1984). Pseudopituitary dwarfism due to resistance to somatomedin: Anew syndrome. Eur. J. Pediatr. 142, 186-188. Blum, W. F., Jenne, E. W., Reppin, F., Kietzmann, K., Ranke, M. B., & Bierich, J. R. (1989). Insulin-like growth factor I (IGF-I) binding protein complex is a better mitogen thanfreeIGF-I. Endocrinology 125, 766-772. Blundell, T. L., Bedarkar, S., & Humbel, R. E. (1983). Tertiary structures, receptor binding, and antigenicity of insulin-like growth factors. Fed. Proc. 42, 2592-2597. Bolinder, J., Lindblad, A., Engfeldt, P., & Amer, P. (1987). Studies of acute effects of insulin-like growth factors I and II in human fat cells. J. Clin. Endocrinol. Metab. 65, 732—737. Bondy, C. A., Underwood, L., Clemmons, D. R., Guler, H. R, Bach, M. A., & Skarulls, M. C. (1994). Clinical uses of insulin-like growth factor I. Ann. Int. Med. 120, 593-601. Bondy, C. A., Werner, H., Roberts, C, & LeRoith, D. (1990). Cellular pattern of insulin-like growth factor I and type I IGF receptor gene expression in early organogenesis; comparison with IGF-II gene expression. Mol. Endocrinol. 4, 1386-1398. Bouillon, R. (1991). Growth hormone and bone. Hormone Res. 36,49-55.

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Boulware, S. D., Tamborlane, W. V, Matthews, L. S., & Sherwin, R. S. (1992). Diverse effects of insulin-like growth factor I on glucose, lipid, and amino acid metabolism. Am. J. Physiol. 262, £130-133. Camacho-Hubner, C, Busby, Jr., W. H., McCusker, R. H., Wright, G., & Clemmons, D. R. (1992). Identification of the forms of insulin-like growth factor binding proteins produced by human fibroblasts and the mechanisms that regulate their secretion. J. Biol. Chem. 267, 1194^11956. Cascieri, M. A., Chicchi, G. G., Hayes, N. S., & Strader, C. D. (1986). (Thr-59)-insulin-like growth factor I stimulates 2-deoxyglucose transport in BC3H1 myocytes through the insulin-like growth factor receptor, not the insulin receptor. Biochem. Biophys. Acta. 886,491^99. Cheetham, T. D., Jones, J., Taylor, A. T., Holly, J., Matthews, D. R., & Dunger, D. B. (1993). The effects of IGF-I administration on growth hormone levels and insulin requirements m adolescents with type 1 (insulin-dependent) diabetes mellitus. Diabetologica 36, 678-681. Christofori, G., Naik, R, & Hanahan, D. (1994). A second signal supplied by insulin-like growth factor II in oncogene-induced tumorigenesis. Nature 369, 414-417. Clemmons, D. R., & Underwood, L. E. (1994). Uses of IGF-I in clinical conditions. J. Clin. Endocrinol. Metab. 79, 4-6. Conover, C. A., Lee, R D. K., Kanaley, J. A., Clarkson, J. T., & Jensen, M. D. (1992). Insulin regulation of insulin-like growth factor binding protein I in obese and nonobese humans. J. Clin. Endocrinol. Metab. 74, 1355-1360. Conover, C. A., & Powell, D. A. (1991). Insulin-like growth factor (IGF)-binding protein-3 blocks IGF-I-induced receptor downregulation and cell desensitization in cultured bovine fibroblasts. Endocrinology 129, 710-716. Cooke, D. W., Bankert, L. A., Roberts, Jr., C. T., LeRoith, D., & Casella, S. J. (1991). Analysis of the human type I insulin-like growth factor receptor promoter region. Biochem. Biophys. Res. Commun. 177, 1113-1120. Daughaday, W. H., & Rotwein, P. (1989). Insulin-like growth factor I and II. Peptide messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endo. Rev. 10, 68-91. de Pagter—Holthuizen, P., Jansen, M., van der Kammen, R. A., van Schaik, F. M. A., & Sussenbach, J. S. (1988). Differential expression of the human insulin-like growth factor II gene. Biochem. Biophys. Acta. 950, 282-295. de Pagter—Holthuizen, P., Jansen, M., van Schaik, F. M. A., van der Kammen, R. A., Oosterjk, C , Van der Brande, J. L., & Sussenbach, J. S. (1987). The human insulin-like growth factor II contains two development-specific promoters. FEBS Lett. 214, 259-264. DeChiara, T. M., Robertson, E. J., & Efstratiadis, A. (1991a). Parental imprinting of the mouse insulin-like growth factor II gene. Cell 64, 849-859. DeChiara, T. M., Efstratiadis, A., & Robertson, E. J. (1991b). A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature (London) 345, 78-80. Delaney, A. M., Pash, J. M., & Canalis, E. (1994). Cellular and clinical perspectives on skeletal IGF-I. J. Cell Biochem. 55, 328-333. Ding, H., Kopple, J. D., Cohen, A., & Hirschberg, R. (1993). Recombinant human insulin-like growth factor-I accelerates recovery and reduces catabolism in rats with ischemic acute renal failure. J. Clin. Invest. 91(5), 2281-2287. Dull, T. J., Gray, A., Hayflick, J. S., & Ullrich, A. (1984). Insulin-like growth factor II precursor gene organization in relation to the insulin gene family. Nature (London) 310, 777-781. Dunger, D. B., Cheetham, T. D., & Crowne, E. C. (1995). Insulin-like growth factors (IGFs) and IGF-I treatment in the adolescent with insulin-dependent diabetes mellitus. Metabolism 44(10 Suppl 4), 119-123. Ebina, Y., Ellis, L., Jamagin, K., Edery, M., Graf, L., Clauser, E., Qu, J., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A., & Rutter, W. J. (1985). The human insulin receptor cDNA: The sttucttiral basis for hormone-activated transmembrane signaling. Cell 40, 747-758. Elgin, R. G., Busby, W. H., & Clemmons, D. R. (1987). An insulin-like growth factor binding protein enhances the biologic response to IGF-I. Proc. Natl. Acad. Sci. Acad. Sci. USA 84, 3245-3258.

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GROWTH HORMONE

Gerhard Baumann

I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Abstract Introduction Genomic Organization of GH Genes GH Gene Expression and Its Products Posttranslational Modifications of GH Structural Properties of GH Biological Actions of GH Regulation of GH Secretion Plasma Transport and Metabolic Fate of GH GH Deficiency and Insufficiency States Conditions with GH Excess Bioinactive GH References

ABSTRACT Growth hormone (GH) is the master anabolic hormone that orchestrates postnatal somatic growth. It is produced in the pituitary gland and in the placenta. A number of

Growth Factors and Cytokines in Health and Disease Volume lA, pages 27-42. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0091-4 27

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GH variants are derived from gene duplication, alternative mRNA splicing and posttranslational modifications. GH is present in all vertebrates, but its structure is species specific. GH acts by binding to a specific, ubiquitously expressed receptor, using two distinct binding interfaces on its surface to form a 2:1 receptor—ligand complex. This GH-induced receptor dimerization process is important for transmembrane signaling and biological action. The extracellular domain of the GH receptor circulates in blood as a soluble GH-binding protein (GHBP). The GHBP forms a complex with plasma GH and thereby delays GH clearance and modulates GH interaction with tissue receptors. The secretion of GH from the pituitary is pulsatile, governed by hypothalamic GH-releasing hormone (GHRH) and somatostatin rhythms. There is marked sexual dimorphism of GH secretion. GH exhibits a wide spectrum of metabolic, cell differentiating and proliferative bioactivities, many of which are mediated by insulin-like growth factor I (IGF-I). IGF-I is produced by many tissues in response to GH. GH deficiency results in dwarfism and may be caused by mutations in the GH gene or in genes controlling GH expression, or by processes which destroy GH- or GHRH-producing cells (e.g., pituitary or hypothalamic tumors). GH excess causes gigantism or acromegaly and results from pituitary somatotroph adenomas or, rarely, from tumors overproducing GHRH.

I. INTRODUCTION Growth hormone (GH) or somatotropin is a pituitary hormone largely responsible for postnatal longitudinal grov^th. GH is present in all vertebrates and exhibits a high degree of species specificity. Generally, lower species respond to GH from higher species, but not vice versa. This is particularly the case with primate (including human) GH, which is biologically active in most other species. This property of the GHs has been termed "one-way species specificity". In addition to the growth promoting (somatogenic) activity, primate GHs have lactogenic activity, owing to their ability to interact with prolactin receptors. This activity is not inherent in nonprimate GHs. The growth promoting activity of GH is in large part, but not exclusively, mediated by insulin-like growth factor I (IGF-I), which is produced in response to GH in liver and some peripheral tissues. In addition to its effect on IGF-I generation, GH also has IGF-independent metabolic activities, such as lipolytic, protein anabolic (amino acid transport), insulin-like, and insulin-antagonistic effects. In some instances, it is difficult to differentiate clearly between the direct and the IGF-mediated effects of GH. The absence or dysfunction of pituitary GH leads to dwarfism; GH excess leads to gigantism or, in adults, acromegaly. Among the GHs of various species, the most detailed information is available for human (h)GH. Because of this, this treatise will primarily focus on hGH. Where warranted, major differences between human and animal GH physiology will be pointed out. Normal biology will be discussed first, followed by examples of pathological conditions.

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II. GENOMIC ORGANIZATION OF GH GENES The human genome contains five GH-related genes in a 55-kb cluster on chromosome 17q22-24. These genes are derived from an ancestral GH/PRL gene by duplication 350-400 million years ago (Miller and Eberhardt, 1983). Each contains five exons and four introns and occupies about 2 kb. The GH-N gene (N for "normal") codes for pituitary GH, the GH-V gene (V for "variant") for a variant GH also known as placental GH. Two other genes (PL-A and PL-B) encode placental lactogen (hPL) or chorionic somatomammotropin (hCS) (the same mature protein is encoded by both PL genes), and the fifth gene (PL-L) is probably a pseudogene. The 5' -> 3' order in this gene cluster is GH-N, PL-L, PL-A, GH-V, PL-B. GH-N is expressed in the pituitary gland, whereas the other members of the cluster are expressed in the placenta (Chen et al., 1989; Parks, 1989). Similar gene duplication in rodents has led to two PL genes as well as to other related genes (proliferin, proliferin-related protein, etc.) (Talamantes, 1990).

III. GH GENE EXPRESSION AND ITS PRODUCTS As indicated, the hGH-N gene is expressed primarily in the pituitary gland. Small amounts are also expressed in lymphocytes. In the pituitary, GH expression is under the control of the transcription factor Pit-1, a member of the POU-homeodomain family of transcription factors (Theill and Karin, 1993). The hGH-N gene gives rise to two alternatively spliced transcripts. The principal gene product is a 191 amino acid, single-chain protein with two disulfide bridges. Based on its molecular weight of about 22,000, it is called hGH22K» or 22K for short. This GH represents the full-length coding sequence of the hGH-N gene and accounts for 90-95% of the primary gene product. A shorter hGH variant with an internal deletion of 15 amino acid residues is derived from the hGH-N gene by alternative pre-mRNA splicing, which skips part of the sequence encoded by exon 3 (DeNoto et al., 1981). This so-called hGH20K» or 20K, represents 5—10% of the primary GH gene product in the pituitary (Lewis et al., 1978). [The term "primary gene products" is used here to differentiate these GH forms from the posttranslationally modified variants (see below)]. Similar 20K forms have been shown for murine and bovine GH (Sinha and Gilligan, 1984; Howland et al., 1987; Sinha 1987). The hGH-V gene is expressed in the placenta; its product is also a 191 amino acid polypeptide that differs in 13 positions from hGH-N (Frankenne et al., 1987; Liebhaber et al., 1989; Chen et al., 1989). A 20K-variant of hGH-V mRNA is not produced because of minor differences in the splice-acceptor site (Estes et al., 1990). Instead, another type of alternative splicing gives rise to a product that retains intron D and, in addition, contains a frameshift (Cooke et al, 1988). Its product, called hGH-V2, is a 230 amino acid protein whose carboxy-terminal half completely diverges from GH. hGH-V2 is present in the trophoblast as a membrane-bound, nonsecreted GH form of unknown function (MacLeod et al., 1992).

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Two other alternative hGH gene transcripts have been described, but the corresponding proteins remain to be convincingly demonstrated. One transcript lacks exon 3, predicting a 17.5-kD protein (LeComte et al, 1987). The other predicts a truncated, frameshifted hOH^"^^ (LeComte et al., 1987). It is not clear whether the corresponding proteins are produced, although a 17-kD hOH-like immunoreactivity has been described in humans and mice (Baumann et al., 1985a; Yoyoka and Friesen, 1986; Sinha and Jacobsen, 1988). The nature of this 17-kD material may correspond to hOH-lacking exon 3, but may also represent hGH fragments or unrelated immunoreactive material. The bovine GH gene yields two transcripts, one coding for 191 amino acid bovine GH, and the other for a frameshifted carboxy-terminal derivative with a predicted molecular weight of 27 kD (Hampson and Rottman, 1987). However, the protein corresponding to the latter has yet to be demonstrated.

IV. POSTTRANSLATIONAL MODIFICATIONS OF GH Several posttranslational modifications of GH have been described. hGH22K can be acylated (probably acetylated) at its amino terminus ("fast GH"), or deamidated at Gln^^"^ or Asn^^^ ("acidic forms") (Lewis et al., 1979,1981). A glycosylated 12-kD form of hGH has also been described in pituitary extracts, though the precise nature of this material remains unknown (Diaz et al., 1993). Posttranslational modifications have not yet been described for hGH20K> but they may well exist for that variant also. Placental hGH (hGH-V) exists both in a nonglycosylated and a glycosylated form (Ray et al., 1989; Frankenne et al., 1990). Glycosylation in hGH-V is most likely N-linked at Asn^"^^, which contains the glycosylation consensus sequence Asn-X-Ser—a feature not present in pituitary hGH-N which contains lysine in position 140. In pituitary extracts, a number of other modified GH forms have been reported. Many of these represent chemically modified forms generated in the process of extraction rather than native variants. Among animal GHs, phosphorylated forms have been demonstrated for several species (e.g., rat, sheep, and chicken) (Liberti and Joshi, 1986; Aramburo et al., 1990), but corresponding phosphorylated human GH forms have not been described. In addition to the these modifications listed for monomeric GH, a series of oligomeric GH forms exist. These have previously been denoted as "big" GH forms. In humans, about two thirds of the oligomers are noncovalently associated, whereas the rest are linked by intermolecular disulfide bridges, and in a small minority, by other, unknown covalent bonds (Stolar et al., 1984). Both homo- and hetero-oligomers have been described, giving rise to a large number of potential aggregates. In human plasma, oligomers up to pentameric hGH have been identified (Stolar et al, 1984). hGH2Qj^ is particularly prone to dimerization (Lewis et al, 1978; Stolar et al, 1984). Bovine GH also exists preferentially as a dimer.

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Figure 1, A comparison of the primary structures of the family of GH-like molecules In the human species. The principal polypeptide chain shown represents unmodified hGH22K (also known as pituitary hGH or hCH-N). Amino acid substitutions in hCH-V (placental GH) and placental lactogen (hPL or hCS) are shown next to the residues involved. Those in hGH-V (13 of 191 residues) are indicated by a three letter amino acid code, those in hPL (29 of 191 residues) by a single letter code. The 15 amino acid sequence that is deleted in hGH20K is denoted by the heavy connecting lines (residues 32-46). Asterisks at Gln^ ^'^ and Asn^ ^^ indicate positions that are known to be naturally deamidated (acidic hGH forms); the dot at the amino terminus represents acylated hGH ("fast hGH"). The tree structure at Asn^^^ depicts the glycosylation site in hGH-V. (Adapted from Endocrine Reviews (1991). 12, 424-449.)

V. STRUCTURAL PROPERTIES OF GH High resolution crystal structures of recombinant porcine and human GH have recently been obtained (Abdel—Meguid et al., 1987; DeVos et al., 1992; Ultsch et al., 1994). The main three-dimensional feature is a twisted bundle of four a-helices, arranged in an antiparallel orientation and connected by flexible loops. In addition, there are minihelices v^ithin some of the loops of hGH (Ultsch et al., 1994). For hGH variants other than hGH22K5 detailed structures are not available. Using the conformational information available for hGH22K5 both its antigenic and receptorbinding epitopes have been mapped (Cunningham and Wells, 1991; Jin etal., 1992). hGH contains two binding sites for the GH receptor on opposite aspects of the hGH molecule (site 1 and site 2), which sequentially bind two GH receptors to form a

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ternary receptor-GH-receptor complex (Cunningham et al., 1991; De Vos et al., 1992). Occupation of site 1 by a receptor is required before a second receptor molecule can bind to site 2. This ligand-induced dimerization of the GH receptor is important for receptor signaling and GH action, as evidenced directly in vitro (Fuh et al., 1992) and indirectly in vivo by a naturally occurring mutant receptor that is incapable of stable dimer formation (Duquesnoy et al., 1994). Despite normal hGH binding, this mutant receptor confers a Laron syndrome (GH resistance) phenotype indistinguishable from that of subjects with GH receptor deletions or truncations (Buchanan et al, 1991). The two interfaces between GH and its receptor and the amino acid residues making contact have been mapped in detail (Clackson and Wells, 1995). In addition to human GH, ligand-induced receptor dimerization has also been demonstrated for bovine GH, although in that species some biological activity appears to be retained even in a 1:1 stoichiometric complex composed of the GH receptor and placental lactogen (Staten et al, 1993).

VI. BIOLOGICAL ACTIONS OF GH GH is responsible for postnatal longitudinal growth and maintenance of normal body composition. To that end, it exhibits a protean, seemingly unrelated, array of biological activities. Many of these are mediated by IGF-I, and they all cooperate in promoting accretion of lean body mass. Among the spectrum of GH bioactivity, the most important are protein anabolism, nitrogen and phosphate retention, differentiation of immature chondrocytes and preadipocytes (Isaksson et al., 1987; Morikawa et al., 1982), generation of IGF-I (as well as IGF-binding protein 3 and the acid-labile subunit of the main carrier complex for IGF in the serum), mitogenesis and clonal expansion of chondrocytes and other cells (an IGF effect), lipolysis, trophic effects on pancreatic (3-cells and insulin production, antagonism of insulin action, and sodium retention. The cumulative in vivo effect of these actions is longitudinal bone growth, and a change in body composition, with increased lean body (muscle) mass, decreased adiposity, and accelerated bone turnover. GH has a stimulatory effect on the immune system, which differs markedly among species. In humans, the immunostimulatory effect is relatively minor, whereas in rodents, it is more pronounced. The considerable redundancy of immune functions makes it difficult to discern any immune deficit in human GH deficiency. GH also exhibits a so-called "insulin-like effect", that is, it can acutely increase glucose uptake by cells and mimic other insulin-mediated effects. The physiological importance of this activity in the intact organism is uncertain. Human GH has the additional property of lactogenic activity, due to its ability to bind to prolactin receptors. The interaction of hGH with the prolactin receptor, which is zinc-dependent, has been examined in detail (Cunningham et al., 1990). The binding epitopes for somatogenic and lactogenic binding overlap, but are not identical. It is not clear to what extent hGH contributes to normal lactation as milk production is primarily a function of prolactin.

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The activities of the different variants of GH fall within the bioactivity spectrum described, but some variants exhibit qualitative and/or quantitative differences. hGH20K has decreased insulin-like and slightly diminished anti-insulin properties compared to hGH22K (Kostyo et al, 1985; Culler et al., 1988). It is fully bioactive as a somatogen in the rat in vivo despite diminished affinity for GH receptors (Lewis et al., 1978; Sigel et al., 1981) — an apparent discrepancy attributed to the longer biological half-life of hGH20K (Baumann et al., 1985b). hGH-V (placental GH) is equipotent with pituitary hGH-N (hGH22K:) ^^ growth promotion, but has relatively weak lactogenic activity (Ray et al., 1990; Baumann et al., 1991). In general, the oligomeric GH forms have diminished receptor-binding affinity (Gorden et al., 1973) and probably diminished bioactivity. In some cases, in vivo bioactivity may not directly reflect receptor-binding affinity because of differences in metabolic clearance (see below). The net effect of the large number of GH forms circulating in human blood (monomeric variants, oligomeric forms, etc.) is difficult to estimate, but must represent the sum of a variety of agonistic and partially antagonistic influences at the GH receptor. Oligomers, for example, may be seen as agonists on the one hand, or as antagonists for more potent monomeric hGH forms on the other. The assessment of the net biological activity of circulating GH is rendered even more difficult by the presence of GH binding proteins (see below).

VIL REGULATION OF GH SECRETION GH is synthesized in the somatotropic cells of the pituitary gland. In the case of placental GH, the production site is the syncytiotrophoblast. Pituitary GH secretion is under dual hypothalamic control by GH-releasing hormone (GHRH; stimulatory) and somatostatin (inhibitory). These hypothalamic peptides are delivered to the pituitary via the portal venous system. They regulate GH synthesis and release through interaction with specific receptors on somatotrophs. IGF-I also participates in GH regulation as an inhibitory factor, representing a classical, long, negative feedback loop between peripheral tissues and the hypothalamo-pituitary system. Additionally, GH can directly inhibit its own secretion via a short feedback loop. Pituitary GH secretion is pulsatile, and specific ultradian neuroendocrine rhythms exist in different species. Pulsatility results from acute GHRH bursts on the one hand, and more gradual fluctuations in somatostatin tone on the other. There is marked sexual dimorphism of GH secretory patterns (Jansson et al., 1985). Women tend to have higher GH pulses and a higher interpulse intervals than men (Winer et al., 1990). This is believed to be an estrogen effect. In the rat, males have high pulses alternating with very low baselines, whereas females have lower pulse amplitudes with higher interpulse activity (Jansson et al., 1985). In humans, the majority of GH secretion occurs at night during slow wave sleep. In contrast to pituitary GH, placental GH (hGH-V) secretion is nonpulsatile (Eriksson et al., 1989). The local mediators of its release from the trophoblast are not known. In the second half of pregnancy, there is a progressive increase of

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hGH-V in the maternal circulation, with a concomitant decrease of pituitary GH (Frankenne et al., 1988). Thus, placental GH supplants pituitary GH during gestation, and sustained high serum GH levels (20-30 ng/ml) cause a "pseudoacromegalic state" in the mother during the third trimester. Maternal pituitary GH secretion is inhibited during this period. At the same time, the fetal circulation contains relatively high GH levels derived from the fetal pituitary, although the high fetal GH levels are largely inconsequential because of the relative paucity of GH receptors during fetal life (Daughaday et al., 1987). It should be noted that, in addition to hGH-V, the maternal circulation contains very high levels of hPL. There is no significant transplacental passage of hGH or hPL in either direction. Pituitary GH secretion is developmentally and nutritionally regulated. Secretion is high in late fetal life and during puberty. GH secretion declines as a function of age in adulthood and becomes markedly depressed in older age (Corpas et al., 1993). IGF-I levels roughly parallel the GH trends in puberty and during adult life. In humans and most other mammals, acute fasting or chronic malnutrition results in enhanced GH secretion, and ovemutrition blunts GH secretion (Hartman et al., 1992; Veldhuis et al., 1991). In rodents, the opposite is the case: GH secretion is inhibited by nutritional deprivation (Tannenbaum et al., 1976). In all species examined to date, undernutrition results in a significant decrease in IGF-I production.

Vm. PLASMA TRANSPORT AND METABOLIC FATE OF GH Upon entering the blood stream, secreted GH binds to at least two circulating GH binding proteins (GHBP). The principal GHBP is a circulating fragment of the GH receptor that encompasses its extracellular domain (for a review, see Baumann, G., 1994). The level of GHBP in blood, and, consequently, the bound GH fraction is highly variable among species; sheep and cattle exhibit low GHBP levels and pig and rabbit high GHBP levels. In humans, with an intermediate GHBP level, approximately 45% of hGH22K in plasma is bound to this GHBP (Baumann et al., 1988). Another 5-10% of hGH22K is bound to one or more low-affinity GHBPs, which is not related to the GH receptor (Baumann and Shaw, 1990a). Both pituitary hGH22K and placental hGH-V interact with equal potency with the high-affinity GHBP (Baumann et al., 1991). On the other hand, hGH20K» binds primarily to the low-affinity GHBP because of its relatively low affinity for the GH receptor and the receptor-related GHBP (Baumann and Shaw, 1990b). Nothing is presently known about binding of the other hGH variants to GHBP. Generally, animal GHs bind to their homologous plasma GHBPs, but usually with lower affinity than hGH (Davis etal., 1992). The principal physiological factors regulating plasma GHBP levels are ontogeny (Daughaday et al., 1987) and nutritional state (Hochberg et al., 1992). A number of pathological conditions attended by altered GH action (Laron and pygmy dwarfism, insulin-dependent diabetes, malnutrition and obesity, liver cirrhosis, renal failure

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and hypothyroidism) exhibit altered plasma GHBP levels (for a review, see Baumann, 1994). In most instances, GH-resistant conditions are associated with decreased GHBP levels. Plasma GHBP levels are generally assumed to reflect GH receptor levels in tissues, based on observation of parallel regulation of GHBP and GH receptor in animal models. However, this tenet remains to be rigorously proven. Binding of GH to GHBPs results in a prolongation of the biological half-life of GH (Baumann et al, 1987; Veldhuis et al, 1993). Both metabolic clearance and chemical degradation of GH are inhibited by complex formation with the GHBPs (Baumann et al., 1989), primarily because in contrast to free GH, the GH/GHBP complex is too large to be filtered at the glomerulus. Glomerular filtration and subsequent degradation in the proximal tubule is the principal route of clearance for GH. The GHBPs functionally serve to maintain a circulating GH reservoir and to provide a dynamic equilibrium between free and bound GH as GH levels fluctuate due to pulsatile secretion. Additionally, the high-affinity GHBP modulates GH action by competing with GH receptors for ligand (Mannor et al., 1991). This inhibits GH action as well as GH internalization and degradation within cells. This latter effect contributes to the GHBP-induced delay of the metabolic clearance of GH. The net in vivo effect exerted by the GHBPs is the sum of prolonging GH half-life and inhibiting receptor binding. At near-physiological GHBP levels, the net effect appears to be an enhancement of GH action (Clark et al., 1991). At pathologically high GHBP levels, their inhibitory effect on GH action is probably dominant, as suggested by the short stature in a familial syndrome with a hundredfold elevation in GHBP levels (Rieu et al., 1993). Different species generate GHBP by different mechanisms. Thus, in humans and rabbits GHBP is produced by proteolytic cleavage of the GH receptor (Sotiropoulos et al., 1993). In murine species, GHBP is an alternative splicing product of the GH receptor gene (Baumbach et al., 1989; Smith et al., 1989), with a unique carboxyterminal tail encoded by a separate exon (Zhou et al., 1994; Edens et al., 1994). This exon is spliced out in the GH receptor mRNA. The metabolic clearance of oligomeric GH forms is slower than for monomeric GH, and there are also differences between the monomeric variants (Baumann et al., 1985b, 1986). The question has been raised whether some of the metabolic degradation products of GH may have biological activities of their own (Lewis et al., 1981). This is an intriguing possibility which merits further exploration. GH fragments, such as hGH^^^ and hGH"^^^^^ with glucoregulatory properties have been found to be naturally occurring in pituitary and plasma (Singh et al., 1983; Sinha and Jacobson, 1994).

IX. GH DEFICIENCY AND INSUFFICIENCY STATES GH deficiency can result from mutations in the GH gene (Phillips and Cogan, 1994), the Pit-1 gene (Parks et al., 1993), or, in the mouse, from a mutation in the GHRH receptor gene (Godfrey et al., 1993). It can also be caused by organic

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hypopituitarism (destructive hypothalamic or pituitary tumors, granulomata etc.). However, frequently the cause of isolated (idiopathic) GH is unknown. The phenotype of GH deficiency is one of pituitary dwarfism, which represents severe but proportionate growth retardation. Body composition is altered, with a relative increase in adipose tissue and a decrease in musculature. Serum IGF-I and IGFbinding protein 3 are low in GH deficiency, and IGF-binding protein 2 tends to be elevated. Hypoglycemia is a common feature of GH deficiency, particularly in young children, perhaps because of the absence of GH as a potent anti-insulin factor. GH insufficiency, that is, an apparent limitation of the amount of GH spontaneously secreted ("neurosecretory dysfunction"), has been postulated as a cause of milder short stature (Bercu and Diamond, 1986), but classification is difficult, and the existence of this nosological entity has not been universally accepted because of the difficulty in quantifying GH secretion rates and the wide variation of GH production—^as well as stature—even among normal persons. Mutations in the GH gene have been subclassified into those resulting in no GH-like protein (Type 1 A), which include gene deletions, nonsense mutations, and "early" frameshifts, and those resulting in some abnormal GH-like material (Types IB, II, and III), caused by splice site mutations or downstream missense mutations. Types IB, II, and III are distinguished by their mode of inheritance (autosomal recessive, autosomal dominant and x-linked, respectively). Because GH is a foreign protein for patients with Type 1A GH deficiency, such patients are immunointolerant against GH and respond to exogenous GH with high titer anti-GH antibodies and immunological GH resistance. This is not the case with the other types, who respond well to GH administration. Mutations in the Pit-1 gene are responsible for the dwarfed phenotypes in the Snell and the Jackson dwarf mouse (Li et al., 1990). Pit-1 mutations have also been shown in several humans families (Parks et al., 1993). These mutations result in a combined GH/prolactin/TSH-deficient state. Depending on the type of mutation, TSH expression and resultant hypothyroidism can vary in severity. Apoint mutation in the extracellular domain of GHRH receptor which inactivates GHRH binding, is responsible for the GH deficiency and dwarfed phenotype of the little (lit/lit) mouse (Godfrey et al., 1993). Corresponding mutations in the human GHRH receptor gene have not yet been described. Also, a search for possible mutations in the gene coding for GHRH itself has not revealed any abnormalities in a considerable number of patients with familial GH deficiency (Perez Jurado et al., 1994). Therefore, for the great majority of patients with isolated GH deficiency, the molecular nature of the defect remains unknown. In contrast to lack or dysfunction of pituitary hGH, the lack of placental hGH (hGH-V) does not appear to have any obvious deleterious consequences. Subjects with a partial deletion of the GH gene cluster, which includes the hGH-V and hPL genes, appear normal (Wurzel et al., 1982). Similarly, pregnancy does not seem to be adversely affected by the absence of hGH-V and hPL (Wurzel et al., 1982; Simon etal., 1986).

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X. CONDITIONS WITH GH EXCESS Excess GH production usually results from somatotroph tumors in the pituitary gland. About one third of such tumors have been shown to harbor constitutively activating mutations of the Gg^ subunit of the G protein which normally couples the GHRH receptor to adenylate cyclase (Vallar et al, 1987; Landis et al, 1990). Rarely, GH overproduction results from pituitary somatotroph hyperplasia secondary to eutopic (hypothalamic hamartoma) or ectopic (carcinoids, various cancers in pancreas, lung, ovary etc.) production of GHRH (Faglia et al., 1992). Ectopic production of GH itself in sufficient amounts to produce systemic effects is very rare; only one such case has been reported to result in clinically apparent GH excess (Melmed et al., 1985). GH overproduction results in accelerated growth in children before epiphyseal closure (pituitary gigantism) and in acral and visceral growth in adults (acromegaly). Transgenic animals overexpressing GH show a corresponding giant phenotype (Palmiter et al., 1983; Hammer et al., 1985). Other manifestations of acromegaly include soft tissue swelling, sodium retention and hypertension, insulin resistance with or without hyperglycemia, dental malocclusion due to enlargement of the jaw, and variable degrees of bony overgrowth with attendant arthritic and nerve entrapment syndromes. Biochemical manifestations include increased serum IGF-I, as well as hyperphosphatemia, hyperinsulinemia and dyslipidemia. Local intracranial symptoms/signs (optic, oculomotor, cavernous sinus manifestations) caused by the presence of a pituitary tumor are commonly seen in patients with somatotroph tumors; they are tumor mass effects rather than GH-specific effects.

XL BIOINACTIVE GH The possibility of a structurally abnormal GH with diminished bioactivity, yet retained immunoreactivity, has been contemplated as a possible cause of unexplained short stature for a number of years (Kowarski et al., 1978; Rudman et al., 1981). The converse, namely, an abnormal GH that is bioactive, but not immunoreactive, has also been postulated (Bistritzer et al., 1988). These postulates have been difficult to prove or disprove at the protein level. However, some of the missense mutations in the GH gene that produce altered GH molecules (Phillips and Cogan, 1994) can now be classified as bona fide "bioinactive" GH. At this writing, these GH forms have not yet been expressed in sufficient amounts to permit detailed assessment of their biological activities. It will be interesting to determine the structure and function of these naturally occurring mutant GH molecules.

REFERENCES Abdel-Meguid, S. S., Shieh, H-S., Smith, W. W., Dayringer, H. E., Violand, B. N., & Bentle, L. A. (1987). Three-dimensional structure of a genetically engineered variant of porcine growth hormone. Proc. Natl. Acad. Sci. USA 84, 6434-6437.

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Aramburo, C, Donoghue, D., Montiel, J. L., Berghman, L. R., & Scanes, C. G. (1990). Phosphorylation of chicken growth hormone. Life Sciences 47, 945-952. Baumann, G., Stolar, M. W., & Ambum, K. (1985a). Molecular forms of circulating growth hormone during spontaneous secretory episodes and in the basal state. J. Clin. Endocrinol. Metab. 60, 1216-1220. Baumann, G., Stolar, M. W., & Buchanan, T. A. (1985b). Slow metabolic clearance of the 20,000-dalton variant of human growth hormone: Implications for biological activity. Endocrinology 117, 1309-1313. Baumann, G., Stolar, M. W., & Buchanan, T. A. (1986). The metabolic clearance, distribution, and degradation of dimeric and monomeric growth hormone (GH): Implications for the pattern of circulating GH forms. Endocrinology 119, 1497-1501. Baumann, G., Ambum, K. D., & Buchanan, T. A. (1987). The effect of circulating growth hormonebinding protein on metabolic clearance, distribution and degradation of human growth hormone. J. Clin. Endocrinol. Metab. 64, 657-660. Baumann, G., Ambum, K., & Shaw, M. A. (1988). The circulating growth hormone (GH)-binding protein complex: A major constituent of plasma GH in man. Endocrinology 122, 976-984. Baumann, G., Shaw, M. A., & Buchanan, T. A. (1989). In vivo kinetics of a covalent growth hormone-binding protein complex. Metabolism 38, 330-333. Baumann, G., & Shaw, M. A. (1990a). A second, lower affinity growth hormone-binding protein in human plasma. J. Clin. Endocrinol. Metab. 70, 680-686. Baumann, G. & Shaw, M. A. (1990b). Plasma transport of the 20,000-dalton variant of human growth hormone (20K): Evidence for a 20K-specific binding site. J. Clin. Endocrinol. Metab. 71, 1339-1343. Baumann, G., Davila, N., Shaw, M. A., Jay, R., Liebhaber, S., & Cooke, N. E. (1991). Binding of human growth hormone variant (hGH-V; placental GH) to growth hormone-binding protein in human plasma. J. Clin. Endocrinol. Metab. 73, 1175-1179. Baumann, G. (1994). Growth hormone-binding proteins: State of the art. J. Endocrinol. 141, 1-6. Baumbach, W. R., Homer, D. L., & Logan, J. S. (1989). The growth hormone-binding protein in rat semm is an altematively spliced form of the rat growth hormone receptor. Genes Devel. 3, 119^1205. Bercu, B. B., & Diamond, F. B., Jr. (1986). Growth hormone neurosecretory dysfunction. Clinics Endocrinol. Metab. 15, 537-590. Bistritzer, T., Chalew, S. A., Lovchick, J. C, & Kowarski, A. A. (1988). Growth without growth hormone: The "invisible" GH syndrome. Lancet 1, 321-323. Buchanan, C. R., Maheshwari, H. G., Norman, M. R., Morrell, D. J., & Preece, M. A. (1991). Laron-type dwarfism with apparently normal high affinity growth hormone-binding protein. Clin. Endocrinol. 35, 179-185. Chen, E. Y., Liao, Y. C, Smith, D. H., Barrera-Saldana, H. A., Gelinas, R. F., & Seeburg, R H. (1989). The growth hormone locus: Nucleotide sequence, biology, and evolution. Genomics 4,479-497. Clackson, T., & Wells, J. A. (1995). A hot spot of binding energy in a hormone-receptor interface. Science 267, 383-386. Clark, R. G., Cunningham, B., Moore, J. A., Mulkerrin, M. G., Carlsson, L. M. S., Spencer, S. A., Wood, W. 1., & Cronin, M. J. (1991). Growth hormone-binding protein enhances the growth promoting activity of GH in the rat. Program 73rd Meeting Endocrine Soc, p. 1611. Cooke, N. E., Ray, J., Emery, J. G., & Liebhaber, S. A. (1988). Two distinct species of human growth hormone-variant mRNA in the human placenta predict the expression of novel growth hormone proteins. J. Biol. Chem. 263, 9001-9006. Corpas, E., Harman, M. S., & Blackman, M. (1993). Human growth hormone and human aging. Endocrine Rev. 14, 20-39.

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Culler, F. C , Kaufmann, S., Frigeri, L. G., & Jones, K. L. (1988). Comparison of acute metabolic effects of 22,000-dalton and 20,000-dalton growth hormone in human subjects. Horm. Metab. Res. 20, 107-109. Cunningham, B. C , & Wells, J. A. (1989). High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244, 1081-1086. Cunningham, B. C , Bass, S., Fuh, G., & Wells, J. A. (1990). Zinc mediation of the binding of human growth hormone to the human prolactin receptor. Science 250, 1709-1712. Cunningham, B. C , Ultsch, M., DeVos, A. M., Mulkerrin, M. G., Clauser, K. R., & Wells, J. A. (1991). Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science 254, 821-825. Daughaday, W. H., Trivedi, B., & Andrews, B. A. (1987). The ontogeny of serum GH-binding protein in man: A possible indicator of hepatic GH receptor development. J. Clin. Endocrinol. Metab. 65, 1072-1074. Davis, S. L., Graf, M., Morrison, C. A., Hall, T. R., & Swift, R J. (1992). Identification and partial purification of serum growth hormone-binding protein in domestic animal species. J. Anim. Sci. 70, 773-780. DeNoto, F. M., Moore, D. D., & Goodman, H. M. (1981). Human growth hormone DNA sequence and mRNA structure: Possible alternative splicing. Nucl. Acids Res. 9, 3719-3730. DeVos, A. M., Ultsch, M., & Kossiakoff, A. A. (1992). Human growth hormone and extracellular domain of its receptor: Crystal structure of the complex. Science 255, 306-312. Diaz, M. J., Dominguez, F., Haro, L. S., Ling, N., & Devesa, J. (1993). A 12-kilodalton N-glycosylated growth hormone-related peptide is present in human pituitary extracts. J. Clin. Endocrinol. Metab. 77, 134-138. Duquesnoy, R, Sobrier, M. L., Duriez, B., Dastot, R, Buchanan, C. R., Savage, M. O., Preece, M. A., Craescu, C. T., Blouquit, Y., Goossens, M., & Amselem, S. (1994). A single amino acid substitution in the exoplasmic domain of the human growth hormone (GH) receptor confers familial GH resistance (Laron syndrome), with positive GH-binding activity by abolishing receptor homodimerization. EMBO J. 13, 1386-1395. Edens, A., Southard, J. N., & Talamantes, F. (1994). Mouse growth hormone-binding protein and growth hormone receptor transcripts are produced fi'om a single gene by alternative splicing. Endocrinology 135, 2802-2805. Eriksson, L., Frankenne, F., Eden, S., Hennen, G., & Von Schoultz, B. (1989). Growth hormone 24-h serum profiles during pregnancy — lack of pulsatility for the secretion of the placental variant. Brit. J. Obstet. Gynaecol. 96, 949-953. Estes, P. A., Cooke, N. E., & Liebhaber, S. A. (1990). A difference in the splicing patterns of the closely related normal and variant human growth hormone gene transcripts is determined by a minimal sequence divergence between two potential splice-acceptor sites. J. Biol. Chem. 265, 1986319870. Faglia, G., Arosio, M., & Bazzoni, N. (1992). Ectopic acromegaly. Endorinol. Metab. Clinics N. Amer. 21,575-595. Frankenne, F., Rentier-Delrue, R, Scippo, M. L., Martial, J., & Hennen, G. (1987). Expression of the growth hormone variant gene in human placenta. J. Clin. Endocrinol. Metab. 64, 635-637. Frankenne, R, Closset, J., Gomez, R, Scippo, M. L., & Hennen, G. (1988). The physiology of growth hormones (GHs) in pregnant women and partial characterization of the placental GH variant. J. Clin. Endocrinol. Metab. 66, 1171-1180. Frankenne, R, Scippo, M-L., Van Beeumen, J., Igout, A., & Hennen, G. (1990). Identification of placental human growth hormone as the growth hormone-V gene expression product. J. Clin. Endocrinol. Metab. 71, 15-18. Fuh, G., Cunningham, B. C, Fukunaga, R., Nagata, S., Goedell, D. V, & Wells, J. A. (1992). Rational design of potent antagonists to the human growth hormone receptor. Science 256, 1677-1680.

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Godfrey, P., Rahal, J. O., Beamer, W. G., Copeland, N. G., Jenkins, N. A., & Mayo, K. E. (1993). GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function. Nature Genetics 4, 227-232. Gorden, P., Lesniak, M. A., Hendricks, C. M., & Roth, J. (1973). "Big" growth hormone components from human plasma: Decreased reactivity demonstrated by radioreceptor assay. Science 182, 829-831. Hammer, R. E., Brinster, R. L., Rosenfeld, M. G., Evans, R. M., & Mayo, K. E. (1985). Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth. Nature 315,413-416. Hampson, R. K., & Rottman, F. M. (1987). Alternative processing of bovine growth hormone mRNA: Nonsplicing of the final intron predicts a higher molecular weight variant of bovine growth hormone. Proc. Natl. Acad. Sci. USA 84,2673-2677. Hartmann, M. L., Veldhuis, J. D., Johnson, M. L., Lee M. M., Alberti, K. G. M. M., Samojlik, E., & Thomer, M. O. (1992). Augmented growth hormone (GH) secretory burst frequency and amplitude mediate enhanced GH secretion during a two day fast in normal men. J. Clin. Endocrinol. Metab. 74, 757-765. Hochberg, Z., Hertz, P, Colin, V., Ish-Shalom, S., Yeshurun, D., Youdim, M. B. H., & Amit, T. (1992). The distal axis of growth hormone (GH) in nutritional disorders: GH-binding protein, insulin-like growth factor-I (IGF-I), and IGF-I receptors in obesity and anorexia nervosa. Metabolism 41, 106-112. Howland, D. S., Farrington, M. A., Taylor, W. D., & Hymer, W. C. (1987). Alternative splicing model for the synthesis and secretion of the 20-kilodalton form of rat growth hormone. Biochem. Biophys. Res. Commun. 147, 650-657. Isaksson, O. G. P., Lindahl, A., Nilsson, A., & Isgaard, J. (1987). Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocrine Rev. 8,426-438. Jansson, J. O., Eden, S., & Isaksson, O. (1985). Sexual dimorphism of growth hormone secretion. Endocrine Rev. 6, 128-150. Jin, L., Fendly, B. M., & Wells, J. A. (1992). High resolution functional analysis of antibody—antigen interactions. J. Mol. Biol. 226, 851-865. Kostyo, J. L., Cameron, C. M., Olson, K. C, Jones, A. J. S., & Pai, R-C. (1985). Biosynthetic 20-kilodalton methionyl-human growth hormone has diabetogenic and insulin-like activities. Proc. Natl. Acad. Sci. USA 82,4250-4253. Kowarski, A. A., Schneider, J., Ben-Galin, E., Weldon, V. V., & Daughaday, W. H. (1978). Growth failure with normal serum RIA-GH and low somatomedin activity: Somatomedin restoration and growth acceleration after exogenous GH. J. Clin. Endocrinol. Metab. 47, 461-464. Landis, C. A., Harsh, G., Lyons, J., Davis, R. L., McCormick, F., & Bourne, H. R. (1990). Clinical characteristics of acromegalic patients whose pituitary tumors contain mutant Gs protein. J. Clin. Endocrinol. Metab. 71, 1416-1420. Lecomte, C. M., Renard, A., & Martial, J. A. (1987). Anew natural hGH variant—17.5 kd—produced by alternative splicing. An additional consensus sequence which might play a role in branchpoint selection. Nucl. Acids Res. 15, 6331-6348. Lewis, U. J., Dunn, J. T., Bonewald, L. F., Seavey, B. K., & VanderLaan, W. P (1978). A naturally occurring variant of human growth hormone. J. Biol. Chem. 253, 2679-2687. Lewis, U. J., Singh, R. N. R, Bonewald, L. F., Lewis, L. J., & VanderLaan, W. P (1979). Human growth hormone: Additional members of the complex. Endocrinology 104, 1256-1265. Lewis, U. J., Singh, R. N. P., Bonewald, L. F., & Seavey, B. K. (1981). Altered proteolytic cleavage of human growth hormone as a result of deamidation. J. Biol. Chem. 256, 11645-11650. Li, S., Crenshaw, E. B., Ill, Rawson, E. J., Simmons, D. M., Swanson, L. W., & Rosenfeld, M. G. (1990). Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene Pit-1. Nature 347, 528-533.

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Liberti, J. P., & Joshi, G. S. (1986). Synthesis and secretion of phosphorylated growth hormone by rat pituitary glands in v/7ro. Biochem. Biophys. Res. Commun. 137, 806-812. Liebhaber, S. A., Urbanek, M., Ray, J., Tuan, R. S., & Cooke, N. E. (1989). Characterization and histological localization of human growth hormone variant gene expression in the placenta. J. Clin. Invest. 83, 1985-1991. MacLeod J. N., Lee, A. K., Liebhaber, S. A., & Cooke, N. E. (1992). Developmental control and alternative splicing of the placentally expressed transcripts from the human growth hormone gene cluster. J Biol. Chem. 267, 14219-14226. Mannor, D. A., Winer, L. M., Shaw, M. A., & Baumann, G. (1991). Plasma growth hormone-binding proteins: Effect on growth hormone binding to receptors and on growth hormone action. J. Clin. Endocrinol. Metab. 73, 30-34. Melmed, S., Ezrin, C , Kovacs, K., Goodman, R. S., & Frohman, L. A. (1985). Acromegaly due to secretion of growth hormone by an ectopic pancreatic islet-cell tumor. N. Engl. J. Med. 312,9-17. Miller, W. L., & Eberhardt, N. L. (1983). Structure and evolution of the growth hormone gene family. Endocr. Rev. 4, 97-130. Morikawa, M., Nixon, T., & Green, H. (1982). Growth hormone and the adipose conversion of 3T3 cells. Cell 29, 783-789. Palmiter, R. D., Norstedt, G., Gelinas, R. E., Hammer, R. E., & Brinster, R. L. (1983). Metallothioneinhuman GH fusion genes stimulate growth of mice. Science 222, 809-814. Parks, J. S. (1989). Molecular biology ofgrowth hormone. Acta Paediatr. Scand.(Suppl.)349,127-135. Parks, J. S., Abdul-Latif, H., Kinoshita, E., Meacham, L. R., Pfaffle, R. W., & Brown, M. R. (1993). Genetics ofgrowth hormone gene expression. Horm. Res. 40, 54-61. Perez Jurado, L. A., Phillips, J. A. Ill, & Francke, U. (1994). Exclusion ofgrowth hormone (GH)-releasing hormone gene mutations in familial isolated GH deficiency by linkage and single strand conformation analysis. J. Clin. Endocrinol. Metab. 78, 622-628. Phillips, J. A. Ill, & Cogan, J. D. (1994). Molecular basis of familial human growth hormone deficiency. J. Clin. Endocrinol. Metab. 78, 11-15. Ray, J., Jones, B. K., Liebhaber, S. A., & Cooke, N. E. (1989). Glycosylated human growth hormone variant. Endocrinology 125, 566-568. Ray, J., Okamura, H., Kelly, P. A., Cooke, N. E., & Liebhaber, S. A. (1990). Human growth hormone variant demonstrates a receptor-binding profile distinct from that of normal pituitary growth hormone. J. Biol. Chem. 265, 7939-7944. Rieu, M., Le Bouc, Y, Villares, S. M., & Postel-Vinay, M-C. (1993). Familial short stature with very high levels ofgrowth hormone binding protein. J. Clin. Endocrinol. Metab. 76, 857-860. Rudman, D., Kutner, M. H., Blackston, R. D., Cushman, R. A., Bain, R. R, & Patterson, J. H. (1981). Children with normal-variant short stature: Treatment with human growth hormone for six months. N. Engl. J. Med. 305, 123-131. Sigel, M. B., Thorpe, N. A., Kobrin, M. S., Lewis, U. J., & VanderLaan, W. P (1981). Binding characteristics of a biologically active variant of human growth hormone (20K) to growth hormone and lactogen receptors. Endocrinology 108, 1600-1603. Simon, P., Decoster, C , Brocas, H., Schwers, J., & Vassart, G. (1986). Absence of human chorionic somatomammotropin during pregnancy associated with two types of gene deletion. Hum. Genet. 74, 235-238. Singh, R. N. P., Seavey, B. K., Lewis, L. J., & Lewis, U. J. (1983). Human growth hormone peptide 1^3: Isolation from pituitary glands. J. Protein Chem. 2, 425-436. Sinha, Y. N., & Gilligan, T. A. (1984). A"20K" form ofgrowth hormone in the murine pituitary gland. Proc. Soc. Exp. Biol. Med. 177,465-^74. Sinha, Y N. (1987). Evidence for a "20K" form ofgrowth hormone in the bovine pituitary gland. Clin. Res. 35, 183A.

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Sinha, Y. N., & Jacobsen, B. P. (1988). Three growth hormone- and two prolactin-related novel peptides of Mr 13,000-18,000 identified in the anterior pituitary. Biochem. Biophys. Res. Commun. 156, 171-179. Sinha, Y. N., & Jacobsen, B. P. (1994). Human growth hormone (hGH)-(44-191), a reportedly diabetogenic fragment of hGH, circulates in human blood: Measurement by radioimmunoassay. J. Clin. Endocrinol. Metab. 78, 1411-1418. Smith, W. C, Kuniyoshi, J., & Talamantes, F. (1989). Mouse serum growth hormone (GH)-binding protein has GH receptor extracellular and substituted transmembrane domains. Mol. Endocrinol. 3, 984-990. Sotiropoulos, A., Goujon, L., Simonin, G., Kelly, P., Postel-Vinay, M-C., & Finidori, J. (1993). Evidence for generation of the growth hormone-binding protein through proteolysis of the growth hormone membrane receptor. Endocrinology 132, 1863-1865. Staten, N. R., Byatt, J. C, & Krivi, G. G. (1993). Ligand-specific dimerization of the extracellular domain of the bovine growth hormone receptor. J. Biol. Chem. 268, 18467—18473. Stolar, M. W., Ambum, K., & Baumann, G. (1984). Plasma "big" and "big-big" growth hormone (GH) in man: An oligomeric series composed of structurally diverse GH monomers. J. Clin. Endocrinol. Metab. 59,212-218. Talamantes, F. (1990). Structure and regulation of secretion of mouse placental lactogens. Prog. Clin. Biol. Res. 342, 81-85. Tannenbaum, G. S., Martin, J. B., «fe Colle, E. (1976). Ultradian growth hormone rhythm in the rat: Effects of feeding, hyperglycemia and insulin-induced hyperglycemia. Endocrinology 99, 720727. Theill, L. E., & Karin, M. (1993). Transcriptional control of GH expression and anterior pituitary development. Endocrine Rev. 14, 670-689. Ultsch, M. H., Somers, W., KossiakofF, A. A., & DeVos, A. M. (1994). The crystal structure of affinity-matured human growth hormone at 2 A resolution. J. Mol. Biol. 236, 286-299. Vallar, L., Spada, A., & Giannattasio, G. (1987). Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas. Nature 330, 566-568. Veldhuis, J. D., Iranmesh, A., Ho, K. K. Y, Waters, M. J., Johnson, M. L. & Lizzaralde, G. (1991). Dual defects in pulsatile growth hormone secretion and clearance subserve the hyposomatotropism of obesity in man. J. Clin. Endocrinol. Metab. 72, 51—59. Veldhuis, J., Johnson, M. L., Faunt, L. M., Mercado, M., & Baumann, G. (1993). Influence of the high-affinity growth hormone (GH)-binding protein on plasma profiles offi-eeand bound GH and on the apparent half-life of GH. J. Clin. Invest. 91, 629-641. Winer L. M., Shaw, M. A., & Baumann, G. (1990). Basal plasma growth hormone levels in man: New evidence for rhythmicity of growth hormone secretion. J. Clin. Endocrinol. Metab. 70, 16781686. Wurzel, J., Parks, J. S., Herd, J. E., & Nielsen, P. V. (1982). A gene deletion is responsible for absence of human chorionic somatomammotropin. DNA 1,251-257. Yoyoka, S., & Friesen, H. G. (1986). Human growth hormone (GH)-releasing factor stimulates and somatostatin inhibits the release of rat GH variants. Endocrinology 119,2097-2105. Zhou, Y, He, L., & Kopchick, J. J. (1994). An exon encoding the mouse growth hormone-binding protein (mGHBP) carboxy terminus is located between exon 7 and 8 of the mouse growth hormone receptor gene. Receptor 4, 223—227.

GROWTH HORMONE RECEPTOR

Lisa S. Smit and Christin Carter-Su

Abstract I. Introduction II. Growth Hormone Receptor (GHR) Gene A. Cloning ofthe GHR Gene B. The Cytokine/Hematopoietin Receptor Superfamily C. Alternative SpHcing of GHR mRNA III. Localization ofGHR and Regulation of GHR Expression A. Localization of GHR B. Developmental Regulation of GHR C. DifferentialExpressionof GHR between Sexes D. GH Regulation of GHR Expression E. Regulationof GHR Expression by Insulin and Glucocorticoids IV. GHR and Growth A. GHR and Conditions Associated with Growth Retardation B. GHR and Dwarfism V. GHR Signal Transduction A. GH Signal Transduction Via GHR B. GH Binding and Receptor Dimerization C. GHR Glycosylation

Growth Factors and Cytokines in Health and Disease Volume lA, pages 43-84. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0091-4 43

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D. GHR Internalization E. GHR Tyrosyl Phosphorylation F. GH Activation of JAK2 Tyrosine Kinase G. GH Activation of MAP Kinases H. GH Induction of IGF-1 Synthesis and Secretion I. GH Induction of c-yc>5 and Other Genes J. GH Activation of Stats 1,3, and 5 K. GH Utilization of Protein Kinase C and G Proteins L. GH Stimulation of IRS-1 and IRS-2 Tyrosyl Phosphorylation M. GH and a Calcium Response VI. Conclusions References

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ABSTRACT The ability of growth hormone (GH) to stimulate body growth and regulate body metabolism results from GH binding to its cell surface receptor (GHR) in a variety of ceU types. This review provides an overview of what is currently known about the GHR, including the cloning of the cDNA encoding GHR, identification of GHR as a member of the cytokine/hematopoietin receptor super-family, expression and regulation of GHR mRNA and protein, signaling pathways initiated by GH binding to GHR, and regions of GHR required for signal transduction. Signaling molecules, that have been implicated in GH action and are discussed in this review, include the GHRassociated JAK2 kinase, the SH2-containing adaptor molecules SHC and Grb2, the latent transcription factors, Stats 1,3, and 5, the mitogen-activated protein kinases designated extracellular signal regulated kinases ERKs 1 and 2, the 90-kDa S6 kinase, the insulin receptor substrates (IRS) 1 and 2, phosphatidyl-inositol-3 (PI-3) kinase, diacylglycerol, protein kinase C, and calcium. These recent studies on GH receptors and actions have given us a new view of the way GH acts in the cell and have begun to provide insight into how GH elicits it diverse effects on body growth and metabolism via binding to its receptor.

1. INTRODUCTION The ability of growth hormone (GH) to promote somatic growth and produce both insulin-like and insulin-antagonistic metabolic effects has long been established (reviewed in previous chapter and Davidson, 1987; Isaksson et al., 1985). The inability to demonstrate direct effects of GH in vitro initially led to the somatomedin hypothesis which proposed that GH acts upon the liver to induce synthesis and secretion of insulin-like growth factor 1 (IGF-1), which in turn is responsible for mediating the effects of GH. However, the identification of GHR in an increasing variety of tissues, the demonstration of the ability of GH to promote longitudinal bone growth (Isaksson et al, 1982), cell proliferation in chondrocytes (Madsen et

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al., 1983) and other cell types (Mercola et al., 1981), differentiation of mouse 3T3-F442Apreadipocytes to adipocytes (Morikawa et al, 1982; Nixon and Green, 1984a), lOTl/2 myoblasts to multinucleated muscle cells (Nixon and Green, 1984b) and prechondrocytes to chondrocytes (Lindahl et al, 1987), lipolytic activity (Goodman, 1984), and insulin-like effects on tissues from hypophysectomized rats (Kostyo and Nutting, 1973) have suggested that GH acts directly on many tissues by binding to GHR. Until recently, knowledge of the cellular effects following GH-GHR interaction has been limited. The cloning of the cDNA encoding GHR (Leung et al., 1987), the classification of GHR as a member of the cytokine/hematopoietin receptor super family (Bazan, 1990), the crystallization of GH-GH binding protein (GHBP, the extracellular domain of GHR) structure (Cunningham et al., 1991; deVos et al., 1992) and the recent identification of JAK2 as a GHR-associated tyrosine kinase activated in response to GH (Argetsinger et al., 1993) has significantly increased our understanding of the signaling mechanisms employed by GH and the role of GHR in transducing these signals. GH/GHR signaling pathways include a variety of molecules, including MAP kinases (Campbell et al., 1992; Winston and Bertics, 1992), the latent transcription factors known as Stats (signal transducers and activators of transcription) (Gronowski and Rotwein, 1994; Meyer et al., 1994; Campbell et al., 1995; Gronowski et al., 1995), the insulin receptor substrates-1 and 2 (IRS-1 and IRS-2) (Souza et al., 1994; Argetsinger et al., 1995a; Argetsinger et al., 1995b) and protein kinase C (PKC) (Smal and DeMeyts, 1987). GH has also been shown to induce transcription of a number of genes including those encoding IGF-1, c-Fos, c-Jun, c-Myc and the serine protease inhibitor (Spi) 2.1 (Murphy et al., 1987; Gurland et al., 1990). Based on mutational studies of GHR and homology to other cytokine receptors, functionally important regions of GHR have been defined, including the intracellular, membrane proximal, proline rich domain (VanderKuur et al., 1994). While the exact details of all of these GH/GHR signaling pathways are not yet known, the studies summarized in this chapter have served to advance our knowledge of the signaling cascades initiated by GH binding to its receptor.

IL GROWTH HORMONE RECEPTOR (GHR) GENE A. Cloning of the GHR Gene GHR was first cloned from a rabbit liver cDNA library (Leung et al., 1987) (Figure 1). Partial amino acid sequence was determined from GHR purified from rabbit liver membranes. An oligonucleotide, based on the amino acid sequence, was then used to screen a rabbit liver cDNA library. The fiill-length GHR cDNA was found to encode a 638 amino acid protein, including a 20 amino acid membrane signal sequence and recognizable extracellular, transmembrane and cytoplasmic domains (Figure 2). The mature form of the receptor is expected to be 620 amino acids, with a predicted molecular weight of 70 kDa. The extracellular domain

46

LISA S. SMIT and CHRISTIN CARTER-SU

human MDLWQLLLTLALAGSSDAFSGSEATAAILSRAPWSLQSVNPGLKTNSSKEP] rabbi t MDLWQLLLTVALAGSSDAFSGSEATPATLGRASESVQRVHPGLGTNSSGKPKF' porcine MDLWQLLLTLAVAGSSDAFSGSEATPAVLVRASQSLQRVHPGLETNSSGKPl bovine MDLWQLLLTLAVAGSSDAFSGSEATPAFLVRASQSLQILYPVLETNSSGNPKF' ovine hlDLWQLLLTLAVAGSSDAFSGSEATPAFFVRASQSLQILYPGLETNSSGNLKF' mouse MDLCQVTLTLALAVTSSTFSGSEATPATLGKASPVLQRINPSLGTSSSGKPRF' rat MDLWRVFLTLALAVSSDMFPGSGATPATLGKASPVLQRINPSLRESSSGKPRF' chicken MDLRHLLFTLALVCANDSLSASDD LLQW PQI;

ISPERETFSC ■IWTDEVHHGTKNLG ISPELETFsb iWTDGVHHGLKSPG ISPELETFSC ^IWTDGVRHGLQSPG ISPELETFSC WTDGANHSLQSPG ISPELETFSC ^WTDGANHSLQSPG ISPELETFSC /WTEGDNPDLKTPG ISPELETFa:SfWTEGDDHNLKVPG ISPELETFSC IfWTDG. . .KVTTSG

human rabbit porcine bovine ovine mouse rat chicken

PIQLFYTRRNTQ .... EWTQEWKE&»DYVSAGENS6/FNSSFTSIWi; SVQLFYIRRNTQ EWTQEWKadPDYVSAGENSCK'FNSSYTSIWi: SIQLFYIRRSTQ EWTQEWKEEpDYVSAGENSCKTNSSYTSIWIF SVQMFYIRRDIQ. . EW KadpDYVSAGENSOlfFNSSYTSVWTF SVQMFYIRRDIQ EW KBqPDYVSAGENSnYFNSSYTSVWT] SIQLYYAKRESQRQAARIAHEWTQEWKBCpDYVSAGKNSafFNSSYTSIWi: SIQLYYARR lAHEVH'PEWKBdpDYVSAGANScfcFNSSYTSIWi: TIQLLYMKRSDE DWKIOPDYITAGENSCpFNTSYTSIWi:

human rabbit porcine bovine ovine mouse rat chicken

lALNV^LLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWKMKDPILTTSVPVYSLKVDKEYEVRVRS IGLNWTLL^A;SLTGIHADIQVRWEPPPNADVQKGWIVLEYELQYKEVNETQWKMMDPVLSTSVPVYSLRLDKEYEVRVRS IGLNWTLUJISLTGIHADIQVRWEPPPNADVQKGWIVLEYELQYKE\mETQWKMMDPVLSTSVPVYSIJ^ VGLNWLLNISLTEIHADILVKWEPPPNTDVKMGWIILEYELHYKELNETQWKMMDPLMVTSVPMYSLRI^ VGLNWTLLNISLTEIHADILVKWEPPPNTDVKMGWIILEYELHYKELNETQWKMMDPLLVTSVPMYSLRLDKEYEVRVRT IGLNV^LLNISLTGIRGDIQVSWQPPPNADVLKGWIILEYEIQYKEVNESKWKVMGPIWLTYCPVYSLRMDKEHEVRVRS IGLNWLLNISLPGIRGDIQVSWQPPPSADVLKGWIILEYEIQYKEVNETKWKTMSPIWSTSVPLYSLRLDKEHEVRVRS VHUJViH'LLNTSQTGIHGDIQVRWDPPPTADVQKGWITLEYEI^YKEVNETKWKELEPRLSTVVPLYSLKMGRDYEIRVRS WSXWS-lUca Trtaummahraxim Donnin Boa^; LYVTLPQMSQ.F.. TCEEDFYrpWLLIIIFGIFGLTVMLFVFLJtSKQQRIKMqiLPPVPVP|KIKGID LYVTLPQMSP.F.. TCEEDFPFPWFLIIIFGIFGLTVMLFVFIFJSKQQRIKMLIILPPVPVPKIKGID LYVTLPQMSP.F., .ACEEDFRFPWFLIIIFGIFGLTVILFLLIFJSKQQRIKMlllLPPVPVPKIKGID LLITFPQMNP.S., . ACEEDFQFPWFLI IMFGIL/3IAVTLFLLireKQQRIKMlllLPPVPVPKIKGID LLITFPQMNP.S., . ACEEDFOT-PWFLII IFGILGLTVTLFLLIFJSKQQRIKMlilLPPVPVPKIKGID LRVIFPQTNI . LEACEEDI^-pWFLIIIFGIFGVAVMLFVVIFtSKQQRIKMlilLPPVPVHKIKGID LRVTFPQMDT. LAACEEDFRFPWFLIIIFGIFGVAVMLFVVireKQQRIKMl|rLPPVPVPKIKGID EILYVSFTQAGIEFVHCAEEIEFPWFLVWFGVCGLAVTAILILLBKOPRLKMIgFPPVPVPiKIKGID Y Y Box 2 PDLLKEGKLEEVNTILAIHDgVtKPEFHSDDSWVEFIELDIDEPD. EKFTEESDTDRLLSSDHEKSHSNLGVKDGDSGRTS PDLLKEGKLEEVNTILAIQDgykpEFtStoDSWVEFIELDIDDPD. EOTEGSDTDRLLSNSHQKSLSVLAAKDDDSGRTS PDLLKEGKLEEVOTILAIHDNYtKHEFWBDDSWVEFIELDIDDPD. EKJTEGSDTDRLLNNDHEKSLTILGAKEDDSGRTS PDLLKEGKLEEVNTILAIHDNYpOffiFjyNDDSWVEFIELDI . EKFTEGSDTDRLLSNDHEKSLNIFGAKDDDSGRTS PDLLKEGKLEEVNTILAIHDNYpffiFWNDDSV^FIELDIDDPD. EKTREGSDTDRLLSNDHEKSLSIFGAKDDDSGRTS PDLLKEGKLEEVNTILGIHDNV^PDF|^DSWEFIELDIDEADVDEK|^^ PDLLKEGKLEEVNTILGIHDNYKPDFtiTroDSVr^ .EKJEEESDTDRLLSDDQEKSAGILGAKDDDSGRTS PDLLKKGKLDEVNSILASHDNaKTQlJaJDDLWVEFIELDIDDSD. . EK^VSDTDRLLSDDHLKSHSCLGAKDDDSGRAS

human rabbi t porcine bovine ovine mouse rat Chicken

IKLTSNGGTVDEKCFSVDEIVQPDPP IKLTNNGGMVDQKCFSVEEIVQPDPP IKLTSNGGTVDQKEFSVEEIVQPDPP IKLTSNGGIVDHKCFSVEDIVQPDPP IKLTSNGGIVDHKppSVEDIVQPDPP IKLTTNGDLLDE#TVDEIVQPDPP lEVFDEKCFSVDEIVLPDPP

80 80 80 80 80 80 80 55 152 152 152 148 148 160 152 123 232 232 232 228 228 240 232 203 310 310 310 306 306 319 311 283 388 388 388 384 384 399 389 361

CCEPDILETDFNANDIKEGTSEVAQPQRLKG . EADLLCLDQKNQNNSPYHDACPATQQPSVIQAEKNK . PQPLPTEGAES

466

466 466 462 462 477 466 441

human rabbit porcine bovine ovine mouse rat chicken

:PDILENDFNASDGCDGNSEVAQPQRLKG . EADLLCLDQKNQNNSPYHDVSPAAQQPEWLAEEDK . PRPLLTGEIES :PDILETDFNANDVCDGTAEVAQPQRLKG . EADLLCLDQKNQNNSPSNDAAPATQQPSVILAEENK . PRPLIISGTDS iPDILEADFHVSDMCDGTSEVAQPQRLKG. EADISCLDQKNQNNSPSNDAAPANQQPSVIHVEENK . PRPLLIGGTES :PDILETDFHVSDMCDGTSEVAQPQRLKG . EADILCLDQKNQNNSPSNDAAPASQQPSVILVEENK . PRPLPIGGTES PDILDTDFHTSDMCDGTLKFAQSQKLNM. EADLLCLDQKNLKNLPYDASLGSLHPSITQTVEENK. PQPLLSSETEA ^ _ PDILDTDFHTSDMCDGTSEFAQPQKLKA . EADLLCLDQKNLKNSPYDASLGSLHPSITLTMED. K. PQPLLGSETES (:JilEPDIPETDFSASDTCDAISDIIX5FKKVTEKEEDLLCLHRKDDVEALQSLA^^I^)TQQPHTSTQSESRESWPPFADS^^ Y Y THQAAHIQLSNPSSLSNIDFJV]AQVSDITPAGSVVLSPGQKNKAGMSQCDMHPEMVSLCQENFLMDNAfi^ TLQAA?SQLSNPNSIJ^IDFmAQVSDITPAGSVVLSPGQKNKAGNSQCDAHPEVVSlX:QTNFIMDNAf^CEADAKKCIAV THQTAHTQLSNPSSIJ^IDFp^QVSDITPAGSVVLSPGQKNKAGISQCDMHLEVVSPCPANFIlClNAf^ TKQAVHHQLSNPSSIANIDFT^QVSDITPAGNVVLSPGQKNKTGNPQCDTHPEVWSCQANFIVD THQAVHTQLSNPSSIJ^IDFTvkQVSDITPAGNVVLSPGQKNKTGNPQCDTHPEVVTPSQADFrTDSAn^ THQLASTPMSNPTSLANIDFjYkQVSDITPAGGDVLSPGQKIKAGIAQGNTQREVATPCQE^^fSMNSAp^CESDAKKCI THQLPSTPMSSPVSIANIDFJmQVSDITPAGGVVLSPGQKIKAGIAQGNTQLEVAAPCQENYSMNSAr^ ANPSVQTQLSNQNSLTNTDFta^QVSDITPAGSWLSPGQKSKVGRAQCESCTE QNFTMDNAOdFCEADVKKCIAV Y Y APHIKVESHIQPSLNQEDI[Y|ITTESLTTAA . GRPGTGEHVPGSEMPVPEfijrSIHIVQSPQGLILNATALPLPDK. EFLSS APHVDVESRVEPSFNQEDMITTESLTTTA. ERSGTAEDAPGSEMPVPEKTTSIHLVQSPQGLVLNAATLPLPDK . EFLSS APHVEVESRLAPSFNQEDDYtrTTESLTTTA. GRSATAECAPSSEMPVPEWTSIHIVQSPQGLVLNATALPLPDK . EFLSS APHVEAESHVEPSFNQEDMITTESLTTTA . GRSGTAEHVPSSEIPVPDYlrSIHIVQSPQGLVLNATALPLPDK. EFLSS APDVEAESHIEPSFNQEDmpTTESLTTTA. GRSGTAENVPSSEIPVPEMTSIHIVQSPQGLVLNATALPLPDK. EFLSS APRMEATSCIKPSFNQEDmllTTESLTTTA. QMSETADIAPDAEMSVPETOTTVHTVQSPRGLILNATALPLPDKKNFPSS APHMEATTCVKPSFNQEDmllTTESLTTTA. RMSETADTAPDAE. PVPnY|rTVHTVKSPRGLILNATALPLPDKKKFLSS ISQEEDEPRVQEQSCNEDTMFTTESLTTTGINIXSASMAETPSMEMPVPqi'SIHrVHSPQGLVLNATALPVPEK. EFNMS

human rabbit porcine bovine ovine mouse rat chicken

VSTDQLNKIMP 7STDQLNKILP VSTDQLNKIMP VSTDQLNKIMP CGfVVSTDQLNKIMP ' VSTDQLNKIMQ VSTDQLNKIMQ VSTDQLNKIMP

human reJobit porcine bovine ovine mouse rat chicken

638 638 638 634 634 650 638 608

546 546 546 542 542 557 546 515 624 624 624 620 620 636 624 594

Growth Hormone Receptor

47

Figure 1. GHR amino acid sequence alignment. The amino acid sequences of human, rabbit, porcine, ovine, murine, rat and chicken GHR are aligned. Gaps have been introduced as necessary to maximize alignment. The conserved extracellular cysteines linked by disulfide bonds, the WSXWS-like motif, the transmembrane domain. Box 1, Box 2 and the conserved intracellular tyrosines are outlined. Amino acids numbers are indicated on the right for each species.

AH

AH

Llgand binding •

JAK2 phosphorylation (box 1) MAP kinase phosphorylation (box 1) Receptor internalization (box 1) Stat activation (box 1) Spi 2.1 induction (box 1) IRS-1 activation Lipid synthesis

WSXWS-like motif hC Transmembrane domain [Box 1 Box 2

Insulin synthesis Spi 2.1 induction Calcium response

GH receptor Figure 2, Growth hormone receptor. The transmembrane domain is shown in black. The extracellular asparagines (A) that are potential N-linked glycosylation sites are shown on the left. The seven extracellular cysteines (C) are shown on the right, with the three pairs of linked cysteines indicated. The position of the WSXWS-like motif is indicated by the striped box. Intracellular Box 1 (proline-rich domain) and Box 2 are shown as gray boxes. Regions of GHR required for various functions are indicated.

48

LISA S. SMIT and CHRISTIN CARTER-SU

contains five potential N-linked glycosylation sites. Glycosylation may explain, in part, the large discrepancy between the predicted molecular weight and the observed value of 130,000. In addition, ubiquitin was found covalently linked to the purified GHR and may account for part of the size difference. Expression of rabbit GHR in COS-7 cells was shown to confer the ability to bind ^^^I-labeled hOH, providing proof that the correct gene had been cloned. The sequence was not related to any receptor with known signaling pathways, including receptors with intrinsic tyrosine kinase activity. Human GHR was then cloned using a rabbit GHR cDNA as a probe to screen a human cDNA library (Leung et al, 1987), followed by rat, mouse, bovine, ovine, porcine, and chicken GHR cDNAs (Baumbach et al., 1989; Mathews et al, 1989; Smith et al, 1989; Hauser et al, 1990; Adams et al., 1990; Cioffi et al., 1990; Bumside et al., 1991). The amino acid sequence identity among species is quite high, approximately 70%. Six intracellular tyrosines are conserved among all cloned GHRs, suggesting the functional importance of these residues. The GHR is reported to be tyrosyl phosphorylated (Foster et al., 1988a), thus, these conserved tyrosines may represent phosphorylation sites. B. The Cytokine/Hematopoietin Receptor Superfamily

Sequence analysis initially revealed that GHR has considerable homology with the prolactin receptor (PRLR) (Boutin et al., 1988). This was not surprising because GH and PRL are themselves homologous and can bind each other's receptors (Niall et al., 1971). Subsequently, GHR and PRLR were found to be members of the recently defined cytokine/hematopoietin receptor superfamily (Bazan, 1989). In addition to GHR and PRLR, this family includes specific binding subunits of receptors for erythropoietin (EPO) (D'Andrea et al., 1989), granulocyte-colony stimulating factor (G-CSF) (Fukunaga et al, 1990), granulocyte-macrophage colony stimulating factor (GM-CSF) (Gearing et al., 1989), ciliary neurotrophic factor (CNTF) (Davis et al., 1991), interleukin (IL)-2 (Hatakeyama et al., 1989), IL-3 (Kitamura et al., 1991), IL-4 (Mosley et al, 1989), IL-5 (Takaki et al., 1990), IL-6 (Yamasaki et al, 1988), IL-7 (Goodwin et al., 1990), IL-9 (Renauld et al., 1992), IL-10 (Ho et al., 1993), IL-11 (Hilton et al., 1994), IL-12 (Chua et al., 1994), thrombopoietin (Vigon et al., 1992), interferons (IFN) a/p (Uze et al., 1990; Novick et al., 1994) and IFNy (Aguet et al., 1988; Soh et al., 1994; Hemmi et al., 1994), as well as gpl30, a receptor component employed by IL-6 (Hibi et al., 1990), IL-11 (Fourcin et al., 1994), oncostatin M (OSM) (Gearing et al., 1992), CNTF and leukemia inhibitory factor (LIF) (Ip et al, 1992), the p subunit of the LIF receptor employed by LIF, CNTF and OSM (Gearing et al., 1991), the common p-chain employed by IL-3, IL-5 and GM-CSF (Itoh et al., 1990; Gorman et al., 1990), and y-chain of the IL-2 receptor utilized by IL-2, IL-4, IL-7, and potentially IL-9 (Takeshita et al., 1992). Receptors for IFN a/p, IFNy and IL-10 are more distantly related and have been classified as class II receptors in this family (Bazan, 1990; Ho et al., 1993) (Figure 3). The family was originally defined on the basis of homologous regions in the extracellular domains of approximately 200 amino

Growth Hormone Receptor

49

acids. Although the level of amino acid identity between receptors in this region is relatively low, two distinctive motifs exist. The first is an amino-terminal set of two pairs of cysteine residues. The extracellular domain of GHR actually contains seven cysteines, three pairs of which are linked by disulfide bonds (Fuh et al., 1990). The class II receptors share only two of the conserved cysteines. The second extracellular motif is more membrane-proximal and defined by the consensus sequence WSXWS (tryptophan, serine, any amino acid, tryptophan, serine). GHR is an exception in the cytokine receptor family, because it contains conservative substitutions within the WSXWS motif The sequence present in hOHR is YGEFS (amino acids 222—226). The F and S are conserved in rabbit, mouse, rat, ovine, bovine, porcine, and chicken; the Y is conserved in all of these species except chicken. Based on GH-GHBP crystallographic studies, it has been postulated that the WSXWS motif is critical in ligand binding (deVos et al., 1992). Mutational studies in the EPO receptor (R), IL-2-R P-chain, and PRL-R have shown that the WSXWS motif is essential for ligand binding and subsequent signal transduction (Miyazaki et al., 1991; Quelle et al., 1992; Watowich et al., 1992; Rozakis—Adcock and Kelly, 1992). More detailed mutational studies have been performed in GHR, in which each amino acid in the motif was replaced with alanine, or the GHR sequence was replaced with WSEWS to match the consensus sequence (Baumgartner et al., 1994). Mutation of Y222, G223 or S226 to alanine was associated with a lower binding affinity; no effect was observed for the other mutations. Of all the mutations made, only Y222Aand S226 A resulted in structural perturbations in the receptor and decreased signal transduction as measured by a decrease in the ability of GHR to stimulate protein synthesis and to transactivate the c-fos promoter. These results suggest that only the first aromatic residue (W or Y) and the last serine residue are critical for receptor structure and function. A mutation in the GHR WSXWS-like motif was also found to be associated with sex-linked dwarfism in chickens. In these growth-deficient, GH-resistant chickens, an isoleucine replaces the final serine in the motif and the encoded receptor displays reduced affinity for GH (Duriez et al., 1993). The identification of this naturally occurring mutation provides additional support for the importance of the WSXWS motif in GHR and other receptors in the cytokine/hematopoietin receptor superfamily. While the initial definition of the cytokine receptor family derived from homology in the extracellular domains, two intracellular regions of homology have been reported. The work of Murakami et al. (Murakami et al., 1991) and O'Neal and Yu—Lee (O'Neal and Yu—Lee, 1993) has defined a conserved intracellular prolinerich sequence motif (yXXXAlPXP, where v|/ represents hydrophobic, X represents any amino acid, Al represents aliphatic and P represents proline), also known as box 1. In hGHR, the sequence of the proline-rich region is ILPPVPVP (amino acids 298—305). This sequence in GHR is conserved among mammalian species although there is a conservative substitution at position-2 in chicken (see Figure 1).

LL CO

O

6

Q.

cc I

Id*) »u »Ndi

»U'<-Ndl

]f^

^4;4>| yj

fcl^Lfl^^

|i]||[gfeSrapH B ^

50

Ju fr-ni

CNTF

LIF

OSM

IL-6

IL-11 IL-12

3

fu

Figure 3. Cytokine receptor superfamily. Members of the cytokine receptor super family are schematically illustrated. The conserved extracellular cysteine motifs are represented by the four thin lines. The extracellular black box represents the WSXWS motifs (a thinner box represents WSXWS-like motifs with conservative substitutions). The intracellular white boxes represent boxes 1 and 2, as indicated.

52

LISA S. SMIT and CHRISTIN CARTER-SU

The second homologous intracellular segment, referred to as box 2, begins with a cluster of hydrophobic amino acids and ends with one or two positively charged amino acids (Murakami et al, 1991). In GHR, box 2 is located approximately 30 amino acids carboxy terminal to box 1 and spans about 15 amino acids (DaSilva et al., 1994). Mutation or deletion of box 1 and/or box2 in GHR, G-CSF-R, I1-2R(3chain, PRL-R, EPO-R, LIF-Rp, or the gp 130 receptor molecule resuhs in defective ligand-mediated cellular growth, suggesting that these regions are critical in receptor-mediated signal transduction (Murakami et al., 1991; Fukunaga et al., 1993; Goldsmith et al, 1994; DaSilva et al., 1994; He et al., 1994; Baumann et al., 1994; Wang and Wood, 1995). C. Alternative Splicing of GHR mRNA

In human, bovine and rabbit liver, Northern blot analysis revealed a single major mRNA transcript of approximately 4.5 Kb (Leung et al., 1987, Hauser et al., 1990). However, in mouse and rat liver, two prominent mRNAs were identified, one of 4.5 Kb and one of 1-1.5 Kb (Mathews et al, 1989; Smith et al., 1989). The smaller mRNA represents an alternatively spliced transcript that encodes the GH binding protein (GHBP), a soluble short form of the liver receptor. GHBP mRNA includes the exons encoding the extracellular domain of GHR, with an additional exon encoding a short hydrophilic tail replacing the exons encoding the transmembrane and cytoplasmic domains (Smith et al., 1989, Baumbach et al., 1989). Because a single transcript of approximately 4.5 Kb is observed in humans, bovines and rabbits, it is hypothesized that, in these species, GHBP is the result of specific proteolysis. Although the function of GHBP has not been well-established, it can bind GH, and it has been hypothesized that GHBP competes with GHR for GH binding, thereby inhibiting the actions of GH (Lim et al., 1990). This chapter will focus on the full-length GHR, encoded by the larger, 4.5-Kb transcript. Another alternatively spliced GHR mRNA, lacking exon 3 (GHRA3), has also been detected. GHRA3 was first identified in human placenta (Urbanek et al., 1992) and subsequently found in other tissues as well (Sobrier et al, 1993; Mercado et al., 1994). The alternatively spliced mRNA encodes a receptor that binds and internalizes GH in a manner identical to full-length GHR (Urbanek et al., 1992; Sobrier et al., 1993); thus, the physiological significance of this form of hGHR is unknown. There is significant differential splicing in the 5'-untranslated portion of the gene as well (Leung et al., 1987).

III. LOCALIZATION OF GHR AND REGULATION OF GHR EXPRESSION A. Localization of GHR

GHR expression was initially thought to be restricted to the liver. While the liver is indeed one of the most abundant sources of GHR, improved detection techniques combined with analysis of mRNA expression, have provided evidence that the

Growth Hormone Receptor

53

tissue distribution of GHR includes adipose tissue, heart, kidney, stomach, small intestine, colon, pancreas, lung, brain, cartilage, muscle, adrenal gland, skin, lymphatic and immune cells, testis, ovary, corpus luteum, and mammary gland (Mathews et al, 1989; Tiong et al., 1989; Lobie et al., 1990; Tiong and Herington, 1992; Burton et al., 1992; Lucy et al, 1993; Delehaye et al., 1994). Detection of GHR in multiple tissues supports the hypothesis that GH acts directly on many target tissues. A significant fraction of GHR in the liver is associated with intracellular structures, compared to the plasma membrane (Hocquette et al., 1989; Picard and Postel-Vinay, 1984). This distribution may reflect rapid GHR synthesis because GHR half-life is short (estimated at 45 minutes in freshly isolated rat adipocytes) (Gorin and Goodman, 1985). B. Developmental Regulation of GHR

The ontogeny of GHR has been studied in a number of different species. Despite high concentrations of circulating GH concentrations during fetal life, GH does not appear to be required for normal birth size in many species, including man (Goodman et al, 1968; Honnebier and Swaab, 1973; Lovinger et al., 1975). This suggested that GHR might be absent in fetal tissues. However, studies have demonstrated that GHR mRNA is present in a number of tissues, including liver, during fetal development in mammalian and avian species (Bumside and Cogbum, 1992; Hill et al, 1992; Tiong and Herington, 1992; Ymer and Herington, 1992; Walker et al, 1992; Klempt et al., 1993). The exact role of GHR during fetal development remains unclear. In rat, sheep, and rabbit, GHR mRNA is barely detectable in early fetal liver. Expression increases with time in utero and with age postnatally, reaching maximal adult levels by 7 days postpartum for sheep, 42 days for rat and 2-6 months for rabbit (Mathews et al., 1989; Adams et al., 1990; Tiong and Herington, 1992; Ymer and Herington, 1992; Walker et al., 1992; Klempt et al., 1993). GHR mRNA regulation varies somewhat in other tissues, but, in general, GHR is expressed at low levels in the fetus, with the level of expression increasing postpartum to reach maximal adult levels. The observation that GH responsiveness increases with age (Albertsson and Isaksson, 1976; Nutting, 1976; Goodman and Coiro, 1981) may reflect the increase in GHR expression. C. Differential Expression of GHR between Sexes

It remains unclear whether a difference exists in GHR expression between the sexes. Female rats have been reported to have higher levels of hepatic GHRs than males, as assessed by GH-binding studies (Baxter et al., 1980; Maes et al., 1983). This difference was not observed in other similar studies (Ranke et al, 1976; Husman et al., 1988). Equivalent levels of GHR mRNA are observed in male and female rat liver, as well as pancreas (Mathews et al., 1989; Moldrup et al., 1993), suggesting that, if a sexual difference in GHR levels exists, it must be at the translational or posttranslational level. Expression of differentially spliced GHR mRNAs appears to exhibit some sex-specificity (Baumbach and Bingham, 1995).

54

LISA S. SMIT and CHRISTIN CARTER-SU

Consistent with a sex hormone dependence, both binding studies and mRNA analysis indicate that GHR expression is increased during pregnancy (Husman et al, 1988; Mathews et al, 1989; Cramer et al., 1992). D. GH Regulation of GHR Expression

GH is involved in the regulation of its own receptor, although its exact role in that regulation is unclear. The magnitude and direction of its effects appear to vary with tissue and/or sex. In female hypophysectomized rats, a 30-35% decrease in liver GHR mRNA levels and a 50% decrease in liver GH-binding sites has been reported (Baxter and Zaltsman, 1984; Maiter et al., 1992). In these studies, GH replacement was able to reverse changes in liver GH binding but not in GHR mRNA abundance, suggesting that GH infusion upregulates GHR via a posttranscriptional mechanism. In contrast, hepatic bGH-binding sites are increased following hypophysectomy of male rats (Baxter and Zaltsman, 1984; Picard and Postel-Vinay, 1984). Other studies in hypophysectomized rats revealed that hepatic GHR mRNA levels are unchanged (Mathews et al., 1989; Frick et al., 1990), suggesting that GH regulates GHR mRNA levels via a posttranscriptional mechanism. In adipocytes isolated from male hypophysectomized rats, GHR mRNA levels and the number of hGH binding sites are decreased in comparison to normal rats (Grichting and Goodman, 1986; Frick et al., 1990; Vikman et al., 1991). GHR expression levels can be restored, in part, by GH treatment. In contrast, an increased level of GHR mRNA in muscle isolated from hypophysectomized rats is observed (Frick etal, 1990). Further evidence for a role of GH in regulating its receptor comes from studies in a number of cell lines. In a human hepatoma cell line, treatment with physiological concentrations of GH results in an increase in GHR mRNA levels within one hour. Steady-state levels are reached after 3-4 hours and maintained for at least 48 hours. Superphysiological concentrations lead to a decrease in GHR mRNA levels during the first 3 hours, followed by an increase. The observed decrease in GHR mRNA levels changes result from decreases in the rate of transcription (Mullis et al., 1991). GH has also been shown to regulate, specifically, GHR mRNA in rat epiphyseal chondrocytes. Physiological concentrations of GH result in increased receptor mRNA within 3 hours and maximal levels of mRNA are observed at 12 hours. The rise in GHR mRNA is due to an increased rate of transcription (Nilsson etal., 1990). E. Regulation of GHR Expression by Insulin and Glucocorticoids

A number of additional hormones are possible regulators of GHR. Insulin appears to be involved in the upregulation of GHR. Insulin therapy reverses the decrease of GHR observed in liver membranes of rats with streptozotocin-induced diabetes (Baxter et al., 1980). In obese Zucker rats an increase in liver GH-binding sites compared to lean litter mates immediately follows the onset of hyperinsulinemia (Postel-Vinay et al., 1990). Insulin also stimulates an increase in GH-binding sites

Growth Hormone Receptor

55

in preadipocytes from obese rats (Landron et al., 1987). Similarly, in fasted/refed rats an increase in GHR parallels the increase in plasma insulin levels (Postel-Vinay etal., 1982). Glucocorticoids inhibit growth and render tissues, such as liver and bone, growth-plate-insensitive to GH, suggesting a possible role for glucocorticoids in GHR regulation. Unexpectedly, the artificial glucocorticoid, dexamethasone, administered to rabbits in growth-suppressing doses, increases GHR mRNA levels in liver and growth plate (Heinrichs et al., 1994). It was also demonstrated that dexamethasone increases GHR mRNA levels to 250% of control in cultured islet cells and to 400-500% of control in insulinoma cells (RIN-5AH) (Moldrup et al., 1993) and increases GH binding to 150% of control in UMRl 06.01 rat osteosarcoma cells (Salles et al., 1994). More consistent with the GH antagonistic effects of glucocorticoids in peripheral tissues, it has been shown that dexamethasone in 3T3-F442 Afibroblastsdecreases levels of GH binding (King and Carter-Su, 1995).

IV. GHR AND GROWTH A. GHR and Conditions Associated with Growth Retardation

The abundance of GHRs is reduced in a number of conditions associated with growth retardation including fasting (Postel-Vinay et al., 1982), streptozotocininduced diabetes (Baxter et al., 1980) and chronic renal insufficiency (Finidori et al., 1980). When female rats are subjected to fasting for 4 days, a 65% decrease in GH binding in liver microsomal membranes is observed compared to control animals and a 75% decrease in liver plasma membrane GH binding. The decrease reflects altered receptor number rather than decreased binding affinity (Postel-Vinay et al., 1982). A similar decrease in GHR mRNA levels occurs in male rats subjected to fasting for up to 3 days (Straus and Takemoto, 1990). Streptozotocininduced diabetes in rats is associated with decreased bovine (b) GH binding to liver membranes, a condition that can be reversed by insulin therapy (Baxter et al., 1980). Nephrectomy-induced chronic renal failure in rats is associated with, approximately, a 60% decrease in GH binding to liver microsomal membranes, plasma membranes and Golgifi-actions.As in the case of fasting rats, the binding affinity of GH is not affected (Finidori et al., 1980). A 50% reduction in hepatic GHR mRNA levels is observed in nephrotic rats compared to pair-fed controls (Tonshoff et al., 1994). B. GHR and Dwarfism

Laron-type dwarfism, an autosomal recessive genetic disorder, is characterized by severe growth failure, low levels of circulating IGF-1 but high levels of biologically active GH (Laron et al., 1966). Some patients with the Laron syndrome have demonstrated mutations in the GHR gene. One mutation involves an unusual deletion of nonconsecutive exons in the region of the gene encoding the extracellular domain (Godowski et al., 1989). A premature stop codon is introduced as a

56

LISA S. SMIT and CHRISTIN CARTER-SU

result of a shift in the readingfi-ameand the predicted protein is severely truncated so that the transmembrane and intracellular portions are lost (Meacham et al., 1993). Another mutation that leads to Laron syndrome is a substitution of serine for phenylalanine at position 96 of the extracellular domain of the receptor (Amselem et al., 1989). The mutant receptor appears to be mislocalized in the cell and is unable to bind GH (Duquesnoy et al., 1991). Two nonsense mutations at codons 38 and 43 in the GHR gene have also been identified (Amselem et al., 1991). The corresponding proteins, if stable, would lack the entire transmembrane and intracellular domains. Sex-linked dwarfism in chickens, a form of GH-resistance that resembles the Laron syndrome in humans, is also associated with mutations in GHR. Two mutations have been identified; a splice donor site mutation and a point mutation that effects the invariant serine in the WSXWS-like motif (Huang et al., 1993; Duriezetal., 1993). The short stature of African pygmies may also be due to a defect in the GHR gene. A number of studies have shown that these pygmies respond poorly to GH (Merimee et al., 1968; Merimee et al., 1982). Although a specific mutation in GHR has not been demonstrated, a restriction fragment length polymorphism in a GHR intron has been detected in pygmies, but not in non-pygmy populations. This polymorphism may be in linkage disequilibrium with a GHR mutation that is responsible for GH insensitivity in pygmies (Merimee et al., 1989).

V. GHR SIGNAL TRANSDUCTION A. GH Signal Transduction Via GHR Although the effects of GH on growth, differentiation, and metabolism have been recognized for years (reviewed in Cheek and Hill, 1974; Davidson, 1987; previous chapter), many of the cellular signaling mechanisms by which GH elicits these responses have only recently begun to be appreciated. The cloned GHR elicits multiple cellular responses, including GH-dependent proliferation of mouse FDCPl cells and Ba/F3 cells (Colosi et al., 1993; Wang and Wood, 1995), insulin production in RIN-5 A cells (Billestrup et al., 1990; Moldrup et al., 1991; Billestrup et al., 1994), and protein and lipid synthesis and sustained lipolysis in CHO cells (Emtner et al., 1990; Moller et al., 1994). Experiments with GHR truncation mutants have demonstrated that the membrane-proximal half of the cytoplasmic domain of GHR is required for proliferation in FDC-Pl cells and Ba/F3 cells (Colosi et al., 1993; Wang and Wood, 1995) and for protein and lipid synthesis and lipolysis in CHO cells (Moller et al., 1994; Billestrup et al., 1994), while the distal half is necessary for insulin production in RIN-5 A cells (Moldrup et al., 1991) (see Figure 2). The mechanisms and pathways by which GHR may mediate the effects of GH will be discussed in the remainder of this chapter.

Growth Hormone Receptor

57

B. G H Binding and Receptor Dimerization

The initial step in GH signaling is the binding of GH to its receptor. Crystallographic studies have demonstrated that the addition of GH to GHBP (a soluble form of GHR containing only the extracellular domain) results in a complex in which two GHBP molecules are bound by a single GH molecule (Cunningham et al, 1991; deVos et al., 1992). The binding appears to be sequential: one GHBP molecule binds to site 1 on GH, and then the second GH site becomes available for a second GHBP molecule to bind. Interestingly, no obvious sequence homology exists between site 1 and 2. Studies in which ^^^I-hGH is covalently cross-linked to full-length and truncated GHR indicate that GHR also forms dimers, although whether GHR dimerization is GH-dependent has not yet been demonstrated (Rui et al., 1996). However, mutant GH, that fails to induce GHBP dimerization, is biologically inactive when added to cells expressing full-length GHR, suggesting that GH-induced dimerization of GHR is required for GH action (Fuh et al., 1992). C. GHR Glycosylation

As predicted by amino acid sequence analysis, GHR is a glycoprotein (Asakawa et al., 1986; Husman et al., 1989). Studies of adipocytes pretreated with tunicamycin, an inhibitor of N-linked glycosylation, demonstrated that N-linked glycosylation is not required for membrane insertion of GHR and GH binding, however the N-linked carboyhydrates are necessary to maintain a high affinity of GHR for GH. N-linked carbohydrates are also required for normal receptor internalization (Szecowka et al., 1990). Pretreatment of adipocytes with swainsonine, which interferes with processing of carbohydrate side chains of glycoproteins, does not affect insertion of GHR into the membrane or GH binding or insulin-like responses to GH. However, the ability of GH to mediate lipolytic responses was compromised (Chipkin et al., 1989). These studies indicate that GHR glycosylation is important for some actions of GH. D. GHR Internalization

After binding, the GH-GHR complex is internalized and GH is quickly degraded (Gorin et al., 1984; Weyer and Sonne, 1985; Roupas and Herington, 1987a). It has been shown that the proline-rich region of GHR is required for receptor-mediated GH internalization (Moldrup et al., 1991). GHR appears to turn over rapidly on the cell surface, as evidenced by its short half-life of approximately 45 minutes in freshly isolated adipocytes (Gorin and Goodman, 1985) and 75 minutes in 3T3 fibroblasts (Murphy and Lazarus, 1984). In contrast to many other receptors, the bulk of GHR does not appear to recycle back into the membrane, but, like GH, is degraded (Roupas and Herington, 1988). GHR is also internalized in the absence of GH as evidenced by that fact that incubation of freshly isolated adipocytes with cyclohexamide for 3 hours abolishes GH binding (Eden et al., 1982). Lobie and colleagues have data suggesting that GHR is subject to ligand-dependent nuclear

58

LISA S. SMIT and CHRISTIN CARTER-SU

translocation by a process requiring the C-terminal half of the cytoplasmic domain of GHR. A physiological role of this nuclear GHR remains to be established (Lobie etal, 1994) E. GHR Tyrosyl Phosphorylation

In response to GH, GHR becomes phosphorylated on tyrosyl residues (Foster et al, 1988a; Argetsinger et al., 1993; Silva et al, 1993). The cytoplasmic domain of human GHR contains six tyrosines that may potentially be phosphorylated (Figure 1). These tyrosines are conserved in all cloned species of GHR. Phosphorylated tyrosine residues in GHR may provide docking sites for Src-homology 2 (SH2) domain-containing proteins. SH2 domain-containing proteins are known to bind to phosphorylated tyrosines in other proteins (Pawson and Schlessinger, 1993). Experiments conducted with truncated and mutated rat (r) GHR expressed in CHO cells have provided insight as to which tyrosines may be phosphorylated. In addition to the six cytoplasmic tyrosines present in hGHR, the cytoplasmic domain of rGHR contains four more tyrosines. Introduction of a stop codon at amino acid 455 of rGHR, so that only the amino-terminal half of the cytoplasmic domain, containing four tyrosines, is expressed, reduces, but does not abolish, tyrosyl phosphorylation of rGHR (VanderKuur et al., 1994). Mutation of the two membrane-proximal cytoplasmic tyrosyl residues in the context of the full-length rGHR reduces tyrosyl phosphorylation, and these same mutations, in the context of the truncated receptor, result in a further, substantial reduction in phosphorylation (VanderKuur et al., 1995b). Taken together, these results suggest that rGHR is phosphorylated on multiple tyrosine residues, including one or more of the six distal tyrosines and one or both of the two most membrane-proximal cytoplasmic tyrosines. F. GH Activation of jAK2 Tyrosine Kinase

Several experimental lines of evidence suggested that a tyrosine kinase is involved in GH signaling cascades. GH promotes the tyrosyl phosphorylation of GHR (Foster et al., 1988a; Argetsinger et al., 1993; Silva et al., 1993) and other cellular proteins (Campbell et al., 1993; Winston and Bertics, 1992; Moller et al, 1992; Silva et al., 1993), and kinase activity capable of phosphorylating GHR is found in highly purified preparations of GHR (Carter-^u et al., 1989). However, the amino acid sequence of GHR bears no resemblance to that of any known tyrosine kinase (Leung et al., 1987). These two disparate facts were reconciled by the recent identification of JAK2, a nonreceptor tyrosine kinase (Harpur et al., 1992; Silvennoinen et al., 1993) as a GHR-associated, GH-activated tyrosine kinase (Argetsinger et al., 1993). JAK2 is a member of the Janus family of tyrosine kinases; the family presently includes JAKl, JAK2, JAK3 and tyk2 (Firmbach-Kraft et al., 1990; Wilks et al, 1991; Harpur et al., 1992; Silvennoinen et al, 1993a; Witthuhn et al., 1994; Takahashi and Shirasaw, 1994; Rane and Reddy, 1994). Although initially, it was not observed that GH activates JAKl in 3T3-F442A fibroblasts or

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IM-9 cells (Argetsingeretal., 1993; Silvaetal., 1994), additional studies performed with a higher affinity JAKl antibody demonstrated that GH induces low level tyrosyl phosphorylation of JAKl in 3T3-F442A fibroblasts (Smit et al, 1996), suggesting that JAKl may also be activated by GH. Although significantly less JAKl than JAK2 is tyrosyl phosphorylated in response to GH, the level of JAKl tyrosyl phosphorylation is similar to that induced by IFNy. Because JAKl has been implicated by genetic complementation experiments (Muller et al., 1993) as critical to the action of IFNy, it seems possible that, despite its low signal, JAKl may mediate some effects of GH. GH has also been shown to stimulate low levels of JAK3 tyrosyl phosphorylation (Johnston et al., 1994). GH does not appear to activate tyk2 (Argetsinger et al., 1993). Tyrosyl phosphorylation and/or activation of JAK kinases has been demonstrated in response to ligand binding to other members of the cytokine receptor family. JAK2 is activated by ligand binding to receptors for EPO, IL-3,6,11, and 12, CNTF, LIF, OSM, GM-CSF, G-CSF, and PRL as well as IFNy (Silvennoinen et al, 1993b; Watling et al., 1993; Witthuhn et al., 1993; Silvennoinen et al., 1993a; Stahl et al., 1994; Quelle et al., 1994; Rui et al, 1994; Campbell et al, 1994; Yin et al., 1994b; Bacon et al., 1995). Other ligands that bind to members of the cytokine receptor family activate different JAKs. For example, JAK3 is activated in response to IL-2 and 4 (Witthuhn et al., 1994). As suggested for GH, a number of other cytokines have been reported to activate multiple JAKs. For example, IFNy stimulates tyrosyl phosphorylation of JAKl as well as JAK2 (Shuai et al., 1993; Silvennoinen et al., 1993b), and LIF, IL-6, CNTF, and OSM stimulate tyrosyl phosphorylation of JAKl, JAK2, and tyk2 (Stahl et al., 1994). At the present time, it has been shown that all receptors identified as members of the cytokine receptor family activate at least one JAK kinase, indicating that members of the cytokine receptor superfamily act by activating JAK kinases. Like GH, the finding that many cytokines activate both JAKl and JAK2 raises the question how GH achieves its specific effects. One explanation may be in the finding that, although multiple ligands stimulate JAK kinases, they do not do so to the same extent. For example, in 3T3-F442A fibroblasts, GH is significantly more effective (greater than 10 fold) in stimulating tyrosyl phosphorylation of JAK2 than LIF or IFNy (Argetsinger et al., 1995a; Smit et al., 1996). The greater ability of GH to activate JAK2 may contribute to some specificity in response. Clearly, other factors, such as the level of GHR expression and contribution of specific sequences in GHR itself, are also likely to contribute to GH-specific responses. Several studies have used truncated or mutated GHRs to identify the region of GHR required for JAK2 association and activation. These studies have identified the membrane-proximal region of the cytoplasmic domain of GHR containing the proline-rich motif (box 1) as necessary for GH-dependent JAK2 association with GHR and JAK2 tyrosyl phosphorylation and activation (VanderKuur et al, 1994; Sotiropoulos et al., 1994; Frank et al, 1994; Wang and Wood, 1995) (see Figure 2). Specific deletion of the eight amino acids that comprise the proline-rich motif

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(residues 298-305 of the rat GHR) destroys GHR-JAK2 association and GHdependent JAK2 phosphorylation. Mutation to alanine of a single proline in this region abolishes the ability of GH to induce JAK2 tyrosyl phosphorylation, suggesting that this is a key amino acid for GH-dependent JAK2 activation (Wang and Wood, 1995). Although the proline rich-region appears to play a primary role in GHR-JAK2 association, more distal regions appear to augment the interaction (Frank et al., 1994; Sotiropoulos et al, 1994). Similarly, membrane-proximal regions of the cytoplasmic domains of EPO-R and PRL-R were implicated in JAK2-receptor association and JAK2 activation (Witthuhn et al, 1993; DaSilva et al., 1994). Consistent with the activation of JAK2 as an initial signaling event for GHR, GH-dependent mitogenesis in FDC-Pl cells and Ba/F3 cells, increased insulin synthesis in RIN cells; induction of the Spi 2.1 gene in CHO cells is not observed with GHR mutants that lack the JAK2 binding site (Moldrup et al., 1991; Billestrup et al., 1994; Colosi et al., 1993; Goujon et al., 1994; Wang and Wood, 1995) (Figure

Figure 4. GHR signaling pathways. Possible signaling pathways initiated by GH binding to its receptor are shown. Many of these pathways may lead to the activation oic-fos, as shown. The dotted lines indicate molecules which have not yet been shown to be involved In GH-dependent signal transduction, but which are likely to be employed by GH because they are utilized by other growth factors that activate MAP kinases.

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4). It is possible then, that a primary role of GH binding to GHR is to facilitate recruitment and activation of JAK2. Subsequent signaling events may require JAK2; and may or may not require GHR itself. G. GH Activation of MAP Kinases

It has been shown that GH activates the mitogen-activated protein (MAP) kinases, designated extracellular signal-regulated kinases (ERKs) 1 and 2 (Campbell et al, 1992; Winston and Bertics, 1992; Moller et al, 1992; Anderson, 1992). MAP kinases are activated by a number of receptor and nonreceptor tyrosine kinases and are believed critical in regulating growth and differentiation (Cobb et al., 1991). Thus, MAP kinases are likely to represent important signaling molecules ofGH. A number of MAP kinase substrates have been identified, including c-Raf-1, MEK, the S6 kinases (rskl and rsk2 (p90rsk)), EGF receptor, phospholipase A2, the microtubule-associated protein, tau, as well as a number of transcription factors, including c-myc, NF-IL6, p62TCF/ELK-l, ATF-2, and c-jun (reviewed in Davis, 1993). Of these, it has been shown that GH activates the S6 kinase, p90''^^ in 3T3-F442A fibroblasts (Anderson, 1992). Presumably some of these other substrates are phosphorylated by MAP kinases in response to GH and are responsible for other actions of GH. One pathway leading from membrane receptor tyrosine kinases to MAP kinases involves SHC, Grb2, son-of-sevenless (sos), ras, raf, and Map/Erk kinase (MEK). It has been shown that ligand-activated tyrosine kinase receptors interact directly with Grb2, a small SH2 and SH3 domain-containing protein, or bind and tyrosyl phosphorylate the SH2 domain-containing SHC proteins, which in turn bind Grb2 (Rozakis-Adcock et al., 1992; Cutler et al., 1993; Ravichandran and Burakoff, 1994; Ohmichi et al., 1994). It has been shown that Grb2 interacts with the mammalian homologue of the drosophila gene product, sos, a guanine nucleotide exchange factor, which activates the small GTP binding protein, ras. Ras, in turn, activates the serine/threonine kinase raf, and raf activates the mixed function serine/threonine/tyrosine kinase MEK. MEK then phosphorylates and activates the MAP kinases (Crews and Erikson, 1993). It has been demonstrated recently that GH promotes rapid tyrosyl phosphorylation of the 66-, 52- and 46-kDa SHC proteins in 3T3-F442A fibroblasts (VanderKuur et al., 1995a). GH.also induces the binding of a GHR-JAK2 complex to the SH2 domain of the 46- and 52-kDa SHC proteins fused to a glutathione S-transferase (GST) and the association of Grb2 with SHC. These results suggest that GH stimulates the association of SHC proteins with JAK2-GHR complexes via SHC SH2 domains; SHC is then tyrosyl phosphorylated, presumably by JAK2, and Grb2 associates with SHC via its SH2 domain. Although it has not yet been shown that GH activates the remaining molecules in this signaling cascade (sos, ras, raf and MEK), it seems likely that the recruitment of SHC and Grb2 are early events in GH activation of ERKs 1 and 2. GH activation of MAP kinases requires the proline-rich region of GHR, the same region impli-

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cated in JAK activation, providing further evidence for a role of JAK2 in the activation of MAP kinases (Moller et al, 1992; VanderKuur et al., 1994) (see Figure 2). H. GH Induction of IGF-1 Synthesis and Secretion

GH treatment regulates the synthesis and secretion of IGF-1, a known activator of somatic growth (reviewed in Daughaday and Rotwein, 1989). The levels of IGF-1 protein in liver and other tissues is increased by GH (Clemmons et al., 1981; D'Ercole et al., 1984). Similarly, GH treatment increases the abundance of IGF-1 mRNA in primary cultures of rat liver cells (Norstedt and Moller, 1987; Kachra et al., 1991) and in cultured adipocytes (Doglio et al., 1987; Vikman et al., 1991). Reduced levels of IGF-1 mRNA are observed in multiple tissues from hypophysectomized rats and GH treatment of these animals reverses these changes (Roberts et al., 1986; Murphy et al., 1987; Roberts et al, 1987; Hynes et al., 1987; Vikman et al., 1991;Bichelletal., 1992;Maiteretal., 1992). The abundance of IGF-1 mRNA begins to increase 2 hours after GH treatment with peak expression between 4 and 16 hours (Murphy etal., 1987;Bichelletal., 1992).IGF-1 has multiple alternatively spliced forms, all of which are induced by GH, although to varying degrees (Lowe et al., 1987; Bichell et al., 1992; Foyt et al, 1992). Taken together, these studies suggest that GH binding to GHR induces a signaling pathway that effects IGF-1 mRNA synthesis, processing, or stability. Several studies have shown that GH enhances IGF-1 expression by predominantly transcriptional mechanisms (Mathews et al., 1986; Bichell et al., 1992), although the mechanism by which GH activates transcription of IGF-1 has proven difficult to delineate. A DNase hypersensitivity site has been mapped to the second intron of IGF-1. The kinetics of induction of the hypersensitive site, in response to GH, mirror the pattern of IGF-1 transcription stimulated by GH (Bichell et al., 1992), suggesting that the hypersensitive site is involved in GH induction of IGF-1. Protein binding sites have been identified in the IGF-1 promoter; however, none of these are affected by GH treatment (Thomas et al, 1994; Thomas et al, 1995). These results imply that GH induces an alteration in nuclear organization rather than a stimulation of protein— DNA binding. The ability of GH to stimulate IGF-1 transcription and a corresponding increase in IGF-1 secretion represents one means by which GH can mediate cell proliferation or differentiation. However, in some situations, GH and IGF-1 have opposite effects clearly indicating that GH-dependent induction of IGF-1 cannot account for all of the actions of GH. For example, GH suppresses long-term glucose oxidation in 3T3-F442A adipocytes although IGF-1 stimulates glucose oxidation (Schwartz et al., 1985). I. GH Induction of c-fos and Other Genes

It is known that GH treatment promotes a rapid increase in the expression of a number of genes in addition to gene encoding IGF-1, including the genes encoding c-Fos, c-Jun, c-Myc, Spi 2.1, lipoprotein lipase and several forms of cytochrome

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p450 (Morgan et al, 1985; Noshiro and Negishi, 1986; Murphy et al., 1987; Gurland et al., 1990; Pradines et al, 1990). Transcriptional activation of these genes in response to GH occurs more rapidly than activation of IGF-1, indicating that GH mediates the increases in expression of these genes directly, rather than indirectly via IGF-1. The proline-rich region of GHR was shown to be important for GH-dependent activation of Spi2.1 (Goujon et al., 1994), suggesting that JAK2 is required for GH induction of Spi2.1. The protein products ofc-fos, c-jun and c-myc are all nuclear phosphoproteins. Fos and Jun, whose expression is induced by a number of growth factors known to stimulate cell growth and/or differentiation, are thought to be important in cellular growth and/or differentiation (Lord et al., 1993; Distel and Spiegelman, 1990; Johnson et al., 1992). Myc is thought to influence cell proliferation by modulating transcription and affecting initiation of DNA replication (Penn et al., 1990). In 3T3-F442Aadipocytes, binding of Fos-containing DNA binding complexes is required for expression of the adipocyte specific gene aP2 (Distel et al., 1987; Rauscher et al., 1988; Herrera et al., 1989), suggesting that c-fos stimulated by GH contributes to cellular differentiation by regulating the expression of tissue specific genes. Recent advances in the study of GH signaling pathways have served to illuminate our understanding of the mechanisms by which GH binding to its receptors leads to the induction of genes such as c-fos and eventually to cell proliferation, differentiation, or changes in metabolism. Several response elements have been identified in the promoter of the c-fos gene. These include the serum-response element (SRE), that mediates transcriptional stimulation by serum, and a variety of growth factors (Visvader et al., 1988; Stumpo et al., 1988; Siegfried and Ziff, 1989), an AP-1 site to which Jun and Fos bind (Fisch et al., 1989), and the sis-inducible element (SIE), which binds a factor induced by sis/PDGF in BALB/c 3T3-cells or by EGF in A431 cells (Hayes et al., 1987; Herrera et al., 1989; Wagner et al., 1990). It has been demonstrated that the SRE is capable of mediating GH induction of c-fos (Meyer et al., 1993). One possible pathway leading to c-fos induction via the SRE exists via MAP kinase. MAP kinases, activated by a number of growth factors, phosphorylate transcription factors, including ELK-1 and p62TCF (Pulverer et al., 1991; Gille et al., 1992). ELK-1 or p62TCF form a ternary complex with the serum response factor (SRF) and the serum response element (SRE). Phosphorylation of p62TCF by MAP kinase is believed to promote formation of this ternary complex, thereby stimulating transcriptional activity (Gille et al., 1992). This suggests at least one pathway (GHR-MAP kinase-p62TCF-Fos) by which GH stimulation is likely to result in nuclear events that may contribute to the ability of GH to promote cellular differentiation. J. GH Activation of Stats 1,'^,and 5 The SIE in the c-fos promoter may also play a role in the induction of c-fos by GH. GH treatment of 3T3-F442A fibroblasts results in three DNA-binding complexes capable of binding to the SIE (Meyer et al., 1994; Campbell et al., 1995).

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Although it has not been shown that the SIE mediates GH induction of c-fos by itself, the binding of GH-activated factors to the SIE suggests that it may contribute to GH-dependent c-fos induction. In addition, mutation of the SIE obliterates normal expression of endogenous Fos in transgenic mice and Fos inducibility in fibroblasts derived from them (T. Curran, personal communication), further suggesting the importance of the SIE in GH-dependent c-fos induction. It has recently been shown that GH induces tyrosyl phosphorylation and the SIE binding activity of cytoplasmic transcription factors, referred to as signal transducers and activators of transcription (Stats) (Meyer et al., 1994; Gronowski and Rotwein, 1994; Campbell et al., 1995; Gronowski et al., 1995; Wang and Wood, 1995). Members of the Stat family of proteins, which possess both an SH2 and SH3 domain, are activated by tyrosine phosphorylation (Darnell et al., 1994). Statl, also referred to as p91, was originally identified as a component of transcription factor complexes induced by IFNs. In response to IFNa, Stat 1, Stat 1 p (p84, an alternatively spliced product of Statl), and Stat2 (pi 13) are tyrosyl phosphorylated and translocated to the nucleus. These three proteins, together with a DN A binding subunit, p48, form the IFN-stimulated gene factor 3 complex which initiates transcription of IFNa stimulated genes (Fu et al., 1992; Kessler et al., 1990). Statl, but not the other proteins in the IFN-stimulated gene factor 3 complex, is activated in response to IFNy. IFNy-activated Statl translocates to the nucleus and binds to IFNy-responsive DNA sequences such as the y response region (GRR) (Shuai et al., 1992). The GRR shows remarkable sequence similarity to the SIE. Recently, tyrosyl phosphorylation of Statl and induction of its DNA-binding activity were observed in response to GH. GH treatment of 3T3-F442A fibroblasts results in tyrosyl phosphorylation of Statl (or an antigenically related protein) and the formation of a Statl-containing, DNA-binding complex that is capable of binding to both a normal and high-affinity, sis-inducible element (SIE) of the c-fos promoter (Meyer et al., 1994; Campbell et al., 1995). Of the three GH-induced binding complexes, the two slower migrating complexes contain Statl. In Ba/F3 cells transfected with GHR, GH induces binding of Stat-1-containing complexes to the SIE and the y-response region (GRR) (Wang and Wood, 1995). Statl is also tyrosyl phosphorylated in hepatocytes from hypophysectomized rats treated with GH (Gronowski and Rotwein, 1994) and CHO cells expressing recombinant GHR (Smit et al., 1996). GH-dependent activation of Statl may be cell-type dependent because it was not observed that GH activates Statl in IM9 cells (Finbloom et al., 1994; Silva et al, 1994). Activation of Statl is also observed in response to LIF, OSM, IL-6, IL-10, CNTF, PRL, epidermal growth factor (EGF), platelet derived growth factor (PDGF), and colony stimulating factor-1 (CSF-1) (Fu, 1992; Bonni et al., 1993; Lamer et al., 1993; Ruff-Jamison et al., 1993; Silvennoinen et al, 1993c; Sadowski et al., 1993; Feldman et al, 1994; David et al., 1994), suggesting that activation of Stat proteins is a common mechanism by which cytokines and other growth factors regulate transcription.

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It has also been shown that Stat3, also known as acute phase response factor, or an antigenically related protein, is activated in response to GH (Campbell et al., 1995; Gronowski et al, 1995). Like Statl, Stat3 isactivatedby a number of different ligands, including IL-6, IL-11, LIF, OSM, CNTF, IFNy, IFNa, EGF,-PDGF, and CSF-1 (Zhong et al, 1994; Wegenka et al, 1994; Akira et al., 1994; Lutticken et al., 1994; Raz et al., 1994). In response to GH, Stat3 is tyrosyl phosphorylated and forms part of a complex that binds the SIE of the c-fos promoter and the APRE of the a2 macroglobulin promoter (Campbell et al., 1995; Gronowski et al., 1995). It has been demonstrated that the two faster migrating SIE-binding complexes contain StatB. Because it has been demonstrated that Statl forms homodiomers through SH2-phosphotyrosine interactions (Shuai et al., 1994), it has been hypothesized that the SIE-binding complexes contain Stat 1 homodimers (upper, slower migrating band), Statl-Stat3 heterodimers (middle band) and Stat3 homodimers (lower, faster migrating band). The binding of Stat-containing complexes to the SIE may represent an important contribution to GH-induction of c-fos expression and suggests a direct pathway (GHR-JAK-Stats-c-fos) by which GH can elicit nuclear events. GH has also been demonstrated to activate Stat5, or mammary gland factor (MGF). Stat5 was initially discovered as a factor which binds to DNA sequences essential for a lactogenic hormone response (Wakao et al., 1994). In response to GH, Stat5 is tyrosyl phosphorylated and can bind to a GAS-like element in the Spi2.1 or P-casein promoters (Wood et al., 1995; Gouilleux et al., 1995; Tourkine etal., 1995). Several lines of evidence suggest that Stat activation may require direct interaction of Stats with cytokine receptors. A peptide corresponding to the amino acids surrounding tyrosine 440 of the IFNyR binds Statl when tyrosine 440 is phosphorylated (Greenlund et al., 1994), presumably through the Statl SH2 domain. Similarly, it has been shown that Stat3 associates with gp 130, the signal transducing receptor component employed by IL-6, IL-11, LIF, CNTF, and OSM (Lutticken et al., 1994). It has been shown that a small tyrosine-containing motif, YXXQ, in gpl30 and LIF-Rp is required for Stat3 activation (Stahl et al., 1995). Similarly, phosphorylated tyrosines in the prolactin and erythropoietin receptors are required for maximal Stat5 activation (LeBrun et al., 1995; Damen et al., 1995), suggesting that Stat5 activation may also require Stat5 binding to a receptor. Studies in which panels of truncated and mutated GHRs were used to examine the regions of GHR required for Stat activation are consistent with activation of Stats 1, 3 and 5 by GH requiring JAK2 activation (Wang and Wood, 1995; Smit et al., 1996). In addition, GH-dependent Stat5 activation requires phosphorylated tyrosines in the C-terminal half of the cytoplasmic domain of GHR, which presumably serve as Stat5 docking sites (Hansen et al., 1996; Smit et al., 1996). Tyrosines in the N-terminal half of the cytoplasmic domain of GHR may also contribute to maximal activation of Stats 1, 3, and 5 by GH (Smit et al., 1996). Although phosphorylated tyrosines in GHR contribute to Stat activation, the observation that Stats 1,3, and 5 can be activated in the absence of any phosphorylated tyrosines in GHR suggests that Stat proteins

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may also bind to sequences in GHR other than phosphorylated tyrosines or to phosphorylated tyrosines in a GHR-associated protein, such as JAK2 (Smith et al., 1996; Wang etal., 1995). K. GH Utilization of Protein Kinase C and G Proteins Several lines of evidence support the hypothesis that protein kinase C (PKC) plays a role in GH-initiated signal transduction. Downregulation of PKC as a result of pretreatment of rat adipocytes with the phorbol ester, PMA, abrogates GHmediated lipogenesis (Smal and DeMeyts, 1987). In some cells (obi771 mouse preadipocytes and freshly isolated rat liver hepatocytes), it has been shown that GH enhances the formation of diacylglycerol (DAG), a known activator of PKC, but not of inositol trisphosphate (InsPg) (Doglio et al., 1989; Johnson et al., 1990). In basolateral membranes from canine kidney, GH has been reported stimulating production of both DAG and InsP3 (Rogers and Hammerman, 1989). The observation that GH stimulates production of DAG, a known activator of PKC, suggests that GH may also activate PKC. Studies of obi 771 preadipocytes suggest that GH mediates DAG production by phosphatidyl choline breakdown by a phospholipase C (PLC) (Catalioto et al., 1990). Pertussis toxin, which destroys the function of Gj and GQ families of GTP-binding proteins, abolishes this GH-induced DAG formation (Catalioto et al., 1990). These results suggest that phosphatidyl choline breakdown by PLC is coupled to GHR via a pertussis toxin-sensitive G-protein. Other experiments, with adipose tissue from ob/ob mice and S-carboxymethylated human GH, suggest that GH may interfere with the ability of a putative Gi-like protein to mediate activation of phosphatidylinositol (PI)-PLC by insulin in adipose membranes (Roupas et al., 1991). Evidence exists for a role of PKC in the activation of MAP kinases and p90rsk and the induction of c-fos by GH. Chronic treatment of cells with phorbol ester 12-myristate 13-acetate (PMA), so that PKC is depleted, reported by one group, markedly reduces the ability of GH to activate ERKs 1 and 2 and S6 kinase (Anderson, 1992) whereas a second group found no effect on GH-dependent activation of ERKs 1 and 2 (Winston and Bertics, 1992). However, inhibitors of PKC, that do not also inhibit tyrosine kinases, do not block GH activation of MAP kinases (Campbell et al., 1992). GH induction ofc-fos was also blocked by chronic preincubation of 3T3-F442A cells with PMA in these cells (Gurland et al., 1990). The protein kinase inhibitors, staurosporine and H7, as well as a more specific PKC inhibitor, l-O-alkyl-2-O-methylglycerol, all abrogated GH-induced expression of c-fos (Slootweg et al., 1991). These observations suggest that c-fos induction by GH may occur via a PKC pathway or at least be subjected to modulation by PKC. Whether PKC activation by GH is downstream of JAK2 or is a JAK-independent pathway remains to be determined.

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L. G H Stimulation of IRS-1 and IRS-2 Tyrosyl Phosphorylation

Although it is well established that GH has long-term anti-insulin-like effects, under certain circumstances, GH is also known to have rapid, transient effects that mimic the actions of insulin (Davidson, 1987). The existence of insulin-like effects in response to GH suggests that GH may utilize some of the same signaling molecules employed by insulin. It has been shown that GH stimulates tyrosyl phosphorylation of the insulin receptor substrate-1 (IRS-1), the principal substrate of the insulin receptor, in primary cultures of rat adipocytes, 3 T3-F442 A fibroblasts, and CHO cells expressing recombinant GHR (Souza et al., 1994; Ridderstrale et al., 1995; Argetsinger et al., 1995a). Recent studies indicate that GH stimulates tyrosyl phosphorylation of IRS-2 as well (Argetsinger et al., 1996). Studies using truncated GHRs expressed in CHO cells revealed that the region of GHR required for GH-induced IRS-1 and IRS-2 tyrosyl phosphorylation is the same region as that required for JAK2 association and activation (see Figure 2). These results suggest that JAK2 kinase is responsible for the phosphorylation of IRS-1 and IRS-2 and that GHR in GH-dependent tyrosyl phosphorylation of IRS-1 and IRS-2 facilitates the activation of JAK2. LIF and IFNy also can stimulate tyrosyl phosphorylation of IRS-1 and IRS-2 in 3T3-F442 A fibroblasts (Argetsinger et al., 1995a; Argetsinger et al., 1996). The ability of GH, LIF, and IFNy to stimulate phosphorylation of IRS-1 follows in the same order as their ability to activate JAK2 (GH > LIF > IFNy), providing additional evidence for the role of JAK2 in IRS-1 tyrosyl phosphorylation in response to cytokines. These data also suggest the possibility that IRS-1 is interacting with JAK2 rather than with GHR. Tyrosyl phosphorylation of IRS-1 in response to insulin or IGF-1 provides docking sites for multiple SH2 domain-containing proteins, including the 85-kDa regulatory subunit of phosphatidyl inositol (PI) 3' kinase (Sun et al., 1991; Sun et al., 1993). GH also stimulated binding of IRS-1 to the 85-kDa regulatory subunit of PI 3'-kinase. PI 3' kinase is believed required for insulin-dependent glucose transport, DNA synthesis, and activation of 70-kDa S6 kinase, an enzyme implicated in cell cycle (Cheatham et al., 1994). PI 3' kinase inhibitor, wortmannin, does inhibit lipogenic and antilipolytic effects of GH in rat adipocytes, suggesting a role for PI 3' kinase in the insulin-like metabolic effects of GH (Ridderstrale and Tomqvist, 1994). These studies provide a biochemical basis for responses shared by insulin and GH, including the well characterized insulin-like metabolic effects of GH observed in a variety of cell types. M. G H and a Calcium Response

Recent evidence suggests that some of the actions of GH may involve a calcium— calmodulin response. GH treatment causes an increase in intracellular free calcium concentrations in freshly isolated adipocytes (Schwartz and Goodman, 1990; Schwartz et al., 1992), IM-9 lymphocytes (Hondo et al., 1994), and CHO cells transfected with GHR (Billestrup et al., 1995). In adipocytes, GH has transient.

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insulin-like effects if the cells have been previously deprived of GH. The insulinlike effects of GH are gone by 3 hours, at which point the cells become refractory to GH, and insulin-like effects cannot be elicited even by high concentrations of GH (Goodman, 1981). Refractoriness is not a result of a change in the number of GHR or GHR binding affinity (Grichting et al, 1983). Blockade of calcium channels with verapamil, inhibition of calmodulin with calmidozolium or trifluoropirazine, or depletion of cellular calcium by incubation in a calcium-free medium restores sensitivity to the insulin-like effects of GH. Conversely, incubation of GH-responsive cells with the calcium ionophore A23187 in the presence of normal extracellular calcium concentrations causes the cells to become refractory, suggesting that refractoriness to insulin-like stimulation by GH depends on a calcium-calmodulin sensitive process (Schwartz and Goodman, 1990). Consistent with the hypothesis that refractoriness to GH involves a calcium-dependent process, intracellular calcium concentrations are twice as high in refractory cells as in sensitive cells (Schwartz et al., 1991). Mutagenesis studies have indicated that the GH-dependent increase in intracellular calcium in CHO cells requires the C-terminal half of GHR but not the prolines in box 1, suggesting that calcium signaling may be independent of JAK2 activation (Billestrup et al., 1995).

VI. CONCLUSIONS Recent work in the field of GH research has provided a wealth of knowledge about the GHR, GH-induced signal transduction cascades, and the role of GHR in these signaling pathways. There is increasing evidence that GH exerts direct effects on multiple tissues by binding to its cell surface receptor. GHR is expressed in a variety of tissues and its expression is regulated by a number of hormones, including GH itself GHR is a member of the cytokine/hematopoietin receptor superfamily. Like other members of this family, GHR lacks intrinsic tyrosine kinase activity. However, GH stimulates the association of GHR with JAK2, a nonreceptor tyrosine kinase and promotes activation of JAK2 by a mechanism that requires GH-induced GHR dimerization. In response to GH, both GHR and JAK2 are phosphorylated on tyrosines. These phosphorylated tyrosines are potential docking sites for SH2 domain-containing signaling molecules. Subsequent signaling events include activation of MAP kinases, presumably via a SHC-Grb2-sos-ras-raf-MEK pathway, activation of the latent transcription factors, Stat 1 and 3, tyrosyl phosphorylation of IRS-1 and IRS-2 and association of IRS-1 with PI 3' kinase, activation of PI-3 kinase, diacyl glycerol and PKC, and increases in intracellular calcium concentrations. The activation of MAP kinases, which phosphorylate transcription factors, and Stats is likely to be indirectly implicated in the GH-dependent induction of transcription of c-fos, an event thought to be important for GH-induced cellular differentiation. GH stimulates transcription of a number of other genes, including insulin and Spi 2.1. Analysis of the regions of GHR, required for activation of these signaling molecules, indicates that JAK2 is required for activating SHC, Grb2, Map

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kinases, Stats 1 and 3, IRS 1 and 2 and PI-3 kinase. Direct interactions of SHC, IRS 1 and 2 with GHR have not been demonstrated, suggesting the possibility that these signaling molecules bind to phosphorylated tyrosyl residues in JAK2 itself or as yet unidentified accessory molecules. In contrast, regions of GHR, other than those required for GH-dependent activation of JAK2, are required for GH-induced increases in intracellular calcium concentrations, GH activation of Stats 1, 3, and 5 and GH stimulated expression of the insulin and Spi 2.1 genes. Thus, the findings described here suggest that signaling mechanisms initiated by GH binding to GHR are complex. Identifying which pathways elicit which responses, how the different signaling cascades intersect, and the further characterization of the role of GHR in initiating these pathways promises to a be a fascinating challenge that should provide a greater understanding of how GH elicits its diverse effects on body growth and metabolism. REFERENCES Adams, T. E., Baker, L., Fiddes, R. J., & Brandon, M. R. (1990). The sheep growth hormone receptor: Molecular cloning and ontogeny of mRNA expression in the liver. Mol. Cell. Endocrinol. 73, 135-145. Aguet, M., Dembic, Z., & Merlin, G. (1988). Molecular cloning and expression of the human interferon-gamma receptor. Cell 55, 273—280. Akira, S., Nishio, Y., Inoue, M., Wang, X.—J., Wei, S., Matsusaka, T., Yoshida, K., Sudo, T., Naruto, M., & Kishimoto, T. (1994). Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gpl30-mediated signalling pathway. Cell 77, 63-71. Albertsson-Wikland, K., & Isaksson, O. (1976). Development of responsiveness of young normal rats to growth hormone. Metabolism 25, 747-759. Amselem, S., Duquesnoy, R, Attree, O., Novelli, G., Bousnina, S., Postel—Vinay, M. C , & Goossens, M. (1989). Laron dwarfism and mutations of the growth hormone-receptor gene. N. Engl. J. Med. 321,989-995. Amselem, S., Sobrier, M. L., Duquesnoy, R, Rappaport, R., Postel-Vinay, M. C , Gourmelen, M., Dallapiccola, B., & Goossens, M. (1991). Recurrent nonsense mutations in the growth hormone receptor from patients with Laron dwarfism. J. Clin. Invest. 87, 1098-1102. Anderson, N. G. (1992). Growth hormone activates mitogen-activated protein kinase and S6 kinase and promotes intracellular tyrosine phosphorylation in 3T3-F442A preadipocytes. Biochem. J. 284, 649-^52. Argetsinger, L. S., Campbell, G. S., Yang, X., Witthuhn, B. A., Silvennoinen, O., Ihle, J. N., & Carter-Su, C. (1993). Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase. Cell 74, 237-244. Argetsinger, L. S., Hsu, G. W., Myers, M. G., Jr., Billestrup, N., Norstedt, G., White, M. R, & Carter-Su, C. (1995). Growth hormone, interferon-gamma, and leukemia inhibitory factor promoted tyrosyl phosphorylation of insulin receptor substrate-1. J. Biol. Chem., manuscript submitted. Argetsinger, L. S., Myers, M. G., Jr., Billestrup, N., Norstedt, G., White, M. R, & Carter-Su, C. (1996). Growth hormone, interferon-gamma, and leukemia inhibitory factor promoted tyrosyl phosphorylation in insulin receptor substrate-2, submitted. Asakawa, K., Hedo, J. A., McEldufif, A., Rouiller, D. G., Waters, M. J., & Gorden, R (1986). The human growth hormone receptor of cultured human lymphocytes. Biochem. J. 238, 379-386.

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EPIDERMAL GROWTH FACTOR: CELLULAR AND MOLECULAR FUNCTION

Douglas K. Tadaki and Salil K. Niyogi

Abstract I. Historical Perspective , . II. Biological Activity A. Developmental Biology B. Wound Healing III. Gene Organization of EGF IV. EGF Precursor V. Primary Sequences of EGF Family Members VI. Solution Structure of EGF VII. EGF Receptor VIII. Mechanism of Receptor Activation IX. Gene Expression in Response to EGF X. MolecularNatureofEGF-Receptor Interaction XI. Determining Receptor Affinity, Kinase Stimulation, Gross Structure, and Mitogenicity of EGF Mutant Proteins XII. Effects of Single-Site EGF Mutations on Receptor-Ligand Association . . . . A. Alteration of Structural Residues

Growth Factors and Cytokines in Health and Disease Volume lA, pages 85-121. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0091-4 85

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B. Substitution of Hydrophobic Residues C. Substitution of Aromatic Residues D. Mutation of Polar Residues E. Substitution of Acidic Residues F. Replacement and/or Alteration of Basic Residues XIII. Cumulative Effect of Multiple Mutations on Receptor Binding XIV. Concluding Remarks Acknowledgments References

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ABSTRACT Epidermal growth factor (EGF) is the prototypical member of a large family of potent cell mitogens. EGF acts by binding with high affinity to its specific cell-surface receptor, the EGF receptor (EGFR), thereby, stimulating the intrinsic protein-tyrosine kinase activity of the receptor. This, in turn, initiates a signal transduction cascade that ultimately leads to DNA synthesis and cell proliferation. A variety of biochemical changes occurs within the cell in response to the signal cascade; these changes include a rise in intracellular calcium levels, increased glycolysis and protein synthesis, and increases in the expression of certain genes including the gene for EGFR (for reviews on the above see Carpenter and Cohen, 1990; Ullrich and Schlessinger, 1990; Carpenter and Wahl, 1990). EGF is a bridge that connects several realms of biology, from molecular biology to cUnical medicine and from normal cell function to carcinogenesis. The current research on EGF is aimed at understanding how it interacts with the EGFR and analyzing the many different branches of the EGF-induced signal transduction pathway. In this treatise, we have attempted to discuss the biological and molecular consequences of EGF action. Recent studies have brought to light several new and exciting observations covering all aspects of signal transduction and receptor-ligand interactions. Understanding the pathways of signal transduction has elucidated the biological action of EGF. Structure-function studies of the EGF ligand have facilitated a detailed analysis of the formation of a stable receptor-ligand complex and possible understanding of the mechanics of receptor activation that leads to signal transduction.

I. HISTORICAL PERSPECTIVE EOF was first found as a contaminant in crude preparations of nerve growth factor. It displayed remarkable activities distinct from those of nerve growth factor. These included precocious eyelid opening and early tooth eruption in neonatal mice (Cohen, 1960; Levi-Montalcini and Cohen, 1960). In 1962, Stanley Cohen isolated this factor and dubbed it "tooth-lid factor" (Cohen, 1962). The name was later changed to "epidermal growth factor" after it was observed that this factor promoted

Epidermal Growth Factor

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hEGF 1 mEGF 1 rEGF 1 gpEGF 1 hTGFo 1 rTGFa 1 hAR 41 HBEGF 30 SDGF 116 VGF 38 MGF 30 SFGF 30 consensus

NSDSECPLSHDGYCLHDGVCMYIEAL DKYACNCWGYIGERCQYRDLKWWELR NSYPGCPSSYDGYCLNGGVCMHIESL DSYTCNCVIGYSGDRCQTRDLRWWELR NSNTGCPPSYDGYCLNGGVCMHIESV DRYVCNCVIGYIGERCQHRDLR QDAPGCPPSHDGYCLHGGVCMHIESL NTYACNCVIGYVGERCEHQDLDLWE WSFHNDCPDSHTQFCFH-GTCRFLVQE DKPACVCHSGYVGARCEHADLLA WSFHNKCPDSHTDYCFH-GTCRFLVQE EKPACVCHSGYVGVRCEHADLLA ..-KKKNPCNAEFQNFCIH-GECKYIEHL EAVTCKCQQEYFGERCGEK ...KKRDPCLRKYKDFCIH-GECKYVKEL RAPSCICHPGYHGERCHGLSLPVEN... .RKKKKNPCAAKFQNFCIH-GECRYIENL EWTCHCHQDYFGERCGEKTMKTQKK .DIPAIRLCGPEGDGYCLH-GDCIHARDI DGMYCRCSHGYTGIRCQHWLVDYQ .IIKRIKLCNDDYKNYCLNNGTCFTVALNNVSLNPFCACHINYVGSRCQFINLITIK .IVLHVKVCNHDYENYCLNNGTCFTIALDNVSITPFCVCRINYEGSRCQFINLVTY -V Cp--y--yClh-G-C-yi--l d C-C--gY-GeRCqh-dl

rNDF HRG-a HRG-Pl HRG-p2 HRG-p3 Pro-ARIA

.GTSHLIKCAEKEKTFCVNGGECFTVKDLSNPSRYLCKCQPGFTGARCTENVPMASKVQT... -GTSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCQPGFTGARCTEhfVPMKVQNQE... -GTSHLIKCAEKEKTFCVNGGECFTVKDLSNPSRYLCKCPNEFTGDRCQNYV-MASFYKH... .GTSHLIKCAEKEKTFCVNGGECFTVKDLSNPSRYLCKCPNEFTGDRCQNYV-MASFYK-... .GTSHLIKCAEKEKTFCVNGGECFTVKDLSNPSRYLCKCPNEFTGDRCQNYV-MASFYST... -GTSHLTKCDIKQKAFCVNGGECYMVKDLPNPPRYLCRCPNEFTGDRCQNYV-MASFYKH...

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Figure 1. Comparison of amino acid sequences of EGF and Heregulin families of ligands. The members of the EGF family are human EGF (hEGF), mouse EGF (mEGF), rat EGF (rEGF), guinea pig EGF (gpEGF), human TGFa (hTGFa), rat TGFa (rTGFa), human amphiregulin (hAR), heparin-binding EGF (HBEGF), schwanoma-derived growth factor (SDGF), vaccinia virus growth factor (VGF), myxoma virus growth factor (MGF), and shope-fibroma growth factor (SFGF). The members of the heregulin family are rat neu differentiation factor (rNDF), heregulin a (HRG-a), heregulin p i , p2, and p3 (HRG-pi, HRG-P2, HRG-p3), and acetylcholine receptor inducing activity (proARIA). The consensus for the EGF family shows all of the conserved residues in capitals, the partially conserved residues in lower case, and the conserved cysteines in bold.

the growth and keratinization of the epidermis (Cohen, 1964). Savage and coworkers were able to isolate and determine the primary sequence of EGF and map the locations of the disulfide bonds (Savage et al., 1972,1973). This work enabled the comparison of urogastrone, a protein isolated from the urine of pregnant women with potent inhibitory activity on gastric acid secretion, to mouse EGF (mEGF) and showed that they were homologous proteins (Gregory, 1975). These investigations eventually led to the isolation of EGF from a variety of other animal species including rat, guinea pig, and dog (Simpson et al., 1985; Kobayashi et al., 1985). In addition to the EGFs, other members of this expanding family have been identified (Figure 1). These include transforming growth factor alpha (TGFa) (Anzano et al., 1983; Marquardt et al., 1983, 1984), amphiregulin (Shoyab et al., 1988, 1989), the pox virus family of growth factors (Todaro and De Larco, 1976; De Larco and Todaro, 1978; Blomquist et al., 1984; Brown et al., 1985; Reisener, 1985), and heparin-binding EGF (HB-EGF) (Higashiyama et al., 1991). These proteins all share a similar primary structure, disulfide linkages, can bind to the EGFR, and have similar biological activities.

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II. BIOLOGICAL ACTIVITY The biological effects of EOF have been a major focus of research ever since its discovery. Several laboratories are examining the in vivo activities of EOF in development and the use of EOF as a therapeutic agent in treating a variety of epithelial traumas. A. Developmental Biology

The role of EOF in development has been of continued interest because of early experiments showing its effects on epithelial growth and differentiation in newborn mice (Cohen, 1962). The effects of growth factors inpreimplantation embryos have been characterized by several groups (see Adamson, 1994, for a review). Although EOF has not been detected at any stage of preimplantation embryos in mice (Rappolee et al., 1988), TGFa and EGFR mRNAs have been detected as early as the one-cell stage (Rappolee et al., 1988; Wiley et al., 1992). The early expression of TGFa and EGFR may be important in the growth and differentiation of the embryo. It has also been suggested that the presence of EGFR on the outer surface of the blastocyst may aid in the implantation of the embryo by interacting with TGFa on the uterine wall in mice (Paria et al., 1993). Other members of the EGF family, EGF, amphiregulin and HB-EGF, are also produced by the uterus at periimplantation and are implicated in implantation of the trophoblast (Cross et al., 1994). The role of EGF in organ development has been examined in several laboratories. EGF is synthesized by a number of tissues of mesenchymal/epithelial origin, for example, sweat glands, salivary glands, breast tissue, and Brunner's glands. Evidence from /« vitro studies suggests that EGF has a role in the development of such tissues (Sundell et al., 1980; Coleman et al., 1988; Blecher et al., 1990). B. Wound Healing

It has been shown that binding and activation of EGFR induces a variety of physiological responses which produce certain biological effects in vivo, such as angiogenesis and keratinocyte growth and differentiation (Cohen and Elliott, 1963; Rheinwald and Green, 1977; Gospodarowicz et al., 1979; Schreiber et al., 1986). Investigating the effects of EGF/TGFa in their roles as mediators of tissue regeneration has been of interest. In addition to the atigiogenic and proliferative ability of these growth factors, it has also been shown that EGF has a chemotactic activity for inducing migration of fibroblasts into the wound area (Sturrock et al., 1989). Currently, EGF is being studied as a treatment of several types of wounds, including wounds to corneal, epidermal, connective tissue, and gastric ulcers (reviewed in Schultzetal., 1991). The ability of EGF to stimulate the proliferation of the corneal epithelium has been explored as a possible treatment of corneal trauma. Two groups have demonstrated that eye drops containing EGF can accelerate corneal epithelial healing in

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primates and humans (Daniele et al, 1979; Brightwell et al., 1985). Cell culture studies have shown that EGF can stimulate DNA synthesis in corneal fibroblasts (Woost et al., 1985). Another study demonstrated that corneal endothelial cells which maintain corneal clarity, although having a low capacity to divide in vivo, can be stimulated to proliferate in culture, thereby, offering a method of regenerating this important cell layer (Couch et al., 1987). It is known that EGF can inhibit gastric acid secretion (Bower et al., 1975; Finke et al., 1985; Dembinski et al., 1986; Shaw et al., 1987) and increase the proliferation of cells in the gastrointestinal tract (Scheving et al., 1979, 1980; Johnson and Guthrie, 1980; Dembinski et al., 1982; Chabot et al, 1983; Ulshen et al., 1986; Finney et al., 1987; Jacobs et al., 1988). These studies have led to the investigation of EGF as a treatment for gastric ulcers by lowering gastric acidity and promoting cell growth. A study using rats with chronic gastroduodenal ulcers showed accelerated healing with EGF treatment (Konturek et al., 1988). It has also been suggested that EGF may act as a cytoprotectant by inducing the production of prostaglandins and stimulation of DNA synthesis in the mucosal cells (Konturek et al., 1981). Induction of other factors, such as somatostatin, by EGF has also been implicated in cytoprotection (Olsen et al., 1984; Sakamoto et al., 1985). The effect of EGF on epidermal proliferation was the hallmark experiment in the initial characterization of this growth factor. Early studies on the use of EGF to promote repair of second-degree scald bums in rats and suction bullae in humans showed no significant increases in the rates of healing (Thornton et al., 1981; Arturson, 1984). It has been demonstrated that fibroblasts in culture require continued exposure to EGF for 6-12 hours to stimulate DNA synthesis (Aharonov et al., 1978). All of the early in vivo repair studies were done with brief applications of EGF. This may explain why no effect was seen in vivo. To test this hypothesis, one group used implants of sponges that continuously released EGF at the site of the injury. The results showed a significant increase in the formation of granulation tissue with the sustained release of EGF (Buckley et al., 1985). Compounding of EGF or TGFa in lanolin or silvadene cream to maintain exposure to EGF at the wound site showed promising increases in tissue regeneration of partial thickness bums and dermatome injury in pigs and partial thickness dermatome injury in humans (Brown et al., 1986, 1989; Schultz et al., 1987; Nanney, 1990). These results show the clinical importance of EGF/TGFa in the treatment of epithelial trauma.

III. GENE ORGANIZATION OF EGF EGF is first synthesized as a large (-1200 amino acids) transmembrane glycoprotein precursor (PreproEGF). The gene encoding the EGF precursor is located on chromosome 4 in humans and chromosome 3 in mice (Brissenden et al., 1984; Zabel et al., 1985; Morton et al., 1986). The human EGF (hEGF) gene consists of 24 exons and 23 introns spanning 120 kilobases. The exons range in size from 79 to 1062

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bp, the two largest being the first and the last exons at 578 and 1062 bp, respectively. The introns range in size from 0.4 to 27 kb and all the introns interrupt the coding sequence. Each of the 8 EGF-like units is coded by an individual exon (exons 6-9, 15, 17-19) and the segment, that codes for mature EGF, is made up of 2 exons (exons 20 and 21) (Bell et al., 1986). Interestingly, the splice junction between exons 20 and 21 is located within the codon for asparagine 32 of the mature EGF peptide, and this residue is important in serving as a "hinge" between the N-terminal and the C-terminal domains of the EGF protein.

IV. EGF PRECURSOR The EGF precursor contains nine EGF-like repeats. Only the one closest to the transmembrane domain forms the mature growth factor. Most of the ligands in the EGF family are produced as membrane-bound precursors (for a review see Massague and Pandiella, 1993). EGFs of different organisms are the only members in this family of growth factors possessing multiple EGF-like units. All other members of this family possess only a single EGF-like unit. Upon reaching the cell surface, PreproEGF is processed by proteolytic cleavage to generate a soluble factor (ProEGF) approximate in size to the membrane-bound form (Mroczkowski et al., 1988). Both PreproEGF and ProEGF have the ability to interact with the EGFR and activate the signal transduction cascade (Mroczkowski et al., 1989). Besides the mitogenic function of the mature protein, there is no known function for the EGF precursor or the other eight EGF-like repeats. There are several groups currently attempting to ascertain a possible biological role for the EGF precursor. One study has implicated the EGF precursor in the differentiation of stratified squamous epithelia independent of EGFR due to the expression of the former in the nonproliferating cell layers of vaginal epithelia, whereas the expression of the EGFR is in the basal cell layer (Sakai et al., 1994). An error in the production of the soluble 165-kDa EGF precursor has been suggested as the cause of a form of congenital polycystic kidney disease (Lakshmanan and Fisher, 1993). Cystic kidney diseases are usually associated with a high incidence of urinary tract infection. Therefore, this group has also suggested that the EGF precursor may function as a pathogenesis-related protein similar to the Tamm—Horsfall protein, a urinary glycoprotein which possesses both antibacterial and antiviral properties. Both proteins possess multiple EGF-like repeats and they are secreted by the same renal tubule cells.

V. PRIMARY SEQUENCES OF EGF FAMILY MEMBERS All members of the EGF family of proteins can be aligned by their six conserved cysteine residues. Another criterion for inclusion in this family is the ability to bind to the EGFR. The comparison of primary amino acid sequences shows approximately 20% sequence identity and 40% functional homology between family members (Figure 1). Most of the sequence identity resides within the C-terminal

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portion of the molecule, but, upon closer scrutiny, a conservation of similar residues within the N-terminal region becomes apparent. In addition, the sequences of several peptide ligands belonging to the Heregulin/NDF (neu differentiation factor) family of growth factors, each containing a functional EGF-like domain, but having affmity for the HER3/c-erbB3 and HER4/c-erbB4 receptors (close relatives of EGFR), have also been determined (Holmes et al, 1992; Wen et al., 1992, 1994; Peles et al, 1992, 1993; Tzahar et al, 1994) (Figure 1). The ligand(s) for HER2/cerbB2/Neu has not been identified. The conservation of sequence among the different species of EGF, in comparison with sequences of both related growth factors and unrelated EGF-like sequences, provides some indication of the degree to which specific residues in the protein are required for structure and/or function. The importance of some residues is easily recognized, such as the highly conserved cysteines and glycines, which enable the protein to assume its stable native tertiary structure. The importance of other residues is often more difficult to resolve from sequence conservation alone. Aside from the six cysteine residues, a high degree of conservation is observed for EGF residues Pro7, HislO, Tyrl3, Leul5, Glyl8, Gly36, Tyr37, Gly39, Arg41, and Leu47, implicating these sites as targets for mutagenesis. Cautious interpretation of information from sequence conservation, combined with judicious use of the predicted EGF structure, have facilitated a directed approach to the analysis of EGF structure/fiinction.

VI. SOLUTION STRUCTURE OF EGF Models of EGF structure derived from 2-D NMR (nuclear magnetic resonance) data(Montelioneetal., 1987,1992; Cooke etal., 1987;Kohdaetal., 1988;Hommel et al., 1992) predict that the protein is composed of two slightly overlapping structural domains (Figure 2). The N-terminal domain (Asnl to Val35) contains a major antiparallel p-sheet structure (residues 19-31). The C-terminal domain (Ala30 to Arg53) contains a double hairpin structure as well as a minor antiparallel P-sheet between residues 37—38 and 44-45. NMR and NOE studies (Mayo et al., 1986) predict a clustering of the aromatic groups in aqueous solution, thereby, providing a hydrophobic surface on the protein molecule which, in concert with the physical constraints imposed by the three intramolecular disulfide bonds, functions to establish an extremely stable protein structure (Holladay et al, 1976). The models for EGF structure portray the growth-factor peptide with its two separate domains in a fixed position, relative to each other. However, these independent reports describe EGF models that differ somewhat in the relative orientations of the two domains. The degree of flexibility and the level of dynamic motion of the individual domains and of the entire molecule have been examined and indicate a significant degree of motion involving each of the various subdomains of the molecule (Ikura and Go, 1993).

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Figure 2. Solution structure of EOF. This model was generated from NMR coordinates provided by Dr. G. T. Montelione (Rutgers University) using MOLSCRIPT (Kraulis, 1991). The arrows indicate p-sheet secondary structures.

VII. EGF RECEPTOR EGFR is a 170-kDa membrane glycoprotein consisting of a large extracellular ligand-binding domain, a single transmembrane domain, and an intracellular protein-tyrosine kinase domain. This receptor is a member of the type I subclass of tyrosine kinase receptors, whose other members include the HER2/Neu/c-erbB2 proto-oncogene, and HER3/c-erbB3 and HER4/c-erbB4 receptors. The extracellular domain can be further divided into four contiguous subdomains numbered 1,2, 3, and 4 from the N-terminal to the C-terminal portion. Subdomains 2 and 4 are cysteine-rich regions that share some homology. Several studies have utilized chemical cross-linking agents to covalently attach the EGF ligand to the EGFR to identify the ligand-binding site. One study used the cross-linking agent disuccinimidyl suberate (DSS) to identify a region of EGFR corresponding to domain 3 as a possible site for ligand binding (Lax et al., 1988). The deletion or exchange of large segments of the human and avian receptors generates receptors with altered affinity for grovv1:h factor and supports the evidence that domain 3 participates in receptor-ligand interactions (Lax et al., 1989). A 40-kDa fragment, isolated from domain 3 of the EGFR extracellular domain by limited proteolysis, binds to the EGF-related ligand, TGFa, about 1/100th as

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strongly as does the intact EGFR (Kohda et al., 1993). This fragment is currently being used in the structural determination of the ligand-binding site by NMR (Kohda et al., 1993). Another group, also using chemical cross-linking agents, has narrowed that region to a 47 amino acid interval flanked by Phe321 and Glu367 (Wuetal., 1990). The evidences above suggest that the major surface of interaction with the ligand occurs in domain 3 of the receptor extracellular domain. However, a study (Woltjer et al., 1992a), using step-wise affinity cross-linking with the heterobifunctional reagent sulfo-N-succinimidyl 4-(fluorosulfonyl)benzoate, has identified a fragment in domain 1 and a single residue, TyrlOl, within thefi-agmentthat is the point cross-linking to the N-terminally modified mEGF (Woltjer et al., 1992b). This suggests that domain 1 may have some role in ligand binding. Recent characterization of a naturally occurring mutation in subdomain 4 of EGFR, in which a lysine is substituted for an arginine at position 497, shows altered binding for TGFa but not for EGF (Moriai et al., 1994). Therefore, it is possible that the cysteine-rich domains may confer some specificity to the different ligands of the EGF family. Upon ligand binding, a signal is transmitted through the receptor from the extracellular milieu to the intracellular environment. The intracellular signal is initiated as an activation of the receptor's intrinsic tyrosine kinase and phosphorylation of a variety of cellular substrates including autophosphorylation of the receptor (for a review, see Fantl et al., 1993). The major interaction between the receptor kinase and the substrates is mediated mainly through SH2 (src homology 2) domains. The individual SH2 domains on the substrates interact with certain phosphotyrosine residues on the receptor providing a receptor-specific signal in terms of which substrates are activated (Koch et al., 1991; Heldin, 1991; Margolis, 1992). Several intracellular substrates for the EGFR have been identified. Phospholipase C-y has been shown to be tyrosine phosphorylated in an EGF-dependent manner (Margolis et al., 1989, 1990; Wahl et al., 1989). Phospholipase C-y hydrolyzes phosphoinositol-4,5-bisphosphate to produce inositol-1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). In turn, IP3 and DAG act by increasing free intracellular Ca^"^ and stimulating protein kinase C, respectively. Phosphatidylinositol 3-kinase (PI3K) phosphorylates the D-3 position of inositol to form phosphatidylinositol-3-phosphate. The p85 subunit, believed to be the regulatory subunit of this enzyme, has been found to be tyrosine phosphorylated in EGF-stimulated cells (Otsu et al., 1991). Recently, a protein termed GRB2 (growth factor receptor bound 2) has been identified in humans (Lowenstein et al., 1992). GRB2 is an adaptor protein that links the receptor to a nucleotide exchange factor, termed Son of Sevenless (SOS) of the Ras pathway (Egan et al., 1993; Li et al, 1993; Rozakis-Adcock et al., 1993; Buday and Downward, 1993). Ras acts upon the kinase cascade involving Raf, mitogen-activated protein kinase kinase (MAPKK), and mitogen-activated protein kinase (MAPK) (Satoh et al., 1992). Lipocortin-1 is heavily phosphorylated upon EGF stimulation and is believed to play a role in

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regulating prostaglandin synthesis by inhibiting phospholipase A2 (Flower and Blackwell, 1979; Blackwell et al., 1980; Hirata et al, 1980).

Vm. MECHANISM OF RECEPTOR ACTIVATION The activation of the receptor's intrinsic kinase activity by the physical association of the ligand is a process not clearly understood. One thought on the mechanism of activation of the receptor tyrosine kinase is the formation of the receptor-ligand complex leading to a ligand-induced conformational change in the receptor. This change is initially in the extracellular domain (Greenfield et al, 1989) and subsequently transmitted to the kinase domain through the single transmembrane region of the receptor protein. The precise structural differences between the latent inactive receptor and the activated receptor-ligand complex have not been established despite extensive attempts to elucidate these alterations using a variety of physical and chemical techniques. Several studies have correlated receptor activation with receptor dimerization (Defize et al., 1986; Schlessinger, 1986, 1988a, 1988b; Yarden and Schlessinger 1987a, 1987b; Yarden and Ullrich, 1988; Kashles et al, 1991; Spaargaren et al., 1992; Spivak-Kroizman et al., 1992). Others have proposed the idea of kinase activation by the dissociation of latent receptor dimers to active receptor monomers (Biswas et al., 1985) or via an intramolecular mechanism without receptor dimerization (Koland and Cerione, 1988; Northwood and Davies, 1988; Cadena et al., 1994). Several studies show that EGFR has increased susceptibility to covalent cross-linking in the presence of the EGF ligand (Fanger et al., 1986, 1989; Cochet et al., 1988; Wada et al., 1990; Lax et al., 1991; Canals 1992), suggesting increased receptor-^-eceptor interaction induced by EGF. The nature and specificity of receptor—receptor interactions have not been established. Formation of the catalytically active receptor-ligand complex requires the proper interaction of the ligand, the extracellular domain of the receptor, and their solvent environment. A recent study has utilized a receptor mutant with a cysteine insertion between residues 618 and 619 in the extracellular juxtamembrane region in an attempt to characterize the dimeric form of the EGFR (Sorokin et al., 1994). Their study suggests that the dimeric form is the high-affinity state of the receptor and that the dimer possesses the active kinase. The significance of the ligand-dependent regulatory influence imposed by the receptor's extracellular domain over the tyrosine-kinase domain was established by the characterization of the w-erbB gene product, a constitutively activated version of EGFR that lacks an extracellular domain and is a member of the src family of oncogenic proteins (Downward et al., 1984). The critical importance of growth-factor control over the receptor kinase activity is exhibited by the serious consequences of unregulated cell growth observed upon the loss of receptor kinase regulatory control. A breakdown in receptor kinase control can occur by several different mechanisms. Mutation and/or overexpression of various components of

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the growth factor-receptor signaling pathway, which includes growth factors, EGFR and the related c-erbB2/neu proto-oncogene, have been correlated with unregulated cell growth (DeLarco and Todaro, 1978; Libermann et al., 1985; Goodwin et al., 1986; Di Fiore et al., 1987; Slamon et al., 1989).

IX. GENE EXPRESSION IN RESPONSE TO EGF The stimulation of gene expression within the nucleus is a major effect of the EGF-induced signal-transduction cascade. Increases in the levels of mRNA have been detected for many genes in response to EGF, including those for prolactin, gastrin, EGFR, c-Fos, c-Jun, and c-Myc (for a review, see Carpenter and Wahl, 1991). The transcriptional regulation of the genes for prolactin and gastrin has been studied in detail. Both of these genes possess a distinct c/5-acting EGF response element. The EGF response element for prolactin is sensitive to protein kinase C activation (Elsholtz et al., 1986); therefore, it is not clear whether the increase in prolactin mRNA is mediated via the phospholipase C pathway. TPA and the calcium ionophore, A23187, can increase the levels of prolactin mRNA (Murdoch et al., 1985). The EGF response element for gastrin is GC-rich and mediates only a weak transcriptional activation by TPA (Merchant et al., 1991). The EGFR mRNA is increased 3-5 times after 2-4 hours of EGF stimulation (Earp et al., 1986). This suggests that the EGFR is an inducible gene that is "upregulated" by EGF stimulation. A 36-base pair proximal element which responds to EGF, phorbol ester, and cAMP stimulation has been identified (Hudson et al., 1990). Experiments using actinomycin D to block transcription have shown that EGFR mRNA has a relatively short half-life and that EGF may be modulating the stability of the message (Clark et al., 1985; Jinno et al., 1988). The c-jun, c-myc, and c-fos genes are heavily regulated at the transcriptional and posttranscriptional levels. EGF increases expression of all three of these protooncogenes (Quantin and Breathnach, 1988; Heldin and Westermark, 1988). c-jun is a proto-oncogene that encodes the AP-1 transcription factor. The c-fos protooncogene encodes a nuclear transcription factor that can complex with c-Jun. This complex, in turn, can bind to the AP-1 enhancer element, which has the sequence TGANTCA, and regulate gene expression. The c-myc proto-oncogene product complexes with another transcription factor called Max. This dimer binds a specific DNA sequence through a basic-helix-loop-helix-leucine zipper motif The oncogenicity of c-Myc requires a complex with Max (Amati et al., 1993). There is no evidence, however, which suggests that EGF can affect the expression of Max. There are multiple EGF-dependent signals which increase the expression of these proto-oncogenes. There is evidence suggesting the involvement of protein kinase C (Greenberg et al., 1985), increased intracellular Ca^"" (Bravo et al., 1985, 1987) and increased cAMP levels (Ran et al., 1986) in stimulating the expression of c-fos and c-myc.

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All of the pathways for the activation ofc-fos, c-Jun, and c-myc have been the end result of a complicated series of events within the EGF-induced signal transduction cascade. Recently, a more "direct pathway" to the nucleus has been elucidated. This "direct pathway" involves the proteins known as STATs (signal transducers and activators of transcription). There are currently 5 members in the STAT family of transcription factors: STAT l a is p91, STATip is p84, a splice variant of p91, STAT2 is pi 13, STAT3 is p92, and STAT4 is p89 (Ihle et al., 1994; Zhong et al, 1994a). Evidence from several laboratories suggests that STAT la, STATip and STATS are directly phosphorylated by the EGFR and, then, translocated to the nucleus (Ruff-Jamison etal., 1993; Silvennoinenetal., 1993; Sadowskietal., 1993; Zhong et al., 1994b). One study has demonstrated the association of STAT l a with EGFR and activation of the c-fos gene promoter (Fu and Zhang, 1993). These studies are important because they provide a pathway for activation of immediate responses to EGF-induced stimulation of the cell.

X. MOLECULAR NATURE OF EGF-RECEPTOR INTERACTION The identification of amino acid residues in EGF critical for high-affinity association with the receptor and, thus, leading to receptor kinase activation, is the necessary first step toward understanding the nature of EGF-stimulated signal transduction. Several approaches have been used to study the molecular nature of EGF-receptor interaction. Domain replacement and proteolytic digestion were used in studies of the N- and C-terminal portions of EGF. These studies showed that residues 1—5 and 48-53 were not required for receptor binding (Savage et al., 1972; Cohen etal., 1975; Matrisian etal, 1982; Burgess etal, 1982,1983,1988; Gregory et al., 1988). The examination of synthetic peptides of EGF showed that peptides corresponding to Cys20-^Cys31 (no disulfide), Tyr-14->Cys31 (one disulfide), and Leul5->Arg53 (two disulfides) have very minor activity, approximately, 0.01% of full-length wild type EGF (Komoriya et al., 1984; Heath and Merrifield, 1986) suggesting the need for all three disulfide loops for significant biological activity. The above studies provided a broad analysis of EGF but a more detailed study was needed to gain a true understanding of EGF-receptor interactions. Site-directed mutagenesis provided a method of scrutinizing individual amino acid residues of EGF to understand their roles in receptor binding and activation. The various codon changes in the EGF and TGFa proteins have been introduced by oligonucleotidedirected mutagenesis (Zoller and Smith, 1983; Botstein and Shortle, 1985; Engler et al., 1988), PCR mutagenesis (Helmsley et al., 1989; Tadaki and Niyogi, 1993), or cassette mutagenesis (Wells et al., 1985; Campion et al., 1993a). In addition, modifications of wild-type and mutant EGF proteins with specific chemical reagents have enhanced several mutagenesis studies by providing an even greater range of protein alterations for analyzing EGF structure and fiinction.

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XL DETERMINING RECEPTOR AFFINITY, KINASE STIMULATION, GROSS STRUCTURE, AND MITOGENICITY OF EGF MUTANT PROTEINS The receptor-binding affinity has been measured on whole cells (Carpenter, 1985; Koide et al., 1988) or with membrane fractions of cells which overexpress the EGFR (Engler et al., 1988). The basis for the many variations of this assay is the competition of a fixed concentration of radiolabeled EGF with increasing concentrations of unlabeled mutant protein for the receptor. The concentration of protein required to displace 50% of ^^^I-EGF is determined for the wild-type and for each mutant EGF analogue. Direct comparison of these values provides a simple means of assessing the relative affinity of each EGF mutant with respect to the wild-type EGF protein and is a valuable measure of the importance of individual amino acid residues in receptor binding. The ability of an EGF analogue to stimulate the receptor tyrosine kinase activity is a measure of its relative agonist activity. A comparison of the concentration of the mutant relative to that of wild-type EGF, required to activate the receptor kinase, also, provides a reliable means of assessing the relative receptor affinity of EGF variants. The stimulation of the EGFR's tyrosine kinase activity is evaluated by measuring the phosphorylation of a synthetic (G\u^,TyY^\ substrate (Akiyama et al., 1985; Engler et al, 1988), using solubilized and lectin-purified EGFRs from A431 cells (Akiyama et al., 1985; Engler et al., 1988). The ability of exogenously added EGF to stimulate receptor kinase activity is rather sensitive to differences in assay conditions, particularly with respect to detergent concentration, ionic strength and metal ion cofactors like Mg^"^ and Mn^"^ in the incubation buffer (Koland and Cerione, 1988). It is important to identify structural differences that might account for the observed changes in growth factor activity. The structure of mutant EGF analogues has been examined at several levels. On a gross scale, the ability to isolate functional EGF protein from E. coli extracts requires that the molecule be processed and folded into the native EGF structure during expression of the recombinant EGF gene product. We have observed that the high performance liquid chromatography (HPLC) elution profile during purification of hEGF proteins is a sensitive indicator of altered conformation. Deviation from the normal EGF molecular folding motif results in protein molecules with significantly altered behavior during purification by reverse-phase HPLC and readily permits identification of non-native EGF proteins. More direct comparisons of the structures of wild-type and selected EGF mutant proteins have been made in attempts to identify differences in protein structure at the molecular level by spectroscopic methods including circular dichroism (CD) (Tadaki and Niyogi, 1993) and ^H NMR (Moy et al, 1989; Campion et al., 1990; Dudgeon et al., 1990; Engler et al., 1990; Matsunami et al., 1991; Hommel et al., 1991; Koide et al., 1992a, 1992b; Campion et al, 1993b). These studies indicate

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that, for the most part and despite possible subtle stuctural changes throughout the EOF molecule, the decrease in receptor binding affinity of the EOF mutants is due to the effect of local changes in the interactions of the EOF molecule with the solvent and/or the receptor. Further structural analysis of EOF mutants, currently underway, is expected to reveal greater detail about the interactions responsible for receptor-ligand association and kinase activation. The mitogenic potential of various hEGF mutants was assessed by their stimulation of DNA synthesis in EGF-responsive cells (Engleretal., 1990,1991,1992). The incorporation of [-^Hlthymidine into acid-insoluble material was used as a measure of DNA synthesis. The stimulation of thymidine incorporation, relative to wild-type EGF, as a function of the concentration of mutant EGF analogues was used a measure of their mitogenicity. Although mitogenesis is a late event in signal transduction, the mitogenic potential of each EGF mutant tested reflects its relative receptor affmity

Xll. EFFECTS OF SINGLE-SITE EGF MUTATIONS ON RECEPTOR-LIGAND ASSOCIATION The effects of mutagenesis and/or chemical modification on the activity of EGF are summarized here and discussed with regard to their sequence conservation and the relative importance of each class of amino acid side-chain functional group. The mutations, relative binding affinities, and relative agonist activities are listed in Table 1. A. Alteration of Structural Residues

Proline, glycine, and cysteine residues are unique in their respective abilities to induce, accommodate, and maintain bond angles and protein conformations not allowable with any other amino acid. The conservation of a proline residue adjacent to the first cysteine residue in EGF and TGFa suggested a potential role for this residue in establishing some critical feature of the native EGF structure. However, substitution of hEGF residue Pro? with threonine resulted in only a slight decrease in receptor affinity (Engler et al., 1988). The structural requirement for repeated tight turns in the C-terminal domain of EGF may be met by residues Gly36 and Gly39. The requirement for each of these residues was evaluated by substitution with valine and leucine, respectively. Introduction of these side-chains resulted in an apparent inability of EGF to fold into its native structure and, therefore, lowered receptor affinity (our unpublished observations). Substitution of any or all of the absolutely conserved cysteine residues of EGF or TGFa results in complete loss of function (Defeo-Jones et al., 1988).

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Table 1. Properties of EGF Analogues

Growth Factor Species

Relative Binding Affinity

Relative Agonist Activity

Reference

Structural residues (hEGF)

(hEGF)

(mEGF)

Pro7->Thr Gly39->Leu Leul5->Tq5 Val Ala Arg Vall9^Gly Met21->Leu Thr Ile23-^Thr Ala Ile23-^Leu Val Phe Trp Ala Asp Leu26^^Ala Gly Val34,35->Ala Ile38^Leu Leu47->Ile His Pro Ala Gly Asp Arg Leu47->Val Ala Glu Asp Leu47^Val Ser

55 53 4 n/a Hydrophobic residues 26 18 2.7 1.6 58 100 36 3 19 61 45 22 11 6 0.1 48 5 23 77 17 7.2 2.3 1.8 1.0 0.09 0.06 14 2.5 2.2 1.9 33 14

n/a n/a n/a n/a 35 n/a 19 4 n/a n/a n/a n/a n/a n/a n/a 10 5 n/a n/a 16.1 9.1 3.4 2.9 2.7 n/a n/a n/a n/a n/a n/a n/a n/a

(Engler et al., 1988)

(*) (Nandagopal et al., 1994)

(Campion et al., 1990) (Sumietal., 1985) (Campion et al., 1990) (Campion et al., 1993) (Koideetal., 1992a)

(Campion et al., 1993;*) (Campion et al., 1990)

(*) (*) (Matsunami et al., 1991)

(Dudgeon et al., 1990)

(Rayetal., 1988) (Rayetal, 1988; Moyetal., 1989) {continued)

DOUGLAS K. TADAKI and SALIL K. NIYOGI

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Table 1, (Continued)

Growth Factor Species

Relative Binding Affinity

Relative Agonist Activity

Reference

Aromatic residues (hEGF)

(hEGF)

Tyrl3-^Phe Leu He Val His Arg Ala Gly Tyrl3->Leu Tyr22->Asp Lys Trp Pro Phe Leu Ala Tyr29->Gly Phe Leu Ala Lys Pro Tyr37-^Phe His Ser Ala Asp Arg Gly

97 78 22 20 16 6 3 0.3 202 8 120 106 85 75 70 56 17 64 56 50 30 16 126 74 62 39 26 10 7 Polar residues

80 75 7 7 2 5 2 n/a n/a 25 57 113 87 82 67 61 n/a 86 63 60 49 n/a 113 65 62 41 25 8.8 5.7

Asn32^Lys Trp Gly Asp Pro His Val Phe Asp Gln43'->Lys

110 100 35 25 <0.02 78 46 29 7 100

n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a

(Tadaki and Niyogi, 1993)

(Hommel et al., 1991) (Campion et al., 1990) (Tadaki et al., 1994)

(Engleretal, 1988) (Tadaki et al., 1994)

(Engleretal., 1990, 1991)

(Campion et al., 1993b)

(Koideetal., 1992b)

(Campion et al., 1992) (continued)

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Table 1, (Continued)

Growth Factor Species

Relative Binding Affinity

Relative Agonist Activity

Reference

Acidic residues (hEGF)

86 48 30 25 23 23 14 4 Basic residues

Glu24-^Gly Asp27->Gly Glu40->Asp Gin Ala Asp46->Ala Tyr Arg

84 71 n/a n/a n/a n/a n/a n/a

(Engler et al, 1988) (Campion et al., 1992)

(Campion et al., 1990) 95 79 180 188 (Engler et al., 1990, 1992) n/a 0.40 n/a 0.20 n/a 0.15 n/a 0.15 n/a 0.15 n/a 0.05 n/a 0.01 n/a 0.01 n/a 0.4 (Hommel et al., 1991) 0.2 n/a n/a 0.1 n/a (Campion et al., 1992) 100 n/a 120 (*) Chemical modifications of basic residues

(hEGF)

Lys28^'Leu Arg Arg41->Lys Gin He Tyr Gly Ala Asp Glu Arg41^Gln His Leu Arg45->Lys Lys48->Arg

(hEGF)

Mutant Ly s41 -^homoarginine Mutant Lys41 ->lysine-amidine Native Lys28^homocitrulline Native Lys48^homocitrulline Mutant Lys45->homocitrulline

100 3.5

95

(Engler et al., 1992)

n/a

100

n/a

100

n/a

100

n/a

(Campion etal., 1992)

{continued)

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Table 1. (Continued)

Growth Factor Species

Relative Binding Affinity

Relative Agonist Activity

Reference

Double-Site mutations (hEGF)

Tyrl3->His/Leu47^Ala Tyrl3-^His/Ile23->Thr Tyr22^Asp/Leu47-^Ala Ile23->Thr/Leu47^Ala Leu26^Gly/Leu47->Ala Ile23-^Ala/Leu26->Ala Lys28^Arg/Lys48->Arg Leu26-Gly/Asn32-Asp Leu47-Ala/Asn32-Asp

0.20 0.18 0.04 0.05 0.05 11 200 2 0.5

nidi

n/a n/a n/a n/a n/a n/a n/a n/a

(Campion et al., 1993a)

(*) (Campion et al., 1993b)

Notes: (*) Unpublished results n/a - not available

B. Substitution of Hydrophobic Residues

In initial mutagenesis studies, the highly conserved Leu47 in the C-terminal domain of hEGF was found to be very important for receptor-ligand association (Engler et al., 1988). The specific requirement for leucine at position 47 appears to be more than a simple necessity for a nonpolar side chain at this site in the molecule. Strong conservation of this leucine residue, specifically among the high-affinity receptor-binding growth factor sequences, suggested that some property unique to the Leu47 side-chain was required. Replacement of Leu47 in hEGF with isoleucine, having a similar chemical character, reduced receptor affinity to 17 percent relative to wild type and confirmed the stringent requirement for the Leu47 side chain for optimal activity (Matsunami et al., 1991). Substitution of Leu47 in either hEGF (Dudgeon et al., 1990; Matsunami et al, 1991) or mEGF (Ray et al, 1988; Ohgai et al., 1989) with a wide spectrum of side-chain functional groups resulted in decreases in relative receptor affinity to 1/5 to 1/2000 of the wild type. The relative mitogenic activity, in general, paralleled the loss in receptor affinity. Substitution with ionic residues led to the most drastic reduction in biological activity (Matsunami etal, 1991). Structural analyses of Leu47 mutants by NMR indicated minimal alterations in protein conformation; rather, the decreased affinities are probably due to disruption of a direct interaction of Leu47 with the aqueous solvent and/or the receptor (Moy et al., 1989; Dudgeon et al., 1990; Matsunami et al, 1991). The growth factor amphiregulin, which contains an EGF-like domain, but lacks the hydrophobic Leu47 found in the C-terminal domain of EGF and TGFa, binds to the EGFR with

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an affinity 1/lOth that of wild-type EGF (Shoyab et al, 1989). However, it is not clear whether the EGFR is the physiological receptor for amphiregulin. As for the N-terminal domain, a cursory examination of the EGF molecule by NMR initially suggested a predominantly structural role for the large p-sheet in the N-terminal domain, acting as a backbone for the EGF protein (Campbell et al., 1989). This concept was further reinforced by the sequence homology data, which suggested that the more highly conserved C-terminal domain is likely the primary molecular determinant involved in forming the active EGF-receptor complex. A closer examination of the EGF residues 19-32 in the N-terminal domain, that are involved in forming the antiparallel P-sheet conformation, revealed that amino acid residues are locked into positions either above or below the plane of the p-sheet due to intramolecular hydrogen bonding of the corresponding peptide backbone. Amino acid side chains on one face of the P-sheet, including two tyrosines, appear to be engaged in intramolecular interactions with other residues in the protein (residues 6 to 13). The clustering of the aromatic side-chains in aqueous solution (Mayo et al., 1986), coupled with the physical constraints imposed by the three internal disulfide bonds, leads to the formation of a very stable protein structure (Holladay et al., 1976) that permits a group of hydrophobic residues to remain in a conformation relatively exposed to the aqueous solvent environment. It is also interesting to note that hydrophobic side-chains are conserved at EGF positions 19, 21, 23, and 26, within the large P-sheet of the N-terminal domain, suggesting possible functional roles for these sites. Similar to results obtained with the C-terminal Leu47, mutation of hEGF residues Ile23 and Leu26, located in the antiparallel P-sheet of the N-terminal domain, showed dramatic decreases in receptor-binding affinity without significant changes in EGF conformation, as revealed by NMR measurements (Campion et al., 1990). A recent study evaluating various substitutions at the Ile23 site, has further demonstrated the importance of this nonpolar side-chain for receptor—ligand association (Koide et al., 1992a). Here again, structural analysis of the mutant proteins by NMR indicated only minor perturbations insufficient to account for the dramatic loss of biological activity (Koide et al., 1992a). Mutation of other hydrophobic residues, namely Val 19, Met21, and Ala25, in the P-sheet, also, led to moderate decreases in receptor affinity (Campion et al., 1990). Studies replacing hydrophobic residues outside the P-sheet, namely Val34, Val35, and Leul 5 of hEGF, have also demonstrated a general requirement for the presence of hydrophobic side-chains on the surface of EGF (our unpublished observations). A recent study of Leu 15 has demonstrated a requirement for a hydrophobic side-chain at this site (Nandagopal et al., 1994). Two-dimensional NMR shows that the EGF structure is not significantly altered, indicating a functional role for Leul 5 (Nandagopal, unpublished observations). (An additional requirement for hydrophobic interaction(s) involving the aromatic side-chain of Tyrl3 is discussed in the next section). It is clear that the major forces involved in receptor-ligand binding

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are hydrophobic interactions of nonpolar, aliphatic amino acid side-chains at critical sites throughout the EOF molecule. In several instances, the decreased receptor binding, observed with hEGF mutants involving critical hydrophobic residues Leul5, Ile23, Leu26, and Leu47, is accompanied by a decreased ability to activate fully the receptor tyrosine-kinase activity, and such mutants act as partial antagonists of the EGF-dependent receptor kinase activity (Matsunami et al, 1990; unpublished results). The identification of those sites within the receptor's extracellular ligand-binding domain, which interact with these important sites on the ligand, may provide some insight into the initial steps of the mechanism of receptor activation. C. Substitution of Aromatic Residues As observed among homologous proteins, the aromatic character of specific sites in the EGF family of proteins is often retained by the presence of alternate residues having aromaticity (i.e., tyrosine, phenylalanine, histidine, and tryptophan). The various EGF species contain no phenylalanine, whereas TGFa contains one or more phenylalanines. All members of the EGF family contain a variable number of tyrosine, histidine, and tryptophan residues. Functional conservation of aromatic character at positions His 10, Tyr 13, His 16, Tyr22, Tyr29, Tyr3 7, and Tyr44 of hEGF appears to be substantial, with complete conservation of the phenolic tyrosine side-chain at position 37. The potential importance of aromatic residues in EGF was also implicated by NMR and NOE studies (Mayo et al., 1986). These studies predicted a clustering of the aromatic side chains on the surface of the protein and suggested that the aromatic residues might be involved in ligand-receptor interactions by providing a hydrophobic surface on the EGF protein. In our earlier studies (Engler et al., 1988; Campion et al., 1990), the individual replacement of Tyr22 and Tyr29 with aspartate and glycine led to decreased receptor affinity lowered to 8 and 17 percent, respectively. In our recent studies, replacement of either of these tyrosine residues with apparently less disruptive (as evident from "normal" elution profiles on reverse-phase HPLC) side-chains including phenylalanine, leucine, and alanine, resulted in only minor decreases in receptor affinity. The Y22W mutant displayed no loss of receptor affinity and the Y22K analogue had an affinity slightly higher than that of wild type (-120%). Interestingly, the Y22P mutant retained -70% receptor affinity, while the Y29P analogue had only 16% of wild-type hEGF activity. The above resuhs, together with computer modeling analysis of hEGF based on NMR coordinates, suggest that Tyr29 probably plays a role in maintaining the native structure of EGF; certain mutations at this site can cause sufficient structural alterations to reduce receptor affinity. Computer modeling also indicates that Tyr22 is located within a pocket of acidic residues, that is, Asp3, Glu5, Glu24, and Asp27. This suggests that the considerably lower receptor affinity of the Y22D mutant is due to local structural perturbations caused by charge repulsion between an existing

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electronegative group(s) and the electronegative aspartate side-chain at position 22. The higher receptor affinity of the Y22K analogue is probably due to the stabilization induced by ion-pairing between lysine at position 22 and an electronegative group in the pocket. In TGFa position 29 is a proline, yet a proline substitution in hEGF leads to a significant reduction in binding affinity suggesting a difference in the way these ligands interact with the receptor or in intramolecular interactions to provide the correct conformation for binding. The importance of the highly conserved tyrosine at position 13 in receptor—ligand association was suggested from NMR studies (Kohda et al., 1988; Montelione et al., 1992) that predicted its close proximity to Arg41, which plays a critical role in the binding of EGF and TGFa to the EGFR, and those that indicated its possible role in providing a hydrophobic surface on the EGF molecule (Mayo et al., 1986). Site-directed mutagenetic studies show that aromaticity at position 13 is not critical for overall binding to the receptor, because the aromatic tyrosine residue can be adequately replaced by an aliphatic leucine residue (Hommel et al., 1991; Tadaki and Niyogi, 1993). The hydrophobic nature of this site appears to be the functional characteristic required to form a stable ligand-^eceptor complex, because substitution with smaller, less hydrophobic or electrostatic residues resulted in significant losses in receptor affinity (Tadaki and Niyogi, 1993). CD spectral analysis of several hEGF mutants (Tadaki and Niyogi, 1993) and an NMR study of the hEGF Leu 13 mutant (Hommel et al., 1991) showed no major structural alterations. The results indicate that the Tyr 13 side-chain plays a critical functional role in receptor binding by contributing to hydrophobic ligand-receptor interactions. Similar to Tyr 13 in hEGF, the mutation of the hTGFa equivalent, Phel5, to alanine resulted in substantially reduced receptor affinity (Defeo-Jones et al., 1988). Substitution of the hEGF residue Tyr37, which is completely conserved among the EGF family, by a variety of amino acids indicated that neither an aromatic nor an aliphatic group is essential at this site for EGF's biological activity (Engler et al., 1990,1991). The corresponding residue, Tyr38, in TGFa appears to be considerably more important for the biological activity of this growth factor (Defeo-Jones et al., 1988). For example, substitution of Tyr38 in hTGFa with alanine decreased relative receptor affinity to 1/30, whereas the equivalent substitution of Tyr37 in hEGF decreased relative receptor affinity to 1/3 (Engler et al., 1990, 1991). The reason(s) for the drastic difference in the effects of mutating the highly conserved amino acid residues Tyr 13 (Phe 15 in TGFa) and Tyr3 7 (Tyr3 8 in TGFa) is not clear at this time. It is possible that Tyr37, as well as Tyr22 and Tyr29, might participate in some function common to EGF-like proteins but unrelated to receptor recognition and high-affinity binding. The high degree of conservation of these aromatic residues remains an intriguing subject. D. Mutation of Polar Residues The highly conserved Asn32, located between Cys31 and Cys33, resides in the "hinge" region of EGF and separates the N- and C-terminal motifs of the EGF

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molecule. Aside from its potential role in receptor—ligand interaction, its unique location suggests a possible role in maintaining the native EOF conformation. Several hEGF analogues were generated at position 32 (Koide et al., 1992b; Campion et al., 1993b). Substitution of the relatively small, neutral, polar Asn32 with the larger and electrostatically charged lysine or the bulky aromatic tryptophan side chain, had no effect on receptor binding affinity, suggesting a fairly high degree of tolerance for replacements at this site (Campion et al., 1993b). Removal of the Asn32 side-chain by substituting glycine resulted in a decrease to 35% in relative receptor affinity, and replacement with aspartate decreased it to 25%. However, no binding of the Pro32 mutant could be detected by radioreceptor competition. NMR analysis indicated gross structural perturbation for the Pro32 analogue. In contrast, the Lys32 and Asp32 mutants exhibited spectra similar to native wild-type EGF. These results suggest the importance of hydrogen-bond donor functionality of the residue at position 32 in forming a fully competent receptor-binding epitope. A similar conclusion was reached independently (Koide et al., 1992b) in studies combining mutagenesis and NMR analysis. The Val32, Phe32, and Asp32 analogues had relative receptor affinities of 46, 29, and 7 percent, respectively, while exhibiting NMR spectra similar to that of wild type. The close proximity of Gln43 to the essential residue Arg41 and the potential for interaction of these side-chains makes Gln43 an attractive target for site-directed mutagenesis. However, the replacement of the neutral, polar Gln43 side chain with the positive lysine amine had no effect on receptor affinity (Campion et al., 1992). E. Substitution of Acidic Residues

Asp 11, Glu24, Asp27, Glu40, and Asp46 are conserved within the various species of EGF. Four acidic residues in hEGF have been substituted with amino acids having nonelectrostatic side-chains. The removal of the side-chain at either Glu24 or Asp27 by replacement with glycine led to little or no decrease in receptor affinity, indicating that these acidic residues are not involved in receptor binding (Engler et al., 1988). The replacement of Glu40 by aspartate, which retains the electrostatic charge while shortening the side-chain by one methylene group, reduced the binding affinity to 30% (Campion et al, 1992). The Glu40->Gln and Glu40^Ala mutations, both of which remove the electrostatic charge, decreased receptor affinity to approximately 25%. This suggests that Glu40 may not be involved in direct receptor interaction because substitutions, which drastically alter the sidechain functionality, do not lower the binding affinity beyond the loss seen with the conservative aspartate mutation. Glu40 may play a role in the stability of the native conformation of hEGF (Campion et al., 1992). The most highly conserved of the acidic residues is Asp46, the substitution of which has led to the most significant decreases in receptor affinity. Replacement with alanine and tyrosine resulted in a decrease in receptor affinity to 23 and 14 percent, respectively (Campion et al., 1992). Replacement of Asp46 with arginine introduced a side-chain with an electrostatic charge opposite in polarity to the native

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aspartate, establishing adjacent positive charges (Arg45-Arg46) that resulted in affinity decreased to 4 percent relative to wild type. The results indicate that Asp46 may be involved in short-distance electrostatic interactions in the sequestered ligand-receptor complex (Campion et al., 1992). Computer models suggest a possible divalent metal-binding pocket comprised of Asp3, Glu5, Glu24, and Asp27. Preliminary evidence from electron spin resonance (ESR) and NMR studies seem to support this hypothesis (Tadaki, Campion, and Niyogi, unpublished data). F. Replacement and/or Alteration of Basic Residues

Human EOF contains five positively charged amino acids (two lysines and three arginines) concentrated predominantly in the C-terminal domain of the protein. Substitution of Lys28, located within a strongly hydrophobic region of the N-terminal domain, with the uncharged hydrophobic leucine had little effect on receptor binding (Campion et al., 1990). Eliminating the charged e-amino groups of Lys28 and Lys48, along with the N-terminal a-amine, by reacting with potassium cyanate converted lysine to uncharged polar homocitruUine with no effect on receptor binding (Campion et al., 1992). The positively charged residue Arg45 in the C-terminal domain was modified by a combination of site-directed mutagenesis and chemical modification. The charge-conservative substitution of Arg45 by lysine followed by conversion to the neutral homocitruUine derivative by reaction with potassium cyanate had no effect on binding affinity (Campion et al., 1992). Examination of sequence conservation data suggested the importance of Arg41, which is retained in all EOF and EGF-like proteins known to bind to the EGFR. Replacement of Arg41 with lysine, which retains the positive charge, reduced the receptor affinity to only 0.4 percent of wild type (Engler et al, 1990). Substitutions which changed the side-chain functionality, by either altering or removing the charge or changing the size of the side-chain, decreased binding affinity even further. Substitution of a homologous residue in hTGFa with lysine also showed a drastic loss in receptor binding affinity (Defeo-Jones et al, 1988). Chemical modification of Lys41 was used to determine what functional characteristic of arginine was required at this site to restore full or partial activity. Methyl acetimidate modifies primary amines to amidines; in the case of lysine, to form lysine-amidine. Lysine-amidine is approximately the same size as arginine and possesses a positive charge, but one of the guanidium nitrogens is replaced by a methyl group. Omethylisourea modifies lysine to homoarginine which restores the guanidinium moiety but is one methylene group longer. The lysine-amidine modification partially restored receptor binding to 3.5% of wild type, whereas the homoarginine modification fully restored receptor affinity (Engler et al., 1992). The stimulation of receptor tyrosine kinase activity, which could not be reliably measured for the Lys41 mutant, was also fully restored to wild-type activity with homoarginine 41 (Engler et al., 1992). The results demonstrate that electrostatic charge alone is not

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sufficient for binding and establish a clear requirement for the guanidinium functional group of the side-chain at position 41 in optimal receptor-4igand interaction.

Xm. CUMULATIVE EFFECT OF MULTIPLE MUTATIONS ON RECEPTOR BINDING As described above, individual mutation of several important EOF residues including Tyrl3, Leul5, Ile23, or Leu26 in the N-terminal domain, or of the highly conserved Arg41 or Leu47 in the C-terminal domain of hEGF, decreases receptor affinity. Computer modeling (D. K. Tadaki, S. R. Campion, and S. K. Niyogi, unpublished observations) based on NMR data indicate that these residues, which probably serve as "contact points" in the interaction of EGF with its receptor, are all located on one face of the EGF molecule in three distinct clusters (Figure 3). Having identified most of the important sites in studies individually replacing single amino acids, two related questions about the potential interaction of these important sites located in different regions of the molecule can be asked. First, does any single-site mutation disrupt the interactions of the receptor with one or more of the other important sites on the ligand, or are the structural effects of any LEU26

LEU47

Figure 3. Computer model of EGF generated from NMR coordinates provided by Dr. G. T. Montelione (Rutgers University) using MOLSCRIPT (Kraulis, 1991). The residues important for receptor binding (Tyr13, Leu15, Ile23, Leu26, Arg41, and Leu47) are depicted in the ball and stick form.

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individual mutation limited, reducing affinity by directly affecting only the region of the mutated site(s)? Secondly, does the association of any single region of the EGF molecule with the receptor influence, by cooperative binding, the subsequent association of any other region of the molecule? These questions can be answered by evaluating the effect(s) of simultaneous mutation of two hEGF residues in various combinations at locations throughout the molecule including most of the amino acids of known importance. Single-site EGF mutations Tyrl3^His, Tyr22^Asp, Ile23-^Thr, Ile23-^Ala, Leu26->Gly, Leu26->Ala, Asn32-»Asp, Arg41->Lys, and Leu47-^Ala were combined in a variety of ways to produce double-mutant gene products with alterations either within the same domain or in separate domains of the EGF molecule (Campion et al., 1993). The relative receptor-affinity values determined for the double-site hEGF analogues are given in Table 1, showing the relationship of the double-mutant proteins relative to each of the corresponding single-site parent mutations. The effect of simultaneous mutation on receptor affinity, in nearly all cases, indicated that mutation at any one site does not substantially alter the effect of mutation at the second site. The cumulative effect of double mutation is the product of the two individual parent mutations. This finding confirms the importance of these individual residues in receptor binding suggesting that each of these separate sites functions essentially independently in the interaction of the EGF molecule with its receptor. Consequently, the overall high affinity of EGF-receptor binding is the result of the cumulative interaction of these individual sites of receptor—ligand interactions.

XIV. CONCLUDING REMARKS We have attempted to discuss the past and present research of the cellular and molecular nature and action of EGF. EGF-induced signal transduction regulates the growth and differentiation of cells. The mechanisms of this cascade are current areas of vigorous research with more pathways being analyzed and defined. To activate the receptor's intrinsic tyrosine kinase, the interaction between EGF and EGFR must be understood. The mechanism for the ligand binding of the receptor has been deciphered, but the forces and interactions necessary for transducing the signal across the cell membrane and activating of the tyrosine kinase have not been elucidated. The detailed studies of receptor—ligand interaction suggest that the complex formation is mediated through five hydrophobic residues, Tyrl3, Leul5, Ile23, Leu26, and Leu47, and one electrostatic residue, Arg41. A study using double-site mutations demonstrate that these residues interact independently of one another and are probably individual contact points for the receptor-binding epitope. The elucidation of the STAT proteins provides a mechanism for the rapid changes in gene expression in response to ligand-induced receptor activation. These obser-

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vations demonstrate that inducing cell proliferation by growth factors is a multifold process where regulation of gene expression also occurs in response to immediate activation by the STAT family. The association of Src homology 2 (SH2) domains with phosphotyrosine residues provides a mechanism for specific substrate interactions with receptor tyrosine kinases and provides continued stimulation by a complex signal-transduction cascade. Future areas of research will probably focus on the ligand precursors for members of the EOF family, the differences in the interaction of the various EOF family members with their cognate receptors, the mechanism of interactions among the EGFR family, and the way all of these relate to human health and disease.

ACKNOWLEDGMENTS Supported by the Office of Health and Environmental Research, United States Department of Energy under contract No. DE-AC05-960R22464 with the Lockheed Martin Energy Systems, Inc. We would like to thank Krishnadas Nandagopal and Stephen R. Campion for insightful discussions. D.K. Tadaki was partially supported by NSF Grant BCS-91-11940 awarded to D. A. Lauffenburger, University of Illinois.

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Scheving, L. A., Yeh, Y. C, Tsai. T. H., & Scheving, L. E. (1980). Circadian phase-dependent stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the duodenum, jejunum, illeum, caecum, colon, and rectum of the adult male mouse. Endocrinology 106, 1498-1503. Schlessinger, J. (1986). Allosteric regulation of the epidermal growth factor receptor kinase. J. Cell Biol. 103, 2067-2072. Schlessinger, J. (1988a). The epidermal growth factor receptor as a multifunctional allosteric protein. Biochemistry 27, 3119-3123. Schlessinger, J. (1988b). Signal transduction by allosteric receptor oligomerization. Trends Biol. Sci. 13,445-447. Schlessinger, J., & Ullrich, A. (1992). Growth factor signalling by receptor tyrosine kinases. Neuron 9, 383-391. Schreiber, A. B., Winkler, M. E., & Derynck, R. (1986). Transforming growth factor-a: A more potent angiogenic mediator than epidermal growth factor. Science 232, 1250-1253. Schultz, G. S., White, M., Mitchell, R., Brown, G., Lynch, J., Twardzik, D. R., & Todaro, G. J. (1987). Epithelial wound healing enhanced by transforming growth factor-alpha and vaccinia growth factor. Science 235, 350-352. Schultz, G., Rotatori, D. S., & Clark, W. (1991). EGF and TGF-a in wound healing and repair. J. Cell. Biochem. 45, 346-352. Shaw, G. R, Hatt, J. F., Anderson, N. G., & Hanson, R J. (1987). Action of epidermal growth factor on acid secretion by isolated rat parietal cells. Biochem. J. 107, 429-436. Shoyab, M., McDonald, V. L., Bradley, J. G., & Todaro, G. J. (1988). Amphiregulin: A bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7. Proc. Natl. Acad. Sci. USA 85, 6528-6532. Shoyab, M., Plowman, G. D., McDonald, V. L., Bradley, J. G., & Todaro, G. J. (1989). Structure and function of human amphiregulin: A member of the epidermal growth factor family. Science 243, 1074^1076. Shum, L., Reeves, S. A., Kuo, A. C, Fromer, E. S., & Derynck, R. (1994). Association of the transmembrane TGF-a precursor with a protein kinase complex. J. Cell Biol. 125, 903-916. Silvennoinen, O., Schindler, C, Schlessinger, J., & Levy, D. E. (1993). Ras-independent growth factor signaling by transcription factor tyrosine phosphorylation. Science 261, 1736-1739. Simpson, R. J., Smith, J. A., Moritz, R. L., O'Hara, M. R., Rudland, P S., Morrison, J. R., Lloyd, C. J., Grego, B., Burgess, A. W., & Nice, E. C. (1985). Rat epidermal growth factor: Complete amino acid sequence. Homology with the corresponding murine and human proteins; isolation of a form truncated at both ends with full in vivo biological activity. Eur. J. Biochem, 153, 629-637. Sinha, N. D., Biemat, J., McManus, J., & Koster, H. (1984). Polymer support oligonucleotide synthesis XVII: Use of P-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucl. Acids Res. 2,4539-4557. Slamon, D. J., Godolphin, W, Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A., & Press, M. F. (1989). Studies of the W^x-llneu proto-oncogene in breast and ovarian cancer. Science 244, 707-712. Sorokin, A., Lemmon, M. A., Ullrich, A., & Schlessinger, J. (1994). Stabilization of an active dimeric form of the epidermal growth factor receptor by introduction of an interreceptor disulfide bond. J. Biol. Chem. 269, 9752-9759. Spaargaren, M., Defize, L. H. K., Boonstraa, J., & De Laat, S. W (1991). Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase. J. Biol. Chem. 266, 1733-1739. Spivak-Kroizman, T., Rotin, D., Pinchasi, D., Ullrich, A., Schlessinger, J., & Lax, I. (1992). Heterodimerization of c-erbB2 with different epidermal growth factor receptor mutants elicits stimulatory or inhibitory responses. J. Biol. Chem. 267, 8056-8063.

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Sturrock, A. B., Woodward, S. C, Senior, R. M., Griffin, G. L., Klagsbrun, M., & Davidson, J. M. (1989). Differential stimulation of collagenase and chemotactic activity in fibroblasts derived from rat wound repair tissue and human skin by growth factors. J. Cell. Physiol. 138, 70-78. Sumi, S., Akira, H., Shintaro, Y, Kenichi, M., Atsushi, K., Shizutoshi, N., & Masanori, S. (1985). Overproduction of human epidermal growth factor/urogastrone in Escherichia coli and demonstration of its full biological activities. J. Biotechnol. 2, 59-74. Sundell, H., Gray, M. E., Serenius, F. S., Escobedo, M. B., & Stahlman, M. T. (1980). Effects of epidermal growth factor on lung maturation in fetal lamb. Am. J. Pathol. 100, 707—726. Tadaki, D. K., &. Niyogi, S. K. (1993). Functional importance of hydrophobicity of the tyrosine at position 13 of human epidermal growth factor in receptor binding. J. Biol. Chem. 268, 1011410119. Tadaki, D. K., Campion, S. R., & Niyogi, S. K. (1994). Analysis of the tyrosine residues at position 22 and 29 of human epidermal growth factor (hEGF) by site-directed mutagenesis. FASEB J. 8, A1459. Thornton, J. W., Hess, C. A., Cassingham, V., & Bartlett, R. H. (1981). Epidermal growth factor in healing of secorid degree bums: A controlled animal study. Bums 8, 156-160. Todaro, G. J., & De Larco J. E. (1976). Transformation by murine and feline sarcoma vimses specifically blocks binding of epidermal growth factor to cells. Nature 264, 26-31. Tzahar, E., Levkowitz, G., Kamnagaran, D., Yi, L., Peles, E., Lavi, S., Chang, D., Liu, N., Yayon, A., Wen, D., & Yarden, Y. (1994). ErbB-3 and erb-B4 function as the respective low and high affinity receptors of all neu differentiation factor/heregulin isoforms. J. Biol. Chem. 269,25226-25233. Ullrich, A., & Schlessinger, J. (1990). Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203-212. Ulshen, M. H., Lyn-Coo, L. E., & Raasch, R. H. (1986). Effects of intraluminal epidermal growth factor on mucosal proliferation in the small intestine of adult rats. Gastroenterology 91, 1134—1140. Wada, T., Qian, X., & Greene, M. I. (1990). Intermolecular association of the pi 85"^" protein and EGF receptor modulates EGF receptor function. Cell 61, 1339-1347. Wahl, M. I., Nishibe, S., Pann-Ghill, S., Rhee, S. G., & Carpenter, G. (1989). Epidermal growth factor stimulates tyrosine phosphorylation of phospholipase C-II independently of receptor intemalization and extracellular calcium. Proc. Natl. Acad. Sci. USA 86, 1568-1572. Wells, J. A., Vasser, M., & Powers, D. B. (1985). Cassette mutagenesis: An efficient method for generation of multiple mutations at defined sites. Gene 34, 315-323. Wen, D., Peles, E., Cupples, R., Suggs, S. V., Bacus, S. S., Luo, Y, Trail, G., Hu, S., Silbiger, M., Ben-Levy, R., Koski, R. A., Lu, H. S., & Yarden, Y (1992). Neu differentiation factor: A transmembrane glycoprotein containing an EGF domain and an immunoglobulin homology unit. Cell 69, 559-572. Wen, D., Suggs, S. V., Kamnagaran, D., Liu, N., Cupples, R. L., Luo, Y, Janssen, A. M., Ben-Bamch, N., Trollinger, D. B., Jacobsen, V. L., Meng, S. Y., Lu, H. S., Chang, D., Yang, W, Yanagihara, D., Koski, R. A., & Yarden, Y (1994). Stmctural and functional aspects of the multiplicity of neu differentiation factors. Mol. Cell. Biol. 14, 1909-1919. Wiley, L. M., Wu, J. X., Harari, I., & Adamson, E. D. (1992). Epidermal growth factor receptor mRNA and protein increase after the four-cell preimplantation stage in murine development. Dev. Biol. 149,247-260. Woljter, R. L., Lukas, T. J., & Staros, J. V. (1992a). Direct identification of residues of the epidermal growth factor receptor in close proximity to the amino terminus of bound epidermal growth factor. Proc. Natl. Acad. Sci. USA 89, 7801-7805. Woljter, R. L., Weclas-Henderson, L., Papayannopoulos, L A., & Staros, J. V. (1992b). High-yield covalent attachment of epidermal growth factor to its receptor by kinetically controlled, stepwise affinity cross-linking. Biochemistry 31, 7341-7346. Woost, P G., Brightwell, J., Eiferman, R. A., & Schultz, G. S. (1985). Effect of growth factors with dexamethasone on healing of rabbit comeal stromal incisions. Exp. Eye Res. 40,47-60.

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Wu, D., Wang, L., Chi, Y., Sato, G. H., & Sato, J. D. (1990). Human epidermal growth factor receptor residue covalently cross-linked to epidermal growth factor. Proc. Natl. Acad. Sci. USA 87, 3151-3155. Yarden, Y, & Schlessinger, J. (1987a). Self-phosphorylation of epidermal growth factor receptor: Evidence for a model of intermolecular allosteric activation. Biochemistry 26, 1434—1442. Yarden, Y, & Schlessinger, J. (1987b). Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor. Biochemistry 26, 1443-1451. Yarden, Y, & Ullrich, A. (1988). Molecular analysis of signal transduction by growth factors. Biochemistry 27, 3113-3119. Zabel, B. U., Eddy, R. L., Lalley, P. A., Scott, J., Bell, G. I., & Shows, T. B. (1985). Chromosomal locations of the human and mouse genes for precursors of epidermal growth factor and the p subunit of nerve growth factor. Proc. Natl. Acad. Sci. USA 82,469-473. Zhong, Z., Wen, Z., & Darnell, J. E. (1994a). Stat3 and Stat4: Members of the family of signal transducers and activators of transcription. Proc. Natl. Acad. Sci. USA 91,4806-4810. Zhong, Z., Wen, Z., & Darnell, J. E. (1994b). Stat3: A STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 264, 95—98. Zoller, M. J., & Smith, M. (1983). Oligonucleotide-directed mutagenesis of DNA fragments cloned into Ml3 vectors. Methods Enzymol. 100, 468-500.

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PLATELET-DERIVED GROWTH FACTOR

Carl-Henrik Heldin, Arne Ostman, and Bengt Westermark

Abstract I. Introduction II. Structure of PDGF A. Isoforms of PDGF B. PDGF Genes C. Sequence of PDGF D. Three-Dimensional Structure of PDGF E. Receptor Binding Epitopes F. Receptor Binding Specificity III. Effects ofPDGF on Cells in Culture IV. Production of PDGF V. In P7vo Function of PDGF A. Embryogenesis B. Wound Healing C. Inhibition of Platelet Aggregation VI. PDGF in Disease A. Malignancies

Growth Factors and Cytokines in Health and Disease Volume lA, pages 123-145. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0091-4 123

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B. Atherosclerosis C. Fibrosis D. Glomerulonephritis VII. PDGF Antagonists VIII. Future Perspectives Acknowledgments References

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ABSTRACT Platelet-derived growth factor (PDGF) has mitogenic or trophic effects on fibroblasts, smooth muscle cells, glial cells, capillary endothelial cells, and neurons. Structurally, PDGF is a family of disulfide-bonded dimers of different combinations of A- and B-polypeptide chains. The PDGF isoforms are stored in the a-granules of platelets and are also produced by a number of other cell types; they exert their cellular effects by binding with different affinities to two related tyrosine kinase receptors. PDGF has a functional role during embryonal development, and also stimulates wound healing in the adult. Another important function of PDGF may be to regulate platelet aggregability. Overactivity of PDGF may be part of the development of several disorders characterized by excessive cell growth, for example, malignancies, atherosclerosis, fibrotic conditions, and glomerulonephritis.

I. INTRODUCTION Platelet-derived grov^th factor (PDGF) is a major mitogen for connective tissue cells. It was originally purified from human platelets (Antoniades et al., 1979; Deuel et al., 1981; Heldin et al., 1979; Raines and Ross, 1982), but has more recently been found to be produced by a number of different cell types (for reviews, see Heldin and Westermark, 1990; Raines et al., 1990). Recent studies have also shown that the target cell spectrum for PDGF is broader than initially anticipated; in addition to the traditional target cells, fibroblasts, smooth muscle cells and glial cells, PDGF also acts on, for example, capillary endothelial cells, mesangial cells, mesothelial cells, and neurons. The localization of PDGF and its target cell specificity suggest that it has functional roles during embryonal development and wound healing in the adult. Available information supports the notion that the effect of PDGF is local, through autocrine or paracrine stimulation. PDGF has also been implicated in certain pathological conditions. The development of certain tumors may involve autocrine or paracrine stimulation by PDGF. Overactivity of PDGF may also be part of the development of certain nonmalignant diseases involving excessive cell proliferation, such as atherosclerosis, rheumatoid arthritis, glomerulonephritis, and fibrotic conditions.

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The present review will focus on the structure of PDGF and its normal function as well as possible involvement in disease. We will also discuss the design and possible clinical use of PDGF antagonists.

11. STRUCTURE OF PDGF A. Isoforms of PDGF

PDGF is a family of isoforms consisting of disulfide-bonded dimers of A- and B-polypeptide chains. The heterodimer, PDGF-AB, is the most common isoform in human platelets, but homodimers also occur (Hammacher et al., 1988; Hart et al., 1990; Soma et al., 1992). Among other cell types, there are examples of cells, which make both A- and B-chains and, thus, produce all isoforms, and of cell types which make only A- or B-chains and, thus, produce only the corresponding homodimer. The different isoforms have overlapping but distinct cellular effects, because they bind with different affinities to two different receptors (see further below). B. PDGF Genes

The human genes for the A- and B-chains of PDGF are localized on chromosomes 7 and 22, respectively (Dalla Favera et al., 1982; Stenman et al., 1992; Swan

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Figure 1, Sequence of PDGF. The sequences of the long and short versions of the PDGF A-chain and B-chain, as deduced from cDNA clones (Betsholtz et al., 1986b; Josephs et al., 1984), are depicted. Processing sites for the removal of the hydrophobic signal sequences, as well as for the removal of N- and C-terminal propeptides, are indicated by arrows. Cysteine residues are indicated by dots and amino acid residues identical in the A- and B-chains are indicated by asterisks. The localization of intron/exon borders are also indicated (▼), as well as the numbers of the exons.

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et al., 1982). They are structurally organized in a similar manner with seven exons each. In each case exon 1 encodes the signal sequence, exons 2 and 3 the N-terminal sequences that are removed during processing, and exons 4 and 5 most of the mature protein (Bonthron et al., 1988; Johnsson et al., 1984; Rorsman et al., 1988) (Figure 1). Exon 7 is mainly noncoding, and exon 6 of the B-chain gene encodes a C-terminal sequence which may be removed during processing. Interestingly, the A-chain gene occurs as two splice variants, that is, with or without exon 6. The form without exon 6 encodes the more common short variant of the PDGF A-chain. Exon 6 contains a 10 amino acid basic motif homologous to a sequence within the C-terminal prepart of the B-chain, which mediates binding to components intracellularly, and in the extracellular matrix (see further below). C. Sequence of PDGF

The B-chain is synthesized as a 241 amino acid primary translational product including a hydrophobic signal sequence (Josephs et al, 1984). After processing in the N- and C-terminals, a mature product of 109 amino acid residues is formed (Figure 1). The primary translational products of the short and long forms of the A-chain are 196 and 211 amino acid residues, respectively, which, after N-terminal processing, is converted to mature proteins of 109 and 124 amino acid residues, respectively, (Figure 1) (Betsholtz et al., 1986b). The amino acid similarity between the mature parts of the A- and B-chains is about 60%, with a perfect conservation of the eight cysteine residues. Similar spacing of cysteine residues is also seen in two other growth factors, vascular endothelial cell growth factor (Keck et al., 1989; Leung et al., 1989) and placenta growth factor (Maglione et al., 1991). D. Three-Dimensional Structure of PDGF

Of the eight cysteine residues in the PDGF chains, the second and fourth from the N-terminus form interchain disulfide bonds; the disulfide bonds bridge the second cysteine residue in one chain to the fourth in the other, and vice versa, giving rise to an antiparallel arrangement of the two subunits in the dimer (Andersson et al., 1992; Jaumann et al., 1991). The three intracellular disulfide bonds have also been localized; the first cysteine from the N-terminus bridges with the sixth, the third with the seventh, and the fifth with the eighth (Haniu et al., 1993; Ostman et al, 1993). The assignments of the disulfide bonds are confirmed by the crystallographic structure of PDGF-BB, which has been determined at 3.0 A resolution (Oefner et al., 1992). The structure of the molecule is an unusual one with the three intrachain disulfide bonds forming a knot-like structure in one end of each subunit. The major part of the molecule consists of two twisted p-sheets which end in two loops pointing in one direction (loop 1 and 3); in the other end of the molecule a short region (loop 2) connects the two P-sheets. Thus, loops 1 and 3 from one subunit will be close to loop 2 of the other subunit in the dimer (Figure 2).

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Figure 2. Schematic illustration of the three-dimensional structure of PDGF-BB. The molecule is an antiparallel disulfide-bonded dimer. The folding of the polypeptide backbone is illustrated with a solid line and disulfide bonds by hatched lines. The receptor binding epitopes encompass loops 1 and 3 from one subunit in the dimer and loop 2 from the other.

E. Receptor Binding Epitopes

Mutational analyses of PDGF have indicated that amino acid residues in loop 1 and loop 3 are important for receptor binding (Clements et al., 1991; LaRochelle et al., 1992; Ostman et al, 199 lb). Moreover, a peptide comprising sequences from these two regions was able to compete well with the binding of ^^^I-PDGF to receptors (Engstrom et al., 1992). The loop 2 region may also be part of the receptor binding epitope together with loops 1 and 3 from the companion subunit (Andersson et al., unpublished data; LaRochelle et al., 1990), but it is likely to be of less importance compared to loops 1 and 3.

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CARL-HENRIK HELDIN, ARNE OSTMAN, and BENGT WESTERMARK PDGF-AA

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stimulation of chemotaxis

stimulation of actin reorganization, including edge ruffling and loss of stress fibres

stimulation of actin reorganization, including edge mffling, loss of stress fibres, and induction of circular ruffles

Figure 3. Cellular effects of the PDGF isoforms. The different dimeric receptor complexes that each PDGF isoforms induces are indicated, as well as the cellular effects that result from the activation of a- and p-receptor homodimers. The specific signals transduced by a heterodimeric receptor complex remain to be determined.

F. Receptor Binding Specificity

The PDGF isoforms exert their cellular effects by binding to two structurally similar protein tyrosine kinase receptors. The A-chain binds to a-receptors with high affinity, whereas the B-chain binds to both a- and P-receptors with high affmities. Given the bivalency of the PDGF molecule, ligand-binding induces dimerization of the receptors. Thus, PDGF-AA induces a a receptor dimers, PDGFAB a a and ap receptor dimers, and PDGF-BB all three combinations of a- and p-receptors (Figure 3).

III. EFFECTS OF PDGF ON CELLS IN CULTURE PDGF has a potent mitogenic effect on cells in culture and also affects cell motility. Thus, PDGF stimulates directed cell migration, chemotaxis, and the rearrangement of actin filaments in the cell. Whereas both a- and P-receptors mediate cell proliferation, only the P-receptor mediates stimulation of chemotaxis. In fact, the a-receptor inhibits chemotaxis, at least in certain cell types, such as human fibroblasts. In the case of the effects of PDGF isoforms on the actin filament system, both receptor types mediate formation of edge ruffles and loss of stress fibers, but only the P-receptor stimulates the formation of circular ruffles on the dorsal surface of the cell (Eriksson et al., 1992). It is an interesting possibility, which remains to

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be elucidated, that the heterodimeric receptor complex has unique properties (Figure 3). The difference in signal transduction between the a- and P-receptors is likely to be due to differences in the interaction with signal-transduction molecules. Ligandinduced receptor dimerization induces autophosphorylation of specific tyrosine residues in the receptors; the autophosphorylated regions interact in a specific manner with SH2-domain containing signal-transduction molecules (chapter by Williams in this volume; Claesson-Welsh, 1994). It is conceivable that differences in autophosphorylation sites and, thereby, in the abilities to interact with and activate different signal-transduction molecules, account for the different effects of the two receptors on cell motility.

IV. PRODUCTION OF PDGF The in vivo function of PDGF is determined by the expression of PDGF isoforms as well as a- and (3-receptors in different tissues. PDGF is present in large quantities in the a-granules of platelets. Moreover, PDGF is produced by endothelial cells (DiCorleto and Bowen-Pope, 1983), smooth muscle cells (Nilsson et al., 1985; Seifert et al., 1984), syncytial trophoblasts (Goustin et al., 1985), macrophages (Shimokado et al., 1985), glial cells (Richardson et al, 1988), fibroblasts (Paulsson et al., 1987), keratinocytes (Ansel et al., 1993), and neurons (Sasahara et al., 1991; Yeh et al., 1991). The PDGF production in these cells is regulated; for example, the PDGF production by endothelial cells is stimulated by thrombin, that of macrophages is increased in conjunction with activation of these cells, and that of fibroblasts occurs in conjunction with mitogenic stimulation of the cells. Interestingly, because PDGF stimulates fibroblasts, the factor induces its own synthesis in a positive feedback manner (Paulsson et al., 1987). As far as is known, PDGF does not have any endocrine function, but acts locally on the producer cell itself in an autocrine manner, or on neighboring cells in a paracrine manner. The exact range of action of the various isoforms of PDGF is restricted in a differential way by interactions with different components at the producer cell and in the matrix. Thus, the B-chain and the long form of the A-chain contain C-terminal basic sequence motifs which bind to components inside the producer cell, at the cell surface, and in the cell matrix (LaRochelle et al., 1991; Raines and Ross, 1992; Ostman et al., 1991a). A specific interaction between the basic sequence and the acidic polysaccharide heparan sulfate has been demonstrated (Pollock and Richardson, 1992). It is possible that the PDGF isoforms with retention sequences have preferentially autocrine effects, whereas the short form of PDGF-AA also has a paracrine effect. It remains to be elucidated whether the association with matrix molecules and other components affect the activity of PDGF, or whether the binding of PDGF to these components only provides a way to store PDGF.

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V. /NV/VO FUNCTION OF PDGF A.

Embryogenesis

PDGF and PDGF receptors show specific spatial and temporal'expressions in the embryo (Morrison-Graham et al, 1992; Orr-Urtreger et al, 1992; Orr-Urtreger andLonai, 1992; Schattemanetal., 1992) and in the placenta (Goustinetal, 1985). A role for PDGF during the very early phases of embryonal growth is suggested by the finding ihsit Xenopus oocytes contain maternally derived mRNA for PDGF and PDGF receptors (Mercola et al, 1988). A stimulatory effect of PDGF on the growth of bovine embryos at the eight cell stage has also been reported (Larson et al., 1992; Thibodeaux et al., 1993). In the mouse embryo, the PDGF A-chain and a-receptor are expressed at the two-cell and blastocyst stages, suggesting an autocrine stimulation. In the early postimplantation embryos, PDGF A-chain expression is restricted to the ectoderm, whereas the a-receptor is expressed in the mesoderm, suggesting paracrine stimulation (Palmieri et al, 1992). In contrast, expression of B-chain and p-receptor are absent or very low in early mouse development (Mercola et al., 1990b), but is induced in specific tissues, such as nervous tissues, at later stages of development (Sasahara et al., 1991; Smits et al., 1991; Yeh et al., 1991). The characterization of the spatial and temporal expression of PDGF and PDGF receptors has given important information about the possible function of PDGF during embryonal development. Moreover, more direct evidence for the functional role of PDGF has come from the recent knockout by homologous recombination of the genes for the A- and B-chains of PDGF. Mice lacking the A-chain gene are bom alive with a rather normal appearance except for a reduction in weight (Betsholtz et al., 1995). However, after 2—3 weeks, the mice die from lung dysftmction. Analysis of the lung tissue revealed a specific loss of smooth muscle cells in the alveolar septa, leading to a generalized lung emphysema. Taken together with earlier studies showing the expression of PDGF and PDGF receptors in the lung (Orr—Urtreger and Lonai, 1992), these findings indicate that PDGF produced by the lung epithelium stimulates the development of the smooth muscle cells needed for the formation of alveolar septa. The phenotype of the mice lacking the B-chain gene is more severe and most of the mice die perinatally (Leveen et al., 1994). The few animals bom alive show subcutaneous bleedings and dilatation of the heart and the large vessels. Similar to the case with the A-chain knockout mice, this suggests a defect in the development of smooth muscle cells, albeit primarily affecting the vessel walls rather than the lung tissue. Another interesting finding was that the kidney glomemli of the B-chain gene knockout mice completely lacked mesangial cells. This leads to a collapse of the capillaries of the glomemli. Anormal function of PDGF, thus, seems to be to stimulate the development of mesangial cells in the kidney. A specific function for PDGF has also been elucidated in controlling the differentiafion of glial cells in the optic nerve (Noble et al., 1988; Richardson et al., 1988).

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PDGF-AA produced by type 2 astrocytes acts on a-receptor expressing 0-2A progenitor cells and prevents premature differentiation into oligodendrocytes. Another important function of PDGF is to prevent cells of the oligodendrocytic lineage from programmed cell death, apoptosis (Barres et al., 1992). PDGF may also be important for growth and regeneration of glial cells of the peripheral nerve system because Schwann cells express functional PDGF a-receptors (Eccleston et al., 1990; Weinmaster and Lemke, 1990). B. Wound Healing The well-characterized effects of PDGF on growth, chemotaxis and matrix production of connective tissue cells have led to suggestions that PDGF is involved in wound healing. This notion is supported by recent studies showing that PDGFBB stimulates the healing of wounds in animal models (Mustoe et al., 1991; Pierce et al., 1988) and the healing of decubitus ulcers of patients (Robson et al., 1992). In order for PDGF to have a role in normal wound healing, it has to be at the wound sites. This is likely to be so, because PDGF is present in large amounts in platelets which release their contents at the wound site. Moreover, PDGF is secreted by activated macrophages (Shimokado et al., 1985), thrombin-stimulated endothelial cells (Harlan et al., 1986), smooth muscle cells of damaged arteries, activated fibroblasts (Paulsson et al., 1987) and by epidermal keratinocytes (Ansel et al., 1993). Consistent with the notion that PDGF is present in a healing wound, the factor has been detected in wound fluid (Matsuoka and Grotendorst, 1989). PDGF stimulates different phases of the healing process. In the initial phase, it stimulates the migration of neutrophils and macrophages (Deuel et al, 1982; Senior et al., 1983; Siegbahn et al., 1990) and fibroblasts (Seppa et al., 1982) into the wounded area through chemotaxis. Moreover, PDGF activates macrophages to produce and secrete other growth factors important for the healing process. One important aspect of the healing process is the production of matrix components. Although other growth factors, in particular, transforming growth factor |3 (TGF-P), stimulate matrix formation more efficiently, PDGF stimulates the production of fibronectin (Blatti et al, 1988) and hyaluronic acid (Heldin et al., 1989) by fibroblasts. Another important aspect of wound healing is vascularization of the newly formed tissue; PDGF-BB has a weak angiogenic effect (Risau et al., 1992). PDGF may also have important roles in the later phases of wound healing. It stimulates the contraction of collagen matrices in vitro (Clark et al, 1989; Gullberg et al., 1990), implicating an effect on wound contraction in vivo. PDGF may also affect the remodeling phase of wound healing, because it stimulates the production and secretion of collagenase by fibroblasts (Bauer et al., 1985). C. Inhibition of Platelet Aggregation Platelets are a major source of PDGF. Interestingly, PDGF inhibits platelet aggregation (Bryckaert et al., 1989). It is, therefore, likely that PDGF stored in the platelet a-granulae serves a negative autocrine feedback role; after platelet aggre-

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gation and release of PDGF and other factors, PDGF binds to a-receptors present in the cell membrane of platelets and reverses the aggregation (Vassbotn et al, 1994).

VI. PDGF IN DISEASE A. Malignancies The finding of a close similarity between the sis oncogene and the B-chain of PDGF (Doolittle et al., 1983; Waterfield et al, 1983) suggested a general mechanism whereby transforming oncogenes act, by causing the production of factors which subvert the mitogenic pathway of growth factors. Consistent with the notion that transformation by the sis oncogene involves autocrine growth stimulation by a PDGF-like factor, ^/^-transformed cells produce a PDGF-like factor and sis was found able to transform only cells carrying PDGF receptors (reviewed by Westermark et al., 1987). More recent studies on human tumors have revealed that overactivity of PDGF and PDGF receptors is common in certain types of tumors, suggesting that autocrine stimulation of growth may contribute to the loss of growth control in these tumors. PDGF and PDGF receptors are often overexpressed in human glioblastoma (Fleming et al, 1992; Maxwell et al., 1990; Nister et al., 1988). Investigations of the expression patterns of PDGF and PDGF receptors in glioblastoma tumors of different degrees of malignancy, revealed, interestingly, an uneven distribution among the different cell types in the tumor tissue (Hermanson et al., 1992; Hermanson et al., 1988; Plate et al., 1992). PDGF-A and a-receptors were mainly expressed by the tumor cells, whereas PDGF-B and (3-receptors were expressed in the stroma compartment of the tumor. Whereas the expression of a-receptors on tumor cells and PDGF B-chain in vessels was seen in both benign and malignant tumors, the expression of PDGF A-chain and P-receptor was higher in the more malignant tumors. These observations suggest that two separate autocrine loops occur in human glioma, one involving the a-receptor in the tumor cell compartment and the other involving the P-receptor in the stroma compartment. These autocrine mechanisms, together with additional paracrine mechanisms involving different isoforms of PDGF, may be important for the balanced growth of different cell types in glioblastoma tumors. The fact that glioma cell growth can be inhibited by expression of a dominant negative mutant of PDGF (Shamah et al., 1993) or by a truncated PDGF receptor acting in a dominant negative manner (Strawn et al., 1994), provides additional support for the notion that PDGF is important for glioma cell growth. Also, fibromas and fibrosarcomas express PDGF and PDGF receptors in a malignancy-dependent manner, suggesting a role for PDGF in autocrine stimulation of growth as connective tissue tumors progress to forms of higher malignancy (Alman et al., 1992; Smits et al., 1992; Wang et al, 1994). Epithelial cells do not normally respond to PDGF, but there are several examples of tumor cells of epithelial origin that produce PDGF (Raines et al., 1990). There

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are cases where autocrine loops have been established by aberrant production of PDGF receptors, for example, certain thyroid carcinoma cell lines (Heldin, N - E . et al., 1991; Heldin et al, 1988), gastric carcinoma cells (Chung and Antoniades, 1992) and lung cancer cell lines (Antoniades et al., 1992). In addition to possible autocrine effects, PDGF produced by the cancer cells may affect stroma formation by paracrine mechanisms (Chaudhry et al., 1992; Lindmark et al., 1993). That PDGF has a potent stroma-inducing effect was shown by Forsberg et al. (1993), who analyzed the histology of tumors formed in nude mice by melanoma cells and compared that with the histology of tumors formed by the same cells after transfection of a PDGF B-chain cDNA. The tumors that arose from the PDGF-producing melanoma cells contained a network of connective tissue with abundant blood vessels. In contrast, the tumor tissue derived from PDGF nonproducing control cells showed less well developed stroma and areas of necroses. B. Atherosclerosis

According to the "response-to-injury" hypothesis, the atherosclerotic process is an excessive repair response to different forms of insults to the endothelium and smooth muscle of the artery wall (reviewed in Ross, 1993). After injury to the endothelial cell layer, macrophages adhere to the subendothelial space, where they accumulate lipids and become foam cells. The foam cells, together with T-cells, form fatty streaks which progress to fibrous plaques. In advanced stages, the plaques also contain smooth muscle cells, which have migrated from the media to the intima layer of the vessel, and platelet-containing thrombi. The atherosclerotic process is likely to involve the action of several growth factors, including PDGF, which stimulate chemotaxis and proliferation of the cells involved. Evidence for the involvement of PDGF in the atherosclerotic process include the observation that PDGF-BB infused into rats after carotid injury caused an increase in the intimal thickening and in the migration of smooth muscle cells from the media of the vessel wall to the intima (Jawien et al., 1992). For PDGF to be involved in the atherosclerotic process it needs to be in the plaques. In fact, PDGF is produced by many cell types in the lesion, including activated macrophages (Shimokado et al., 1985), smooth muscle cells (Nilsson et al., 1985; Seifert et al., 1984) and endothelial cells (DiCorleto and Bowen-Pope, 1983), and is also released from sequestered platelets. Interestingly, the expression of PDGF by smooth muscle cells and macrophages increased in atherosclerotic lesions compared to normal controls (Ross et al., 1990; Wilcox et al., 1988). Moreover, intimal smooth muscle cells in the lesions express increased amounts of PDGF p-receptors, and may therefore be more responsive to PDGF (Rubin et al., 1988b). Consistent with a role for PDGF in the atherosclerotic process, infusion of neutralizing PDGF antibodies inhibit the intimal thickening of rat carotid arteries that follows balloon angioplasty (Ferns et al., 1991).

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The fact that PDGF is a major mitogen for connective tissue has led to speculations that overactivity of PDGF is involved in the development of fibrotic diseases. There is now accumulating evidence that PDGF is involved in several such diseases. Liver cirrhosis is characterized by a dedifferentiation and proliferation of fat-storing cells (Ito cells) to myofibroblast-like cells. It is possible that PDGF is involved in this process, because dedifferentiated Ito cells respond to PDGF (Heldin, P. et al., 1991;Pinzanietal., 1989;Pinzanietal., 1991) and activated macrophages from patients with liver disease secrete large amounts of PDGF (Peterson and Isbrucker, 1992). PDGF may also have a role in the development of idiopathic pulmonary fibrosis, because alveolar macrophages from patients with this disease produce significantly higher amounts of PDGF than those of healthy subjects (Martinet et al., 1986). PDGF and other cytokines are produced by macrophages (Vignaud et al., 1991) and by the alveolar epithelium (Antoniades et al, 1990) of patients with idiopathic pulmonary fibrosis. PDGF may also have a role in the development of other fibrotic conditions in the lung, like those following hypoxid pulmonary hypertension (Katayose et al., 1993), breathing of high concentrations of oxygen (Han et al., 1992; Powell et al., 1992), and obliterative bronchiolitis after lung transplantation (Hertz et al., 1992), because PDGF is overexpressed in these conditions. PDGF is present at high concentrations in the chronic inflammation in the joints of patients with rheumatoid arthritis (Sano et al, 1993). Moreover, the expression of PDGF P-receptors is upregulated in the synovium of inflamed joints (Reuterdahl et al., 1991; Rubin et al., 1988a). It is thus possible that PDGF stimulation contributes to the increased cellularity of chronically inflamed joints. A role for PDGF in scleroderma (systemic sclerosis), a disease characterized by progressive fibrosis in the skin and in visceral organs, has also been proposed based on the observation that PDGF-p receptors, absent on fibroblasts of normal skin, are expressed on skin cells of scleroderma patients (Klareskog et al, 1990), and that PDGF immunoreactivity is present in scleroderma skin (Gay et al., 1989). The possibility that PDGF and the precursor cell of platelets, the megakaryocyte, are involved in myelofibrosis has been discussed (Castro-Malaspina et al., 1981; Groopman, 1980). Consistent with this possibility, the levels of PDGF in circulating plasma and urine are significantly elevated in patients with myelofibrosis (Gersuk et al., 1989) and the concentration of PDGF in platelets is correspondingly decreased (Katoh et al., 1988). D.

Glomerulonephritis

Glomerulonephritis is an inflammatory process which involves remodeling and repair of damaged kidney glomeruli. Mesangial cells are important in this process.

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Given the importance of PDGF in developing mesangial cells, it is not surprising that PDGF has been implicated in the development of glomerulonephritis. Mesangial cells produce PDGF and respond to PDGF in vitro (Shultz et al., 1988; Silver et al., 1989), and the expression of PDGF increases in conjunction with glomerulonephritis in patients and in animal models (Gesualdo et al, 1991; lida et al., 1991). Administration of neutralizing PDGF antibodies decreased the mesangial cell proliferation four days after injury in a rat model for glomerulonephritis, suggesting a causative role of PDGF in the process (Johnson et al., 1992). Moreover, infusion of PDGF-BB in normal rats induced a mild mesangial proliferation, and a massive mesangial proliferation in rats with a mild glomerulonephritis (Floege et al., 1993). Apart from the mesangial cells themselves, platelets and macrophages can be the source of PDGF in the diseased kidney; PDGF may, therefore, stimulate mesangial cell proliferation through autocrine as well as paracrine mechanisms. It is likely that other growth factors in addition to PDGF are important in the development of glomerulonephritis. In particular, there is evidence for the involvement of TGF-P (Border et al., 1990), whereas TGF-p appears to affect primarily the extracellular matrix accumulation. PDGF primarily affects mesangial cell proliferation (Isaka et al., 1993).

Vll. PDGF ANTAGONISTS As discussed above, there is accumulating evidence that PDGF is involved in a number of serious diseases. Agents that inhibit PDGF action in vivo could, therefore, be of clinical utility. In the design of PDGF antagonists, two aspects deserve consideration. One is specificity: the antagonist should preferentially interfere only with PDGF action and leave other signal-transduction pathways intact. The other is that the administration of PDGF antagonists should not interfere with important normal fiinctions of PDGF. The known functions of PDGF are regulating the embryonal development, stimulating wound healing and controlling platelet aggregability. The role of PDGF during development is of no concern clinical for the use of PDGF, except maybe during pregnancy. Given the fact that many different factors stimulate wound healing, it is not likely that inhibition of PDGF would significantly impair the healing process. A potentially more serious complication could arise from blocking the feedback inhibiting function of PDGF on platelet aggregation, which could lead to an increased risk of thrombosis. Because platelets have only a-receptors, whereas many cell types involved in disorders associated with PDGF overactivity have mainly P-receptors, such as smooth muscle cells, fibroblasts and mesangial cells, a possible strategy would be to develop antagonists that inhibit PDGF action via the P-receptor without affecting the a-receptor. In theory, it is possible to interfere with the PDGF signal-transduction pathway at any level, for example inhibition of ligand binding to the receptor, receptor dimerization, the kinase activity of the receptor, or components of intracellular signaUransduction pathways. Because many intracellular signal-transduction path-

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ways are shared among different growth factors, specific inhibition by antagonists acting extracellularly is more likely. One possibility achieving intracellular inhibition specifically affecting PDGF-induced pathways would be to inhibit the kinase activity of the receptors or their autophosphorylation and, thereby, their interaction with downstream signal-transduction molecules. Potentially interesting inhibitors are the low molecular weight inhibitors of tyrosine kinases denoted tyrphostins; recently, tyrphostins which inhibit certain tyrosine kinases, but not others, have been described (Bryckaert et al., 1992; Kovalenko et al., 1994). A possible strategy for inhibiting PDGF action would be to sequester the factor and thereby prevent its binding to the receptor. Due to the conservation of PDGF, it has been difficult to obtain high titer antisera against the factor. However, neutralizing antisera made in rabbits or goats inhibit autocrine stimulation in ^^-transformed cells (Huang et al., 1984; Johnsson et al., 1985), as well as the atherosclerotic process that occur after deendothelialization of carotid arteries in rats (Ferns et al., 1991) and mesangial proliferation in a rat model for glomerulonephritis (Johnson et al., 1992). For clinical use, monoclonal antibodies against PDGF (Vassbotn et al., 1990) would be more useful, as they can be "humanized" to avoid complicating immune reactions. Another possibility of sequestering PDGF would be by administering soluble PDGF receptors, comprising only the extracellular ligand-binding part. A soluble variant of the PDGF P-receptor can be produced and retains ligand binding activity (Duan et al., 1991). Another approach is to use low molecular weight compounds interfering with the interaction between PDGF and its receptors. One example of such a compound is suramin which displaces PDGF from its receptor (Williams et al., 1984). However, suramin is not specific for PDGF, and interferes with the interactions of a number of other ligand-receptor pairs (Betsholtz et al, 1986a). Another example is neomycin which inhibited the binding of PDGF-BB to the a-receptor, but interestingly, did not affect binding to the p-receptor (Vassbotn et al., 1992). Although, the concentrations of neomycin needed were too high to be obtainable in vivo, the results are encouraging because they demonstrate that PDGF interacts with slightly different epitopes on the a- and P-receptors; receptor specific antagonists should, therefore, be possible to find. A key event in signal transduction via PDGF receptors is ligand-induced receptor dimerization. An antagonistic effect would, therefore, be expected if one could interfere with the dimerization process. The fact that kinase negative PDGF receptor mutants act in a dominant negative fashion (Strawn et al., 1994) supports this notion. Because PDGF is a symmetric dimer, it is likely to induce receptor dimerization by simultaneously binding two receptors. PDGF mutants, which retain only one receptor binding epitope, may, thus, have antagonistic effects. Simply preventing interchain disulfide bonding in PDGF-BB by mutating the cysteine residues forming these bridges did not lead to a product with antagonistic properties; rather, it was found to be an agonist (Andersson et al., 1992), probably because the PDGF molecule occurs as a dimer, also, in the absence of interchain

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disulfide bonds (Kenney et al., 1994). A possible strategy for achieving an antagonist may be to combine a wild-type PDGF chain with a mutant chain which is unable to bind to receptors and actively prevents receptors from dimerizing.

V m . FUTURE PERSPECTIVES PDGF is one of the more well-characterized growth factors. Research over more than 20 years has provided a wealth of information regarding its structural and functional properties. The three-dimensional structure of one of the PDGF isoforms has now been solved. Of importance for the future will be solving the structure of the complexes between PDGF isoforms and their receptors and defining the epitopes in ligands and receptors that interact. Such information will aid in developing efficient and specific antagonists. Recent reports on the disruption of the PDGF A- and B-chain genes have provided important information about the role of PDGF during development. However, because the knockout mice die pre- or perinatally, other approaches are needed to evaluate the role of PDGF in specific tissues and in the adult animal. Expression of dominant negative forms of PDGF (Mercola et al., 1990a; Vassbotn et al., 1993) in transgenic mice under the control of either inducible or tissue-specific promoters, will provide additional valuable information about the physiological role of PDGF. Procedures for preparing large quantities of recombinant PDGF with full biological activity will make it possible to further explore the clinical utility of PDGF. The initial trials with PDGF-BB to stimulate wound healing are promising, but the choice of isoform and the method of application remain to be optimized.

ACKNOWLEDGMENTS We thank Ingegard Schiller for valuable help in preparing this review.

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FIBROBLAST GROWTH FACTORS

Ann Logan and Andrew Baird

I. II. III. IV.

V. VI. VII. VIII. IX. X. XL

Abstract Introduction FGF Genes Target Cells and Distribution Molecular Regulation of FGF Gene Transcription, FGF Isoforms, and Peptide Localization A. FGF-1 B. FGF-2 C. FGF-3 D. FGF-5 E. FGF-7 Molecular Properties of the FGF Family FGF Secretion FGF Localization Heparan Sulphate Proteoglycans FGF High-Affinity Receptors FGF Signaling Proteoglycans and the Regulation of FGF Bioactivity

Growth Factors and Cytokines in Health and Disease Volume lA, pages 147-178. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0091-4 147

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XII. Physiology and Pathology of the FGFs A. Limb Development B. Lung Development C. Development ofthe Nervous System D. FGFs in Neurodegenerative Conditions E. FGFs as Angiogenic Factors F. FGFs in Tumorigenesis XIII. Conclusions References

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ABSTRACT Recent advances in the field of fibroblast growth factors (FGF) have led to a better understanding of their physiological functions and their roles in the pathogenesis of disease. In reviewing this field, we have concentrated on the numerous recent advances suggesting that these molecules are pivotal in embryonic development, reproduction, growth, transformation, and injury repair. However, these advances have raised as many new unanswered questions as they have addressed.

I. INTRODUCTION In the last few years the field of fibroblast growth factors (FGFs) has been transformed. Once restricted to one activity mediated by one growth factor, it now consists of at least nine gene products, each with a multitude of activities depending on the target cell examined, the context of its evaluation, and the endpoint evaluated. Once thought to be mediated by a high-affinity receptor, FGFs are now known to interact with any of four high-affinity receptors, each of which can exist in numerous isoforms, and all of which may heterodimerize at the cell surface in response to ligand activation. Once thought to be simply sequestered by heparan sulfate proteoglycans (HSPG) in the extracellular matrix, cell surface HSPGs also deliver FGFs to the signal-transducing receptor complex. In this review, we have consolidated current concepts describing the action and regulation of FGFs. Although it is incomplete, we have incorporated other reviews by reference. The accumulated data evaluated here are a starting point for further studies aimed at understanding the roles FGFs play in normal and pathophysiological cell growth.

II. FGF GENES Nine distinct FGF genes, named FGF-1 through FGF-9, have been identified to date. Because of their similar organization, it is most probable that they arose from duplication of a common ancestral gene. Each consists of three exons, separated by introns of variable length. Typically the second exon is very short, and, in many cases, the third exon includes a long (2—3 kb) 3'-untranslated sequence. The FGF

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Table 1, Chromosomal Location of Human FGF Genes FGF

Chromosomal Location 15q31-33 4q25 llql3

FGF-1 FGF-2 FGF-3 FGF-4 FGF-5 FGF-6 FGF-7 FGF-8 FGF-9

— 4q21 12pl3 llql3

— —

Reference Jayeetal., 1986 Fukushima et al., 1990 Huebner et al, 1988

— Nguyen etal., 1988 Maries etal., 1989 Nguyen etal., 1988

— —

genes map to several chromosomes. The known locations in the human are shown in Table 1.

III. TARGET CELLS AND DISTRIBUTION One of the most perplexing observations about FGFs is the breadth of target cells that are responsive to the growth factors. This should question the physiological significance of these molecules. After all, how can molecules, virtually ubiquitous in their distribution (see below), also have target cells in all tissues? This is best illustrated in Table 2, where (in the case for FGF-2) the tissue localization and the potential target cells are listed. However, this widespread distribution does not apply to all FGFs, only to FGF-1 and FGF-2. Other FGFs are much more restricted and found either in the central nervous system or during fetal development.

Table 2. Distribution and Target Cells for FGF-2 Tissue Sources ofFGF-2 brain pituitary adrenal ovary kidney thyroid muscle heart bone cartilage

Potential Target Cells neurons, astrocytes, glia, endothelium, fibroblasts thyrotrophs, lactotrophs, endothelium, fibroblasts chromaffin cells, adrenocortical cells, endothelium, fibroblasts granulosa cells, luteal cells, endothelium, fibroblasts mesangial cells, endothelium, fibroblasts follicular cells, C-cells, endothelium, fibroblasts smooth muscle cells, myocytes, myoblasts, endothelium, fibroblasts cardiocytes, cardioblasts, endothelium, fibroblasts osteocytes, osteoblasts, endothelium, fibroblasts chondrocytes, endothelium, fibroblasts

In all tissues where it is found, there are large numbers of potential target cell types.

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Most remarkably, each of the in vitro targets for FGF-2 observed in vitro can be validated in vivo. Its ability to stimulate angiogenesis reflects its activity on endothelial cells, cartilage repair on chondrocytes, fracture repair on bone cells, neurotrophic activity on neurons, and limb regeneration on mesenchymal stem cells. These activities have served as the focal point for understanding FGF action. By identifying the mechanisms that confer target cell specificity on FGFs, it should be possible to design strategies promoting and preventing their action.

IV. MOLECULAR REGULATION OF FGF GENE TRANSCRIPTION, FGF ISOFORMS, AND PEPTIDE LOCALIZATION The cis and trans acting elements involved in regulating FGF gene transcription are only just beginning to be elucidated. Most of the information currently available is limited to FGF-1, 2, 3, 5, and 7. Whatever mechanisms regulating FGF gene expression emerge, the range of FGF ligands available in tissues is broadened by the generation of multiple isoforms of individual FGFs. These can each be localized to different subcellular and extracellular pools. Each of these isoforms can be generated in a number of ways, including alternative splicing of their mRNAs and transcriptional regulation. Additional variation is achieved in the case of FGF-2 and FGF-3 by alternate translation initiation codons leading to N-terminal extended isoforms. Further diversity in FGF ligands may be generated by posttranslational modifications. A. FGF-1

FGF-1 was originally isolated as a 155 amino acid, 18-kDa protein (Thomas et al., 1984), but truncated 140 and 134 amino acid forms were subsequently identified (Esch et al., 1985). FGF-1 has an in-frame translation termination codon just upstream from its AUG initiation codon (Jaye et al., 1986), precluding the possibility of N-terminally extended forms. The existence of alternate 5'-untranslated exons in FGF-1 RNAhas been described (Chiu et al., 1990; Crumley et al., 1990). The role of these untranslated sequences is unknown, but they may be involved in differentially regulating translation of the molecule. Endogenous and exogenous FGF-1 has been shown to localize to the nucleus of some cells (Sano et al., 1990; Speir et al., 1991). A putative nuclear localization sequence has been identified in residues 21-27 of the protein (Imamura et al., 1990; Imamura et al., 1992), and it is suggested that this sequence is required for full mitogenic activity of the molecule. However, this contention has been disputed by others (Cao et al., 1993), who assert that passage into the nucleus of endogenous FGF-1 is achieved by free diffusion and exogenous FGF-1 does not translocate to this site. Others have shown that an alternative splice of its mRNA, that removes coding exon 2, results in a truncated isoform that antagonizes FGF-1 (Yu et al., 1992), suggesting a putative

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regulatory role for this alternative FGF-1. The significance of this isoform, however, is unclear. B. FGF-2

The human FGF-2 promoter does not appear to have a TATA box, but contains several potential SPl and one API binding site (Shibata et al., 1991). Recently, the FGF-2 gene was shown to be responsive to p53, a nuclear phosphoprotein which regulates a variety of genes via sequence specific DNA binding and/or direct protein-i)rotein interactions (Ueba et al., 1994). Negative regulatory elements, whose deletion increases gene expression, also appear to be present 5' of the promoter region (Shibata et al., 1991). Several "small" and "large" forms of FGF-2 protein are detected in adult tissues. It has been shown by mutagenesis of FGF-2 cDNA that they result from alternative initiation of translation at an AUG codon or at three in-frame CUG codons upstream, leading to synthesis of a small 18-kDa (155 amino acids) form or to large forms of 22, 22.5, and 24 kDa, respectively (196, 201, and 210 amino acids) (Florkiewicz and Sommer, 1989; Prats et al., 1989). It is known that regulation of global or alternative initiation of translation is governed by the cooperation of five CIS acting RNA elements in the FGF-2 mRNA leader, two in the 5' untranslated region and three in the alternatively translated region. Each initiation site can be specifically regulated by trans acting factors yet to be identified. An interesting mechanism of regulation of FGF-2 expression has been reported in Xenopus oocytes (Kimelman and Kirschner, 1989). In addition to a transcript encoding FGF-2, an antisense transcript is present in large excess. The antisense transcript hybridizes to FGF-2 mRNA, but, surprisingly, does not appear to inhibit its translation and could be involved in regulating FGF-2 mRNA stability. Alternative splicing also generates multiple FGF-2 isoforms. For example, alternative exon I sequences of the open reading frame of the avian FGF-2 gene canbe spliced with exons 2 and 3 to provide a predicted FGF-2 isoform with up to eight novel amino terminal domains of the protein during late embryonic development at the time of mesoderm induction and morphogenesis (Borja et al., 1993). In the adult, only the canonical transcripts predominate, suggesting a role for the novel isoforms during late embryonic development. All isoforms of FGF-2 lack a typical signal sequence for secretion, yet the 18-kDa form seems to localize preferentially to the cytoplasm and is also the only form found outside of cells, although the higher molecular weight forms predominate in the nuclear and ribosomal fractions (Renko et al., 1990; Bugler et al., 1991; Florkiewicz et al., 1991). These results suggest that large FGF-2 isoforms contain a nuclear localization sequence (Quarto et al., 1991). The extended N-termini contain several stretches of alternating glycine and arginine residues, some methylated (Sommer et al., 1989), as has been described for other nuclear proteins. Interestingly, even larger forms of FGF-2 have been reported in some tissues, such as the 33-kDa isoform seen in anaplastic thyroid carcinoma (Shingu et al., 1994).

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These have been linked to metastatic activity. This contention is supported by the observation of a potent transforming activity of abnormal 35-kDa FGF-2 (Blam et al., 1988). The significance of the different subcellular pools of FGF-2 remains to be elucidated, but they may be functionally related. It has been shown that the CUG and AUG initiated isoforms of FGF-2 can have completely independent activities within the same cell type in vitro (Couderc et al., 1991; Takayama et al., 1994). In vivo evidence supports specific functional roles for these FGF pools. For example, during development, FGF-2 proteins translocate from the nucleus to the cytoplasm of epiblast and hypoblast cells, during formation of the primitive streak, and relocate to the nucleus of specific differentiating cells during organogenesis (Dono and Zeller, 1994). In the brain, the FGF-2 protein that is found predomitiantly in the nucleus of mature quiescent astrocytes is also found in the cytoplasm of reactive astrocytes after injury. Whether these changes result from peptide translocation or localization after de novo synthesis remains to be determined. Once translated, FGF-2 molecules can be further modified in several ways to generate isoforms of variable activity. In addition to glycosylation of the molecule, unique membrane-bound ectokinases and novel nuclear kinases can phosphorylate serine residues of FGF-2, thereby, changing the heparin-binding characteristics of the molecule and providing a target cell specific means of modulating FGF-2 activity (Fiege et al., 1991; Vilgrain et al., 1993). Similarly, recent work suggests that FGF-2 can undergo nucleotidylation, ribosylation, and methylation with biological consequences. C. FGF-3

Regulation of FGF-3 transcription appears to be coordinated by three distinct promoters and two alternative polyadenylation sites. They can generate six different RNA species all with the same coding capacity (Grinberg et al., 1991). FGF-3 has two alternative upstream initiation codons, but, in this case, CUG is the major start site for FGF-3 translation, which gives rise to an amino-terminal extended, 31.5kDa protein; a downstream AUG codon initiates translation of a shorter 30.5-kDa protein (Acland et al., 1990). The N-terminal extended form of FGF-3, like the N-terminally extended forms of FGF-2, localizes to the nucleus, and the nucleolus is the primary nuclear site for FGF-3 accumulation. A signal peptide for entry into the secretory pathway is positioned adjacent to the AUG start site of the FGF-3 gene, which continues to function when embedded within the CUG-initiated extended protein. A number of replacement and deletion mutations have shown that the amino terminal extension is crucial for nuclear import, although the nuclear targeting signals are located elsewhere in the protein. The decision to enter the secretory pathway or nucleus appears to depend on a balance of competing signals involving the amino terminus, signal peptide, and the nuclear localization sequence. The relative position of the signaling motifs is also an important factor in estab-

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lishing the proportion of FGF-3 destined for the different intracellular compartments. D. FGF-5

The human FGF-5 gene has ahemative polyadenylation sites which transcribe two main RNA species of 1.6 and 1.4 Kb (Zhan et al., 1988). The regulatory elements of transcription have yet to be identified, but there is evidence for translational control of FGF-5 expression. The FGF-5 mRNA contains a short out-of-frame open reading frame (ORF) upstream of the ORF coding for the growth factor. Deletion of the upstream ORF enhances FGF-5 translation and transforming ability (Bates et al., 1991). FGF-5 is secreted as a glycosylated protein. E. FGF-7

There is a canonical TATA box 30 nucleotides upstream of the transcription initiation site. Physiological FGF-7 transcription is regulated developmentally by an enhancer element located in the 3'-untranslated region of exon 3 (Curatola and Basilico, 1990). This enhancer contains a series of consensus binding sites for a number of known transcription factors, including SP1 and AP1. Although probable that the specific trans factors for the FGF-7 gene will belong to the family of octamer-binding proteins (some known to be developmentally regulated), there is no evidence for posttranscriptional or translational control of FGF-7. The mature glycosylated protein is secreted via the Golgi-endoplasmic reticulum, like all "secreted" peptides.

V. MOLECULAR PROPERTIES OF THE FGF FAMILY The FGF family of proteins currently comprises nine structurally related members in mammals, which have now been assembled into a numerical nomenclature loosely based on the chronological order of their identification (Figure 1). The name FGF derived from the initial observations of in vitro mitogenic activity of pituitary extracts for 3T3 fibroblast cells, although, of course, the name is totally unrepresentative of the range of FGF activities. All members of the family share a common core sequence, and, characteristically, all possess two invariant, conserved, cysteine residues and exhibit a high affinity for heparan sulphate proteoglycans and heparin, an activity that resides in a defined heparin-binding domain within the core sequence of the ligand, and a property which has been used extensively in their purification. Six of the family members (FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, and FGF-8) are synthesized with an N-terminal signal sequence that targets the proteins to the secretory pathway. In contrast, FGF-1 and FGF-2 lack a classical secretory sequence, but, nevertheless, appear to be exported from cells.

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VI. FGF SECRETION As indicated, FGF-1 and FGF-2 lack a classical leader sequence to direct their secretion, and there has been considerable debate whether these factors are released from cells under normal physiological conditions. However, because these FGFs are found outside of the cell in almost every peripheral tissue (often complexed to sequestering proteoglycans), a mechanism for regulated release must be invoked. It is established, for example, that FGF-2 is released from cells via a pathway independent of the endoplasmic reticulum-Golgi complex (Mignatti et al., 1992). The balance of evidence now suggests that they are released from cells by novel secretory pathways. For example, FGF-1 is released from NIH 3T3 cells in response to heat shock (Jackson et al., 1992), and export is abrogated following treatment with either actinomycin D or cyclohexamide. This suggests only those molecules synthesized de novo can enter the pathway. Furthermore, FGF-1 is released as a dimer that is inactive in mitogenesis assays and cannot bind heparin. Clearly novel secretory pathways for these molecules will soon be characterized. Overall, it seems that there may also be novel mechanisms for regulating the release of those FGFs normally secreted by the classical pathway. In some situations FGF-3, which has a leader sequence to direct secretion, seems to be retained in the Golgi complex of cells. The existence of a Golgi retention sequence in the molecule's N-terminus has been postulated, which may regulate its secretion (Kiefer et al., 1993). However, precisely which mechanism achieves this release remains to be determined.

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VII. FGF LOCALIZATION Although FGFs may be released to the extracellular compartment by a variety of routes, specific isoforms may be retained in the cytoplasm or translocated to the nucleus. Hence, within tissues, FGFs are localized to different intracellular and extracellular compartments. The differential distribution of FGF-2 within central (intracellular) and peripheral (extracellular) tissues is the best characterized example, but very little is known yet of the intra- and extracellular distribution of the other FGFs. In normal adult peripheral mesenchymal tissues, little or no FGF-2 mRNA can be detected, but significant quantities of the 18-kDa isoform of FGF-2 is localized to glycosaminoglycans in the cell membrane and extracellular matrix, especially the basement membranes underlying epithelia. Little intracellular peptide is apparent (Gonzalez et al., 1990). These matrix interactions inactivate and protect the extracellular FGF-2 from proteolytic degradation. Consequently, the depots outside of cells form long-term stores of functionally inert peptide which can be called upon by responsive tissues at any time. In contrast, FGF-2 mRNA is readily detectable in the central nervous system, and three isoforms of the protein (18 kDa, 21 kDa, and 22.5 kDa) are found in the cytoplasm or nucleus of neurons and glia, depending on the area examined, with no apparent extracellular 18-kDa FGF-2 present at all under normal physiology (Gonzalez et al., 1995). These normal patterns of distribution in central and peripheral tissues may differ during development and under specific physiological and pathological conditions. It is now clear that the different pools of FGFs may have dramatically different cellular functions.

VIII. HEPARAN SULPHATE PROTEOGLYCANS A characteristic shared by all members of the FGF family is their affinity for heparan sulphate (HS). This polysaccharide is an abundant component of cell surfaces and the extracellular matrix, including basement membranes. Here, it is usually found covalently linked to a protein, in the form of a heparan sulphate proteoglycan (HSPG). One important class of cell surface HSPGs that binds FGFs are the syndecans, a family which contains a transmembrane core protein (Bemfield et al., 1992). FGF-binding HSPGs in the extracellular matrix and basement membranes include the large proteoglycan called perlecan (Murdoch et al., 1992). In some circumstances, HSPGs may be shed to the extracellular fluids, where they retain their FGF-binding activities and may act to sequester any FGFs that escape HSPG regulation. The relationship between the different core proteins and FGF binding is unknown, but it is known that FGFs bind to oligosaccharide units of the glycosaminoglycan. In common with all glycosaminoglycans, HS is composed of a linear sequence of disaccharide repeat units, with sulfation on clusters of sugar residues (Gallagher et al, 1992). The pattern of sulfation within the HS chains suggests a great deal of polymorphism, and, in general, the chains are organized into widely

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spaced, highly sulfated structural motifs. This property is important for their differential protein binding activities. In regions of high sulfation, the main repeat unit is N-sulfated glucosamine (GICNSO3) and iduronic acid (IdoA). In regions of low sulfation, the glucosamine is N-acetylated (GlcNAc) and linked to glucoronic acid (GlcA). Arrays of up to ten of these N-acetylated glucosamine-glucoronic acid repeats are interspersed between the sulfated domains. Specific oligosaccharide sequences within the HS molecules, such as Oligo-H, bind FGF-2 with higher affinity than others. It is probable that each FGF requires different HS sulfation patterns for high-affinity binding. HSPG molecules bind FGFs with a lower affinity than the FGF receptors responsible for signaling. This has led to terming them low affinity receptors, a misnomer because HS binding to the ligand does not itself generate a signal and the affinities are quite reasonable ('^l nM).

IX. FGF HIGH-AFFINITY RECEPTORS High-affinity FGF receptors (FGFRs) belong to the tyrosine kinase family and are encoded by four distinct genes (for a review, see Johnson and Williams, 1993). Each has now been assigned the name FGFRl-4, according to the order of their identification (Table 3). The structure of the FGFRs is that of a typical tyrosine kinase transmembrane receptor of the immunoglobulin (Ig) superfamily. The extracellular domain of the FGFRs is characterized by two or three immunoglobulin-like loops and a stretch of acidic residues between Ig loops I and II, the acid box, which is unique to the FGFRs. The intracellular region of the FGFRs possesses a long, juxtamembrane domain and a tyrosine kinase domain split by a kinase insert. The C-terminal tails of the FGFRs are relatively divergent in sequence, and because this region is thought to interact with cellular substrates, may represent a means of receptor specificity (Jaye et al., 1992). Alternative splicing of the FGFRl and FGFR2 RNAs can give rise to multiple isoforms, and this is dictated by the exon structure of the genes encoding the receptors. This exon structure, which is conserved between FGFRl and FGFR2, includes three alternative exons for the Ig III loop. Variants of the FGFRl include

Table 3. Nomenclature of the FGF Receptors FGFRl

FGFRl

FGFRS

FGFR4

flg bPGFR Cekl N-bFGFR h2,h3 h4,h5 FGFRl

bek Cek3 K-sam TK14 TK25 KGFR FGFR2

Cek3 FGFRS

FGFR4

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those that lack just two amino acids in the Ig I loop, those with a complete deletion of the Ig I loop, those with no secretory signal sequence, and those with deletion of the entire intracellular domain, transmembrane region, and part of the Ig II loop. This latter variant is a secreted and soluble form of the receptor. Another form of the FGFRl has a truncated tyrosine kinase domain. Available evidence suggests that the expression of the different FGFRs and their isoforms is regulated in a tissue-specific manner (Patstone et al., 1993; Yazaki et al., 1993), and that coexpression of several types of the receptor on any given cell is common (Bernard et al., 1991). Some degree of specificity is imparted to the FGF-FGFR binding reaction by the alternative RNA splicing discussed above. This is reflected in observations that the two Ig loop isoforms of FGFRl appear to be associated with transformed and malignant phenotypes (Yamaguchi et al., 1994), and the secreted and kinase truncated isoforms of FGFRl may act as FGF antagonists (Duan et al., 1992). Different ligand binding specificities have been reported for isoforms of FGFR1 and FGFR2 that vary in the second and third Ig loop regions. Hence, the secreted form of FGFRl, which uses a different exon for the Ig III loop to the membrane-bound forms, binds FGF-2, but not FGF-1, with high affinity. In contrast, the membrane-bound forms of FGFRl may bind either FGF-1 or FGF-2 with equal affinity, or only bind FGF-1 with high affinity depending on which of the remaining two exons encoding the Ig III loop is used (Werner et al., 1992). Similar observations have been made for FGFR2 where mutually exclusive alternative splicing of exons encoding the C-terminal regions of Ig loop III generates the Illb and IIIc isoforms. This results in proteins that are differentially responsive to FGF-7 (Illb) and FGF-2 (IIIc); FGF-1 is equally active through both isoforms. Thus, one of the three splice variants binds FGF-7 and FGF-1, but not FGF-2, with high affinity, and a second splice variant binds FGF-1 and FGF-2, but not FGF-7, with high affinity. A further splice variant in the Ig III loop binds FGF-1, but not FGF-2, with high affinity (Dell and Williams, 1992). The implied promiscuity of this class of receptors makes for a highly regulated and specific system of regulation by multiple FGFR isoforms of differential activity. A unique cysteine-rich (9%) FGFR (CFR) of unknown function has also been described (Burrus et al., 1992). CFR is an integral membrane protein that contains an intracellular domain of 13 amino acids. Two additional proteins are reported to be critical for CFR function as they bind CFR near the carboxy terminus and are intracellular (Burrus and Olwin, 1989). However, the relationship of this complex to FGF signaling remains to be established.

X. FGF SIGNALING Intracellular signaling via the high-affinity receptor requires that FGF first associate with low-affinity HSPGs (Yayon et al., 1991; Klagsbrun and Baird, 1991). This complex then associates with the ligand binding site of the FGF receptor via domains which are distinct from the HSPG binding domain (Springer et al., 1994),

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thereby, forming a dual receptor signaling system. The balance of evidence at present suggests, at least for FGF-1 and FGF-2, that the binding reaction with the FGFR is of low affinity unless HS is present (Yayon et al, 1991; Rapraeger et al, 1991; Omitz and Leder, 1992; Pantoliano et al, 1994; Roghani et al, 1994). It is probable that the oligosaccharide sequence in the HS molecule, active during facilitation of FGF-2/FGFR signaling, is oligo-H (Walker et al., 1994). The increase in affinity upon binding may be achieved by a reduction in the dissociation of the ligand-receptor complex (Moscatelli, 1992; Wordinger et al., 1992), and partly by inducing a conformational change in the FGF molecule which reveals reactive sites (Prestrelski et al., 1992). Further evidence suggests that some FGFs dimerize upon binding to HS (Omitz and Leder, 1992; Spivak-Kroizman et al., 1994). Thus, the HS induced oligomerization of FGF provides a template whereby two molecules of FGF have the potential to juxtapose two molecules of the FGFR, thereby facilitating receptor dimerization. Current models suggest that activation of the high-affinity receptor is achieved by HS modifying the conformation of and/or the intermolecular association of FGFs and that the structure of the HS chains attached to core proteins determines the binding/activation sequence for a specific FGF. Thus, sulfation of cellular HS will be a major factor in determining whether a cell responds to each of the FGFs. Thus there exists a subtle means of achieving specificity between the FGFs and their receptors. FGF ligands bind to the extracellular Ig-like domains of the high affinity receptor. Although loop I appears to be dispensable for binding and receptor activation, Ig loops II and III are both implicated. But loop I may have a role and be important in determining the binding affinity of FGFs and HS. Individual domains in Ig loops II and III are highly specific in their interactions with the different FGFs, and alternative splicing of exons encoding the C-terminal regions of loop III generate receptor isoforms that are differentially responsive to FGF ligands. As with other receptor tyrosine kinases, key steps in the intracellular signaling pathway have been identified. Ligand-induced dimerization of the receptors, including the formation of hetero-FGFR dimers, activates the cytoplasmic tyrosine kinase domain of the receptor through transphosphorylation. This, in turn, activates the subsequent tyrosine phosphorylation of adapter proteins, leading to the activation of c-Ras and the MAP kinase pathway. The subsequent kinase cascades, which probably involve Raf and MAP kinase, culminate in de novo gene transcription (see the review by Jaye et al., 1992). Considerable evidence is now accumulating that some FGFs can also generate intracellular signals independently of the classical cell membrane FGFR signaling pathway. After binding to HSPG and the tyrosine kinase receptor, exogenous FGF-1 and FGF-2 are rapidly internalized. Several investigators have suggested that internalization is mediated by the HSPGs (Quarto and Amalric, 1994). Once internalized, FGF is extremely stable, with a half-life of up to 10 hours. At that time, the FGF may undergo limited proteolysis to smaller fragments while being translo-

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cated to the nucleus. Here it accumulates in the nucleoli and modulates ribosomal gene transcription (Bouche et al, 1987; Amalric et al, 1994) by mechanisms which may yet again involve the kinase activity of its receptor (Quarto and Amalric, 1994; Wiedlocha et al., 1994). This nuclear translocation is cell cycle dependent, occurring during the transition between GQ and Gj. This process is distinct from the nuclear translocation of the extended isoforms of endogenous, intracellular FGF-2 and FGF-3. In the case of these high molecular weight forms of FGF-2, the translocation of FGF-2 is thought to be mediated by an endogenous and intracellular binding protein, homologous to its high-affmity receptor (Kilkenny and Hill, 1995). Once localized to the nucleus, the ligand may act directly as a transcription factor. These observations led to the proposal of an intracrine mechanism of action for some FGFs, whereby the actions of specific isoforms of the growth factor are achieved without them ever being exported from the cell (Logan, 1990).

XL PROTEOGLYCANS AND THE REGULATION OF FGF BIOACTIVITY It is becoming clear that not all FGF binding sequences in HS-GAGs participate in the dual receptor system (Aviezer et al., 1994), suggesting that the storage function of the HSPGs may be more subtle than originally envisaged. Hence, varying the structure of the oligosaccharide chains within HS can provide a means of precisely regulating which FGF ligand is able to bind and is capable of being presented to the FGFR. This depot storage system lends itself to very tight regulation of ligand bioactivity, an absolute requirement for such potent molecules whose synthesis is defined by a constitutive rather than a regulated pathway. Understanding how FGF activity is regulated came with the realization that it is bioavailability rather than biosynthesis that is controlled. During the rapid growth and remodeling of early development, expression of FGF-2 and FGFRl mRNAs are relatively high in most mesenchymal tissues. In contrast, expression of FGF-2 is negligible in normal adult life, except in a few tissues, such as the brain. In most peripheral tissues, FGF-2 is found outside target cells associated with HSPGs in what is presumed to be a functionally inert form. Because the binding sites for FGF-2 on HSPGs outnumber those on FGFRs, the balance of affinity leans towards extracellular matrix molecules, thereby sequestering FGF-2 from its signaling receptor. This interaction constitutes a depot store of FGF-2 and protects the peptide from proteolytic denaturation, providing a means of regulated release from cell surfaces. More recently, Hanneken et al. (1995) described the presence of immunoreactive FGF receptors in the matrix as well. These molecules, generated from the extracellular portion of the intact receptor (presumably by proteolysis) or encoded by a "secreted variant" of the FGFRl gene, presumably bind the FGF that is sequestered in the matrix. If this is correct, then the ligand would be devoid of any activity.

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These observations raise the possibility that FGFs in the matrix may also be biologically active if not "capped" by a soluble receptor. In such an instance, one particular activity may be its ability to promote adhesion. Early studies from our laboratories (Schubert et al., 1987; Baird et al, 1988) established that FGF-2 is a potent adhesion factor. This characteristic served as the basis for the expression cloning of its low-affmity receptor (Kiefer et al., 1990). In this paradigm, the ligand could play a significant role, even though it is physically restrained by the matrix. Its inability to dissociate from the matrix could serve as an indicator to the target cell that local homeostasis is "normal". Similarly, upon physical injury and/or damage to the matrix, the ligand-matrix interaction is disrupted, and the FGF can be internalized to elicit a biological response. Under these conditions the acidic environment of normal wound fluid (pH <4) would suffice to remove soluble receptors from sequestered ligands in the matrix and, thus, make them bioavailable for neighboring target cells as well. Reactivation of FGF-2 responsiveness in differentiated tissues can be achieved in a number of ways (Feige and Baird, 1991; Logan and Hill, 1992). Upregulation of high-affinity receptor expression or chemical modification of FGF-2 leads to altered kinetics between binding sites and a consequently altered interaction with the existing receptor pools. Evidence also suggests that phosphorylation of FGF-2 by a cell surface protein ectokinase produced by target cells achieves just this modification of the molecule's, binding affinity thereby, releasing the factor from its sequestration and allowing bioactivation and interaction with its signaling receptor (Vilgrain and Baird, 1991; Fiege et al., 1991; Vilgrain et al., 1993). In addition, regulated enzymatic degradation of the extracellular matrix by proteolytic and glycolytic enzymes (plasmins, cathespins, collagenases, and heperanitases), either derived locally or released from invasive cells (macrophages), is a further mechanism of FGF release from the matrix. It should be noted that cell surface and extracellular GAGs may also act in concert to regulate the bioavailability of FGF to their signaling receptor (Ruoslahti and Yamaguchi, 1991). Extracellular matrix HSPGs, such as perlecan, can act as long-term depots to replenish ligand-depleted cell-surface syndecan. Hence, the local expression and modification of perlecan, and the balance between different classes of HSPGs, will determine the FGF-induced cellular response: a stimulating response can be modified by the local overexpression of perlecan or the FGF-2 inhibitory HSPG syndecan-1 (Mali et al., 1993). Alternatively, reduction or altered HSPG expression may lead to ligand activation by shifting the balance of affinities to the signaling receptor.

Xll. PHYSIOLOGY AND PATHOLOGY OF THE FGFS Many FGFs are constitutively and coincidentally expressed by mesenchymal tissues. When considering the physiological actions of FGFs, a key question presents itself: How is specificity of action upon target cells ensured? The answer

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is emerging as the precise and regulated expression of multiple FGF and FGFR isomers during specific physiological processes is determined. FGF and FGFR isomers are expressed very precisely in many tissues throughout embryonic development and organogenesis. Studies inXenopus suggest that FGF-2 acts at one of the earliest stages in the vertebrate embryo to induce formation of the ventral mesoderm (see review by Slack, 1994). Observations with dominant-negative FGFRl mutant mice support this contention, because these embryos undergo defective gastrulation and lack posterior and ventral structures (Amaya et al., 1991; Amaya et al., 1993). Perhaps one of the best illustrations of the balanced specificity in FGF/FGFR interactions is seen during organogenesis of the limb. A. Limb Development There is now good evidence to suggest an interactive role for individual FGFs in the spatial patterning of the limb bud and its later differentiation. FGF-4 is expressed by the posterior half of the apical ectodermal ridge of the limb bud as soon as it is formed. This structure maintains the growth of underlying cells, and FGF-4 may act as a signal between the epithelium and the underlying mesenchyme to ensure outgrowth of the developing limb (Niswander et al., 1993). In this posterior position, FGF-4 maintains the polarizing region of the limb bud, hence, influencing the number and organization of the developing skeletal elements. In contrast, FGF-8 is expressed over the entire apical ectodermal ridge from earlier to later stages than FGF-4, suggesting a different function, perhaps as a general inhibitor of differentiation of underlying mesenchymal cells (Ohuchi et al., 1994), At later stages, FGF-2 is expressed by proliferating chondrocytes and localizes to the perichondrium (Gonzalez et al., 1990). This expression is spatially and temporally organized in the epiphyseal growth plate as differentiation proceeds, so that FGF-2 levels are highest in the proliferative chondrocyte zone, decrease during differentiation, and are absent from hypertrophic chondrocytes. FGF-5 is also expressed by limb mesenchymal cells ventral to the presumptive femur as it undergoes cartilage formation. These observations suggest a mitogenic role for FGF-2 and FGF-5 in mesenchymal proliferation and a possible morphogenic role for both during cartilage formation. The highly specific role of different FGFs in spatial patterning of limb buds and subsequent organogenesis is underlined by observations of discrete patterns of FGFR expression. From the earliest stages of limb bud formation, FGFRl is expressed diffusely within the limb bud mesenchyme (Peters et al., 1992; Patstone et al., 1993). In contrast, FGFR2 is first expressed in the mesenchyme, with a concentration gradient increasing in a posterior and proximal direction. This expression then becomes focused to mesenchymal aggregates that will form the future bones and also to the surface ectoderm of the limb, being strongest in the interdigital web. Consolidation of expression is seen at later stages in the chondrification centers and to the bodies of the distal bones. This temporal pattern of expression strongly suggests that FGFR2 mediates FGF actions on chondrogenic

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pathways, and FGFRl may mediate FGF actions in the surrounding undifferentiated mesenchyme. At even later stages, FGFR4 mRNAmaps to areas of cartilage condensation, and FGFR3 mRNAto resting cartilage during endochondrial ossification (Stark et al, 1991; Peters et al., 1993; Patstone et al., 1993). FGF-2, FGF-4, FGF-5, FGF-6, and FGF-7 are also strongly implicated as regulators of myogenesis within the developing limb (reviewed by Olwin et al., 1994). FGF-2 has widespread expression within newly formed myotomes, where it stimulates myoblast proliferation, inhibits terminal differentiation (Clegg et al., 1987), and suppresses the transcription of at least four myogenic regulatory transcriptional activators (myogenin, myf-5, MRF-4, and myoDl) in an autocrine manner (Vaidya et al., 1989; Brunetti and Goldfme, 1990). This suggests an overall role of repressing terminal differentiation in the developing tissue system, thereby determining the number of cells entering the myogenic lineage. FGF-4,5,6, and 7 expression is switched on much more specifically in muscle just prior to activation of the muscle regulatory genes (Niswander and Martin, 1992), with FGF-5 expression continuing as myotomal cells migrate ventrally and laterally (Haub & Goldfarb, 1991). These observations suggest that FGF-5 may selectively suppress the differentiation of these cell lineages as they migrate. Thus, within the limb, different FGFs are key determinants of the whole skeletal/muscle system, coordinating amplification of myoblasts and chondrocytes, their subsequent migration, and their eventual differentiation. The fundamental role of the FGF axis in ordering chondrogenesis both in limbs and other skeletal elements has been recently emphasized by the demonstration that humans with a variety of genetic bone defects have mutations in genes of the FGFR family. Patients suffering from achondroplasia of the limbs invariably exhibit mutations in the FGFR3 receptor (Rousseau et al., 1994). Jackson-Weiss, Crouzon, Apert, and Pfeiffer syndrome are distinct forms of craniosynostosis (premature fusion of several sutures of the skull), which may be associated with syndactyly (cutaneous and bony fusion of the digits) of the hands and feet, that have been shown to be associated with mutations in the FGFR2 gene (Jabs et al., 1994; Rutland et al., 1995). The origin of Pfeiffer syndrome may be heterogeneous, because others have reported patients of unrelated families who show mutations in the FGFRl gene (Muenke et al., 1994; Lajeunie et al., 1995). B. Lung Development Both FGFR 1 and its ligand FGF-2 have been localized by immunohistochemistry to the airway epithelia of the fetal rat lung (Han et al., 1992). The Illb isoform of FGFR2 is expressed by epithelia at the branch points of the bronchioles. In contrast, FGF-7 (an FGFR2 ligand), is present in lung mesenchyme during the period of epithelial development (Mason et al., 1994). A morphogenic role for FGFs in the development of this organ has been demonstrated by studies with dominant-negative FGFR2 Illb transgenic mice (Peters et al., 1994). In such homozygous animals.

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no branching of the bronchi occurs, and the animals die at birth with an absence of lung development. C. Development of the Nervous System

The in vitro activities of the FGFs for glia and neurons have been well studied. In cultured cells, the activities of FGF-1 and FGF-2 are very similar, and FGF-2 has been the best studied. The ability of FGF-2 to stimulate neurite outgrowth, survival, and differentiated function of, particularly, cholinergic, embryonic, and postnatal cultured neurons is well described (Walicke et al., 1986; Walicke, 1988; Walicke and Baird, 1988; Abe et al., 1992; Brill et al., 1992; Kushima et al., 1992; Ishikawa et al., 1992; Miyagawa et al., 1993; Beck et al., 1993; Mayer et al., 1993; Ray et al., 1993). This factor is also known to regulate astrocyte and oligodendrocyte growth, differentiation, and function in culture (Engele and Bohn, 1992; Wolswijk and Noble, 1992; Yoshida and Gage, 1992; Pechan et al., 1993; Vijayan et al., 1993; McKinnon et al., 1993). Presumably, the activities demonstrated in vitro may relate to different FGF ligands in vivo. However, there are very few studies yet published that relate the neurotrophic activities of other FGFs, although FGF-5 is a potent muscle-derived survival factor for cultured spinal motor neurons (Hughes et al., 1993). Within the nervous system, FGFs are suggested to be particularly important in determining growth, differentiation, and function of both neurons and glia. The adult brain is one of the richest sources of FGFs during development and also in adult life. Despite the early identification of the FGFs within nervous tissue, rigorous mapping studies to show the differential distribution of individual FGFs and their receptors are only just beginning to be described. For example, in the rodent central nervous system (CNS), endogenous expression of FGF-2, 3, 4, and 8 has been demonstrated in embryos from the earliest stages of neural crest and neural tube development to maturation (Gonzalez et al., 1990; Kalcheim and Neufeld, 1990; Mason et al., 1994; Ohuchi et al., 1994). FGF-1 is found in glia and neurons in multiple areas of the brain during development and at maturity (Wilcox andUnnerstall, 1991; Faucheux et al., 1992; Stock etal., 1992; Fallon etal., 1992). Gonzalez et al. (1995) have recently completed the most systematic study to date of FGF-2 expression in the adult brain. This reveals the pattern of FGF-2 synthesis in the adult is unique to this ligand and very precise; there are specific anatomical foci of mRNA expression (for example, the CA2 hippocampus), and the peptide localizes to the nucleus and/or cytoplasm of multiple cell types including neurons, astrocytes, and endothelial, ependymal, and choroid cells. This precise pattern of distribution clearly indicates a primary function of FGF-2 within the brain, but it is not understood. Even less is known about FGFRs in the developing and adult CNS. FGFRl, FGFR2, and FGFR3 are coexpressed in the germinal epithelium of the neural tube in the mouse and, at later stages, become differentially associated with glioblasts and neuroblasts, with the patterns dissociating even more in the adult brain. Here,

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FGFR3 seems to be associated with glia (Peters et al., 1993), whereas FGFRl expression occurs predominantly in defined populations of neurons, although the peptide is also found localized to the sheaths of all myelinated axons (Gonzalez et al., 1995). A further mechanism to generate diversity of FGF action is seen in chick embryos where FGFRs alternate sequentially with nerve growth factor receptors on differentiating neural cells (Heuer et al, 1990). Accordingly, FGF-2 enhances the promoter activity of the NGF receptor gene in human neuroblastoma cells (Taiji et al, 1992). Such mechanisms can represent a highly dynamic means of inducing growth, differentiation, or migration depending on the spatio-temporal program of receptor expression. The clues to the CNS functions of FGFs in fetal and adult life lie in future studies that systematically define the distribution of FGF ligands and receptors. Notwithstanding the lack of systematic information about the in vivo functions of FGFs in the mature CNS, specific roles for individual factors during development are now becoming apparent; for example, FGF-3 and FGF-4 may be involved in the spatial patterning processes of this tissue. During its development, the hindbrain is transiently subdivided into a series of morphological segments called rhombomeres, each of which is a lineage-restricted compartment with a unique molecular identity. This segmentation is subsequently reflected in the disposition of motor neuron populations, the exit points of branchial nerves, and the production of neural crest cells. FGF-3 expression localizes to a population of boundary cells between the rhombomeres, thought to prevent cell movement to adjacent rhombomeres and to encourage the preferential accumulation of axons of certain neuron classes (Mason, 1994). An intriguing new mechanism of regulating developmental neurite outgrowth by the FGF axis has been defined recently. Cell adhesion molecules (CAMs) on the surface of neurons help growing axons navigate towards their defined target cells by linking up with similar molecules in the matrix or on other cells. CAMs themselves are not endowed with an intracellular signaling capacity; therefore, this interaction was thought to provide only passive structural support for the extending neurite. However, evidence now suggests that CAMs act to direct outgrowth via growth factor signaling pathways. Williams et al. (1994) noted that a 20 amino acid sequence within the extracellular portion of the FGFRl molecule has multiple CAM-related motifs (termed the CAM homological domain) and that CAM activity is FGFR-dependent. Further, they demonstrated that certain CAMs can activate the FGFRs, thereby, triggering the intracellular kinase cascade that initiates neurite growth (Doherty et al., 1995). D. FGFs in Neurodegenerative Conditions

The neurotrophic activities of endogenous FGF-1 and FGF-2 within the CNS are well-characterized. Because diffuse or localized loss of viable neurons characterizes diseases of neurodegeneration, such as Parkinson's disease, Huntington's chorea, Alzheimer's disease, and amyotrophic lateral sclerosis, it is not surprising

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that perturbation of the FGF axis has been implicated as a factor contributory to their progression. Studies of patients with Alzheimer's disease have suggested that astrocytic expression of FGF-2 is increased by (i-amyloid and that the peptide is sequestered to HS-GAGs contained in the P-amyloid of senile plaques (Stopa et al, 1990; Yasuhara et al., 1991; Cotman and Gomez-Pinilla, 1991). The observation that exogenous FGF-2 can attenuate the neurodegenerative effects of p-amyloid (Mattson and Cheng, 1993) supports the contention that FGF-2 sequestration contributes to disease progression. Huntington's chorea is characterized by a dramatic gliosis within the diseased and degenerating tissue. The cellular response is accompanied by a concerted increased expression of FGF-1 and FGF-2 (Tooyama et al, 1993a), both known gliogenic factors (Logan et al, 1994). With Parkinson's disease there is a loss of dopamine-containing neurons in the substantia nigra, and this is accompanied by a loss of FGF-2 expression (Tooyama et al., 1993b). Transplantation of embryonic dopamine-rich neuronal tissue into the brains of patients, whilst seeming a satisfactory strategy in theory, has proved to be disappointing in practice due to poor survival of the grafted cells. However, recent work suggests the viability of such grafted neurons can be significantly enhanced by co-infusion of FGF-2 or by co-transplanting cells genetically modified to produce FGF-2 (Takayama et al., 1995). Whilst the potential gliogenic and fibrogenic activities of endogenously produced FGF-2 might be envisaged as a problematic side effect of such treatments, in practice there is little evidence to suggest that this is so (Otto and Unsicker, 1994; Takayama et al., 1995). A significant body of evidence suggests that the neurotrophic activities of FGF-2 may be usefully exploited in clinical treatment of patients after ischemic or traumatic neuronal damage. Damaged CNS neurons do not regenerate after they have been damaged, and ftirthermore, may undergo a process of targeted degeneration (Logan et al., 1994). Expression of FGF-2 is upregulated in damaged CNS tissue (Logan et al., 1992; Gomez—Pinilla and Cotman, 1992; Gomez—Pinilla et al., 1992; Grothe and Unsicker, 1992; Kumon et al., 1993), and inftision of wounds with recombinant FGF-2 protein is reported to facilitate directly and/or indirectly neuronal repair and survival (Blottner and Baumgarten, 1992; Cummings et al., 1992; Gumey et al., 1992; Araujo et al., 1993; Nakata et al., 1993; Mayer et al., 1993). E. FGFs as Angiogenic Factors

FGFs are potent angiogenic factors found in most normal tissues complexed to HSPGs in the sub endothelial extracellular matrix and to the basement membranes of blood vessels (Vlodavsky et al., 1987; Folkman et al., 1988). Early in vitro experiments established the angiogenic activities of FGF-2 by showing that the peptide stimulates proliferation (Folkman et al., 1979), migration, and production of proteases in capillary endothelial cells (Moscatelli et al., 1986), and that its application leads to angiogenesis in chick embryo chorioallantoic membrane and

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cornea (Folkman and Klagsbrun, 1987). Further, the expression of FGF-2 in various tissues reveals a typical accumulation of the peptide in areas of enhanced capillary growth, consistent with angiogenesis. The close proximity of FGF-2 with its receptors to sites of both high and low angiogenic activity suggest that local growth factor—receptor interactions are subject to tight regulatory controls, which maintain the resting state in the adult vasculature, as well as rapid recruitment to an active angiogenic state when required. During embryogenesis, the development of the vascular system and the vascularization of organs tissues is regulated by a panel of angiogenic factors, including FGF-2. For example, neovascularization of the renal anlage is a paracrine process mediated in part by FGF-2 (Folkman, 1995). In the adult, new blood vessel growth occurs only at a few restricted sites where tissue growth and remodeling occurs, for example, angiogenesis in cycling reproductive tissues. Growth of the ovarian follicle and its corpus luteum may be governed by increased angiogenesis in the dominant follicle, where expression of heparin binding FGFs have been demonstrated (Reynolds et al., 1992). The angiogenic activities FGF-2 are implicated in the vascularization of scar tissue that occurs during wound healing of all tissues. Studies in the injured brain have established an upregulation of FGF-2 and FGFRl expression in the endothelial cells of the brains microvasculature and in the surrounding neuronal parenchyma (Logan et al., 1992). The increased synthesis of peptide at the wound site is accompanied by the appearance of soluble FGF-2, both within the wound and circulating in the cerebrospinal fluid (Logan et al., 1994). These observations suggest that FGF-2 may act in a paracrine and/or autocrine fashion initiating the new capillary growth that occurs in damaged neural tissue. Furthermore, this activity is substantiated by the observation that infusion of CNS wounds with recombinant FGF-2 leads to increased an angiogenic response at the injury site (Logan etal., 1994). Several diseases of the gastrointestinal tract are also characterized by abnormal regulation of angiogenesis. Human gastric ulcers show a 23fold decrease in FGF-2 content compared to normal mucosa where levels are relatively high (Hull et al., 1994). In experimental animals, oral administration of FGF-2 leads to a ninefold increase in microvessel density and an accelerated healing of ulcers (Folkman et al., 1991). Phase 1 clinical trials are presently underway to orally treat patients refractory to conventional treatments with an acid-resistant form of FGF-2. In the multistep pathway to tumorigenesis, tumor progression, and metastasis, angiogenesis is an early and essential step with which FGF-2 has been linked (Folkman and Klagsbrun, 1987). For example, FGF-2 is overexpressed in infantile hemangiomas during the proliferative phase, and this expression declines during tumor involution (Takahashi et al., 1994). Also, the transition from poorly vascularized benign fibromatosis to the highly angiogenic state typical of malignant fibrosarcoma is associated with a shift in the export and/or release of FGF-2 from extracellular stores (Kandel et al, 1991), which may be accompanied by an

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increased synthesis of the factor by endothelial cells of the tumor vasculature (Gross et al., 1992). Such a release of FGF-2 from its depot stores results in bioactivation of the solubilized protein, and the potential for enhanced tumor angiogenesis. Such an increase in soluble FGF-2 has been reported in the plasma of patients with multiple endocrine neoplasia (Zimering et al., 1990; Zimering et al., 1993) and in the urine of patients with infantile hemangiomas. In this situation, the dysregulated activity of the soluble FGF-2 may promote tumor growth and facilitate metastasis to certain tissues. The tissues, which become sites of secondary tumor growth, may themselves be targeted for metastatic seeding as a result of their production of angiogenic factors, such as FGF-2 (McCarthy et al., 1991). A number of clinical trials are underway with a variety of antiangiogenic therapies to inhibit tumor growth and metastasis. F. FGFs in Tumorigenesis

In addition to promoting angiogenesis in neoplastic tissues, altered production or activity of growth factors and their receptors may also directly stimulate tumor cell growth. The unregulated synthesis of a growth factor by a tumor cell results in an autocrine mechanism of growth, and alteration in the structure or function of a receptor system produces a similar result by creating a growth factor system that is permanently switched on, even in the absence of exogenous ligand. A growing body of evidence exists suggesting a role for FGFs in tumorigenesis of many tissue systems. In fact, many of the FGFs including FGF-3 (int-2), FGF-4 (hst-1/Kaposi's sarcoma), FGF-5, FGF-6 (hst-2), FGF-8 (androgen-induced growth factor), and FGF-9, were identified as a result of their transforming activities. Perhaps the best studied carcinoma in abrogated FGF activity is that of the mammary gland. Because FGFs are mitogens important in epithelial-mesenchymal interactions in many tissues, particular attention has been placed on their roles in the mammary gland. FGF-1, FGF-2, FGFRl, and FGFR2 are localized within normal human mammary glands and their synthesis is increased in tumorous tissues (Luqmani et al., 1992;Peyratetal., 1992; Hughes and Hall, 1993; Smith etal., 1993; Jacquemier et al., 1994; Anandappa et al., 1994). In humans, the FGFRl gene is located in region pi 2 of chromosome 8, a region amplified in some subsets of breast tumors (Adnane et al., 1991). FGFR2 and FGFR4 are also amplified in some breast tumor samples (Adnane et al., 1991; Jaakkola et al., 1993). However, it is not yet clear whether FGF ligands are synthesized by myoepithelial or epithelial cells (Barraclough et al., 1990; Gomm et al., 1991; Li and Shipley, 1991; Rudland et al., 1993), and, similarly, it seems that the receptors may be present at either site (Takahashi et al., 1989; Femig et al., 1990). The precise physiological and pathological role of FGFs in this tissue is not established. It is likely that the oncogenic activity of FGFs relates to FGF ligands other than FGF-1 and FGF-2. In particular, the oncogenes FGF-3 (Moore et al., 1986), FGF-4 (Jacquemier et al., 1994), and FGF-9 (Tanaka et al., 1992) are activated by mammary virus insertion in some

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mouse mammary tumors and can act as potent mitogenic factors in cellular or transgenic murine models. Whether abrogated expression of FGF related genes is coincidental or contributes to the tumorigenic process in human tissues remains to be established. Furthermore, mechanisms of dysregulation of the FGF axis in addition to changed levels of expression must also be considered. For example, in pancreatic and prostate tumors, structural alterations in FGFRs, such as C-terminal truncations and variations in receptor affinity, have been found which may contribute to tumorigenesis (Kobrin et al., 1993; Yan et al., 1993). This shift in receptor expression and ligand specificity may allow the cell to escape normal regulatory processes by enabling it to access alternative FGFs in the local environment. Clearly, understanding the mechanisms of tumor growth and, in particular, the molecular subclassification of tumors will help in designing new types of therapy based on growth inhibitors, such as growth factor antagonists, mitotoxins, antisense oligonucleotides, or antibodies.

XIII. CONCLUSIONS Now that so much is known regarding the components of the FGF-FGF receptor axis, the challenge will be to understand their role in normal physiology and the pathogenesis of disease. With at least nine ligands interacting with receptor complexes of any combination of four high-affmity receptors (in various isoforms) and several potential low-affmity receptors, the challenge is not insignificant. Major questions remain, and our knowledge has just scratched the surface. For example, what mechanisms regulate the bioavailability of FGFs. What intracellular functions do these molecules play, and what is the significance of their nuclear localization (and that of their receptors)? What molecular processes permit the export across the plasma membrane of molecules (FGF-1, FGF-2) that have no signal peptide? How can ubiquitous distribution, pluripotent growth factors like FGF-1 and FGF-2 have specificity? How can target cells with identical cell surface receptor profiles respond so differently to the same trophic signal? Finally, how do the FGFs alter the biological response to other growth factors, and how do other cytokines alter their responsiveness to FGFs? Although it is unlikely that any of these outstanding issues will be resolved soon, they can be the template for measuring progress. Resolving these questions will enable us to understand the molecular basis of disease, thus, helping develop novel, innovative, and unique approaches to control the pathophysiological response to growth factors. REFERENCES Abe, K., Ishiyama, J., & Saito, H. (1992). Effects of epidermal growth factor and basic fibroblast growth factor on generation of long-term potentiation in the dentate gyrus of fimbria-fomix-lesioned rats. Brain Res. 593, 335-338. Acland, P., Dixon, M., Peters, G., & Dickson, C. (1990). Subcellular fate of the int-2 oncoprotein is determined by choice of initiation codon. Nature 343, 662-665.

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Bugler, B., Amalric, F., & Prats, H. (1991). Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor. Mol. Cell. Biol. 11, 573-577. Burrus, L. W., & Olwin, B. B. (1989). Isolation of a receptor for acidic and basic fibroblast growth factor from embryonic chick. J. Biol. Chem. 264, 18647-18653. Burrus, L. W., Zuber, M. E., Luddecke, B. A., & Olwin, B. B. (1992). Identification of a cysteine-rich receptor for fibroblast growth factors. Mol. Cell. Biol. 12, 5600-5609. Cao, Y., Ekstrom, M., & Pettersson, R. F. (1993). Characterization of the nuclear translocation of acidic fibroblast growth factor. J. Cell Sci. 104, 77-87. Chiu, L. M., Wang, W. P., & Lehtoma, K. (1990). Alternative splicing generates two forms of mRNA coding for human heparin-binding growth factor 1. Oncogene 5, 755-762. Clegg, C. H., Linkhart, T. A., Olwin, B. B., & Hauschka, S. D. (1987). Growth factor control of skeletal muscle differentiation: Commitment to terminal differentiation occurs in Gl phase and is repressed by fibroblast growth factor. J. Cell Biol. 105, 949-956. Cotman, C. W., & Gomez—Pinilla, F. (1991). Basic fibroblast growth factor in the mature brain and its possible role in Alzheimer's disease. Ann. NY Acad. Sci. 638,221-231. Couderc, B., Prats, H., Bayard, F., & Amalric, F. (1991). Potential oncogenic effects of basic fibroblast growth factor requires cooperation between CUG and AUG-initiated forms. Cell Regul. 2, 70^718. Crumley, G., Dionne, C. A., & Jaye, M. (1990). The gene for human acidic fibroblast growth factor encodes two upstream exons alternatively spliced to the first coding exon. Biochem. Biophys. Res. Commun. 171,7-13. Cummings, B. J., Yee, G. J., & Cotman, C. W. (1992). bFGF promotes the survival of entorhinal layer II neurons after perforant path axotomy. Brain Res. 591, 271-276. Curatola, A. M., & Basilico, C. (1990). Expression of the K-^proto-oncogene is controlled by 3' regulatory elements which are specific for embryonal carcinoma cells. Mol. Cell. Biol. 10, 2475-2484. Dell, K. R., & Williams, L. T. (1992). A novel form of fibroblast growth factor receptor 2: Alternative splicing of the third immunoglobulin-like domain confers ligand binding specificity. J. Biol. Chem. 267, 21225-21229. Doherty, P., Williams, E., & Walsh, F. S. (1995). A soluble chimeric form of the LI glycoprotein stimulates neurite outgrowth. Neuron 14, 57-66. Dono, R., & Zeller, R. (1994). Cell-type specific nuclear translocation of fibroblast growth factor-2 isoforms during chicken kidney and limb morphogenesis. Dev. Biol. 163, 316-330. Duan, D.—S. R., Werner, S., & Williams, L. T. (1992). A naturally occurring secreted form of fibroblast growth factor (FGF) receptor 1 binds basic FGF in preference over acidic FGF. J. Biol. Chem. 267, 16076-16080. Engele, J., & Bohn, M. C. (1992). Effects of acidic and basic fibroblast growth factors (aFGF, bFGF) on glial precursor cell proliferation: Age dependency and brain region specificity. Dev. Biol. 152, 363-372. Esch, F., Baird, A., Ling, N., Ueno, N., Hill, F., Denoroy, L., Klepper, R., Gospodarowicz, D., Bohlen, P., & Guillemin, R. (1985). Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine acidic FGF. Proc. Natl. Acad. Sci. USA 82, 6507-6511. Fallon, J. H., Di Salvo, J., Loughlin, S. E., Gimenez-Gallego, G., Seroogy, K. B., Bradshaw, R. A., Morrison, R. S., Ciofi, P., & Thomas, K. A. (1992). Localization of acidic fibroblast growth factor within the mouse brain using biochemical and immunocytochemical techniques. Growth Factors 6, 139-157. Faucheux, B. A., Cohen, S. Y, Delaere, P., Tourbah, A., Dupuis, C, Hartmann, M. P., Jeanny, J. C, Hauw, J. J., & Courtois, Y (1992). Glial cell localization of acidic fibroblast growth factor-like immunoreactivity in the optic nerve of young adult and aged mammals. Gerontology 38,308-314.

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Tanaka, A., Miyamoto, K., Minamino, N., Takeda, M., Sato, B., Matsuo, H., & Matsumoto, K. (1992). Cloning and characterization of an androgen-induced growth factor essential for the androgendependent growth of mouse mammary carcinoma cells. Proc. Natl. Acad. Sci. USA 89, 89288932. Thomas, K. A., Rios-Candelore, M., & Fitzpatrick, S. (1984). Purification and characterization of acidic fibroblast growth factor from bovine brain. Proc. Natl. Acad. Sci. USA 81, 357-361. Tooyama, I., Kawamata, T., Walker, D., Yamada, T., Hanai, K., Kimura, H., Iwane, M., Igarashi, K., McGeer, E. G., & McGeer, P. L. (1993a). Loss of basic fibroblast growth factor in substantia nigra neurons in Parkinson's disease. Neurology 43, 372—376. Tooyama, I., Kremer, H. R H., Hayden, M. R., Kimura, H., McGeer, E. G., & McGeer, R L. (1993b). Acidic and basic fibroblast growth factor-like immunoreactivity in the striatum and midbrain in Huntington's disease. Brain Res. 610, 1-7. Ueba, T., Nosaka, T, Takahashi, J. A., Shibata, R, Florkiewicz, R. Z., Vogelstein, B., Oda, Y., Kikuchi, H., & Hatanaka, M. (1994). Transcriptional regulation of basic fibroblast growth factor gene by p53 in human glioblastoma and heptocellular carcinoma cells. Proc. Natl. Acad. Sci. USA 91, 9009-9013. Vaidya, T. B., Rhodes, S. J., Taparowsky, E. J., & Konieczny, S. F. (1989). Fibroblast growth factor and transforming growth factor p repress transcription of the myogenic regulatory gene MyoD 1. Mol. Cell. Biol. 9, 3576-3579. Vijayan, V. K., Lee, Y. L., & Eng, L. F. (1993). Immunohistochemical localization of basic fibroblast growth factor in cultured rat astrocytes and oligodendrocytes. Int. J. Dev. Neurosci. 11,257-267. Vilgrain, I., & Baird, A. (1991). Phosphorylation of basic fibroblast growth factor by a protein kinase associated with the outer surface of a target cell. Mol. Endocrinol. 5, 1003-1012. Vilgrain, I., Gonzalez, A. M., & Baird, A. (1993). Phosphorylation of basic fibroblast growth factor (FGF-2) in the nuclei of SK-Hep-1 cells. Fed. Eur. Biochem. Soc. Lett. 331,228-232 (Abstract). Vlodavsky, I., Folkman, J., Sullivan, R., Fridman, R., Ishai-Michaeli, R., Sasse, J., & Klagsbrun, M. (1987). Endothelial cell-derived basic fibroblast growth factor: Synthesis and deposition into subendothelial extracellular matrix. Proc. Natl. Acad. Sci. USA 84,2292-2296. Walicke, P. A. (1988). Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions. J. Neurosci. 8, 2618-2627. Walicke, P. A., & Baird, A. (1988). Trophic effects of growth factor on neural tissues. Prog. Brain Res. 78, 333-338. Walicke, R A., Cowan, W. M., Ueno, N., Baird, A., & Guillemin, R. (1986). Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension. Proc. Natl. Acad. Sci. USA 83, 3012-3016. Walker, A., Tumbull, J. E., & Gallagher, J. T. (1994). Specific heparan sulphate saccharides mediate the activity of basic fibroblast growth factor. J. Biol. Chem. 269, 931—935. Werner, S., Duan, D.-S. R., De Vries, C , Peters, K. G., Johnson, D. E., & Williams, L. T. (1992). Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligand-binding specificities. Mol. Cell. Biol. 12, 82-88. Wiedlocha, A., Falnes, R O., Madshus, I. H., Sandvig, K., & Olsnes, S. (1994). Dual mode of signal transduction by externally added acidic fibroblast growth factor. Cell 76, 1039-1051. Wilcox, B. J., & Unnerstall, J. R. (1991). Expression of acidic fibroblast growth factor mRNA in the developing and adult rat brain. Neuron 6, 397-409. Williams, E. J., Fumess, J., Walsh, F. S., & Doherty, P. (1994). Activation of the FGF receptor underlies neurite outgrowth stimulated by LI, N-CAM, and N-Cadherin. Neuron 13, 583-594. Wolswijk, G., & Noble, M. (1992). Cooperation between PDGF and FGF converts slowly dividing Q_2^«^w^ progenitor cells to rapidly dividing cells with characteristics of 0-2A^^'''"''^''' progenitor cells. J. Cell Biol. 118, 88^900.

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Wordinger, R. J., Moss, A. E., Lockard, T., Gray, D., Chang, I.-F. C, & Jackson, T. L. (1992). Immunohistochemical localization of basic fibroblast growth factor within the mouse uterus. J. Reprod. Fertil. 96, 141-152. Yamaguchi, T. R, Harpal, K., Henkemeyer, M., & Rossant, J. (1994). FGFR-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Develop. 8, 3032—3044. Yan, G., Fukabori, Y, McBride, G., Nikolaropoulous, S., & McKeehan, W. (1993). Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)-FGF receptor genes in prostate epithelial cells accompany stromal independence and malignancy. Mol. Cell. Biol. 13, 4513-4522. Yasuhara, O., Tooyama, I., Akiyama, H., Akiguchi, I., Kimura, J., McGeer, R L., Hara, Y, & Kimura, H. (1991). Reactive astrocytes express acidic fibroblast growth factor in Alzheimer's disease brain. Dementia 2, 64—70. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, R, & Omitz, D. M. (1991). Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64, 841-848. Yazaki, N., Fujita, H., Ohta, M., Kawasaki, T., & Itoh, N. (1993). The structure and expression of the FGF receptor-1 mRNA isoforms in rat tissues. Biochim. Biophys. Acta Gene Struct. Express. 1172,37-42. Yoshida, K., & Gage, F. H. (1992). Cooperative regulation of nerve growth factor synthesis and secretion in fibroblasts and astrocytes by fibroblast growth factor and other cytokines. Brain Res. 569, 14-25. Yu, Y L., Kha, H., Golden, J. A., Migchielsen, A. A. J., Goetzl, E. J., & Turck, C. W. (1992). An acidic fibroblast growth factor protein generated by alternate splicing acts like an antagonist. J. Exp. Med. 175, 1073-1080. Zhan, X., Bates, B., Hu, X., & Goldfarb, M. (1988). The human FGF-5 oncogene encodes a novel protein related to fibroblast growth factors. Mol. Cell. Biol. 8, 3487-3497. Zimering, M. B., Brandi, M. L., DeGrange, D. A., Marx, S. J., Streeten, E., Katsumata, N., Murphy, R R., Sato, Y, Friesen, H. G., & Aurbach, G. D. (1990). Circulating fibroblast growth factor-like substance in familial multiple endocrine neoplasia type 1. J. Clin. Endocrinol. Metab. 70, 149-154. Zimering, M. B., Katsumata, N., Sato, Y, Brandi, M. L., Aurbach, G. D., Marx, S. J., & Friesen, H. G. (1993). Increased basic fibroblast growth factor in plasmafrommultiple endocrine neoplasia type 1: Relation to pituitary tumor. J. Clin. Endocrinol. Metab. 76, 1182-1187.

PDGF AND FGF RECEPTORS IN HEALTH AND DISEASE

Wendy J. FantI, Kevin G. Peters, and Lewis T. Williams

I. Introduction 180 II. PDGF and FGF Ligands 181 A. PDGF Ligands and their Biological Effects 181 B. FGF Ligands and their Biological Effects 182 III. Genomic Organization and Structural Features of PDGF and FGF Receptors . 183 A. PDGF Receptors 183 B. FGF Receptors . 183 IV. Receptor Activation and Signal Transduction 184 A. Dimerization, Transphosphorylation and Signaling Complex Formation . 184 B. SH2 Domains 185 C. PTB Domains 186 D. SH3 Domains 186 E. PH Domains 186 F. Signaling Pathways Used by PDGF and FGF Receptors 187 G. p21ras and its Downstream Targets . 188 H. The Role ofPI-3 Kinase in Cell Signaling 190

Growth Factors and Cytokines in Health and Disease Volume lA, pages 17^228. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0091-4 179

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WENDY J. FANTL, KEVIN G. PETERS, and LEWIS T. WILLIAMS

I. Raf is Downstream of Ras J. Downstream of Raf K. Ras-Independent Pathways L. Interaction of FGF Receptors with Heparin Sulfate Proteoglycans . . . . V. PDGF and FGF Receptors During Embryonic Development A. PDGF Receptors in Development B. FGF Receptors in Early Embryonic Patterning C. FGF Receptors During Organogenesis D. PDGF and FGF Receptors in Blood Vessel Development E. PDGF and FGF Receptors in Nervous System Development and Maintenance VI. PDGF and FGF Receptors in Human Disease A. PDGF and FGF Receptors in Tumor Biology B. PDGF and FGF Receptors in Genetic Diseases C. PDGF and FGF Receptors in Vascular Disease VII. Therapeutfc Strategies to Modulate PDGF and FGF Receptor Signal Transduction in Human Disease Summary References

192 193 194 196 197 197 198 200 202 203 204 204 206 209 210 211 212

I. INTRODUCTION The phenotypic state of the cell is under the influence of a variety of extracellular signals. Polypeptide growth factors represent a group of extracellular signaling molecules that bind high affinity transmembrane receptors to activate intercellular signaling pathways that control cell growth, differentiation, and survival. A large group of structurally diverse polypeptide growth factors that include platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) mediate signal transduction via high affinity cell surface receptors which characteristically transduce the signals by catalyzing the phosphorylation of signal transduction molecules on specific tyrosine residues. The overall structure of these receptor tyrosine kinases (RTKs) is similar. RTKs are transmembrane receptors that have an extracellular ligand binding domain, a single transmembrane domain and an intracellular kinase domain. The extracellular ligand binding domain has critical cysteine residues, characteristic structural motifs (Ig-like domains), and 0-linked and N-linked sugar moieties. The transmembrane domain is comprised of a stretch of hydrophobic amino acids that link the extracellular domain with the cytoplasmic portion of the receptor. Upon ligand binding, the cytoplasmic portion which contains a tyrosine kinase catalytic domain becomes "autophosphorylated" after which it associates with and activates a variety of intracellular signaling molecules. Although many of the same signaling molecules are activated by different tyrosine kinase receptors, different responses are elicited in different cell types. Numerous studies over the past decade have revealed that despite the overall similarities between tyrosine kinase receptors they may be subdivided based on distinct structural features (Figure 1). This discussion will focus on receptors for PDGF and FGF and elaborate on their roles in health and disease.

PDGF and FGF Receptors in Health and Disease I

1

II

III

IV

V

181 VI

VII

VIII

IX

i

I T EGFR HER2/neu/c-erbB-2 HER3/c-erbB-3

InsulinR Insulin relatedR IGF-1R

PDGFaR PDGFpR MCSF-1R c-kil

FGFR1 FGFR2 FGFR3 FGFR4

Flt1A/EGFR Flk/XDR

Met/HGFR

TrkA TrkB TrkC

Eph Elk Eck Eek Erk Cek4/Mek4/HEK Cek5

Figure 1. Vertebrate receptor tyrosine kinases arranged into subfamilies based on their structural motifs. The following structural features are shown; tyrosine kinase domains (stippled boxes), transmembrane domains {solid boxes), cysteine-rich domains (striped boxes), immunoglobulin-like domains (semi-circles), acid box domain (open box), and fibronectin type 111 domains (checkeredboxes).

II. PDGF AND FGF LIGANDS A. PDGF Ligands and their Biological Effects

The PDGFs are comprised of disulfide linked dimeric molecules made up to tw^o different gene products, the A-chain and the B-chain which reside on human chromosome 7 and 22 respectively. Both chains are highly homologous to each other and the B-chain is functionally identical to p28^^'^ the transforming protein of Simean Sarcoma virus (Heldin, 1992). Three PDGF isoforms exist, AA, BB, and AB which differ in their biological activities as well as in their differential binding affinities to the two known PDGF receptor subunits, type a and P receptors. The A-chain of PDGF binds the a receptor with high affmity whereas the B-chain can bind either the a or P receptor subunits (Table 1). In adult tissues, platelets are the most abundant source of the AB heterodimer and the BB homodimer whereas PDGF AA has been identified as a product of osteosarcoma, melanoma, and glioblastoma (Heldin, 1992; Heldin and Westermark, 1990; Nister et al., 1988). PDGFs are also produced by a number of other cell types including macrophages, mesangial cells, placental cytotrophoblasts, smooth muscle cells, endothelial cells, fibroblasts, neurons, glial cells, and a number of embryonic cells (Williams, 1990). In adult tissues, only a small proportion of cells are responsive to PDGF. They include connective tissue cells, smooth muscle cells and glial cells. Further, the ability of PDGF to stimulate growth and chemotaxis of mesenchymal cells, its

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Table 1. Growth Factors and their Signaling Receptors Family

Growth Factors

Platelet-derived Growth Factors

Fibroblast Growth Factors

PDGFAA PDGF BB PDGFAB FGFl (aFGF) FGF2 (bFGF) FGF3 (hstFGF) FGF4 (Int2) FGF5 FGF7 (KGF) FGF8 (AIGF) FGF9 (GAF)

Receptors

PDGFRaa PDGFRaP PDGFRpp FGFRl (Fig) FGFR2 (Bek) FGFR3 FGFR4 FGF6

ability to stimulate chemotaxis of inflammatory cells, and stimulation of the production of extracellular matrix proteins has suggested a role for PDGF in a number of human diseases including fibroproliferative disorders, atherosclerosis, and cancer The major biological roles for PDGF are most likely during embryonic development. During development, both the PDGF ligand and receptor are expressed in a variety of embryonic tissues. Considerable progress has been made in determining the function of PDGF in development from studies using mice deficient in the genes for either of the two receptors or the PDGF B-cham ligand (see below). B. FGF Ligands and their Biological Effects

To date, 9 FGFs have been described (Table 1). The first members of the family to be purified and characterized were acidic FGF (aFGF) and basic FGF (bFGF) (Johnson and Williams, 1993). Although acidic and basic FGF were initially purified from pituitary and brain tissue based on their mitogenicity toward fibroblasts, these polypeptides were subsequently found in a wide variety of tissues. These FGFs are encoded by separate single copy genes that have a similar structural organization. There is a 55% identity at the amino acid level between acidic and basic FGF and both proteins are highly conserved across species. Over the past few years, 7 additional members of the FGF family have been described with 30-45% homology to acidic and basic FGF. They include the Hst oncogene or FGF-3, Int-2 oncogene, FGF-4, FG-5, FGF-6, and FGF-7 (keratinocyte growth factor/KGF) (Johnson and Williams, 1993). More recently, androgen induced growth factor (AIGF) or FGFS was purified from an androgen-dependent mouse mammary carcinoma cell line (Tanaka et al., 1992), and glial activating factor (GAF) or FGF9 was isolated from a human glial cell line (Miyamoto et al., 1993). FGFs exert their biological effects by binding to high affinity receptor tyrosine kinases expressed on responsive cells. However, heparin sulfate proteoglycans are obligatory co-factors. There are 4 distinct receptor genes with multiple alternatively spliced forms of each receptor. The 9 FGF family members bind to different receptor isoforms with distinct specificities.

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Biologically, FGFs play diverse roles in many cell types. In addition to their mitogenic activity, FGFs display potent neurotrophic and angiogenic activities. They can stimulate, inhibit, or maintain the differentiated phenotype of certain cells in culture. Like PDGFs, the broad distribution of FGF ligand and receptor expression and their pleotrophic activities in cell culture suggest multiple important roles during embryonic development and during the development and progression of human disease.

III. GENOMIC ORGANIZATION AND STRUCTURAL FEATURES OF PDGF AND FGF RECEPTORS A. PDGF Receptors

The PDGF receptor belongs to a tyrosine kinase receptor subfamily that includes the receptors for macrophage colony stimulating factor, stem cell factor (also referred to as steel factor, mast cell growth factor and kit ligand) and flt3 ligand (Fantletal., 1993; Ullrich and Schlessinger, 1990; van derGeeretal., 1994; Yarden et al., 1986). The most distinct structural features of these receptors is the presence of five Ig-like repeats in the extracellular ligand binding domain. In the intracellular portion of the molecule the tyrosine kinase catalytic domain is divided by a long insert region ranging from about 60 to 100 amino acids. The insert region occurs at a position corresponding to a surface loop in protein kinase A and is expected to protrude out of the globular kinase domain (Hanks and Quinn, 1991). The kinase insert regions of the different family members are important in determining substrate specificity by two criteria; first they are extremely divergent at the amino acid level and second they contain tyrosine autophosphorylation sites. It is noteworthy that while Flkl, FLTl and FLT4 are also structurally related, these family members have seven Ig domains in the extracellular portion of the molecule. All members of this subfamily are organized as three chromosomal clusters indicating gene duplication from a single ancestral gene (van der Geer et al., 1994). For example, the receptors for stem cell factor, PDGFa and Flkl are organized together on chromosome 4ql and the receptors for colony stimulating factor, PDGFp and FLT4 on chromosome 5q3 (van der Geer et al., 1994). Moreover, the large promoters of the PDGF-a and P-receptors contain the structural elements necessary to direct their expression in relevant cell types (Shinbrot et al., 1995; Wang and Stiles, 1994). B. FGF Receptors

The first of the four FGF receptors to be described was FGF receptor 1 (FGFRl) purified from chick embryos (Lee et al., 1989). Subsequent to its isolation came the isolation of three additional FGF receptors encoded by separate genes (Johnson and Williams, 1993; van der Geer et al., 1994). The four FGF receptors are structurally similar and are highly homologous to each other at the amino acid level. FGF receptors comprise a receptor tyrosine kinase subfamily that, although related, is distinct from

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the subfamily that is represented by the receptors for PDGF. There are four structural differences between these two receptor subfamilies. First, the extracellular domain of the FGF receptor is comprised of three Ig-like domains. Second, between the first and second Ig domain FGFRs have a stretch of eight acidic amino acids, referred to as the "acid box." The third and fourth structural differences, within the cytoplasmic portion of the receptor, are the unusually long juxtamembrane region and the short kinase insert (14 amino acids) compared to the receptors for PDGF. A comparison of the different domains of the four human FGF receptors reveals, not unexpectedly, that the most highly conserved regions are the kinase 1 and kinase 2 regions, 75% and 85% respectively. The domains of least conservation are the signal peptide region, 20%, the first Ig domain, 19%, the membrane proximal region, 13%, the transmembrane domain, 24%, and the kinase insert, 7%. A striking and unique feature of the FGF receptor family is the large number of alternatively spliced forms of FGFR 1 and 2 and more recently of FGFR 3 (Avivi et al., 1993; Chellaiah et al., 1994; Johnson and Williams, 1993). No alternatively spliced forms are known for FGFR 4. Several studies demonstrated overlapping affinities of FGFRs for different FGF ligands. So given the fact that nearly all cells express FGF receptors, how is specificity achieved? Further studies demonstrated that there are differences in binding affinities between different alternatively spliced receptor forms and the different ligands. This results in a large combinatorial repertoire of ligand-receptor interactions which have been shown to result in ligand-specific responsiveness (Weng et al., 1995). Expression of FGFR splice variants is regulated in a cell and tissue specific manner suggesting alternative splicing can determine the responsiveness of cells to specific FGF ligands (Johnson and Williams, 1993).

IV. RECEPTOR ACTIVATION AND SIGNAL TRANSDUCTION A. Dimerization^ Transphosphorylation and Signaling Complex Formation

Binding between ligand and receptor results in receptor activation. This activation involves dimerization, transphosphorylation on tyrosine residues and a conformational change such that it can now interact with intracellular signaling molecules. The kinase activity of the receptor is essential for transmitting the mitogenic signal (Escobedo and Williams, 1988). In the case of PDGF, the ligand is dimeric and receptor dimerization may in part be driven by each ligand subunit binding to a different receptor molecule (Heldin, 1995; Lemmon and Schlessinger, 1994). In the case of FGF, heparin oligomerizes the ligand (Spivak-Kroizman et al., 1994) and this event may serve to dimerize receptor molecules. Data based on dominant negative kinase-inactive receptor mutants have shown that receptor dimerization of both PDGF and FGF receptors is necessary for autophosphorylation of tyrosine residues (Ueno et al., 1991; Ueno et al., 1993; Ueno et al., 1992) Coexpression of wild type and dominant negative receptors results in formation of

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dimers between wild type and dominant negative receptors such that, although the wild type receptor can phosphorylate the dominant negative receptor, the mutant receptor cannot phosphorylate its wild type partner. This partially phosphorylated receptor heterodimer is unable to associate with intracellular signaling molecules. Autophosphorylation sites serve as highly selective, high affinity (subnanomolar) binding sites for specific cytoplasmic signaling molecules resulting in the formation of "signaling complexes." Formation of signaling complexes allows the initial binding event between ligand and receptor to be transmitted to the cell nucleus via several cytoplasmic signaling pathways. The crucial protein-protein interactions that occur among the components of the signaling complex are mediated by four known modular units; Src homology 2 (SH2), phosphotyrosine binding (PTB), Src homology 3 (SH3) and Pleckstrin homology (PH) domains (Pawson, 1995). Different combinations of these domains are frequently found in the same protein thus allowing the formation of highly specific protein complexes. Proteins containing these modules can be classified based on their function as enzymes, adaptors and docking proteins, structural proteins and transcription factors. B. SH2 Domains

SH2 domains which are about 100 amino acids in length, were first identified as regions of homology between Src and Fps that were distinct from the tyrosine kinase domain of these two proteins (Pawson, 1995). The finding that SH2 domains bind to phosphotyrosine residues came from studies where v-Crk SH2 domains were shown to bind directly to tyrosine-phosphorylated proteins in transformed cells (Matsuda et al, 1991). Interactions between SH2 domains and phosphotyrosine may be mono-, homo- or heterodimeric (Fantl et al., 1993; Pawson, 1995; Rosen et al., 1995; van der Geer et al., 1994). Subsequently, SH2 domains were found to bind to tyrosine-phosphorylated receptors where specificity was achieved by amino acids C-terminal to the phosphotyrosine (Escobedo et al., 1991; Fantl et al., 1992; Songyang et al., 1993). The structures of several SH2 domains have been determined by nuclear magnetic resonance or X-ray crystallography (Booker et al., 1993; Eck et al., 1993; Pascal et al., 1994; Waksman et al., 1993). These analyses showed that there is a highly conserved positively-charged phosphotyrosine binding pocket, containing the sequence FLVRES (phenylalanine, leucine, valine, arginine, glutamic acid, serine), and a second variable binding pocket for the amino acids C-terminal to the phosphotyrosine. This latter binding pocket is most likely responsible for specificity. The functional consequences of the associations between receptors and SH2-containing signaling molecules are not entirely clear. It is possible that these associations could increase enzymatic activity of signaling molecules (Sugimoto et al., 1994), relocalize them within the cell (Sabe et al., 1994) or position them so that they are better substrates for tyrosine kinase domains (Rotin et al., 1992). These same functional consequences may also be applicable to protein-protein interactions involving SH3, PH and PTB domains.

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WENDY J. FANTL, KEVIN G. PETERS, and LEWIS T. WILLIAMS C. PTB Domains

Recently, PTB domains were described which, although they bind phosphotyrosine residues, are structurally unrelated to SH2 sequences (Blaikie et al., 1994; Bork and Margolis, 1995; Kavanaugh et al., 1995; Kavanaugh and Williams, 1994a). The PTB domain was first identified in the signaling protein She, which also contains an SH2 domain (Kavanaugh and Williams, 1994a). The PTB domain is about twice the length of an SH2 domain (about 200 amino acids), has no functional FLVRES sequence and has been identified in several other proteins across species (Bork and Margolis, 1995; Gustafson et al, 1995; Kavanaugh et al., 1995) It is therefore likely to be of general importance in protein-protein interactions. The PTB domain of She recognizes residues amino-terminal to the phosphotyrosine within the recognition motif, asparagine-X2-X2-phosphotyrosine, where X is any amino acid (Kavanaugh et al, 1995). The solution structure of the She PTB domain has recently been determined and has revealed a striking similarity to pleckstrin homology domains (Zhou et al., 1995). This suggests that these two domains may have overlapping functions. D. SH3 Domains

As mentioned above, many of the proteins involved in the transmission of signals from the receptor to the nucleus contain in addition to SH2 domains, another conserved module of 50—75 residues, the SH3 domain. In contrast to SH2 domains, SH3 domains recognize proline-rich sequences of approximately 10 amino acids (Ren et al., 1994). Structural and mutagenic analyses have shown that despite rather limited sequence homology, the tertiary structures of the folded SH3 domains are rather similar. At the level of secondary structure, all the SH3 domains studied have between five and eight (3 strands which surround a well-conserved hydrophobic pocket which could function as the binding site for cellular ligands (Pawson, 1995; Pawson and Schlessinger, 1993). The amino acids surrounding the pocket could determine the specificity of the interactions and indeed distinct binding preferences have been seen for different SH3 domains (Chen et al., 1993; Rickles et al., 1994; Yuetal., 1994). E. PH Domains

The PH domain, a module of about 100 amino acids was first identified as an internal repeat in pleckstrin, the major substrate for protein kinase C in platelets (Musacchio et al., 1993; Pawson, 1995; Riddihough, 1994). Molecules that contain SH2 and SH3 domains such as phospholipase Cy (PLCy), GTPase Activating Protein (GAP), 3BP2, Grb7 and spectrin also contain the pleckstrin homology domain and although its presence in signaling molecules is well represented, the physiological ligand for the pleckstrin domain is as yet undiscovered. Its overall topology is a sandwich of p sheets (Riddihough, 1994). It will most likely turn out

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to be yet another domain that faciHtates the interactions between receptor tyrosine kinases and signaling molecules. F. Signaling Pathways Used by PDGF and FGF Receptors

As described above, activated tyrosine kinase receptors interact with intracellular signaling molecules to form signaling complexes. The first clue that signaling molecules associate directly with receptor tyrosine kinases came from the observation that the enzyme phosphatidylinositol-3-kinase (PI3-Kinase) was able to bind directly to the PDGF receptor. This enzyme is a heterodimer formed between an 85 kDa subunit that binds to a tyrosine-phosphorylated receptor and links it with the enzymatic component of 110 kDa (Escobedo et al., 1991; Kapeller and Cantley, 1994; Ruiz et al., 1992). Further, a PDGF receptor in which the kinase insert had been deleted failed to associate with this enzyme suggesting that this domain of the receptor was the site of its interaction with PI3-Kinase (Coughlin et al., 1989). Peptide studies and mutational analyses of PDGF and FGF receptors have subsequently identified specific tyrosine residues that interact other with signaling molecules (Coughlin et al., 1989; Escobedo et al, 1991a; Fantl et al, 1992; Peters et al., 1992b; Ronnstrand et al., 1992; Valius and Kaslauskas, 1993a) and Figure 2. These and other analyses demonstrated that the association of PDGF and FGF receptors with signaling molecules involves interactions between short, highly specific peptide motifs on the receptors with SH2 domains on signaling molecules. These motifs invariably contain a phosphotyrosine residues but residues C-terminal of the phosphotyrosine vary and to a large degree determine the specificity of the individual autophosphorylation sites for specific SH2 containing signaling molecules. For example the SH2 domain of PI3-Kinase recognizes the motif pY-Xj-X2M that is C-terminal to tyrosines 708 and 719 of the murine type P receptor (Escobedo et al., 1991; Fantl et al., 1992). This motif is also present in the insulin receptor substrate (IRS-1) and mediates the interaction with PI3-kinase (Backer et al., 1992). It is likely that the compliment of signaling molecules that associate with activated PDGF or FGF receptors determines the cellular response after receptor activation. In order to assess which signaling molecules are important for cellular responses such as proliferation, alterations in gene expression, or cell shape and locomotion, tyrosine residues in both PDGF and FGF receptors were mutated so that the association with a particular signaling molecule was abolished (Courtneidge et al., 1991; Fantl et al., 1995; Fantl et al., 1992; Kashashian and Cooper, 1993;Kashashianetal., 1992;Kundraetal., 1994; Ronnstrand etal., 1992;Seedorf et al., 1992; Valius and Kaslauskas, 1993a). It is worth noting the finding that PI3-kinase contributes a major role to the mitogenic response in fibroblasts. The role of PI3-kinase will be elaborated further below.

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WENDY J. FANTL, KEVIN G. PETERS, and LEWIS T. WILLIAMS G. p21 ras and its Downstream Targets

As described above many signaling molecules have been identified that associate with activated PDGF and FGF receptors (Figure 2). How does this early event transmit information from the receptor to the nucleus? In the past couple of years several components of the puzzle have been pieced together. That the p21 Ras family of proto-oncogenes plays a crucial role in mediating growth factor-stimulated (including PDGF and FGF) cellular proliferation is well established (Boguski and McCormick, 1993; Bollag and McCormick, 1991; Bourne et al., 1991; Fantl et al., 1993; Pronk and Bos, 1994). The p21 Ras proteins were shown to be the cellular counterparts of the transforming genes of Harvey (H) and Kirsten (Ki) sarcoma viruses. Three closely related Ras proteins (80% homology), H, Ki, and N are found in most mammalian cells and are members of a much larger family of small GTPases that are involved in many processes such as vesicle sorting and actin reorganization (Boguski and McCormick, 1993; Bourne et al., 1991; Hall, 1992; Ridley, 1994; Ridley et al., 1992). The importance of p21 Ras is further emphasized by its conservation in Drosophila melanogaster, Caenorhabditis elegans, Xenopus laevis and Dictyostelium discoideum (Boguski and McCormick, 1993; Bollag and McCormick, 1991). The proteins encoded by the Ras genes have a molecular weight of 21 kDa, and are posttranslationally modified at their carboxy terminus to allow anchorage to the inner leaflet of the plasma membrane. Ras proteins are GTPases and cycle between an active GTP-bound form and an inactive GDP-bound state. Forms of Ras that are PDGF-R

Qp)-

FGF-R

KYIYV -KYEYEL

>(P)-|
[ G A P JP)-[
fSyp/PLCYP^ ■

^-k

Figure!. Interaction sites on PDGF and FGF receptors with SH2-containing signaling molecules.

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oncogenic usually have a point mutation so that there is an increase in the GTP-bound form. In quiescent cells Ras is predominantly in the GDP-bound form. In response to a growth factor there can be small to large increases in the amount of Ras-GTP (Satoh et al., 1990). There are about 20,000 copies of the Ras protein in a quiescent cell (Scheele et al., 1995) and growth factor treatment causes a 5-10% increase in proportion of Ras-GTP from the level in the resting state. Importantly, inspection of Ras protein sequence shows this molecule to be devoid of any of the protein modules discussed above and so not unexpectedly Ras fails to associate directly with receptors. How then does growth factor treatment of cells result in activation of Ras? Initially, it was thought that the enzyme GAP might provide the link between the tyrosine kinase receptor and Ras. GAP stimulates the GTPase activity of Ras and is tyrosine-phosphorylated in response to growth factors. This tyrosine phosphorylation does not change GAP activity and so it was likely that other molecules would be involved. Elucidation of the alternatives came with the help of genetic studies in yeast, Drosophila and C. elegans (Fantl et al., 1993; Pawson and Schlessinger, 1993; Rubin, 1991; Schlessinger, 1993; Simon et al., 1991; Simon et al., 1993; van der Geer et al., 1994). In yeast, CDC25, SDC25, and ste6 have been identified as exchange factors for Ras, whereas in Drosophila, the signaling pathway that leads to differentiation of the R7 photoreceptor cells involves signaling through a pathway in which a receptor tyrosine kinase (sevenless) activates Ras by a mechanism that involves a nucleotide exchange factor, SOS (son of sevenless) (Bonfmi et al., 1992; Simon et al., 1991). Given the conservation of sequence between yeast and Drosophila exchange proteins, several groups were able to isolate and characterize the vertebrate homologues of exchange factors by PCR cloning (Bowtell et al., 1992; Chardin et al., 1993). Another protein that links growth factor receptors with Ras was identified from a genetic study with C elegans. Let-60, which has homology to Ras plays a key role in vulval development, with the participation of two additional molecules, sem-5 and the tyrosine kinase receptor let-23. From its sequence, sem-5 appeared to be an adaptor protein as it is composed of an SH2 domain flanked by two SH3 domains (Clark et al., 1992). The human counterpart, Grb2 (growth factor receptor-bound protein 2) or Ash (abundant Src homology) was cloned independently based on its ability to associate with tyrosine-phosphorylated proteins through its SH2 domain (Lowenstein et al., 1992; Matuoka et al., 1992). Like its counterpart in C. elegans and Drosophila, Grb2 was shown to couple receptor tyrosine kinases to a ras exchange factor, Sos (Buday and Downward, 1993; Downward, 1994). The proline-rich sequence of Sos associates with the SH3 domain of Grb2 (Egan et al., 1993; Rozakis-Adcock et al., 1993). Several additional studies showed that other adaptor proteins She and Nek could associate with Sos, indirectly through Grb2 or directly, respectively (Rozakis-Adcock et al., 1992). To complicate matters further, She also associates, through its PTB domain with two tyrosine-phosphorylated proteins of molecular weight 130 and 145 in response

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to treatment of cells with growth factors (Kavanaugh et al., 1994). These two proteins were identified as signaling inositol phyphosphate 5-phosphatates (SIP 130 and SIP 143). These proteins are members of a new family of molecules. A splice variant, SIP 110 associates with the Grb2 SH3 domains (Kavanough et al., 1996). The association of the SIP's with She and Grb2 suggests a connection with Ras signaling. Further, a preference for phosphatidylinositol phosphorylated in the 3' position suggests a link to PI3 kinase. Given the number of exchange factors for Ras (mSosl, mSos2, Guanine nucleotide releasing factor, GRF, Vav, and C3G (Adams et al., 1992; Bowtell et al., 1992; Shou et al., 1992; Tanaka et al., 1994) as well as several adaptor proteins, Grb2, Nek, and Crk (Downward, 1994) the situation is likely to be fairly complex and the exact mechanism by which these molecules link tyrosine kinase receptors into the Ras pathway is still unresolved. Recent publications have proposed that translocation of Sos to the plasma membrane is necessary for stimulation of Ras nucleotide exchange activity (Aronheim et al., 1994; Quilliam et al., 1994). Perhaps the combination of adaptors and exchange factors employed is dependent on cell type and the receptor system being used. As an example, NGF and EGF activate Ras in PCI2 cells by alternative pathways (Qui and Green,). Until a few years ago, the downstream targets of Ras were unknown, although several reports postulated that GAP and the neurofibromatosis gene product, NFl were candidates (Martin et al, 1990; Martin et al, 1992). Recently, several groups showed that Raf, Ral-GDS and PI3-Kinase can associate with Ras directly (Avruch et al., 1994; Finney et al., 1993; Hallberg et al., 1993; Hofer et al., 1994; Kikuchi and Williams, 1994; Kodaki et al., 1994; Moodie et al., 1993; Rodriguez-Viciana etal., 1994; Sjolanderetal., 1991; Van Aelstetal., 1993; Vojteketal., 1993; Wame et al., 1993). From these studies, the data are clear that Raf is downstream of Ras. However, PI3-kinase has been shown to act both upstream and downstream of Ras (Hu et al., 1995a; Rodriguez-Viciana et al., 1994; Wame et al., 1993). Moreover, Ras interacts with diverse targets in organisms that are distinct from an evolutionary standpoint. Further, Ras can interact with different effectors within the same cell (White et al., 1995). To illustrate this point, in PC12 cells, activated Ras induces expression of neurite-specific genes which are not expressed in response to activated Raf (D'Arcangelo and Halegoua, 1993; Lange-Carter and Johnson, 1994; Lange-Carter et al, 1993; Minden et al., 1994). In 3T3 LI cells, transforming Ras could activate MAPK (a molecule downstream of Raf and MEK, discussed below), whereas transforming Raf failed to do so (Porras et al., 1994). H. The Role of PI-3 Kinase in Cell Signaling

PI-3 Kinase was originally described as an activity that was associated with pp60vsrc and middle T/pp60csrc (Whitman et al., 1987) and the functional interaction of both of these molecules correlated with their transforming activity. Later studies showed that activation of growth factor receptor tyrosine kinases stimulated the recruitment to receptor and activation of PI3-Kinase, by a mechanism possibly

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involving a conformational change and tyrosine phosphorylation (Backer et al., 1992; Carpenter etal., 1993; Kavanaughetal., 1992). PI3-kinase can phosphorylate phosphotidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol4,5-(phosphate)2 in the D-3 position of the inositol ring. Although their exact function is as yet undetermined, the rapid increase in these 3-phosphorylated lipids after growth factor stimulation suggests that they could be important second messengers. In addition to its role as a lipid kinase, PI3-kinase is also a protein kinase (Hu et al, 1995). As described above, the heterodimeric PI3-Kinase was the first signaling molecule to be identified that interacted directly with activated PDGF receptors. Subsequent studies showed that PI3-Kinase was necessary for PDGF-mediated cell proliferation (Fantl et al., 1992; Roche et al., 1994). However, its role in FGF-mediated signal transduction is as yet poorly defined. In addition to a crucial role in PDGF-mediated mitogenesis, PI3-kinase seems to be required for a large array of responses. It has been implicated in actin reorganization (Fantl et al., 1995; Kapeller et al., 1993; Nobes et al., 1995; Wennstrom et al., 1994; Wennstrom et al., 1994a). After its association with activated PDGF receptors, PI3-kinase may play a role in receptor internalization and receptor downregulation (Joly et al, 1994). It has also been implicated in glucose uptake after insulin stimulation (Cheatham et al., 1994; Kamohara et al., 1995). There is increasing evidence to suggest that PI3-kinase may participate in the PDGF-dependent activation of S6 kinase activation (Cheatham et al., 1994; Chung et al., 1994; Ming et al., 1994; Weng et al., 1995), inducing the activation of the proto-oncogene, PKB/Akt (Burgering and Coffer, 1995; Klippel etal., 1996). pi 10 is homologous to the yeast protein, vps34, that is involved in vesicular trafficking. This observation suggests that PI3-Kinase may be associated with a similar role in mammalian cells (Kapeller and Cantley, 1994; Schu et al., 1993). Moreover, the isolafion of more homologous isoforms to both p85 and pi 10 suggests the presence of a complex network of molecules involved in the production of 3'-phosphorylated inositol lipids (Hu et al., 1993; Otsu et al., 1991; Stephens et al., 1994; Stephens et al., 1994a). To date the PI3-Kinase pathway is one of two phosphoinosifide pathways used by activated growth factor receptors. The second is phosphoinositide turnover involving the activation of the enzyme phospholipase C (PLCy). This pathway generates two second messengers inositol 1,4,5-trisphosphate and diacylglcerol and is a pathway activated by both PDGF and FGF (Fantl et al., 1993; van der Geer et al., 1994). Importantly, the products of PI3-kinase are poor substrates for PLCy, indicating that they are second messengers distinct from the products of PLCy. The role PLCy in FGF-mediated signal transduction will be discussed later in this review. As mentioned above, PI3-Kinase is composed of an 85 kDa regulatory subunit (p85) and a 110 kDa catalytic subunit (pi 10). The p85 subunit contains an N-terminal SH3 domain and two SH2 domains. Other homologies within p85 are sequences related to the break-cluster region and to two proline-rich sequences that

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flank this domain. The inter-SH2 domain that separates the two SH2 domains has been shown to bind to the amino terminus in the pi 10 subunit and to suffice to activate the pi 10 enzymatic activity. A constitutively active PI3-kinase molecule was generated in which pi 10 was tethered at its amino terminus to the inter-SH2 domain of p85 (Hu et al, 1995a; Klippel et al., 1993). This construct allowed PI3-kinase responses to be tested in a growth factor-independent manner (see below). As stated above, PDGF receptors unable to bind PI3-Kinase could not mediate a proliferative response in fibroblasts. Further studies showed that these mutant receptors also failed to activate Ras in response to PDGF (Satoh et al., 1993). In subsequent studies, expression of pi 10* was shown to activate Ras in Xenopus oocytes (Hu et al., 1995a) and induce fos promoter activation in NIH3T3 cells in a Ras-dependent manner (Hu et al., 1995). The involvement of PI3-kinase in actin reorganization was corroborated by a study in which pi 10* was also able to stimulate the formation of ruffling lamellipodia in fibroblasts. This phenotype was abrogated by coexpression of dominant negative Ras and Rac (Fantl et al., 1995). In contrast to these data, Rodriguez-Vicinia et al. suggest that there can be a direct interaction between Ras and the pi 10 subunit of PI3-Kinase and that v-Ras expression increased the production of 3'-phospholipid products (Rodriguez-Viciana et al., 1994). It is very likely, depending on the experimental system under study, that PI3-Kinase can be both upstream and downstream of Ras. I. Raf is Downstream of Ras

Several studies showed that the protein encoded by the c-raf-1 proto-oncogene functions downstream of p21 Ras since disruption of Raf blocks signaling by Ras in a number of systems (Bruder et al., 1992; Dickson et al., 1992; Kolch et al., 1993). These observations led to a flurry of experiments that showed that Raf is directly downstream of Ras. Experiments in vitro showed a direct interaction between Raf-1 and Ras (Moodie et al., 1993; Van Aelst et al., 1993; Vojtek et al., 1993; Wame et al., 1993; Zhang et al., 1993). Experiments in vivo showed complex formation between Ras and Raf (Finney et al., 1993; Hallberg et al., 1993). Raf-1 is a serine/threonine kinase expressed in all cell types examined and, like Ras, is essential for growth and development in worms, flies, frogs and mammals (Daum et al., 1994). The serine/threonine kinase activity of Raf is activated in cells upon treatment with PDGF, FGF and other growth factors. Moreover, oncogenic, constitutively active Raf abrogates the requirement for growth factors. A comparison between the sequences of cellular and transforming Raf (v-Raf) revealed that the amino terminal half of the molecule has been deleted in v-Raf The implication of this finding is that the amino terminal sequence is a negative regulator of Raf kinase activity. As will be discussed below two proteins, Ras and 14-3-3, have so far been found to interact with the amino terminal portion of Raf and are most likely necessary, but not sufficient for activation of Raf kinase. The three mammalian Raf isozymes, c-Raf-1, A-Raf, and B-Raf share three conserved regions (CR-1, CR-2 and CR-3) that lie within variable domains (Figure 2). The CR-1 domain in the

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amino terminal half of the molecule consists of the Ras binding domain and a cysteine-rich domain, reminiscent of the zinc finger motif which may be a binding site for lipid. The CR-2 domain is rich in serine and threonine residues, some of which are regulatory phosphorylation sites. The CR-3 domain contains the serine threonine kinase activity and is homologous to the consensus kinase domain. It is impossible to activate Raf kinase activity by incubating Raf with Ras in vitro and so the activation step must necessarily involve the participation of additional molecules. The use of yeast genetics, biochemistry and the yeast two-hybrid system by several groups identified two isoforms of the 14-3-3 protein family as candidates for those required molecules (Aitkin, 1995; Fantl et al., 1994; Freed et al., 1994; Fu et al., 1994; Li et al., 1995; Morrison, 1994). However, addition of 14-3-3 to a mixture of Raf and Ras also fails to activate Raf, suggesting the involvement of yet further component(s) of this system. Significantly, a kinase-defective Raf molecule is phosphorylated on its major sites in Sf9 cells when coexpressed with activated PDGF receptors thereby invoking the involvement of an as yet unidentified kinase (Morrison et al., 1993). Various candidates kinases have been suggested, PKCa and PKC8(Caietal., 1993;Daumetal., 1994;Tokeretal., 1994), as well as the recently discovered molecule, kinase suppressor of Ras (Komfeld et al., 1995; Sundaram and Han, 1995; Therrien et al, 1995). The putative kinase most likely activates Raf at the plasma membrane. This can be inferred from two studies in which targeting Raf to the plasma membrane negated the requirement for Ras in activating the Raf kinase activity (Burgering and Bos, 1995; Leevers et al., 1994; Stokoe et al., 1994). Complete activation of a membrane-bound form of Raf, however, was achieved only after growth factor treatment, suggesting that a second Ras-independent signal is required for the activation process (Leevers et al., 1994; Williams et al., 1992). Other studies have shown that heat-shock proteins, whose role is often to act as molecular chaperones, are also complexed with Raf (Wartmann and Davis, 1994), although their role is unclear. Moreover, inhibition of Raf activity and therefore of the molecules that act downstream of Raf is controlled by activated cyclic AMPdependent protein kinase A. This provides a mechanism whereby PDGF and FGF receptors signaling could be coupled to that of G-protein-coupled receptors activation of which results in elevations of cyclic AMP levels. The reader is referred to more detailed discussions of this topic (Burgering and Bos, 1995; Hafner et al., 1994). J. Downstream of Raf

So far, Raf substrates Mekl and Mek2 (MAP or ^rk Ainase) have been identified. The binding site for Mek on Raf was found to be in CR-3. This is consistent with the truncated form of Raf (v-Raf) being able to signal continuously, presumably through activation of Mek (Avruch et al., 1994). Other kinases that are known to phosphorylate Mek are Mos and Mek kinase. It is, however, unclear whether these latter two kinases are regulated by growth factor receptor tyrosine kinases. The substrates for Mek 1 and 2 are Erk 1 and 2, a subgroup of the MAP

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CR2

CR3 WT cRaf-1

Ras Zn binding finger domain

vRaf-1

K375M NAF Regulatory Domain

Catalytic Domain

Figure 3. Structural organization of wild type c-Raf-1 (W.T), transforming Raf-1 (vRaf-1) and dominant negative Raf-1 (NAF, Not A Functional).

Kinase family (see Figure 3). Meks are dual specificity kinases that when themselves are activated by serine phosphorylation, will phosphorylate and activate Erks on threonine and tyrosine within the motif TEY. From here the pathway ramifies even further since Erks 1 and 2 have numerous substrates that include, the ribosomal S6 kinase, (p90'"^^) (Chen et al., 1993a), cytoplasmic phospholipase A2 (CPLA2) (Lin et al., 1993) and the ternary complex factor, TCF, which is a transcription factor involved in the transcriptional activation of the c-fos proto-oncogene (Cano and Mahadevan, 1995; Gille et al, 1992). In addition to ERKs, the MAP Kinase family includes the c-Jun-N-terminal kinase or stress activated protein kinase (Jnk and SAPK respectively) and p38 kinase subgroups (Marshall, 1995). In addition to their ability to phosphorylate c-Jun, these kinases also phosphorylate the transcription factor ATFl in vitro. These MAP Kinase isoforms are activated by tyrosine and threonine phosphorylation potently in response to ultraviolet radiation, osmotic stress, and inflammatory cytokines and weakly in response to growth factors. These molecules are differentially activated by upstream signaling components. So the recently cloned Mek3 and Mek4 both activate p38, whereas only Mek 4 activates Jnk. The murine counterparts of these molecules were also reported recently (Davis, 1994; Derijard et al., 1994; Derijard et al, 1995; Lin et al., 1993; Minden et al., 1994; Sanchez et al., 1994; Yan and Templeton, 1994) (see Figure 3). K. Ras-lndependent Pathways

The multiple Ras-dependent pathways described above all stimulate transcription by means of reversible phosphorylation of transcription factors. As an alternative, treatment of cells with both cytokines and growth factors, including PDGF,

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activates the Janus Kinase/Signal transducer and activator of transcription (JAK/STAT) pathway in a Ras-independent manner. The STAT transcription factor, once activated, travels from the cytoplasm to the nucleus where it binds to the interferon-stimulated regulatory element. STAT proteins have one SH2 domain and one SH3 domain, but the mechanism of their activation is at present unclear (Hill and Treisman, 1995; Shuai et al., 1993a; Silvennoinen et al., 1993). Recently, a role in transcriptional regulation was described for p70/p85'^'^ (de Groot et al., 1994). This kinase was originally known for its ability to phosphorylate a 40S ribosomal protein called S6 (see below). However, pTO^^*^ has also been shown to phosphorylate an alternative substrate, the cyclic AMP responsive element modulator (CREM) and in so doing, stimulate the ability of this factor to activate transcription by binding to the CRE. In addition to its ability to stimulate transcription, by phosphorylation of CREM, phosphorylation of the S6 ribosomal protein stimulates translation of a family of messenger RNAs that encode proteins needed for cell cycle progression from G1 into S phase (Downward, 1994a; Jefferies et al., 1994; Stewart and Thomas, 1994). Microinjection of quiescent cells with neutralizing antibodies to plO/pS5^^^ blocks the ability of cells to enter S phase in response to serum (Reinhard et al., 1994). This data is corroborated by treatment of cells with rapamycin, which blocks both cell cycle progression and activation of pTO/pSS^^*^. Recent data suggests, that activation of s6k by growth factors is Ras-independent, and that upstream mediators are PI3-Kinase and PKC. Further work will identify the additional members of this pathway (Chung et al., 1994; Downward, 1994a; Ming et al., 1994) (see Figure 4).

lllUllll!illliTl«fia?ifffiWOTlMiiimif{K!if)iri?}!iT«i?m?m!i!i?iTm?mTiii?i ,--^p145(SIP)

Figure 4, Intracellular signaling pathways that are recruited by tyrosine kinase growth factor receptors.

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The emerging picture is that multiple pathways are activated by treatment of cells with growth factors. The implementation of one pathway versus another is a cell-type specific phenomenon. For example, in PC 12 cells, activated Mek is necessary and sufficient for neurite outgrowth in these cells (Cowley et al., 1994) and does not require activation of s6 kinase. Over the past few years tremendous progress has been made in identifying the participants of growth- factor stimulated pathways. The next challenge will be to try and decipher the factors that influence activation of each particular pathway. L. Interaction of FGF Receptors with Heparin Sulfate Proteoglycans

Members of the FGF ligand family bind tightly to heparin, a characteristic which greatly facilitated their initial isolation. Early on it was noted that heparin could enhance cellular responses to FGFs in tissue culture suggesting that this interaction may have important biological consequences. More recently, studies from a number of laboratories have identified low affinity cell surface binding sites for FGF as cell surface bound heparin sulfate proteoglycans (Klagsbrun and Baird, 1991; Ruoslahti and Yamaguchi, 1991). Removing heparin sulfate proteoglycans from the cell surface by enzymatic digestion or by inhibiting their production completely abrogates or strongly suppresses FGF binding to high affinity receptors and resultant cellular responses (Mansukhani et al., 1992; Omitz and Leder, 1992; Omitz et al., 1992a; Rapraeger et al., 1991; Roghani et al., 1994; Yayon et al., 1991). Addition of exogenous heparin restores high affinity binding and FGF responsiveness. An important feature of the interaction between FGF and heparin sulfate proteoglycans may be the heparin-induced oligomerization of FGF molecules. This oligomerization appears to provide a mechanism by which FGF receptors are dimerized and activated and provides at least a partial explanation for the enhanced high affinity binding of FGFs in the presence of heparin sulfate proteoglycans (Omitz et al., 1992a; Spivak-Kroizman et al., 1994). Moreover, non-sulfated di- or tri-saccharides can bind FGF, mediate FGF oligomerization and enhance the biological activity of FGF suggesting that FGFs recognize specific structural features of the carbohydrate backbone of heparin sulfate proteoglycans, independent of ionic interactions with highly charged sulfate groups (Omitz et al, 1995). Other evidence suggests that the direct interaction of heparin sulfate proteoglycans with FGF receptors is also critical for the high affinity binding of FGF (Kan et al., 1993). Thus, heparin sulfate proteoglycans may act to stabilize members of the FGF family and their receptor tyrosine kinases in high affinity ternary complexes and because of their ability to bind multiple ligand molecules may mediate receptor dimerization and activation. While heparin sulfate proteoglycans are cmcial for acidic and basic FGF interactions with high-affinity FGFRs, recent evidence suggests that heparin might actually inhibit KGF (FGF7) binding to the KGF receptor (FGFR2-IIIb), while promoting the binding of acidic FGF at similar concentrations (Reich-Slotky et al., 1994). The differential effects of proteoglycans on individual FGFs may be, in part, dictated by specific differences in proteoglycan sulfation (Guimond et al., 1993). In this way, interactions of FGFs with specific sulfated proteoglycans may be an

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important determinant of ligand binding specificity and an important regulatory mechanism for coordinating cellular responses to individual FGFs.

V. PDGF AND FGF RECEPTORS DURING EMBRYONIC DEVELOPMENT A. PDGF Receptors in Development By in situ hybridization, PDGF P and a receptor subunits are expressed in the mesenchyme of a variety of embryonic tissues (Orr-Urtreger et al., 1992; Schatteman et al., 1992; Shinbrot et al., 1994) Although these receptors are expressed in facial mesenchyme, limb bud mesenchyme, and somitic mesenchyme, perhaps the most interesting pattern of expression is the apparent co-expression of these receptors in the subepithelial mesenchyme of tubular organs such as the esophagus, intestines, stomach, trachea, and blood vessels. Although the co-expression of the a and P receptors in these mesenchymal populations suggests overlapping functions, the severe defects in mice homozygous for a chromosomal deletion (Patch mutation) which includes most of the a receptor demonstrates that signaling downstream of the a receptor is an absolute requirement for normal development of connective tissue (Orr-Urtreger et al., 1992; Schatteman et al., 1992) Embryos homozygous for the Patch mutation usually die before Ell with characteristic defects in mesenchymal cell populations including absent dermis of the skin and reduced and disorganized connective tissue in many organs such as the esophagus and trachea. The requirement for a receptor signaling in mesenchymal differentiation may be dictated by the production of the PDGF A-chain by adjacent epithelial tissues (Orr-Urtreger and Lonai, 1992a), Since PDGF AA binds exclusively the a receptor, the absence of the a receptor in the patch mutant mice would render these mesenchymal components unresponsive in this apparently important mesenchymal/epithelial interaction. Consistent with the notion that a receptor signaling is sufficient for the development of most mesenchymal elements, "knockout" of the PDGF P receptor failed to produce defects in most embryonic organs (Soriano, 1994). Rather homozygous animals died in the late prenatal period apparently of severe anemia and dysgenesis of the kidney. The kidney defect is characterized by a severe deficiency in glomerular capillaries and mesangial cells. Whether the anemia was caused by a primary defect in erythropoiesis or was secondary to microangiopathic hemolysis remains to be determined. The expression of the P receptor in the embryonic micro vasculature and presumpive bone marrow suggests that either or both explanations are feasible (Shinbrot et al., 1994). Similar defects were reported after inactivation of the PDGF B-chain gene, indicating that they are most likely attributable to the the absence of PDGF BB or AB signaling through the P receptor (Leveen et al., 1994). Other severe defects, such as the hypertrabeculation of the heart and the dilatation of blood vessels, occurred in the B-chain knockout but not in p receptor deficient embryos suggesting loss of signaling by PDGF BB or AB through the a receptor may be responsible for these defects.

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FGF receptors are expressed throughout embryonic development suggesting roles for FGFs in all stages of development from early embryonic pattern formation to organogenesis (Orr-Urtreger et al, 1991; Peters et al., 1993; Peters et al, 1992b; Stark et al, 1991). Several FGF ligands are expressed during early embryonic development in Xenopus and mouse embryos, and addition of exogenous FGF to primitive embryonic ectodermal cells isolated from Xenopus embryos (animal caps) induces the expression of mesodermal markers such as muscle-specific cardiac actin (Green et al., 1992; Jessell and Melton, 1992; Moon and Christian, 1992; Smith and Howard, 1992). Expression of a dominant negative FGF receptor in Xenopus animal caps blocked mesoderm induction by FGFs but had no effect on mesoderm induction by activin, a member of the TGF-P family (Amaya et al., 1991). When the dominant negative receptor was expressed in Xenopus embryos, a specific defect was observed in posterior and ventral mesodermal tissues. These results suggested that FGFs play a role in the induction and patterning of specific mesodermal populations during vertebrate embryonic development and that signaling via other receptors such as activin receptors probably drives induction and the formation of other mesodermal tissues (specifically those destined to become anterior and dorsal mesoderm). To begin to decipher the signaling pathways downstream of the FGF receptor which mediate mesoderm induction, a dominant negative form of the raf kinase termed naf (A^ot A Functional raf) was expressed in Xenopus embryos and animal caps after RNA injection (MacNicol et al., 1993). In the animal caps, expression of naf blocked the induction of muscle specific actin by FGF but had no effect on induction of muscle-specific actin by activin. Xenopus embryos injected with naf RNA had a phenotype that was essentially indistinguishable from the phenotype of embryos expressing the dominant negative FGF receptor. Similarly, a dominant negative form of SH-PTP2 blocked FGF induced mesoderm induction in Xenopus animal caps and in Xenopus embryos produced posterior truncations essentially identical to those produced by the dominant negative FGF receptor and naf (Tang et al., 1995). Interestingly, an earlier study demonstrated that a dominant negative ras (p21 [AsnlT]^^'''^^) blocked mesoderm induction by both FGF and activin suggesting a common pathway for mesoderm induction by these polypeptides (Whitman and Melton, 1992). However, results obtained with naf clearly demonstrate that signaling by raf-1 is required for FGF-induced mesoderm but not for activin-induced mesoderm. Oddly, the dominant negative SH-PTP2 produced an embryonic phenotype more consistent with blocking FGF receptor signaling but apparently blocked both FGF and activin-induced mesoderm in animal caps. One explanation for this apparent discrepancy is that FGF mediates part of the activin signal (Amaya et al., 1993; Cornell and Kimelman, 1994; LaBonne and Whitman, 1994; Tang et al., 1995). The fact that dominant negative ras also blocks both basic FGF and activin-induced mesoderm induction in animal caps is consistent with the placement of SH-PTP2fiinctionupstream of ras activation in other developmental and

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cell culture systems (Bennett et al., 1994; der Hertog et al, 1994; Feng and Pawson, 1994; Li et al, 1994a; Neel, 1993; Noguchi et al, 1994; Yamaguchi et al, 1994a). Current evidence suggests that PI turnover plays a critical role in vertebrate embryonic axis formation and in mesoderm induction (Berridge, 1993; Kao and Elinson, 1988; Kao and Elinson, 1989). To explore the role of PLC-y activation by FGF receptors in mesoderm induction a chimeric receptor consisting of the PDGF receptor ligand binding domain coupled either with the wild type FGF receptor or an FGF receptor with a point mutation rendering it unable to activate PLC-y (Y766F) were constructed (Muslin et al., 1994). When expressed in Xenopus oocytes, only the wild type chimera was able to mediate PLC-y phosphorylation, PI turnover, and calcium flux in response to PDGF stimulation. However, both receptors mediated mesoderm induction in Xenopus animal caps. These results demonstrate that, unlike the ras pathway, direct activation of PLC-y by the FGF receptor is not required for mesoderm induction. Recently, two groups have "knocked out" FGFRl in embryonic stem cells and produced transgenic mice lacking functional forms of FGFRl (Deng et al., 1994a; Yamaguchi et al., 1994a). As might have been predicted, homozygous mice had severe defects in mesodermal patterning resulting in embryonic lethality. Like Xenopus embryos expressing dominant negative FGF receptors, homozygous mice frequently exhibited truncations and/or severe disorganization of posterior embryonic structures. Unlike Xenopus however, homozygous mouse embryos also had significantly abnormal head structures. Moreover, these embryos were able to form many extraembryonic mesodermal structures such as the allantois, amnion, and yolk sac (complete with blood and blood vessels) which are thought to be derived from the posterior primitive streak regions. FGFR 1 is apparently also not required for differentiation of embryonic muscle or other mesodermal cell types since embryoid bodies formed from FGFRl deficient ES cells contain a variety of mesodermal populations (Deng et al., 1994a). Based on these observations, it seems likely that FGFRl signaling is more critical for mesodermal patterning than mesoderm induction. FGFR2 is also expressed at high levels in the epiblast before and during gastrulation and can perhaps compensate for the loss of FGFRl signaling (Orr-Urtreger et al., 1991). In addition to early embryonic pattern formation, FGF receptors may also play a role in later patterning events such as limb formation (Martin, 1995; Slack, 1995). Expression studies have shown that several of the FGFs are expressed in embryonic limbs in specific regions suggesting specific roles for these receptors in limb patterning. A recent barrage of studies looking at the effects of FGFs on limb formation in chick, mouse, and newt embryos, demonstrate that FGF signaling is of fundamental importance in this process. For example, FGFs placed into the flank of a chick embryo can induce the formation of a new limb bud (Anderson et al., 1993; Cohn, 1995; Fallon, 1994; Niswander et al., 1993). After induction of the limb bud, outgrowth occurs by the progressive addition of more distal elements and depends on signals from cells at the tip of the limb bud known as the apical ectodermal ridge. Members of the FGF family can substitute for the apical ectoder-

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mal ridge to mediate limb outgrowth. Other studies showing that limb outgrowth and patterning are closely linked suggest that signaling by FGF receptors is not only important for induction of limb formation and outgrowth, but for normal patterning of the limb (Laufer et al, 1994; Niswander et al., 1994; Yang and Niswander, 1995). The precise roles of individual FGF receptors and the signal transduction pathways downstream of these receptors have yet to be addressed. C. FGF Receptors During Organogenesis

During organogenesis expression patterns of the four FGFR genes are largely non-overlapping suggesting that individual receptor genes perform specific functions which are dictated at least in part by tissue-specific expression (Orr-Urtreger et al., 1991; Peters et al., 1993; Peters et al., 1992b; Stark et al., 1991). The differential expression of FGFR 1 compared to FGFR2 provides perhaps the most startling example of this differential regulation, with FGFRl being expressed principally in the mesenchyme and FGFR2 being expressed principally in the epithelium of differentiating embryonic tissues (Orr-Urtreger et al., 1991; Peters et al., 1992b). This distribution of FGF receptors is consistent with the broad range of cultured cells that are responsive to members of the FGF family and suggest that these receptors may be important mediators of epithelial/mesenchymal interactions known to be crucial for tissue differentiation. In order to explore the roles of FGF receptors during organogenesis, dominant negative forms of FGFR 1 and FGFR 2 have been targeted to the skin and developing lung (Peters et al., 1994; Werner et al., 1994; Werner et al., 1993). Unlike receptor knockouts, this approach does not interfere with FGF signaling during the patterning of the early embryo or during the differentiation of nontargeted embryonic organs and thus allows the dissection of FGF receptor function in a single organ system. In the skin, a human keratin-14 promoter was used to direct expression of a dominant negative KGF receptor (FGF2-IIIb) to basal cells of the epidermis and to the outer root sheath of hair follicles, known sites of expression of this form of receptor (Werner et al, 1994). Transgenic mice expressing this mutated receptor had characteristics abnormalities of skin development and wound healing. The transgenic mice had an atrophic and disorganized epidermis and a 60-80% reduction in the number of hair follicles, probably a direct effect of KGF receptor blockade in basal keratinocytes. The dermis of these mice was characterized by severe thickening and a gradual replacement of adipose tissue by connected tissue. Since the transgene was not expressed in the dermis, indirect mechanisms must account for this observation. When skin wounds were made, non-transgenic litter mates' wounds were almost completely re-epithelialized by day 5 after injury. In contrast, only a small percentage of the wound was covered by migrating and proliferating keratinocytes in the transgenic animals. This decrement probably reflects a more than 90% reduction in the proliferation rate of keratinocytes at the wound edge in the transgenic mice. Thus, signaling via the KGF receptor appears essential for the normal differentiation of skin and for the prolif-

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eration of basal keratinocytes during the normal renewal of the epidermis and also during wound healing. Besides the skin, FGFR2 is also expressed in the epithelium of a number of parenchymal organs including the lung. The expression pattern of FGFR2 in the embryonic lung begins prior to the onset of branchmg morphogenesis suggesting a role for this receptor in lung development (Peters et al., 1992b). To address this hypothesis, a human surfactant C promoter was used to direct expression of a dominant negative FGFR2-IIIb exclusively to the developing airway epithelium of transgenic mice (Peters et al., 1994). Mice expressing this transgene had no gross developmental abnormalities, but died in the perinatal period. Examination of these mice revealed a marked reduction or complete absence of lung tissue suggesting perinatal asphyxia as the cause of death. Histologic studies revealed that instead of normal lungs these animals formed long, unbranched "lung tubes" that extended bilaterally from the bifurcation of the trachea down to the diaphragm and ended as blind sacks. The epithelium lining the lung tubes consisted entirely of columnar epithelium that expressed the large airway marker CCIO. The lung tubes were completely devoid of cells resembling type 1 alveolar cells or cells expressing endogenous SPC, a molecular marker confined to type 2 alveolar cells. Thus, signaling by FGFR2 in the embryonic lung is required for airway branching and for epithelial differentiation but not for airway outgrowth demonstrating a specific role for this receptor during branching morphogenesis of the embryonic lung. In Drosophila, the tracheal organ is formed by a process analogous to branching morphogenesis of the vertebrate lung. Recently, a Drosophila FGF receptor was cloned and found to be expressed in the epithelium of the tracheal organ (Glazer and Shilo, 1991). In Drosophila strains with mutations in this FGF receptor gene, the tracheal pits form, but tracheal cells fail to migrate from the pits and the tracheal tree fails to develop (Glazer and Shilo, 1991; Klambt et al., 1992; Reichman-Fried et al., 1994). Considering these results together with the effect of disrupting FGF receptor signaling during vertebrate lung development suggests that branching morphogenesis is a primitive and highly conserved morphogenetic process in which FGF receptor signaling appears to play a central role. Several lines of evidence link FGF signaling to skeletal muscle differentiation (Olson, 1992; Olwin et al., 1994; Olwin et al, 1994a). In cultured myoblasts, FGFs promote proliferation while suppressing differentiation into multinucleate myotubes. Myoblast cell lines express predominantly FGFRl and differentiation following removal of exogenous growth factors correlates with a decrease in FGF responsiveness and downregulation of FGF receptor expression (Moore et al., 1991; Templeton and Hauschka, 1992). Current evidence suggests that the suppression of differentiation by FGFs is mediated through signal transduction mechanisms that inhibit the expression of myogenic HLH proteins such as myogenin or by pathways that result in post-translational modification of these proteins such as phosphorylation by protein kinase C, a modification which results in a loss of DNA binding activity (Li et al., 1992; Olson, 1992). Interestingly, embryod bodies

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composed of embryonic stem cells incapable of producing a functioning FGFRl are capable of producing mature skeletal muscle (Deng et al., 1994a). Similarly, Xenopus embryos expressing a dominant negative FGF receptor produce normal appearing, mature skeletal muscle (Amaya et al., 1991). These findings suggest that although FGFs may be involved in controlling the mass and patterning of skeletal muscle, other pathways may be sufficient for its differentiation. Another FGF receptor, FGFR4, is expressed in the myotome of the somite, the site of the earliest skeletal muscle differentiation (Stark et al., 1991). Expression of FGFR4 in the myotome appears restricted to cells that have not yet undergone differentiation consistent with a role for FGFs and FGF receptors in expansion of the myoblast population. In mature skeletal muscle, cells lying between the muscle fiber and the basal lamina, satellite cells, escape the process of differentiation and upon muscle injury can regenerate an entire muscle fiber. In MDX mice, a mouse model of muscular dystrophy, the phenotype is very mild compared to the severe dystrophic changes that occur in human skeletal muscle from patients possessing the same X chromosome gene mutation (Duchenne muscular dystrophy). A potential explanation for this discrepancy is the high level of basic FGF found in skeletal muscle from MDX mice (DiMario and Strohman, 1988). Satellite cells cultured from MDX mice are stimulated to proliferate in the presence of low levels of FGF suggesting that FGF may drive satellite cell proliferation and parallel muscle fiber regeneration enabling MDX mice to escape the fatal phenotype seen in the related human disease (DiMario et al., 1989). These findings suggest that effective gene therapy approaches to deliver FGF to diseased skeletal muscle might enhance muscle regeneration in patients with muscular dystrophy. Recent studies suggest that FGF receptors, particularly FGFRl, might play a similar role in cardiac development as they do in skeletal muscle development (Schneider et al., 1994). Although FGFs are not efficient inducers of cardiac muscle, they do effectively stimulate the proliferation of fetal cardiocytes (Engelmann et al., 1993; Muslin and Williams, 1991). Adult cardiocytes are refractory to mitogenic stimuli suggesting that there may be a window of time during cardiac development when FGF signaling is important for expanding the number of fetal cardiocytes. This idea is supported by a recent study demonstrating that retroviral mediated expression of a dominant negative FGFRl in embryonic chick hearts effectively inhibits the proliferation of cardiocytes (Mima et al., 1995). Understanding the molecular events that control FGF responsiveness during embryonic heart development could lead to novel strategies for the augmentation of cardiac function in patients with congestive heart failure. D. PDGF and FGF Receptors in Blood Vessel Development

PDGF-BB stimulates endothelial proliferation and chemotaxis and promotes angiogenesis in vivo and in vitro (Battegay et al., 1994; Risau et al., 1992). Moreover, the PDGF (3 receptor is expressed by cultured endothelial cells and in specific subsets of extraembryonic and embryonic microvascular endothelial cells (Holmgren et al, 1991; Shinbrot et al., 1994). PDGFs are also potent mitogenic,

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chemotactic, and morphogenetic regulators of vascular smooth muscle cells (Casscells, 1992; Ross, 1993; Schwartz and Liaw, 1993). During embryonic development, both a and p receptors are expressed by a mesenchymal cell population situated between the embryonic endothelium and the development vascular media (Orr-Urtreger et al., 1992; Orr-Urtreger and Lonai, 1992a; Shinbrot et al., 1994). This location of PDGF receptors in cells from which vascular smooth muscle precursors may be derived, suggests a role for these factors in the expansion of smooth muscle precursors and potentially in their differentiation. This idea is supported by the marked vascular dilation observed in mice lacking PDGF B-chain expression (Leveen et al., 1994). The absence of this phenotype in mice lacking a functional (3 receptor suggests that the a receptor is able to provide the compensatory signals required for normal vessel differentiation (Soriano, 1994). PDGF ligands and PDGF receptors are expressed during the development of pathophysiologic states such as atherosclerosis and neoplastic disease (see below). Considering the responses engendered by PDGF signaling in cultured endothelial and smooth muscle cells it is possible that signaling by PDGF receptors may be important for the pathologic vascular growth that drives the progression of these important diseases. Numerous studies have identified fibroblast growth factors as one of the most potent angiogenic substances known (for review see Folkman and Klagsbrun, 1987; Klagsbrun and D'Amore, 1991a). Like PDGFs, FGFs stimulate "angiogenic" responses in cultured endothelial cells. Moreover, during embryonic development, FGF receptors (particularly FGFRl) are expressed by both vascular endothelial cells and by vascular smooth muscle cells implicating them in the growth and differentiation of the embryonic vasculature (Orr-Urtreger et al., 1991; Peters et al., 1992b). The ability of mice lacking a functional FGFRl gene to produce a relatively normal appearing extraembryonic vasculature suggests, however, that signaling by FGF receptors may not be required at least for the early phases of vascular growth and development (Deng et al., 1994a; Yamaguchi et al, 1994a). Although the role of FGF receptors in embryonic vascular growth is not clear, the production of FGFs and the expression of FGF receptors in pathologic tissues such as atherosclerotic plaques and tumors suggests a role for FGF signaling in the development of these disease states (see related sections herein). E. PDGF and FGF Receptors in Nervous System Development and Maintenance

Although PDGF and FGF were first isolated as growth factors for cells of mesenchymal origin, PDGFs and FGFs as well as their receptors are expressed in the nervous system and both PDGF and FGF promote specific responses in cell lines derived from the nervous system. The PDGF a receptor is expressed in the brain, spinal cord, and retina during the late stages of neurogenesis (E15-P14) (Mudhar et al., 1993; Pringle et al., 1992; Yeh et al., 1993). In the brain, PDGF a receptor positive cells are first detected outside the subventricular zone and thereafter are distributed in a pattern most consistent with glial expression. The recent

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discovery that PDGF A-chain is expressed by mammalian neurons during development suggests a paracrine mechanism by which neurons producing PDGF A-chain could influence the migration and proliferation of glial precursors (Yeh et al, 1991). In support of this notion, oligodendocyte progenitors, 0-2A cells, express the PDGF a receptor and the PDGF A-chain enhances the proliferation and survival of these progenitors in vitro (Barres and Raff, 1994; Collarini et al., 1991). Moreover, increasing PDGF A-chain expression in the developing optic nerve prevents normal oligodendocyte death by up to 90% producing a doubling of the number of oligodendocytes in a period of 4 days (Barres et al., 1992). Recent in vitro studies have shown that PDGF is only one of several factors that promote survival of these cells perhaps accounting for the apparent variation in the phenotypes in the CNS of mice carrying the patch mutation (Barres et al., 1993; Morrison-Graham et al., 1992). In contrast to PDGF receptor expression, FGF receptors (FGFRl, FGFR2, and FGFR3) are expressed in the nervous system in all stages of development and in adulthood. FGFRl, R2, and R3 are expressed in the germinal epithelium of the central nervous system and spinal cord suggesting a role for these receptors in the generation and/or maintenance of common neuronal/glial precursors (Orr-Urtreger et al., 1991; Peters et al., 1993; Peters et al., 1992b). Later, FGF receptors can be detected outside the germinal epithelium in differentiating neurons and glia. Of note, FGFRl is expressed primarily in neuronal populations whereas FGFR2 and R3 are expressed primarily in glia (Asai et al., 1993; Peters et al., 1993; Peters et al., 1992b; Yazaki et al., 1994). These findings suggest that FGFs might participate in the growth and differentiation of both neurons and glia. Consistent with this idea, cell lines of glial and neuronal origin are responsive to FGFs and basic FGF has been shown to regulate the proliferation of bipotent progenitor cells that give rise to both neurons and astroglia (Claude et al., 1988; DeHamer et al., 1994; Ip et al., 1994; Vescovi et al., 1993). Consistent with a role as a neurotrophic factor stimulation of FGF receptors results in neurite outgrowth from a variety of neuronal cell lines in vitro, an effect which may rely on the activation of PLC-y (Obermeier et al., 1994; Williams et al., 1994). In vivo, activation of FGF receptors has been shown to enhance the survival of neurons damaged by chemical or physical means suggesting that stimulation of FGF receptors might provide a useful target for the treatment of neurodegenerative diseases (Anderson et al., 1988; Otto and Unsicker, 1993; Otto and Unsicker, 1994).

VL PDGF AND FGF RECEPTORS IN HUMAN DISEASE A. PDGF and FGF Receptors in Tumor Biology

The potential for PDGF and FGF receptors to modulate cell growth, differentiation and chemotaxis suggests their potential to participate in all stages of tumor development including transformation, tumor growth, and tumor metastasis. The v-sis oncogene, the transforming protein of simean sarcoma virus (SSV), is functionally identicle to the PDGF B-chain and when administered to primates, SSV

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produces a variety of tumors (Deuel et al., 1987; Robbins et al., 1981; Westermark and Heldin, 1991). Transformation by SSV apparently involves autocrine activation of PDGF receptors since only cells that express PDGF receptors can be transformed by v-sis (Garrett etal., 1984; Leal etal., 1985). Moreover, in v-sis transformed cells, PDGF receptors are activated in intracellular compartments (Bejeek et al., 1989; Keating and William, 1988a). This may be an important feature of neoplastic transformation mediated via PDGF receptor activation since continual stimulation of PDGF receptor expressing cells by exogenous PDGF does not confer a transformed phenotype. Recently, a subgroup of chronic myelomonocytic leukemia patients have been shown to possess a translocation resulting in the fusion of the kinase domain of the PDGF receptor P on chromosome 5 to a novel ets-like gene, tel, on chromosome 12 (Golub et al., 1994). The tel domain of the fusion protein might serve to autodimerize the PDGF receptor and activate it intracellularly to mediate transformation in this disease. A similar mechanism for oncogenic transformation has already been described for a fusion protein consisting of a leucine zipper dimerization motif to activate the oncogenic potential of the met receptor tyrosine kinase (Rodrigues and Prk, 1993). PDGF ligand and receptor expression has been demonstrated in a variety of human tumors and human tumor cell lines (Antonaides et al, 1992; Chung and Antoniades, 1992; Henriksen et al., 1993; Ponten et al., 1994). Notably, in gliomas, the presence of a PDGF autocrine loop (expression of both PDGF and PDGF receptor) appears to correlate with tumor progression presumably by stimulating growth of tumor cells (Guha, 1991; Hermanson et al., 1992; Vassbotn et al., 1994). Supporting this idea^ expression of a dominant negative PDGF P receptor in a rat glioma cell line inhibits the growth of these cells and reduces their ability to grow as xenografts in nude mice (Strawn et al., 1994). However, there are many other examples of human tumors and tumor cell lines which express elevated levels of PDGF but lack PDGF receptor expression. In these tumors, it is likely that PDGF plays an auxiliary role in tumor progression by stimulating the growth of the connective tissue stroma as well as the tumor vasculature (Chaudhry et al., 1992; Forsberg et al., 1993). Tumor angiogenesis in particular has been recently recognized as a critical factor in the development and progression of many tumor types and the expression of PDGF p receptors on microvascular endothelial cells in tumors and in other settings in which angiogenesis is occurring suggests an important role for the p receptor in blood vessel growth in tumors and other diseases. Taken together, these findings suggest that'inhibition of PDGF receptor signal transduction in tumors could have multiple beneficial effects including the primary inhibition of tumor cell growth as well as inhibition of supporting structure such as the tumor stroma and vasculature. FGF receptors have also been implicated in the development of human tumors particularly human breast and prostate carcinoma (Hattori et al., 1992; Hattori et al, 1990; lida et al., 1994; Lehtola et al., 1992; Peyrat et al., 1991; Story, 1991; Thompson, 1990; Yamanaka et al., 1993). FGF receptor genes have been shown to be amplified in human breast tumors and the amplification of FGFRl correlated

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with spread of the tumor to the lymph nodes and the expression by tumor cells of steroid hormone receptors (Adnane et al, 1991; McLesky et al, 1994). In a variety of cell lines, FGFs and FGF receptor expression has been documented suggesting an autocrine mechanism of tumor cell stimulation. Moreover, forced expression of secreted forms of FGF ligands in cells expressing endogenous FGF receptors confers a transformed phenotype (Forough et al., 1993; Talarico et al., 1993). Interestingly, cells transformed in this manner produce aggressive, highly vascularized tumors in vivo (Kurebayashi et al., 1993). This result suggests that like PDGF receptors, activation of FGF receptors during tumor development not only could contribute to growth of tumor cells but may also contribute to the development of the tumor matrix and tumor vasculature. During development, most embryonic tissues express predominantly a smgle splice variant of a single FGF gene thus restricting the FGF ligands to which they are responsive. Normally, non-malignant rat tumor cells grown in the absence of stroma progress to undifferentiated malignant tumors, a process which is accompanied by a switch from exclusive expression of FGFR2-IIIb to exclusive expression of FGFR2-IIIC (Yan et al., 1993). This switch results in the loss of responsiveness to FGF 7 (KGF) and an acquisition of responsiveness to other members of the FGF ligand family such as FGF2. This switch likely contributes to the malignant phenotype of these cells by an autocrine mechanism since it is accompanied by activation of FGF2 and FGF3 genes. A similar phenomenon has been reported to occur in human astrocytomas where FGFR2 was expressed abundantly in normal white matter and in low grade astrocytomas; whereas, malignant astrocytomas displayed a loss of FGFR2 expression and an increase in FGFRl expression which correlated with the increasing histologic grade of the tumor (Yamaguchi et al., 1994a). Since FGF receptors, like PDGF receptors, appear to be involved in the development and progression of multiple neoplasms, rational therapeutic agents designed to inhibit FGF signaling by FGF receptors might be extremely effective anti-neoplastic agents. For example, forced expression of a dominant negative FGF receptor in an FGF4 transformed NIH 3T3 cell line reverted the transformed phenotype (Li et al., 1994b). It is possible with the advent of more effective gene delivery strategies that dominant negative receptors could be employed to treat a variety of tumors. Another strategy which has evolved is to create FGF ligands tethered to a cellular toxin, i.e. saporin. An FGF-saporin conjugate was found to effectively inhibit tumor growth and decrease metastasis of melanomas in mice (Beitz et al., 1992; Ying et al., 1994). Although this approach may be relatively specific for tumor cells expressing high levels of FGF receptors, binding of the FGF toxin conjugate to FGF receptors on normal tissues may may limit the usefulness of this approach. B. PDGF and FGF Receptors in Genetic Diseases

Based on recent gene knockout experiments, it might be predicted that heritable defects of FGF or PDGF receptors might result in embryonic lethality and thus not

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present a significant clinical problem (see above discussion). In fact, PDGF receptor mutations have not been implicated in any human genetic disease. However, mutations in three of the four known FGF receptor genes (FGFRl, FGFR2 and FGFR3) have been shown to cause a number of related genetic syndromes. These syndromes are characterized by specific skeletal abnormalities that are the result of single point mutations involving either the transmembrane domain or the third Ig domain of FGFRl, 2, or 3 (Figure 5). Achondroplasia, the most common genetic form of dwarfism, is caused by a mutation (G380R) in the hydrophobic transmembrane domain of FGFR3 (Rousseau et al, 1994; Shiang et al., 1994). To date, this mutation has been found in all cases of genetic or sporadic achondroplasia. The effect this mutation has on the

Crouzon Jackson-Weiss TM ^^ Pfeiffer

Figures. Localization of point mutations in FGFR-1, FGFR-2, and FGFR-3 known to be involved in craniofaciaI/skeletal displaslas. Note that analagous mutations in FGFR-1, FGFR-2, and FGFR-3 involving the N-terminal portion of the 3rd Ig-domain are associated with different phenotypes. Also note that mutations in distinctly different regions of FGFR-3 result in a similar phenotype (thanatophoric dysplasia type I).

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function of FGFR3 during cartilage development is not known, however, mutations in other receptor tyrosine kinases involving the transmembrane domain have had a neutral, activating or inhibitory effect depending on their position in the transmembrane domain (Johnson and Williams, 1993; Ullrich and Schlessinger, 1990). Hypochondroplasia is an autosomal dominant disorder with skeletal features similar to but generally milder then those seen in achondroplasia. Genetic evidence demonstrating the potential linkage of achondroplasia and hypochondroplasia led to the recent discovery of a single point mutation of FGFR3 in 50% of patients with hypochondroplasia (Bellus et al, 1995). The point mutation resulted in an Asn 554Lys substitution in the n-terminal kinase domain. Similarities in the phenotypes of achondroplasia and hypochondroplasia suggest that the consequences of the point mutation in the transmembrane domain that causes achondroplasia and the mutation in the n-terminal kinase domain causing hypochondroplasia are functionally similar. Thanatophoric dysplasia (TD) is a lethal skeletal dysphasia with features that are similar to those seen in homozygous achondroplasia. Two distinct forms of this disorder, TDl (curved femurs with variable degrees of cloverleaf skull) and TD2 (straight femurs and severe cloverleaf skull) like achondroplasia are caused by specific mutations in FGFR-3 (Tavormina et al., 1995). In 16 cases of TD2, affected individuals were heterozygous for a Lys650Glu mutation, adjacent to an autophosphorylation site in the FGFR-3 kinase domain. Individuals with TD 1, however, had mutations in the extracellular domain; 22 individuals had an Arg248Cys substitution between Ig domains 2 and 3, and 1 individual had a Ser371Cys substitution between Ig domain 3 and the transmembrane domain. These findings demonstrate that mutations in the extracellular ligand binding domains and the intracellular kinase domain may have similar functional consequences. Four other genetic diseases involving cranial facial abnormalities and variable degrees of distal extremity malformation are caused by mutations encompassing the third Ig domain of FGFRl and FGFR2. Crouzon syndrome, an autosomal dominant syndrome characterized by craniosynostosis without digital abnormalities, has been shown to be caused by at least eight different point mutations in the FGFR2-IIIC exon (Jabs et al., 1994; Reardon et al., 1994). A different mutation of the FGFR2-IIIC exon was found in patients with Jackson-Weiss syndrome which is characterized by cranial synostosis accompanied by specific digital abnormalities (broad big toes and occasional syndactyly) (Jabs et al., 1994). Pfeiffer syndrome is associated with craniosynostosis as well as broad, medially deviated great toes and thumbs with or without syndactyly of other digits. This syndrome can apparently result from point mutations in either FGFRl or FGFR2IIIc (Muenke et al., 1994; Rutland et al., 1995). Interestingly, the mutations in FGFR2-IIIC are identical to those found to cause Crouzon syndrome (Rutland et al., 1995). Since these phenotypes are never seen together in the same family, other inherited traits, acting in cis can evidently modulate the phenotypic expression of the mutant allele. Apert syndrome, the most severe of these syndromes comprising craniosynostosis and severe syndactyly of both hands and feet, has been shown to involve a

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single point mutation in either of two adjacent amino acids within the region adjoining the second and third Ig-like domain of FGFR2 (Wilkie et al, 1995). One of these mutations (Pro253 Arg) of FGFR2 in Apert syndrome corresponds precisely with a mutation of FGFRl (Pro252Arg) that causes Pfeiffer syndrome suggesting that perturbations in this region of FGF receptors has a specific effect perhaps on ligand binding affinity or specificity. The precise mechanism of these mutations in causing these extremely similar syndromes has not been established. However, the fact that each involves specific effects on skeletal development and limb formation is consistent with the expression of these receptors during embryonic development and with recent studies demonstrating roles for FGF ligands during limb formation (Orr-Urtreger et al., 1991; Peters et al, 1992). Moreover, the autosomal dominant nature of these syndromes suggests either that these mutations are activating or in some way produce a dominant negative effect on the function of the wild type allele. Further study to determine the effect of these mutations on FGF receptor function in cultured cells and in transgenic animals and will no doubt lead to a greater understanding of FGF receptors in health and disease. C. PDGF and FGF Receptors in Vascular Disease

Blood vessels respond to a variety of injurious stimuli (hypertension, hyperlipidemia, and mechanical trauma) in a very similar manner (Ross, 1993; Schwartz and Liaw, 1993). Smooth muscle cells are stimulated to proliferate in the muscular media and then migrate into the intima where they produce abundant extracellular matrix resulting in the thickening of the intima and narrowing of the vascular lumen. Inflammatory cells, especially monocyte/macrophages, are also induced to migrate into the intima from the circulation where they proliferate and produce cytokines such as PDGF. As this vascular plaque matures, microvessels form within the plaque probably facilitating plaque enlargement and eventually leading to weaknesses in the vessel wall which allow plaque rupture and thrombosis. FGF and PDGF receptors are expressed on both vascular endothelial cells and smooth muscle cells. Stimulation of cultured smooth muscle cells with PDGF or FGF elicits mitogenesis, cell migration, and in the case of PDGF, a more rapid transition from the "contractile" phenotype to the "synthetic" phenotype, a transition thought to be important for mitogenic response and matrix production by smooth muscle cells (Bell and Madri, 1989; Casscells, 1992; Klagsbrun and Edelman, 1989; Thybert et al., 1983). In a rat model of balloon angioplasty, mechanical trauma to the rat carotid stimulates mitogenesis of medial smooth muscle cells and the subsequent migration into the intima where they produce extracellular matrix and continue to proliferate, albeit at a reduced rate. In this model, antibodies to either PDGF or FGF ligand inhibit intimal thickening, and addition of exogenous PDGF or FGF accelerates intimal thickening (Cuevas et al., 1991; Ferns etal, 1991; Jawienetal., 1992; Lindner and Reidy, 1991a). Given that PDGF and FGF receptors have now been shown to be expressed in human atheroma, it is likely that signaling by these receptors plays a role in the develop-

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ment of primary atherosclerosis as well as a number of other vasculopathies associated with neointima formation (Brogi et al., 1993; Hughes et al., 1993; Rubin et al., 1988; Wilcox et al, 1988). Thus, the development of specific FGF and PDGF receptor antagonists may provide rational therapy for this extremely common pathologic process.

VII. THERAPEUTIC STRATEGIES TO MODULATE PDGF AND FGF RECEPTOR SIGNAL TRANSDUCTION IN HUMAN DISEASE The bulk of available evidence suggests that the most important physiologic roles for PDGF and FGF receptor signaling are played out during embryonic development. During adulthood, PDGF and FGF receptor expression are frequently associated with the development and progression of pathologic states. Signaling by PDGF and FGF receptors likely contributes to the progression of these diseases. Thus, inhibition of signaling by these receptors represents an opportunity for the development of new therapeutic agents. Inhibition of signaling by receptor tyrosine kinases could occur at any level between ligand binding and the activation of specific signaling pathways. One approach to develop inhibitors of FGF signaling has been to create ligands conjugated to cellular toxins such as saporin or pseudomonas exotoxin (Beitz et al., 1992; Biro et al., 1992; Casscells, 1992; Kreitman and Pastan, 1994; Lappi et al., 1994; Lindner et al., 1991; Ying et al., 1994). These growth factor-toxin conjugates have demonstrated activity in animal models of human vascular disease, however, their use may be limited by the toxicity of these agents for normal cells expressing FGF receptors (i.e., endothelial, glial, and neuronal populations). Another approach to block receptor signaling is to create a secreted extracellular domain. Recombinant soluble extracellular domains of FGF receptors have been used extensively to study ligand binding specificity and heparin dependence of FGF receptor/FGF ligand interactions (Gheon et al., 1994; Omitz and Leder, 1992; Omitz et al., 1992a). It has also been shown that recombinant soluble extracellular domains of FGF or PDGF p receptors can inhibit signal transduction by endogenous, cell surface expressed receptors presumably by competing for binding to the Hgand (Duan et al., 1991; Duan et al., 1992; Kiefer et al., 1991). Whether or not these soluble extracellular domains will be effective inhibitors in vivo is not known. However, one recent report demonstrating the effective blockade of tumor necrosis factor activity by a soluble form of the TNF receptor fused to a mouse IgG heavy chain supports the extension of this approach to other receptors such as PDGF and FGF receptors (Kolls et al, 1994). Dominant negative receptors with deleted or inactivated kinase domains represent an additional approach to blocking signal transduction by FGF and PDGF receptors. A dominant negative PDGF p receptor has recently been shown to inhibit the growth of a rat glioma (Strawn et al, 1994). The eventual utility of this approach

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will hinge upon the development of efficient techniques for gene transfer since the dominant negative receptor must be overexpressed in the target cells. Recently, natural inhibitors of protein tyrosine kinases have been discovered (Levitzki and Gazit, 1995). These compounds inhibit receptor autophosphorylation, signaling molecule activation and growth factor-dependent proliferation by competing for ATP binding or substrate binding or both. Naturally occurring protein kinase inhibitors have served as excellent starting points for the design of synthetic inhibitors. Improved characteristics of these synthetic inhibitors include greater specificity for particular kinases, enhanced ability to inhibit both ATP binding and substrate binding and enhanced ability to penetrate the cell membrane. One class of these agents, has been shown to effectively inhibit proliferation of smooth muscle cells in response to PDGF or FGF and so may so may serve as the basis to develop inhibitors of vascular disease progression as well as other diseases such as cancer (Bilder et al., 1991; Kovalenko et al., 1994). In addition to inhibitors that impede receptor function directly, it is also likely that effective inhibitors will be developed to target specific downstream signaling pathways. For example, the recent development of potent inhibitors of ras lipidation may provide effective therapy by blocking ras activity in response to PDGF or FGF receptor activation (Gibbs et al., 1994; James et al., 1993). Similarly, effective approaches for the inhibition of protein kinase C, PI3 kinase, and other downstream signaling pathways will likely add to an ever increasing armamentarium designed to effectively inhibit PDGF and FGF receptor signaling on multiple levels.

SUMMARY PDGF and FGF receptors are closely related receptor tyrosine kinases that initiate complex signaling cascades after binding of their cognate polypeptide ligands. The distribution of these receptors in embryonic and adult tissues as well as the cellular responses engendered after receptor activation suggests important functions in embryonic development with more limited roles in adults. The development and progression of pathophysiologic states such as atherosclerosis and neoplastic diseases are likely mediated in part by inappropriate expression and signal transduction via FGF and PDGF receptors. Expanding knowledge of mechanisms of signal transduction downstream of activated PDGF and FGF receptors provides the basis for development of new therapeutic agents.

ACKNOWLEDGMENTS The authors wish to thank Drs. Mike Cardone, Steve Harrison, Mike Kavanaugh, Anke Klippel and Bert Pronk for their critical reading of this manuscript and invaluable comments. The authors also wish to thank Katie H. Gunter for her expert help in the preparation of this manuscript. Due to the scope of this review, we wish to apologize to those people whose work was not cited.

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THE NERVE GROWTH FACTOR FAMILY

Mari Oshima, Yoko Hirata, and Gordon Guroff

Abstract I. Introduction and Overview II. Nerve Growth Factor A. Early Experiments B. Strucnire C. Gene and Gene Expression D. Biosynthesis E. Occurrence F. Target Tissues G. Effects on Target Tissues H. Mechanism of Action I. CHnical Perspectives III. Brain-Derived Neurotrophic Factor IV. Neurotrophin-3 V. Neurotrophins-4/5 and -6 VI. Conclusion References

230 230 232 232 .232 233 234 235 236 237 239 242 244 246 248 248 249

Growth Factors and Cytokines in Health and Disease Volume lA, pages 229-258. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0091-4 229

230

MARI OSHIMA, YOKO HIRATA, and GORDON GUROFF

ABSTRACT The neurotrophin family now containsfivemembers. The properties of the prototype, nerve growth factor, studied for more than 40 years, are well known and reasonably well-understood. The structure of nerve growth factor has been solved by X-ray diffraction and its gene has been cloned. Nerve growth factor is readily available through the recombinant route. The mechanism by which it acts, while not completely clear, is known in outline and is the subject of detailed research interest. A robust debate is in progress over the clinical use of nerve growth factor to ameliorate some of the most catastrophic and costly human diseases. This debate has been fueled by some early indications of clinical utility. Molecular techniques have accelerated the discovery and exploration of several new neurotrophins, brain-derived neurotrophic factor and neurotrophms 3,4/5, and 6. These molecules share substantial homology and are of the same general physical character as nerve growth factor, but with complementary and sometimes overlappmg target specificities. Information about thefirsttwo makes it likely that they will also share several aspects of their action mechanisms. Their clinical utility is, as yet, a matter of speculation.

1. INTRODUCTION AND OVERVIEW The original studies on what came to be called nerve grov^th factor (NGF) were done more than 40 years ago. Since then, the work on this small protein has been paradigmatic for the studies of the now large number of growth factors known. Growth factors for virtually every organ in the body have been identified, and those acting on the nervous system number in the dozens. It has recently been found that nerve growth factor is simply the first discovered member of a family of neurotrophic agents. Brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) have been cloned. Like nerve growth factor, these are small, basic peptides with about 50% overall homology, very similar physical properties, and, probably, structures. The finding that there are multiple, related neurotrophins has been accompanied by the description of multiple, related receptors, the trks B and C, serving as binding sites for BDNF and NT-3, respectively. Interest in this field has increased dramatically over the last few years, due to a number of observations. First, the understanding that nerve growth factor had actions in the central nervous system indicated that its role was not limited to specific portions of the peripheral nervous system. Then, the discovery that there is a family of neurotrophins broadened the field to include virtually all areas of the nervous system and aspects of neural development. Finally, the realization that these peptides could have clinical significance in disease states, such as Alzheimer's, and in environmental insults, such as ischemia, has attracted significant commercial activity.

Table 7. Characteristics of the Members of the Neurotrophin Family ~~~~

Molecular Weight Monomer

pl

Major Transcript (kb)

nerve growth factor (NGF)

13,259

9.3

1.35

brain-derived neurotrophic factor (BDNF)

13,511

9.99

1.45

neurotrophin-3 (NT-3)

13,625

9.3

I .4

neurotrophins-4 and -5 (NT-415)

13,917

10.3

I .35

neurotrophin-6 (NT-6)

15,968

10.8

1.4

Neurotrophin

N

W A

~

Sources

Target Tissues

Knockouts

mature male mouse sympathetic and ptosis, insensitivity to salivary gland, sensory neurons, pain, small size, prostate, seminal basal forebrain normal motor cholinergic activity, death by fluid, snake venom neurons, adrenal four weeks medulla, mast cells, certain tumors pig brain, culture sensory neurons, few sensory neurons, medium motor neurons, ataxia, normal hi ppocampa I, motor neuron cerebellar, basal activity, death by forebrain, nigra, four weeks and retinal ganglia neurons recombinant sensory neurons, sensory and nodose ganglia sympathetic neurons, glial cells deficits, muscle abnormalities, normal enteric and central nervous system development recombinant sensory and unknown sympathetic neurons, PC 12 cells, motor neurons recombinant sympathetic and unknown sensory neurons

Clinical Perspectives

Alzheimer’s, Parkinson’s, peripheral neuropathies

Parkinson’s, motor neuron disease

peripheral neuropathies

motor neuron degeneration

unknown

232

MARI OSHIMA, YOKO HIRATA, and GORDON GUROFF

II. NERVE GROWTH FACTOR (NGF) A. Early Experiments

It was known in the late 1940s that the fate of specific portions of the peripheral nervous system was determined by the state of the "field" that portion was destined to innervate. Thus, removal of a limb from a chick embryo resulted in a decrease in the vigor with which nerve fibers grew toward that part of the embryo and addition of a limb next to an existing limb significantly increased that vigor. Elmer Bueker asked, experimentally, what would happen if the limb, a very complex structure, was replaced by a more homogeneous collection of cells, namely a fragment of a tumor. The result of this experiment, in which the tumor chosen was Sarcoma 37, was that the portions of the peripheral nervous system near the tumor grew more robustly toward the implant than they did on the opposite side of the embryo (Bueker, 1948). Analyzing Bueker's data, Rita Levi—Montalcini observed that there was no physical contact between the tumor and the advancing fibers and postulated that the tumor was elaborating a diffusible substance that she called nerve growth factor. She proved this postulate in a study in which she repeated Bueker's experiment, but placed the tumor fragment on the outside of the chorioallantoic membrane so that physical contact between the tumor and the peripheral nerves was precluded (Levi-Montalcini and Hamburger, 1953). The resulting increased growth of the proximate ganglia, similar to that seen in Bueker's original experiment, validated her concept of a diffusible factor. Using a tissue culture assay based on this ganglionic response, Stanley Cohen purified the factor from the tumor and, subsequently, from a much richer source, the salivary gland of the mature male mouse (Cohen, 1960). The availability of the purified protein permitted the design of an experiment that unequivocally proved the physiological relevance of the factor. In this experiment (Levi—Montalcini and Booker, 1960), rabbits were actively immunized with the protein, and the resultant antiserum was used to passively immunize newborn rodents. The result was that these latter animals were permanently and almost totally deprived of their sympathetic nervous systems. This "immunosympathectomy" has been taken as incontrovertible evidence that nerve growth factor is required, virtually moment-to-moment, for the survival of sympathetic neurons in immature animals. B. Structure

The amino acid sequence of nerve growth factor has been available for several years (Angeletti and Bradshaw, 1971), as has information about its secondary structure (Argos, 1976). The monomer is a basic protein with a molecular weight of about 13,000, containing three disulfide bonds. The active molecule is a noncovalently bound dimer, held together with a dissociation constant of some 10"^^ M. Since the molecule is active at concentrations on the order of 10"^—10~^^ M, it is most certainly a dimer under physiological conditions. Indeed, if the dimer is cross-linked with dimethylsuberimidate so it cannot dissociate, it is still active. The

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crystal structure of nerve growth factor was solved in 1991 (McDonald et al., 1991) by X-ray analysis revealing a molecule with a flat surface made up of three antiparallel pairs of p-strands, four loop regions, and a cluster of positive charges that might interact with an acidic receptor. Early suggestions, based on modest homologies, that nerve growth factor might be a member of the insulin-like growth factor family (Frazier et al., 1972) have been superseded by its reclassification as a member of a structural superfamily called cystine knot (McDonald and Hendrickson, 1993). It has been shown, through studies of crystal structure, that transforming growth factor P2 and platelet-derived growth factor BB are topologically very similar to nerve growth factor although there is little sequence identity between the three proteins (Murray—Rust et al., 1993). Nerve growth factor is found in the mouse salivary gland as a component of a larger molecule called the 7S because of its sedimentation value (Varon et al., 1967a; 1967b). The 7S is composed of three different peptide subunits, one of which is the active dimer, called P when it occurs in this form. The other two are the a and the y, of which there are two each. Thus the 7S is composed of 6 protein chains and 2 atoms of zinc, which help hold the chains together. The molecule is quite stable at neutral pH, but dissociates into its components above pH 8 or below pH 5. The y subunit has a molecular weight of some 26,000 and, when separated from the 7S, is an active serine esteropeptidase. It had been suggested that the y subunit is involved in the processing of nerve growth factor from a larger precursor, but recent data indicate that the y subunit plays a minor role at best. The a subunit has substantial sequence homology with the y subunit, and a similar molecular weight, but no enzymatic activity. It appears to be an inactive serine esteropeptidase. The function of the 7S is something of a mystery. It must dissociate for the p subunit to be active (Stach and Shooter, 1980) and there is no apparent 7S complex in other organs, such as guinea pig prostate, that also have high levels of nerve growth factor (Harper et al., 1979; Harper and Thoenen, 1980). Thus, although it may be present in the salivary gland to store or protect nerve growth factor, its absence in other tissues that contain nerve growth factor makes it unlikely that it has any general importance. C. Gene and Gene Expression The nerve growth factor gene was identified and characterized simultaneously by two groups (Scott et al., 1983; Ullrich et al., 1983). The data obtained indicate that the mouse nerve growth factor gene covers more than 43 kb and is comprised of five exons and four introns (Selby et al, 1987). Nerve growth factor is coded as a single-copy gene and there is substantial homology across species. Indeed, from mouse to man the sequence of nerve growth factor is more than 85% conserved. Genes from many other species have been cloned. The regulatory region of the nerve growth factor gene has been mapped, and it has been shown that the nucleotide sequences of the human and mouse gene are greater than 90% similar near their promoters (Cartwright et al., 1992). Both stimulatory and inhibitory

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sequences upstream from the promoter have been deUneated, and nuclear proteins binding to several upstream segments have been described (D'Mello et al., 1991). There are at least four nerve growth factor transcripts, two major and two minor (Selby et al., 1978). The major transcripts appear to be the products of alternative splicing (Edwards et al, 1986), and there is some evidence that their appearance is tissue-specific. The transcripts differ in length and the difference is found in the 5' region, the longer one coding for a 34-kDa precursor containing a hydrophobic signal peptide, the shorter one coding for a precursor of 27 kDa. The availability of DN A probes has allowed studies on the expression of the gene, and these studies have led to the concept that nerve growth factor synthesis in various tissues is proportional to, and probably determines, the amount of innervation a given tissue is destined to receive from nerve growth factor-sensitive neurons (Shelton and Reichardt, 1984). This work has made clear, however, that nerve growth factor synthesis is developmentally programmed and does not require innervation in order to trigger expression. This concept has been validated in several systems (Rohrer et al., 1988). Although innervation, per se, does not seem to influence the expression of the nerve growth factor gene, a number of other things do. Brain glia in culture express nerve growth factor mRNA and do so at eightfold higher levels if they are rapidly growing than if they are confluent and quiescent (Lu et al., 1991). Transection of the sciatic nerve increases the levels of nerve growth factor mRNA up to 15-fold in the surrounding support cells (Lindholm et al., 1987), due, apparently, to the migration of mast cells to the site of the lesion and their elaboration of interleukin-1. The effect of interleukin-1 in this system is primarily on nerve growth factor mRNA stability. The action of another cytokine, transforming growth factor-p, in increasing the levels of nerve growth factor mRNA in cultured rat astrocytes, is largely transcriptional (Lindholm et al., 1990). Hippocampal neurons also contain nerve growth factor mRNA and its levels are controlled by the balance between the glutamate and the GAB A pathways; glutamatergic influences increase mRNA levels while GABAergic influences lower them (Zafra et al., 1991). Moderate alterations in nerve growth factor mRNA levels have been produced by catechols (Furukawa et al., 1993), dexamethasone (Saporito et al., 1994), and 1,25-dihydroxyvitamin D3 (Saporito et al., 1994). Although these changes have been modest, the effort to regulate nerve growth factor synthesis in appropriate sites in the nervous system may ultimately have important clinical implications (Carswell, 1993). D.

Biosynthesis

The details of nerve growth factor biosynthesis are not known and not too much work is currently directed toward this problem. Perhaps this is so because the overall outlines of nerve growth factor biosynthesis make it appear similar to that of several other peptide ligands. At least in the mouse submaxillary gland, nerve growth factor is synthesized as a large precursor that is cleaved at both the N- and C-terminals to

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produce the mature active molecule. It was shown some years ago (Berger and Shooter, 1977; 1978) that explants of salivary glands produce a molecule of about 22,000 daltons recognized by anti-nerve growth factor antibody. It was further shown that this molecule can be cleaved by the y subunit of the 7S complex, and by other proteases, to the active nerve growth factor monomer. In a later study, Darling et al. (1983) have identified precursors of 35, 29, 22, 19, and 13 kDa. Gene level studies predict a series of processing steps. Scott et al. (1983) obtained evidence for a prepro nerve growth factor of 33,800 daltons, and a pro nerve growth factor of between 30,000 and 32,000 daltons. Ullrich et al. (1983), using other assumptions about initiation sites, predict a prepro nerve growth factor of 27,000 daltons and a pro nerve growth factor of 25,000 daltons. Although all of these precursors are larger than the 22,000-dalton molecule found by Berger and Shooter, an Arg-Arg cleavage site is present at a point in the sequence appropriate to yield a 22,000-dalton precursor. E. Occurrence Nerve growth factor can be isolated in milligram quantities from gram quantities of the salivary glands of the mature male mouse. As noted above, the biologically active nerve growth factor is found in the 7S form in this organ, a form of somewhat uncertain biological function. The reason that this specific tissue contains so much material is unclear, because comparable glands from female mice, or from mature male rats, are not nearly so rich a source. Other rich sources include the prostate of the guinea pig (Rubin and Bradshaw, 1981), the seminal fluid of the bull (Harper and Thoenen, 1980), and the salivary gland of Mastomys (Aloe et al., 1981), an African rodent that is a hybrid between mouse and rat. The findings with Mastomys are somewhat different than with the mouse; both male and female animals have high levels of nerve growth factor, and it is found in a high molecular weight complex that has a sedimentation of about 5S, but does not contain y subunits (Darling and Fahnestock, 1988). Amaterial having nerve growth factor-like activity has been found in placenta (Goldstein et al., 1978), but complete characterization of this substance has been difficult due to the limited quantities present in this source. Snake venoms of all kinds contain nerve growth factor-like materials (Hogue-Angeletti et al., 1976), that differ somewhat from the mouse protein immunologically and in activity as well, are generally conserved in the nerve growth factor-like region at the carboxy terminus, but are less well conserved in the prepro regions of the molecule. Cells of all kinds make small amounts of nerve growth factor in culture (Bradshaw and Young, 1976), but it is not clear that this synthesis has physiological meaning. The nerve growth factor-like material found in prostate may deserve special mention here. Although detailed characterization has not been completed, such a protein occurs in the conditioned media of cultured stromal cells from the human prostate (Graham et al., 1992). Precursor forms of nerve growth factor, with molecular masses of 65,000, 61,000, and 42,000, have been observed in this

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medium by western blotting with antibody to mouse nerve growth factor (Djakiew et al., 1991), but there are some data indicating that this material may be a prostate-specific form of nerve growth factor. These observations could have substantial clinical significance because the nerve growth factor appears to stimulate the growth of prostate epithelial cells and of tumors arising from them (Djakiew et al., 1993). Thus, nerve growth factor-like materials may play a role in the etiology of benign prostate hyperplasia and of prostate cancer, as well. F. Target Tissues The classic targets for nerve growth factor action are the sensory and sympathetic neurons of the peripheral nervous system, for the survival of which nerve growth factor is absolutely essential. More recently it has been shown that nerve growth factor acts on the chromaffin cells of the adrenal medulla, changing them into sympathefic neurons (Unsicker et al., 1978; Aloe and Levi-Montalcini, 1979), on certain tracts in the central nervous system, producing an enhancement in their neuronal phenotype (Hefti et al., 1985; Martinez et al, 1985; Mobley et al., 1985), and on the kidney during morphogenesis (Sariola et al., 1991). A number of tumor lines respond to nerve growth factor, generally, with morphological and biochemical differentiation. Prominent among the tumors affected by nerve growth factor in culture are the human neuroblastomas, including the IMR32 (Reynolds and Perez-Polo, 1981) and the SH-SY5Y (Perez-Polo et al., 1979). Certain anaplastic gliomas also appear to differentiate in response to nerve growth factor (Vinores and Koestner, 1980), and there is evidence that the Wilms' tumor, a neoplasm originating in the kidney, can also respond to nerve growth factor (Donovan et al., 1994). The most informative of these tumor lines continues to be the PC 12 pheochromocytoma, a tumor that arose spontaneously in the adrenal medulla of the highly irradiated New England Deaconess strain of rats and was put into culture in the late 1970s (Greene and Tischler, 1976; Dichter et al., 1977). PC 12 cells are small, round, and fluorescent and grow readily in standard culture, in the absence of nerve growth factor, with a doubling time of about 48 hours. The fluorescence arises from the high concentrations of catecholamines, primarily dopamine, that are in the cells. The addition of nerve growth factor in nanomolar quantities produces dramatic changes in the phenotype of the cells within a few days. The cells elaborate neurites, become electrically excitable, stop dividing, and will synapse with appropriate muscle cells in culture (Schubert et al., 1977). To all intents and purposes, they go from a chromaffin-like cell to a mature sympathetic neuron within 3 to 5 days. The cardinal difference between them and normal sympathetic neurons is that the differentiation is reversible; removal of nerve growth factor causes the neurites to disintegrate, the cells to round up, and cell division to resume. These cells have become the foremost tool for the study of nerve growth factor action, because, unlike normal sympathetic and sensory neurons, they do not require nerve growth factor for survival. Accordingly, one can compare the biochemistry and molecular

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biology of cells that have and those that do not have nerve growth factor in the medium. In addition, the cells are heavily studied models for such important processes as neurotransmitter release, neuronal survival, cell cycle control, and the signal-transduction pathways serving differentiation and cell division. One other surprising target for nerve growth factor action, that has been the focus of recent studies, is the mast cell, first shown to be a target of nerve growth factor by Aloe and Levi-Montalcini (1977). Nerve growth factor will cause these cells, whether from rats or humans, to release histamine in the presence of the lipid cofactor lysophosphatidylserine (Horigome et al, 1993). This release is dependent on the presence of extracellular calcium and is similar to the secretagogue action of nerve growth factor seen in PC12 cells (Nikodijevic et al., 1990). There is also evidence that nerve growth factor has some action on mast cell survival (Horigome et al., 1994). The finding that nerve growth factor acts on cells of the immune system has been taken by some as evidence for the hypothesis that this molecule plays a role in neuroendocrine-immune interactions. G. Effects on Target Tissues

Nerve growth factor elicits a number of different responses from its target cells, but not all of the responses are seen in all of the cells. In sensory and sympathetic neurons from young animals, as shown by the administration of anti-nerve growth factor antibody (Levi-Montalcini and Booker, 1960), nerve growth factor is required for outright survival. The situation is not so clear in comparable studies with adult animals; administration of anti-nerve growth factor antibody causes sympathetic and sensory neurons to become impaired, but the neurons survive, and the damage appears reversible (Angeletti et al., 1971). Nerve growth factor also produces rapid changes in the structure of the neuronal membrane (Connolly et al., 1981), increases the transport of nutrients (Horii and Varon, 1977), encourages neurite outgrowth (Levi-Montalcini and Hamburger, 1953), produces cellular hypertrophy (Hendry, 1976), increases the expression of enzymes involved in neurotransmitter synthesis (Thoenen et al., 1971), and can act on the advancing neurite in a chemotactic sense (Gundersen and Barrett, 1979). While not requiring nerve growth factor for survival, the chromaffin cells of the adrenal medulla, given nerve growth factor at an early stage, elaborate neurites and assume an altered phenotype, much like that of sympathetic neurons (Aloe and Levi-Montalcini, 1979). The cholinergic neurons of the basal forebrain, the most studied of the target populations in the central nervous system, show enhanced levels of neurotransmitter-synthesizing enzymes (Hefti et al., 1985; Martinez et al., 1985; Mobley et al., 1985) and enhanced survival after axotomy or axonal crush (Hefti, 1986; Williams et al., 1986). PC 12 cells, the widely used model for the study of nerve growth factor responses, show a bewildering number of changes, including exceedingly rapid alterations in membrane structure (Connolly et al., 1979), increases in ion transport (Boonstra et al., 1981; Nikodijevic and Guroff, 1991), induction of several immediate early genes (Greenberg et al., 1985), elevations in ornithine decarboxylase

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levels (Greene and McGuire, 1978), both short and long-term increases in the activity of neurotransmitter-synthesizing enzymes (Greene and Rukenstein, 1981; Hatanaka, 1981), increases in structural proteins (Lindenbaum et al, 1987), changes in the repertoire of ion channels on the cells (Kongsamut and Miller, 1986), cellular hypertrophy (Dichter et al, 1977), decreases in mitogen receptors (Huff et al., 1981), cessation of cell division (Dichter et al., 1977), elaboration of neurites (Dichter et al., 1977), and, in short, global changes in phenotype. Many of these changes are seen in other tumor cells upon nerve growth factor treatment, and certain of these responses happen in normal cells as well. The most useful categorization of the many things that nerve growth factor does to its target cells was presented by Bradshaw some years ago (1978) when he pointed out that nerve growth factor actions fall into two general categories. There are rapid, membranebased events that do not require transcriptional adjustments, such as the very rapid changes in membrane structure or the alterations in ion uptake. Then there are the longer term changes, such as increases in neurotransmitter-synthesizing enzymes and alterations in channel composition, that require the activation of specific gene elements. Perhaps the most distinctive of the effects of nerve growth factor, the elaboration of neurites on many of its target cells, appears to require both transcriptional change and membrane-based events (Burstein and Greene, 1978). The question of the effects of nerve growth factor on its target cells and, in turn, on the whole organism has recently been approached using the knockout methodology (Crowley et al., 1994). Mice homozygous for the disrupted nerve growth factor gene were smaller at birth by 5-25%, but displayed normal motor activity. They did not, however, ingest normal quantities of milk. In spite of efforts to increase their food intake, most animals died within the first three days after birth. Those that survived gained weight more slowly and were delayed in many developmental milestones. Marked ptosis was observed, indicating a lack of sympathetic innervation to the eye. None survived for more than four weeks. These animals were insensitive to pain and their dorsal root ganglia contained less than 30% as many neurons as seen in controls. Their sympathetic ganglia were also very small and, by postnatal day 14, were not visible upon dissection. In contrast, the cholinergic neurons of the basal forebrain were largely unaffected and maintained their phenotype throughout the life of the animals. The reduced life span of these animals, caused, apparently, by the lack of sensory neurons, limits the information available about the development of the central nervous system. To circumvent this sensory deficit, the nerve growth factor gene under the control of the keratin promoter has been introduced into the knockouts. The strategy has been to express nerve growth factor in the periphery, thus, curing the sensory deficits, but to have it remain excluded from the central nervous system by virtue of its inability to cross the blood-brain barrier. Although these data have been presented only in preliminary form (Phillips et al., 1994), the ability of these mice to respond to pain, grow to maturity, and breed normally, promises to provide

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unique information about the influence of nerve growth factor, or the lack of it, on the central nervous system. H. Mechanism of Action

The mechanism by which nerve growth factor acts on its target neurons appears to involve retrograde transport up the axon to the cell body. This conclusion is implicit in the observation that the levels of nerve growth factor in the circulation (Suda et al., 1978) are lower than those known to be required to support sympathetic and sensory neurons. Clearly, then, nerve growth factor must be reaching the neurons by some other route. The retrograde transport of nerve growth factor was demonstrated directly by Hendry et al. (1974) and described in full by Thoenen and his colleagues (Stockel et al., 1974; 1975; 1976; Paravicini et al., 1975). The basic experiment involved the administration of radioactive nerve growth factor to the anterior chamber of the eye, a locus innervated in the rat by the superior cervical ganglia. Observation after several hours showed that, although there was radioactivity on both sides, there was more radioactivity in the ganglia on the side of the injection than on the other side. That the transport was specific to nerve growth factor was shown by the finding that proteins of similar size and charge, for example, cytochrome C, were not transported (Stockel et al., 1974). That the transport was physiologically meaningful was shown by the observations that the nerve growth factor arriving at the ganglia was chemically intact (Stockel et al., 1976), immunologically reactive (Stockel et al., 1976), biologically active (Paravicini et al., 1975), and found in a select population of the neurons, presumably those innervating the eye and not those innervating the salivary gland, heart, etc. Thus, it is generally believed that nerve growth factor is elaborated into the extracellular space by organs that are the targets of nerve growth factor-sensitive neurons, is sensed by the advancing axons of those neurons, is bound to specific nerve growth factor receptors at the synaptic ending, and,finally,transported back to the cell body by retrograde transport up the axon. The nature of the nerve growth factor receptors at the synaptic ending has not been determined, except that they are specific for nerve growth factor. There is no reason to believe that they are any different than the plasma membrane receptors found on the cytoplasmic surface of the neurons and on the membranes of the non-neuronal cells, such as chromaffin cells, mast cells, and tumors, on which nerve growth factor acts. Binding to those cell surface receptors leads to the multitude of actions, both membrane and nuclear, referred to above. But it is clear that nerve growth factor does not elicit nuclear events directly. First, it has been shown in many laboratories that, although there are a small number of receptors on the nucleus of nerve growth factor responsive cells, little or no nerve growth factor reaches the nucleus. Equally persuasive are the data resulting from experiments in which nerve growth factor has been introduced directly into the cell. When turkey erythrocyte ghosts were loaded with nerve growth factor and fused with PC 12 cells, spilling their contents into the cell and giving the protein direct access to the

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nucleus, the cells did not respond (Heumann et al., 1981). When the ghosts contained anti nerve growth factor antibody, thus, presumably creating a barrier to the access of nerve growth factor to the nucleus, the cells responded to nerve growth factor in the same ways they did in the absence of the antibody. These experiments have been supported by studies in which nerve growth factor was introduced into the cells by other means. Thus, it is generally held that, although nerve growth factor is clearly internalized (Levi et al., 1980), probably, in order to inactivate it by lysosomal breakdown, nerve growth factor need not enter the cells to act on them. That being the case, there has been substantial research interest in the intracellular signaling pathways initiated by the combination of nerve growth factor and its receptor(s). This has proved a difficult and complex endeavor, because it has been found that virtually all the known second messengers are influenced by nerve growth factor. Thus it has been shown that there are increases in cAMP (Nikodijevic et al., 1975), cGMP (Laasberg et al., 1988), glycosylphosphatidylinositol metabolites (Chan et al., 1989), Ca""^ (Nikodijevic and Guroff, 1991), phospholipid turnover (Contreras and Guroff, 1987), arachidonic acid release (Fink and Guroff, 1990), and, more recently, in elements of the sphingomyelinase cycle (Dobrowsky et al., 1994). The observation that cyclic nucleotides were involved led to the finding that nerve growth factor altered the phosphorylation state of a number of proteins in the cell (Halegoua and Patrick, 1980), including certain proteins in the nucleus (Yu et al., 1980). Now it is clear that any number of proteins in all of the cellular elements have altered phosphorylation levels and that these are caused, in turn, by the ability of nerve growth factor to alter the activity of a number of different cellular kinases. The sum of these changes in the phosphorylation and the attendant changes in function of key proteins in the cell is widely believed to be the mechanism by which nerve growth factor acts. By exactly which chain or chains of kinases these alterations take place is presently under intense research scrutiny. Clearly, the high-affmity nerve growth factor receptor, trk, is the proximate kinase, the tyrosine kinase action leading to the binding to the receptor of several signaling molecules, among them phospholipase Cy (Ohmichi et al., 1991), phosphatidylinositol-3-kinase (Soltoff et al., 1992), and, probably, the GTPase activating protein, as well (Li et al., 1992). There have been several attempts to determine the sequence of signaling molecules that follow, the most forthright of which (Kremer et al., 1991; Thomas et al., 1992; D'Arcangelo and Halegoua, 1993) implicates the proto-oncogenes, src, ras, raf, and the MAP kinases, in that order. This approach has allowed the conclusion that certain of the cellular effects of nerve growth factor are served by this pathway in its entirety and others only by the initial portions of the pathway. Yet others, such as the induction of sodium channels, have been found independent of the pathway. Exactly how these cytoplasmic elements transmit the nerve growth factor signal to the nucleus is not yet obvious. Clearly, there are nerve growth factor-induced phosphorylations of nuclear elements. Among these are the phosphorylations of the transcription factors NGFI-B (Hirata et al., 1993) and CREB (Ginty et al., 1994),

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the proto-oncogene c-fos (Taylor et al., 1993), and a protein called SNT (Rabin et al., 1993) that is phosphorylated on tyrosine as well as on serine and threonine, but only in response to agents that differentiate the cells. Whether specific kinases enter the nucleus or phosphorylate proteins in the cytoplasm, which then enter the nucleus, is not known. The nuclear signal caused by nerve growth factor leads to changes in the transcription of a number of different genes. However, the data obtained so far does not lead to the identification of any specific concensus sequence for nerve growth factor. For example, the induction of c-fos by nerve growth factor involves two promoter elements 20 bases apart in the linear sequence, both responsive to serum (Visvader et al., 1988). The induction of tyrosine hydroxylase is quite complex and depends on the prior induction of c-fos, which, in turn, binds to an element designated TH-FSE on the tyrosine hydroxylase gene (Gizang-Ginsberg and Ziff, 1990). There is some evidence that nerve growth factor induction of ornithine decarboxylase might also be under the control of c-fos (Wrighton and Busslinger, 1993), but, however it is controlled, some of the regulatory sequences necessary for nerve growth factor-mediated induction appear to be, uniquely, at an extremely long distance from the transcriptional start site (Muller et al., 1993). The gene sequence involved in the nerve growth factor-dependent induction of NGFI-A contains, as does the comparable sequence from the fos gene, more than one serum response element (Changelian et al., 1989). Also in this region are a TATA box, a cAMP response element, and an Spl binding site. The induction of VGF, one of the first of the nerve growth factor responsive genes to be identified, appears to be mediated by a single cAMP response element (Hawley et al, 1992). The induction of TGF-pi has been shown to depend on an Egr-1 site (Kim et al., 1994). Although these data are somewhat unsatisfying, they are consistent with the concept that nerve growth factor activates any number of different pathways leading to the activation of many different transcription elements. It must be remembered that regulation of transcription is probably only one way in which nerve growth factor controls the levels of different proteins in the cell. There is reasonable evidence that nerve growth factor alters translational mechanisms as well; it has been shown to increase the phosphorylation of initiation factor 4E (Frederickson et al, 1992) and the ribosomal protein S6 (Halegoua and Patrick, 1980), and to decrease the phosphorylation of elongation factor 2 (Koizumi et al., 1989). Interestingly enough, the signaling elements serving nerve growth factor are the same as those serving signal transduction by any number of mitogens. This has been shown most clearly in the PC 12 cells, which respond both to nerve growth factor and to the mitogen, epidermal growth factor (Huff et al., 1981). Both ligands increase phosphoinositide turnover, c-fos induction, MAP kinase activation, p70 S6 kinase activation, CREB kinase activation, etc. In fact, although differences in the chemistry of signal transduction have been sought, none have been found, save for the phosphorylation of SNT (Rabin et al., 1993). This has engendered a search for other mechanisms for the deciphering of such signals. Recent work from several

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laboratories (Qui and Green, 1992; Traverse et al, 1992) has now suggested that the differences in signal transduction are temporal, not chemical. That is, specific elements of the signal transduction pathway are activated for a much longer time by nerve growth factor than they are by epidermal growth factor. It is possible, then, that differentiation occurs when the signals persist, and mitogenesis when the signals are transient. In spite of the increasing information about signal transduction, the mechanisms by which nerve growth factor produces such global changes in phenotype are still obscure. There is some suggestion that nerve growth factor promotes the survival of its target neurons by adjusting calcium levels in the cells. It is clear that the survival of neurons, at least in culture, depends upon their ability to maintain adequate intracellular calcium levels (Collins et al., 1991). It has also been shown that, at least in culture, the presence of adequate levels of intracellular calcium, obviates the cell's need for nerve growth factor to survive (Koike et al, 1989). The observation that nerve growth factor can alter both calcium uptake (Nikodijevic and Guroff, 1991) and calcium levels (Kozak et al., 1992) suggests that maintaining adequate calcium levels is at least part of the mechanism by which nerve growth factor keeps neurons alive. Nerve growth factor also has been shown to protect neurons against damage from conditions, such as hypoglycemia and ischemia, that seem to inflict their damage largely by permitting calcium levels to rise too high (Cheng and Mattson, 1991). The ability of nerve growth factor to adjust calcium levels in either direction (Kozak et al., 1992) may suggest that the protective effect of nerve growth factor is due, at least in part, to its ability to lower calcium levels when they become intolerably high. The mechanism by which nerve growth factor can slow or stop cell division is also unknown. One possible clue is in the finding that nerve growth factor treatment can downregulate the levels of epidermal growth factor receptors on PC 12 cells (Huff et al., 1981). Although epidermal growth factor is only a mild mitogen for PC 12 cells under standard culture conditions, the ability of a differentiating agent to control the levels of mitogen receptors may suggest a mechanism by which nerve growth factor instructs the cells to stop dividing and differentiate. Clearly the cells cannot divide when they are blinded to the mitogens that normally control their growth. I. Clinical Perspectives

The possible use of nerve growth factor to therapeutic advantage has been widely discussed for many years. Surely there are a number of clinical conditions that might involve malfunction of nerve growth factor or its receptor. Thus, a number of earlier experiments were designed to uncover changes in nerve growth factor levels in patients with neuroblastoma (Burdman and Goldstein, 1964), disseminated neurofibromatosis (Schenkein et al., 1974), acoustic neurinoma (Siggers et al., 1975), or familial dysautonomia (Siggers et al., 1976). Although changes in levels

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were reported, none of these observations could be confirmed in other laboratories. In any case, all such reports must be viewed with skepticism because the methods available for the measurement of nerve growth factor were generally not sensitive enough to measure the amounts in human serum. Somewhat more recent studies exploring the ability of cells, from patients with familial dysautonomia, to make nerve growth factor (Schwartz and Breakefield, 1980) also have not yielded clarifying information, and there is no evidence that the gene for nerve growth factor is aberrant in either familial dysautonomia (Breakefield et al., 1984) or neurofibromatosis (Darby et al., 1985). Suffice it to say that there is presently no published correlation between the biology of nerve growth factor and any specific disease. However, in spite of the lack of such direct evidence, interest in the clinical and the commercial potential of nerve growth factor and of the entire neurotrophin family is exceedingly high. This interest focuses on four different conditions, Alzheimer's disease, Parkinson's disease, motoneuron degenerative diseases, such as amyotrophic lateral sclerosis, and peripheral neuropathies, such as those accompanying diabetes or treatment with antitumor chemotherapies. Although the original suggestion that alterations in nerve growth factor mechanisms are responsible for the pathological changes seen in Alzheimer's disease (Hefti, 1983) has never found experimental validation, there is substantial support for the therapeutic use of nerve growth factor in this disease. That support is based on the facts that Alzheimer's patients suffer losses of cholinergic innervation of the cortices, that nerve growth factor stimulates cholinergic neurons of the type known to be lost, and that nerve growth factor treatment counteracts both the cholinergic and the cognitive deficits produced when such neurons are damaged in experimental models. The administration of a molecule the size of nerve growth factor to the central nervous system poses something of a problem, because it will not cross the blood-brain barrier. So, a number of different strategies have been tried in experimental animals: nerve growth factor infusion directly into the brain, the implantation of slow-release biodegradable nerve growth factor-containing pellets, carrier-mediated transport using the transferrin receptor to capture anti-transferrin antibody-nerve growth factor conjugates, the grafting of nerve growth factor-producing cells, the enhancement of endogenous nerve growth factor production, and the development of small molecule nerve growth factor mimics. The therapeutic use of nerve growth factor has been widely debated because there are some who feel that such use could be detrimental if, for example, it encouraged inappropriate sprouting of affected or unaffected neuronal populations. Nevertheless, direct application of purified nerve growth factor to an Alzheimer's patient has been reported (Seiger et al., 1993). This report indicates that infusion of nerve growth factor over a period of three months led to increases in cortical blood flow, increases in the binding of [^^CJnicotine as evaluated by PET scan, improvements in EEG characteristics, and enhanced performance in at least one psychological parameter, verbal episodic memory. No harmful effects were noticed, and antibodies against

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nerve growth factor were not detected. These hopeful data will undoubtedly encourage an increased use of nerve growth factor pharmacologically against the now hopeless course of Alzheimer's disease. The use of nerve growth factor in Parkinson's disease has a completely different thrust. One approach to treating this disease has been adrenal-to-brain transplantation. The ability of the adrenal tissue to survive in the neural environment has been of crucial concern. The direct administration of nerve growth factor or the cotransplantation of glia or fibroblasts transfected with the nerve growth factor gene has seemed to improve the survival of the adrenal transplants in experimental animals. Adrenal-to-brain transplants in humans with Parkinson's disease have yielded modest and transient functional improvement. Nerve growth factor has been administered directly to the brains of patients receiving adrenal transplants to provide trophic support for the transplant (Olson et al., 1991), but the methodology has been tried on too few patients to determine if any lasting benefits accrue. There are suggestions that some of the other members of the neurotrophin family might have more direct effects in this disease (Lindsay et al., 1993), as will be discussed below. There is no reason to believe that nerve growth factor per se has any effect on motor neurons or any role in motoneuron diseases such as amyotrophic lateral sclerosis, because nerve growth factor has not been shown to act in any way on motor neurons in animals. However, the interest in the neurotrophic factors, broadly defined, as beneficial agents in this disease is exceedingly high. The possible effects of the other formal members of the neurotrophin family will be discussed below. One particularly promising clinical use of nerve growth factor appears to be in ameliorating or preventing peripheral sensory neuropathies. Small fiber sensory neuropathy is one of the most common and debilitating concommitants of diabetes mellitus. It has been reported (Apfel et al., 1994) that nerve growth factor administration prevents the biochemical and behavioral manifestations of sensory neuropathy in streptozocin-induced diabetes in the rat. Another very important use may be in the prevention of toxic sensory neuropathies induced by antitumor chemotherapeutics. Sensory neuropathy is frequently the dose-limiting factor in treatment for malignancies and, because it is widely believed that the efficacy of these drugs is dose-dependent, the ability to prevent these neuropathies may increase the amount and duration of acceptable doses in such treatments. It has been reported that nerve growth factor can prevent the neuropathies produced in mice by the widely used antitumor agents taxol (Apfel et al., 1991) and cisplatin (Apfel et al., 1992). Clinical trials are presently underway.

111. BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) The second member of the neurotrophin family was isolated in 1982 from massive quantities of pig brain (Barde et al., 1982). It was shown to increase the survival of dorsal root ganglia in culture, but to have little or no activity on sympathetic

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neurons, and, thus, to be distinct from nerve growth factor. The molecule has been sequenced and cloned (Leibrock et al., 1989) and much effort has gone into determining the tissue specificity of its action. Brain-derived neurotrophic factor is a protein of about 13 kDa with an isoelectric point of 9.9. The deduced sequence of 252 amino acids has some 51 identities with the sequence of nerve growth factor, and these identities include the six cysteines making up the three disulfide bridges. The overall homology is on the order of 55%. Although the crystal structure of brain-derived neurotrophic factor has not yet been presented, hydrophilicity plots and immunological cross-reactivity (Murphy et al., 1993), as well as the fact that both nerve growth factor and brain-derived neurotrophic factor bind to the low-affinity neurotrophin receptor, suggest that the three-dimensional structures will be quite similar. The rat gene for brain-derived neurotrophic factor consists of four short 5'-exons linked to separate promoters and one 3'-exon encoding the mature protein (Timmusk et al., 1993). Eight different mRNAs with four different 5' ends and two alternative polyadenylation sites are transcribed from the gene. The gene is expressed very widely in the central nervous system, transcripts being observed in the hippocampus, olfactory cortex, neocortex, amygdala, and ventromedial hypothalamus (Phillips et al., 1990; Isackson et al., 1991), with by far the highest levels in hippocampus. There are also reports of transcripts occurring in the visual system of frogs (Cohen-Corey and Fraser, 1994) and chicks (Herzog et al., 1994) and in the cardiovascular system of the rat (Scarisbrick et al., 1993). The level of expression is altered by any number of different influences, including seizures (Izackson et al., 1991), injections of kainic acid (Zafra et al., 1990; Ballarin et al., 1991), and ischemia or hypoglycemia (Lindvall et al., 1992; Kokaia et al., 1994). In contrast to the rather limited number of cell types showing sensitivity to nerve growth factor, brain-derived neurotrophic factor promotes, at least in vitro, the survival and/or differentiation of many classes of neurons. Included among these are certain sensory (Davies et al., 1986) and motor neurons (Wong et al., 1993), populations of neurons from the hippocampus (Ip et al., 1993), the cerebellum (Segal et al., 1992), the basal forebrain (Knusel et al., 1991), and the nigra (Hyman et al., 1991), and neurons from the retinal ganglia (Johnson et al., 1986). In view of the many brain-derived neurotrophic factor-sensitive cells, it was not unexpected that BDNF knockout mice suffer many deficits. Indeed, mice homozygous for the null mutation usually die within 2 days after birth, but a small number survive up to 4 weeks (Jones et al., 1994). These mice have markedly reduced numbers of sensory neurons and develop symptoms of nervous system dysfunction, including ataxia. They do not show obvious structural abnormalities in the central nervous system, but the expression of several neuronal markers, including neuropeptide Y and calcium-binding proteins, is reduced. Surprisingly, the motor neurons in these mice appear normal. Signal-transduction pathways initiated by brain-derived neurotrophic factor have not been extensively investigated, but initial reports suggest that they are similar to

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those activated by nerve growth factor. Thus, treatment of hippocampal cells produces tyrosine phosphorylation of the brain-derived neurotrophic factor receptor, trkB, and activation of MAP kinases (Marsh et al., 1993). c-Fos induction was also seen, and increased uptake of calcium has been reported by others (Beminger et al, 1993). Fetal brain cultures show enhanced phosphoinositide turnover upon treatment with the factor (Widmer et al., 1993). It seems likely that the pathways activated by brain-derived neurotrophic factor in its target cells will be, at least grossly, the same as those activated by nerve growth factor in its target cells. In spite of the observation that the levels of mRN A for brain-derived neurotrophic factor are decreased in hippocampal tissue of individuals afflicted with Alzheimer's disease (Phillips et al., 1991) using brain-derived neurotrophic factor in this disease has not been, as yet, substantially considered. More attention has been given to the possible use of brain-derived neurotrophic factor in Parkinson's disease because brain-derived neurotrophic factor receptors are localized in areas normally affected in Parkinson's disease and because the factor has been found neurotrophic for such neurons in culture (Hyman et al., 1991). But, clearly, the preclinical evidence for using brain-derived growth factor in Parkinson's disease is equivocal (Lindsay et al., 1993). The reported effects of brain-derived neurotrophic factor on motor neurons are also incomplete. Several groups have found that brain-derived neurotrophic factor enhances the survival of motor neurons in different in vivo situations (Koliatsos et al., 1993; Sendtner et al., 1992; Yan et al., 1992; Oppenheim et al., 1992). Reports of the effects of brain-derived neurotrophic factor on motor neuron survival in vitro have been less frequent.

IV. NEUROTROPHIN-3 (NT-3) Neurotrophin-3 was identified by molecular biological techniques, based on the similarities between nerve growth factor and brain-derived neurotrophic factor (Hohn et al., 1990; Maisonpierre et al., 1990). Thus, in one such strategy, conserved sequences in the two proteins were identified, oligonucleotides representing these sequences were made, these oligomers were used to prime the amplification of mouse genomic sequences using the polymerase chain reaction, and the resulting DNA fragments were digested with restriction enzymes that recognized the sequences for nerve growth factor and brain-derived neurotrophic factor. The uncleaved fragments were sequenced, expanded, and cloned. One of these clones predicted a protein of 258 amino acids that was named neurotrophin-3. The deduced amino acid sequence consists of 119 amino acids, representing a protein with a molecular mass of 13,625 and a pi of 9.3. There are 54 identities with nerve growth factor and brain-derived neurotrophic factor, and all six cysteine residues, comprising the three disulfide bonds, are conserved. Overall, almost 50% of the structures of the three proteins are the same. The three proteins have similar, but not identical secondary structures (Narhi et al., 1993). This similarity accounts for the ability of these three dimeric molecules to form heterodimers (Arakawa et

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al., 1994), but the physiological significance of this heterodimer formation is as yet unknown. Crystals of neurotrophin-3 have been obtained (Kelly et al., 1994), so structural information should be forthcoming. The gene for neurotrophin-3 has been localized to human chromosome 12p and to mouse chromosome 6 (Ozcelik et al., 1991), but little structural information has been presented. The gene codes for an mRNA of some 1.4 kb (Hohn et al., 1990), found in highest levels in the brain, but occurring in different sites outside the nervous system. This distribution is quite different than that of the mRNAs of either brain-derived neurotrophic factor, which are found primarily in the brain, or nerve growth factor, which are hardly detectable in either liver or skeletal muscle. The widespread distribution of neurotrophin-3 mRNA is consistent with the widespread distribution of the protein itself in the brain (Zhou and Rush, 1994) and in sites, such as kidney, spleen, liver, and adrenal medulla (Zhou and Rush, 1993). The original observations on neurotrophin-3 showed that it had a broader target population than either brain-derived neurotrophic factor or nerve growth factor (Hohn et al., 1990). In some cases that specificity is overlapping, as in the case of the nodose ganglion, where neurotrophin-3 and brain-derived neurotrophic factor seem to support different subpopulations of neurons. Neurotrophin-3 has also been shown to support the survival of sensory neurons in culture (Hory-Lee et al., 1993) and the development of sensory neurons in the chick (Gaese et al., 1994). In addition, there is evidence for effects of neurotrophin-3 on the development of glial populations (Barres et al., 1994), consistent with the observation that neurotrophin3 immunoreactivity is present in many glial cells (Zhou and Rush, 1994). The widespread distribution of neurotrophin-3 would predict catastrophic consequences when the gene is deleted. Indeed, targeted disruption of the neurotrophin-3 gene (Farinas et al., 1994; Emfors et al., 1994) gave rise to a group of homozygotes that died within 24 hours of birth. Those few that survived, however, lived as long as 3 weeks. The survivors had severe deficits in sensory and sympathetic populations, lacked muscle spindles and Golgi tendon organs, and displayed abnormal limb postures and athetotic walking movements. Motor neurons, the enteric nervous system, and, grossly, the central nervous system seemed to develop normally. The biochemical pathways activated by neurotrophin-3 have not been explored in any detail. Some initial studies (Knusel et al., 1992) showed that neurotrophin-3 treatment of embryonic rat brain cells results in a robust tyrosine phosphorylation of its receptor and the phosphorylation of phospholipase-Cy and the ERKs. Increased phosphoinositide turnover has been reported in hippocampal neurons (Ohsawa et al., 1993) and also in rat cortical neurons (Widmer et al, 1993). The clinical role of neurotrophin-3 is just now being considered, but the information available from the knockout mice has suggested a role in treating inherited or acquired peripheral neuropathies (Emfors et al., 1994).

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V. NEUROTROPHINS-4/5 AND -6 (NT-4/5; NT-6) Two groups (Hallbook et al., 1991; Berkemeier et al, 1991) independently cloned the same member of the neurotrophin family. Now known as neurotrophin 4/5, its properties are just being explored. Early work indicated that this neurotrophin is expressed in embryonic and adult tissues. It promotes the survival of sensory and sympathetic neurons and induces the morphological differentiation of PCI 2 cells. Neurotrophin 4/5 is a protein of 123 amino acids, 50-60% homologous with the other members of the neurotrophin family. Crystals suitable for X-ray analysis have been prepared (Fandl et al., 1994). The gene is located on human chromosome 19 (Berkemeier et al, 1991), but no structural information is yet available. Clear evidence for the trophic properties of neurotrophin 4/5 for mammalian facial motor neurons has been presented (Koliatsos et al., 1994), and it has been suggested that therapeutic use in motor neuron degenerative disease should be explored. Most recently, a new member of the neurotrophin family, neurotrophin-6, has been cloned from the teleost fish Xiphophorys (Gotz et al, 1994). This molecule has a spectrum of actions similar to that of nerve growth factor on sympathetic and sensory neurons, but is not as potent. Distinct from the other neurotrophins, it is not found in the medium of producing cells, but occurs on the cell surface, from which it can be released by adding heparin. The properties of this newest member of the neurotrophin family await exploration.

VI. CONCLUSION The prediction that each class of neural cell would have a factor or factors that control its development seemed unlikely for several decades, because nerve growth factor remained the only representative of this hypothetical family. Molecular methodology has recently given substance to that prediction in the discovery of new members of this family. It appears that different classes of neuronal cells will have unique neurotrophins controlling their development and that single classes of cells may respond to different neurotrophins at different stages of development. It also seems likely that the array of neurotrophic factors involved in the survival of damaged neural cells differs from those controlling their normal development and survival. The basic information available about the new members of the neurotrophin family is accumulating at a remarkable rate. One can predict that even newer members of this family will be forthcoming; the genes for several have, in fact, already been observed (Berkemeier et al., 1992). Because of the clinical and commercial enthusiasm for these molecules, continuing and accelerating interest in this exploding field is expected.

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Williams, L. R., Varon, S., Peterson, G. M.. Wictorin, K., Fischer, W., Bjorklund, A., & Gage. F. H (1986). Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria-fornix transection. Proc. Natl. Acad. Sci. USA 83, 9231-9235. Wong, v., Arriaga, R., Ip, N. Y, & Lindsay, R. M. (1993). The neurotrophms BDNF, NT-3 and NT-4/5. but notNGF, upregulate the cholinergic phenotype of developmg motor neurons. Eur. J. Neurosci. 5, 466-^74. Wrighton, C , & Busslinger, M. (1993). Direct transcriptional stimulation of the ornithine decarboxylase gene by Fos in PC12 cells but not in fibroblasts. Mol. Cell. Biol. 13, 4657-4669. Yan, Q., Elliott, J., & Snider, W. D. (1992). Brain-derived neurotrophic factor rescues spinal motor neurons from axotomy-induced cell death. Nature 360, 753-755. Yu, M., Tolson, N., & GurofF, G. (1980). Increased phosphorylation of specific nuclear proteins in superior cervical ganglia and PC 12 cells in response to nerve growth factor. J. Biol. Chem. 255, 10481-10492. Zafra, F., Hengerer, B., Leibrock, J., Thoenen, H., & Lindholm, D. (1990). Activity-dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by non-NMDA glutamate receptors. EMBO J. 9, 3545-3550. Zafra, F., Castren, E., Thoenen, H., & Lindholm, D. (1991). Interplay between glutamate and y-aminobutyric acid transmitter systems in the physiological regulation of brain-derived neurotrophic factor and nerve growth factor synthesis in hippocampal neurons. Proc. Natl. Acad. Sci. USA 88, 10037-10041. Zhou, X. F., & Rush, R. A. (1993). Localization of neurotrophin-3-like immunoreactivity in peripheral tissues of the rat. Brain Res. 621, 189-199. Zhou, X. F., & Rush, R. A. (1994). Localization of neurotrophin-3-like immunoreactivity in the rat central nervous system. Brain Res. 643, 162-172.

NGF RECEPTORS

Mariano Barbacid

I. Introduction II. TRK Receptors: Structural Features A. TrkA Receptors B. TrkB Receptors C. TrkC Receptors III. The p75 Receptor: Structural Features IV. Signal Transduction A. TrkA Tyrosine Kinase Receptors B. TrkB and TrkC Tyrosine Kinase Receptors C. TrkC Tyrosine Kinase Receptor Isoforms D. Noncatalytic TrkB and TrkC Receptors E. p75 Receptor V. The Role of the TRK Receptors 7« Fivo A. TrkA Defective Mice B. TrkB Defective Mice C. TrkC Defective Mice VI. The Role ofthep75 Receptor/« Vivo References

Growth Factors and Cytokines in Health and Disease Volume lA, pages 259-27^. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0091-4 259

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I. INTRODUCTION The development and survival of the mammalian nervous system is largely dependent on the existence of soluble neurotrophic factors (Levi-Montalcini, 1987). Whereas some of these factors, such as PDGF and FGF, have pleiotropic activities in many cell types, others act only on cells of neural lineage. Among the latter, the most intensively studied are the members of the nerve growth factor family, now known as "neurotrophins" (Barde, 1994). To date, four different neurotrophins have been identified from a variety of vertebrate species. They include the well-characterized nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), also known as NT-5, A new member of this gene family, designated as neurotrophin-6 (NT-6), has been recently isolated from the teleost fish Xephophorus (Gotz et al., 1994). Whether there are NT-6 analogues in other vertebrate species, such as birds or mammals, is currently under investigation. Neurotrophins recognize two distinct classes of receptors, the Trk family of tyrosine protein kinases (Barbacid, 1994) and p75, a molecule generally known as the low-affmity NGF receptor (Chao, 1994) (Figure 1). The Trk receptors are encoded by three highly related, but distinct, loci designated as trkA (also known as trk), trkB and trkC. To date, more than a dozen Trk receptors (or at least their corresponding transcripts) have been identified (Table 1). Some of these receptors including two TrkA, one TrkB and four TrkC isoforms are tyrosine protein kinases. They interact specifically with each neurotrophin and are primarily responsible for mediating their trophic activities (Figure 1). Whereas the TrkA kinase isoforms mediate NGF signaling (Kaplan et al., 1991; Klein et al., 1991a), TrkB serves as the signaling receptor for BDNF (Klein et al., 1991b; Soppet et al., 1991; Squinto etal., 1991) and NT-4 (Berkermeieretal., 1991;Ipetal., 1992; Klein etal, 1992). The TrkC tyrosine kinases are the primary receptors for NT-3 (Lamballe et al., 1991). However, this neurotrophin can also bind and activate TrkA and TrkB receptors (Cordon-Cardo et al., 1991; Soppet et al., 1991; Squinto et al., 1991). Whether the recently discovered NT-6 also signals trough Trk receptors remains to be determined. In addition to these tyrosine kinases, the trkB and trkC genes encode several noncatalytic receptor isoforms of an, as yet, unknown function (Klein et al., 1990; Middlemas et al., 1991; Tsoulfas et al., 1993; Valenzuela et al., 1993). The p75 receptor (Chao et al, 1986; Radeke et al, 1987) is a member of the TNF receptor superfamily and recognizes all the neurotrophins with similar low affinity (Chao, 1994) (Figure 1). The precise role of p75 in mediating neurotrophin activity is still a matter of debate (Barbacid, 1993a; Chao, 1994). Recent evidence, mostly derived from the analysis of mice carrying a targeted p75 gene (see below), suggests that p75 may facilitate the interaction of NGF with TrkA, possibly by increasing the concentration of NGF in the vicinity of TrkA receptors. To date there is no evidence supporting a role of the p75 receptor in either BDNF, NT-3 or NT-4 activity in vivo (Figure 1).

NGF Receptors

NGF

TrkA

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BINDING SPECIFICITY

FUNCTIONAL SPECIFICITY IN VIVO

P75

NGF Recruitment ? p75

BDNF

NT-4

TrkB

NT-3

NQF

TrkC

jrkA

BDNF

NT-4

TrkB

NT-3

TrkC

Signal Transduction Figure 1, Schematic representation of the biochemical (left panel) and functional (right panel) interactions between the NGF family of neurotrophins and their receptors. Left Panel: All neurotrophins bind with similar low affinity to the p75 receptor (thin arrows). However, they interact specifically with each of the Trk tyrosine kinases (thick arrows). NT-3 has pleotropic activity and can also activate the TrkA and TrkB receptors, albeit with limited efficiency (thin arrows). Right Panel: The thick arrow between NGF and p75 represents their functional relationship primarily based on the defects-NGFdependent sensory and sympathetic neuronal deficiencies of p75 null mice. The absence of neuronal defects in these mice corresponding to those observed in BDNF and NT-3 "knockout'' mice suggests that p75 does not play a significant role in mediating the activity of these neurotrophins in vivo (dotted arrows). The functional relationship between neurotrophins and the Trk tyrosine kinase receptors is the same as in the right panel. However, there is no definitve evidence as yet whether NT-3 signals through TrkA and/or TrkB receptors in vivo.

II. TRK RECEPTORS: STRUCTURAL FEATURES The Trk family of receptors was first identified in 1986 when a human oncogene was found containing the transmembrane and cytoplasmic domains of a previously unknown tyrosine kinase receptor gene (Martin-Zanca et al., 1986). Molecular characterization of this oncogene, designated trk, led to the isolation of its normal allele, the ^rA:proto-oncogene (now known as trkA) (Martin-Zanca et al., 1989) and, subsequently, to the identification of the related trkB (Klein et al., 1989) and trkC (Lamballe et al, 1991) genes. The Trk family of receptors has all of the basic features characteristic of other tyrosine kinase cell-surface receptors (Figure 2). They include a 32 amino acid-long signal peptide followed by distinct extracellular motifs presumably responsible for specific ligand recognition, a single transmembrane domain, and a cytoplasmic region which encompasses the tyrosine kinase

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p75 Receptor SIGNAL PEPTIDE CYSTEINE REPEATS

TRANSMEMBRANE CYTOPLASMIC DOMAIN

Trk Tyrosine Kinase Receptors SIGNAL PEPTIDE CYSTEINE CLUSTER 1 LEUCINE-RICH MOTIFS CYSTEINE CLUSTER 2 Ig-LIKE DOMAINS TRANSMEMBRANE JUXTAMEMBRANE KINASE DOMAIN COOH TAIL

TrkAi TrkAji

TrkB

TrkC TrkC TrkC TrkC K14 K25 K39

Trk Non-Catalytic Receptors SIGNAL PEPTIDE CYSTEINE CLUSTER 1 LEUCINE-RICH MOTIFS CYSTEINE CLUSTER 2 Ig-LIKE DOMAINS TRANSMEMBRANE UNIQUE CYTOPLASMIC SEQUENCES

TrkB TK" T1 T2

[158]

TrkC TK"" [143] [113] [108]

Figure 2. Neurotrophin receptors. Schematic diagram of p75 (top), Trk tyrosine kinase (middle) and Trk noncatalytic (bottom) receptors. Structural domains are indicated to the left. The six amino acid residues (VSFSPV) unique to the neuronalspecific TrkAii receptor are indicated by a small dotted box. Additional sequences present in the TrkC tyrosine kinase TrkC K14, TrkC K25 and TrkC K39 receptors are indicated by hatched boxes. Sequences unique to the Trk noncatalytic receptor isoforms are depicted by boxes with various shadings.

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catalytic domain (Figure 2). However, these receptors have unique motifs in their extracellular (mostly) and cytoplasmic domains that clearly define them as a unique subfamily. For instance, the extracellular domain of all Trk receptors contains three tandem leucine-rich motifs (LRM) of 24 amino acid residues flanked by two cysteine clusters which include eight of the twelve cysteine residues shared by these receptors (Schneider and Schweiger, 1991) (Figure 2). The carboxy half of the Trk extracellular region contains two immunoglobulin (Ig-like) domains of the C2 type (Schneider and Schweiger, 1991). Ig-like domains have been previously identified in other cell-surface tyrosine protein kinases, such as the receptors for PDGF, CSF-1, SF and FGFs (Hardie and Hanks, 1995). Interestingly, the Ig-like domains of the Trk receptors appear to be more closely related to those of cell adhesion molecules, such as N-CAM. The cytoplasmic region of the Trk kinase receptors can be divided into three domains: a 76 to 90 amino acid long juxtamembrane region, a 264 to 267 amino acid long catalytic domain, and a characteristically short (15 residues) carboxyterminal tail (Barbacid et al., 1993b) (Figure 2). The catalytic domain of the Trk receptors is most closely related to those of several novel orphan receptors including the mammalian RORl, R0R2, DDR (also known as Ptk-3, CAK, MEK-10, Nep and TrkE), Tyro-10 (also known as TKT and CCK-2) and the Torpedo RTK tyrosine kinases (Hardie and Hanks, 1995). These receptors however, have rather distinct extracellular domains and it is unlikely that they recognize members of the NGF neurotrophin family. The Trk receptor family also includes a series of isoforms that lack a catalytic kinase domain (Klein et al., 1990; Middlemas et al, 1991; Tsoulfas et al., 1993; Valenzuela et al., 1993) (Table 1). These receptors, so far only encoded by the trkB

Table 1, The Trk Family of Neurotrophin Receptors Gene trkK trkB

trkC

Receptor

Other Designations

Size

Structure

TrkAj (non-neuronal) TrkAii (neuronal) TrkB TrkB.Tl TrkB.T2 TrkC

Trk/gpl40^'"^^ Trk/gpl40^''^^ TrkB^^^gpl45^''^^ TrkB'^^"/gp95'''^^ TrkB^^~ TrkCKl/TrkC^^"^/

790 aa. 796 aa. 821 aa. 476 aa. 414 aa. 825 aa.

Tyrosine kinase Tyrosine kinase Tyrosine kinase Noncatalytic Noncatalytic Tyrosine kinase

TrkC K14 TrkC K25 TrkC K39 TrkC^^~(158) TrkC^*^"(143) TrkC^^~(113) Trkc'''^~(108)

TrkCK2/Trkc'^^'^(14) TrkC K3 / TrkC^^"^(25) TrkCK4/Trkc'^^"'(39)

839 aa. 850 aa. 864 aa. 686 aa. 671 aa. 641 aa. 636 aa.

Tyrosine kinase Tyrosine kinase Tyrosine kinase Noncatalytic Noncatalytic Noncatalytic Noncatalytic

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and trkC genes, possess the same signal peptide, extracellular domain, and transmembrane region as the TrkB and TrkC tyrosine kinase receptors. However, their cytoplasmic domains are short and contain distinct sequences likely to be encoded by alternatively spliced transcripts (Figure 2). A. TrkA Receptors The trkA gene encodes two tyrosine protein kinase isoforms of 790 and 796 amino acid residues, designated as TrkAj (also known as Trk or gpl40^''^) (MarinZanca et al., 1989) and TrkAjj (Barker et al, 1993; Horigome et al., 1993), respectively. These isoforms differ from each other by the six amino acid residues (VSFSPV) located in the extracellular domain near the transmembrane region (Figure 2). Whereas the TrkAjj molecule is expressed in neuronal cells, the TrkAj isoform has been found primarily in cells of non-neuronal origin. The response of both of these receptors to NGF appears to be rather similar, if not identical. However, a systematic comparison of their binding affinities and biochemical properties has not yet been carried out. Recently, it has been reported the the neuronal TrkAjj receptor is more efficient than the TrkAj isoform in mediating the activity of NT-3, at least in PC12 cells (Clary and Reichardt, 1994). Mutated forms of the TrkA receptors have been identified in several human tumors, particularly in thyroid papillary carcinomas (Barbacid, 1993b). These transforming molecules have lost their signal peptide as well as most of their extracellular sequences. Therefore, these oncogenic variants cannot serve as NGF receptors. Instead, they contain sequences derived from structurally unrelated proteins, such as tropomyosin and the product of the tpr gene, among others (Barbacid, 1993b). These ectopic sequences favor the formation of stable homodimers resulting in the constitutive activation of their respective tyrosine kinase domains (see below), a feature likely to be responsible for their oncogenic activity. A variety of other transforming TrkA proteins have also been isolated in vitro (Coulier et al., 1990). One of these molecules, designated Trk5, carries a 51 amino acid deletion in the second Ig-like domain which includes a conserved Cys residue (Cys345). Replacement of this residue with Ser also results in malignant activation of the TrkA receptor, thus, suggesting that this domain may play an important role in the dimerization process (Coulier et al., 1990). B. TrkB Receptors The TrkB tyrosine kinase receptor is a heavily glycosylated molecule of 821 amino acid residues that contains all of the structural motifs described above for the TrkAreceptor (Klein et al., 1989; Middlemas et al, 1991) (Figure 2). The overall homology between the extracellular domains of the human TrkA and the mouse TrkB tyrosine kinases is 57% (38% identity). However, this homology is not randomly distributed. Most of the homologous residues map within the second and third LRM motifs and, particularly, within the second Ig-like domain (Schneider and Schweiger, 1991). In addition, each of the twelve extracellular cysteines in

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TrkA are conserved in TrkB. As expected, the highest degree of homology between TrkB and TrkA (88%) occurs in their catalytic domains. TrkB also possesses a short, highly conserved tail of 15 residues which include a conserved Tyr shared by the TrkA and TrkC tyrosine kinases (Klein et al., 1989; Middlemas et al, 1991). The trkB gene also encodes two noncatalytic receptors designated as TrkB.Tl (also known as gp95'''^^) and TrkB.T2 (Klein et al., 1990; Middlemas et al, 1991) (Table 1). TrkB.T 1 has only 23 cytoplasmic residues of which the last 11 are unique. This receptor is expressed in adult mouse brain at levels comparable to those of the signaling TrkB tyrosine kinase receptor (Klein et al., 1990). TrkB.T2 has a 21 amino acid long cytoplasmic domain of which the last nine residues are unique and unrelated to those in TrkB.Tl (Middlemas et al., 1991) (Figure 2). So far, TrkB.T2 has not been identified at the protein level. C. TrkC Receptors

The trkCgQUQ encodes as many as four TrkC tyrosine kinase isoforms (Lamballe et al., 1991, 1993; Tsoulfas et al, 1993; Valenzuela et al., 1993) (Table 1). One of these isoforms, TrkC (also known as TrkC Kl and gp\45^^^), displays the same structural features as the related TrkA and TrkB tyrosine kinase receptors (Figure 2). The overall homology of the porcine TrkC kinase compared to the human TrkA and mouse TrkB receptors is 67% (54% in the extracellular domain and 87% in the kinase region) and 68% (53% in the extracellular domain and 87% in the kinase region), respectively. The other tyrosine kinase isoforms differ from TrkC by the 14 (TrkC K14), 25 (TrkC K25), and 39 (TrkC K39) additional amino acid residues located after the conserved sequence YSTDYYR which encompasses the putative autophosphorylation site of TrkC (Lamballe et al., 1993; Tsoulfas et al., 1993; Valenzuela et al., 1993) (Figure 2). The unique 14 and 25 amino acid long sequences of TrkC K14 and TrkC K25 receptors are unrelated to each other as well as to other known sequences and do not display informative structural motifs. TrkC K39 contains the combined 25 and 14 amino acid long sequences (in this order) of TrkC K25 and TrkC K14, respectively (Tsoulfas et al., 1993) (Figure 2). Recent studies indicate that these additional residues are encoded by alternatively spliced exons (our unpublished observations). Immunoprecipitation studies using specific antisera against these sequences have demonstrated low levels of expression of the TrkC K14 receptor isoform in various structures of the adult mouse brain (Lamballe et al, 1993). To date, cDNAs encoding four different TrkC noncatalytic receptor isoforms have been described (Tsoulfas et al, 1993; Valenzuela et al., 1993) (Table 1). They have been designated as TrkC^^-(158), TrkC^^-(143), TrkC'^^-(l 13) and TrkC^^" (108), based on their respective number of cytoplasmic residues. The first 74 cytoplasmic residues of each of these TrkC^*^~ receptors are derived fi'om the juxtamembrane region of the catalytic TrkC receptors. However, sequences starting at residue 529 are encoded by four distinct alternatively spliced exons designated as A, B, C, and D (Tsoulfas et al., 1993; Valenzuela et al, 1993). The TrkC'^^^l 58)

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receptor contains exons B (46 residues) and C (38 residues); TrkC^^~(143) has exons B and D (23 residues) and TrkC^^~(l 13) possesses only exon C. The cDNA clones encoding the TrkC^^~(108) isoform contain exons A, B, and C. However, exon A encodes only a short peptide of 34 amino acid residues which is followed by an in-frame terminator codon that prevents translation of the downstream sequences contained in the B and C exons (Tsoulfas et al., 1993; Valenzuela et al., 1993) (Figure 2). To date, it is not known whether any of these TrkC^^~noncatalytic receptors are expressed in vivo.

III. THE p75 RECEPTOR: STRUCTURAL FEATURES The human p75 receptor consists of a single transmembrane polypeptide of 427 amino acid residues of which 399 correspond to the mature protein (size and number of residues vary slightly for the rat and chicken receptors) (Johnson et al., 1986; Radeke et al., 1987; Large et al., 1989) (Figure 2). The 222 amino acid long extracellular domain has four cysteine repeats [CXJ2_I5CXQ_2CX2CX9CX7C] characteristic of a loose family of cell-surface receptors that includes the type I and II receptors for tumor necrosis factor (TNF), the lymphocyte surface antigens CD30, CD40 and OX40, and the apoptosis-mediating Fas cell-surface antigen (Chao, 1994). These conserved cysteine repeats are responsible for ligand recognition because a secreted 168 amino acid long receptor lacking all sequences carboxy terminal to this domain retains NGF binding (Welcher et al., 1991; Yan et al, 1991). None of the other receptors containing this conserved cysteine motif bind NGF or any of the NGF-related neurotrophins, indicating that, whereas these cysteines are likely to provide the necessary secondary structure, other residues within this motif are responsible for ligand recognition. The role of the 155 amino acid long cytoplasmic region of p75 is still unclear. This domain does not have recognized structural motifs nor shares homology with other known molecules, including the above p75-related receptors.

IV. SIGNAL TRANSDUCTION A. TrkA Tyrosine Kinase Receptors

As indicated above, the TrkA tyrosine kinase receptor is responsible for mediating NGF signaling (Kaplan etal, 1991; Klein etal., 1991a)(Figure 1). For instance, PC 12 cells lacking TrkA receptors do not respond to NGF despite abundant p75 receptors (Loeb et al., 1991). Transfection of TrkA into these cells restores full NGF responsiveness. TrkA kinase receptors become activated by a two-step process involving their ligand-mediated dimerization followed by autophosphorylation of their tyrosine residues (Jing et al., 1992), a mechanism common to other tyrosine kinase receptors (Schlessinger and Ullrich, 1992). To date, several TrkA substrates have been identified (Kaplan and Stephens, 1994). They include PLC-y, PI-3 kinase and the adaptor protein, SHC (Stephens et al., 1994; Obermeier et al., 1994). Other potential substrates for the Trk receptor are the Ras GTPase activating protein

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(GAP) and the MAP kinase, ERKl. However, it is not known whether these two proteins are directly phosphorylated by the Trk receptors (Kaplan and Stephens, 1994). PLC-y binds to a conserved Tyr residue (Tyr785) located in the short carboxyterminal tail characteristic of the Trk receptor family. PI-3 kinase interacts with a neighboring Tyr residue (Tyr751) located at the carboxy terminus of the kinase domain. The physiological significance of these phosphorylations remains obscure because Trk receptors carrying Tyr-^Phe mutations in these residues retain their ability to transform NIH3T3 cells and to differentiate PC 12 cells (Stephens et al., 1994; Obermeier et al., 1994). The She binding site has been mapped to a conserved Tyr residue (Tyr 490) located in the juxtamembrane domain. Mutation of this residue significantly decreases the mitogenic (NIH3T3 cells) and differentiating (PC 12 cells) activity of the TrkA receptors. However, complete ablation of TrkA signaling requires at least mutation of the Tyr residue responsible for PLC-y binding (Stephens et al., 1994; Obermeier et al., 1994). These observations, taken together, indicate that neurotrophin activity may require activation of multiple signaling pathways. Accumulating evidence indicates that one of these pathways includes the wellcharacterized Ras/Raf/MAP kinase signaling cascade (Kaplan and Stephens, 1994). Addition of NGF to wild type, but not to TrkA-deficient PC 12 cells, results in the rapid activation of Ras and the downstream Raf and MAP kinases. Because this pathway is clearly implicated in mitogenesis, it has been postulated that differentiation of PCI2 cells, and possibly neuronal differentiation and survival, requires the activation of unique signaling pathways. A component of such a putative pathway might be the recently identified SNT protein (Rabin et al., 1993). SNT is a 90-kDa polypeptide that binds to pl3, a subunit of the cell cycle regulatory complex that includes the cdc2 kinase and cyclin. SNT is rapidly phosphorylated on tyrosine residues upon treatment of PC 12 with NGF, but not with mitogenic factors, such as EGF (Rabin et al., 1993). More recently, it has been observed that TrkA receptors lead to a more robust and sustained activation of MAP kinase than EGF receptors (Dikic et al., 1994; Traverse et al, 1994). This pattern of MAP kinase activation has been been observed in all cases in which PC 12 cells undergo differentiation, including PC 12 cells that overexpress EGF and insulin receptors. These observations have led to the proposal that prolonged activation of MAP kinase might be sufficient to mediate PCI2 cell differentiation. Whether TrkA receptors utilize additional signaling elements (SNT?) to potentiate MAP kinase activation remains to be determined. B. TrkB and TrkC Tyrosine Kinase Receptors

The TrkB tyrosine kinase is the signaling receptor for BDNF (Klein et al., .1991b; Soppet et al., 1991; Squinto et al., 1991) andNT-4 (Berkermeier et al., 1991; Ip et al., 1992; Klein et al., 1992). Likewise, the TrkC tyrosine kinases are the receptors forNT-3 (Lamballeetal., 1991,1993; Tsoulfasetal., 1993; Valenzuelaetal, 1993). TrkB can also serve as a receptor for NT-3, at least in cell culture (Klein et al., 1991b; Soppet et al., 1991; Squinto et al., 1991). However, the biological responses

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induced by NT-3 through TrkB (or TrkA) receptors are much more attenuated than those elicited by BDNF and NT-4 or those induced by NT-3 through its cognate receptor, TrkC (Figure 1). To date, there is limited information regarding the signal-transduction pathway utilized by the TrkB and TrkC tyrosine protein kinases. However, it is very likely that these receptors use the same signaling elements as TrkA. Indeed, all Tyr residues known to play a role in TrkA signaling are conserved in the TrkB and TrkC kinases. Moreover, they have similar biological properties. For instance, ectopic expression of these receptors in PC 12 cells induces differentiation into neuron-like cells when incubated in the presence of their cognate ligands BDNF (or NT-4) and NT-3, respectively. Likewise, coexpression of TrkB and BDNF (or NT-4) and of TrkC and NT-3 in proliferating NIH3T3 cells results in their morphologic transfor-. mation with the same kinetics and potency observed upon expression of NGF and TrkA receptors (Barbacid, 1994). C. TrkC Tyrosine Kinase Receptor Isoforms

The TrkC tyrosine kinase isoforms TrkC K14 and TrkC K25 have rather distinct biological properties. Neither of these TrkC receptor isoforms induces morphological transformation of NIH3T3 cells or mediates neuronal differentiation of PC 12 cells (Lamballeetal., 1993;Tsoulfasetal., 1993; Valenzuelaetal., 1993). However, they mediate downstream signaling. Binding of NT-3 to TrkC K14 and TrkC K25 induces their rapid autophosphorylation on tyrosine residues and activates downstream signaling pathways as determined by their ability to induce resting cells to initiate DNA synthesis (Lamballe et al., 1993; Tsoulfas et al., 1993; Valenzuela et al., 1993). It is possible that the additional sequences in TrkC K14 and TrkC K25 result in a unique pattern of phosphorylated tyrosines that allows these receptor isoforms to engage with specialized signaling pathways distinct from those of the other Trk kinases. In support of this hypothesis, neither TrkC K14 or TrkC K25 activate PLC-y and PI-3 kinase, despite their anchoring tyrosine residues (Lamballe et al., 1993). However, additional studies will be necesary before we can assign a physiological role to these TrkC receptor isoforms. D. Noncatalytic TrkB and TrkC Receptors

There is little information regarding the role, if any, of the TrkB and TrkC noncatalytic receptor isoforms in signal transduction. It is possible that these receptors engage with cytoplasmic signaling elements (i.e., nonreceptor tyrosine kinases) in a manner similar to certain lymphocytic cell-surface molecules, such as the T-cell receptor, CD4/CD8 or gpl30, the signaling subunit of the CNTF, and IL6 and LIF receptors (Patterson, 1992). Alternatively, these noncatalytic Trk receptors may act as dominant negative inhibitors of their tyrosine kinase isoforms, at least in those cells in which they are coexpressed. However, there is no experimental evidence supporting these two hypotheses. Instead, it is more likely that the TrkB and TrkC noncatalytic receptors play a role in functions other than signaling. Indirect evidence based on the abundant expression of the TrkB.Tl receptor in the

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ependymal layer of the ventricles and in the choroid plexus led us to the hypothesis that it might be involved in ligand clearance and/or transport (Klein et al., 1990). Likewise, induction of this receptor upon neuronal injury has led to the proposal that it might be involved in ligand recruitment and/or presentation during axon growth and/or regeneration (Beck et al., 1993). E. p75 Receptor

The precise role of the p75 receptor remains to be elucidated. A significant body of evidence indicates that p75 does not mediate neurotrophin signaling, at least as it relates to neuronal survival and/or differentiation (Barbacid, 1993a; Chao, 1994). For instance, PC 12 cells, expressing high levels of p75 receptors in the absence of TrkA, do not differentiate in response to NGF. Moreover, p75 is expressed in many cell types that do not respond to neurotrophins. There are several reports, however, suggesting that p75 mediates some aspects of signal transduction. For instance, ectopic expression of wild type, but not mutant p75 receptors in PC 12 cells lacking NGF receptors, led to the induction of tyrosine phosphorylation and c-fos expression (Hempstead et al., 1989,1990; Berg et al., 1991). In addition, wild-type PC 12 cells expressing a chimeric EGF-p75 receptor whose cytoplasmic sequences are derived from p75, undergo neuronal-like differentiation in the presence of EGF (Yan et al., 1991). More recently, it has been reported that p75 mediates activation of the sphingomyelin cycle (Dobrowsky et al., 1994), the glycosyl-phosphatidylinositol/inositol phosphoglycan pathway (Represa et al., 1991), Schwann cell migration (Anton et al., 1994), and matrix invasion by melanoma cells (Herrman et al., 1993). Finally, two recent reports indicate that ligand-free p75 mediates signal transduction in apoptotic pathways (Rabizadeh et al., 1993; Barrett and Bartlett, 1994). According to these studies, NGF binding blocks apoptotic signaling, thus, contributing to the known effect of NGF in neuronal survival. A more generally accepted view is that p75 may facilitate and/or potentiate NGF signaling through TrkA receptors. For instance, it has been shown that transfection of p75 into TrkA-expressing MAH sympathoadrenal cells enhances their response to NGF (Verdi et al., 1994). Likewise, blocking p75 binding sites in PC 12 cells with a p75 monoclonal antibody or with BDNF decreases their response to NGF (Baker et al., 1994). Perhaps more importantly, mice carrying a targeted mutation that eliminates expression of p75 receptors display deficiencies in a subset of NGF/TrkA-dependent neurons (see below).

V. THE ROLE OF THE TRK RECEPTORS IN VIVO The generation of mice carrying germ line mutations in the catalytic domains of each of the Trk kinase receptors has provided a unique opportunity to study the role of these receptors in vivo (Klein et al., 1993,1994; Smeyne et al., 1994). Moreover, the phenotypes of mice defective for each Trk receptor have turned out to be strikingly similar to those of similar "knockouf mice lacking their cognate neurotro-

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phins (Snider, 1994). These observations represent the most compelling evidence that the Trk receptors mediate most, if not all, neurotrophin activities in vivo. A. TrkA Defective Mice

Mice defective for TrkA receptors (Smeyne et al., 1994) display severe sensory defects characterized by a complete loss of nociceptive activity (Table 2). These mice fail to react to deep pinpricks in their whisker pads and rear paws. In addition, they exhibit deficiencies in thermoception because they can stay on a 60 °C hot plate for at least 10 seconds. Neuroanatomical examination of these targeted mice revealed extensive neuronal cell loss in trigeminal, dorsal root (DRG), and sympathetic ganglia (Table 2). In the DRGs, the vast majority of the missing neurons correspond to those of small size, a population known to be NGF-dependent. The sympathetic ganglia are severely shrunken and only contain a few neurons (Table 2). Essentially identical defects have been observed in mice lacking the gene encoding NGF (Crowley et al., 1994). Expression of trkA gene in the central nervous system (CNS) is restricted to a small subset of cholinergic neurons in the striatum and in the basal forebrain complex, two brain structures that have been previously shown to be targets for NGF (Holtzman et al., 1992). Disruption of the trkA gene does not appear to cause the loss of these neurons, although limited neuronal cell death cannot be excluded at this time. However, adult trkA null mice exhibit a severe decrease in those cholinergic fibers that project from the medial septum to the hippocampus and from the nucleus basalis to the cerebral cortex (Smeyne et al., 1994). Whether TrkA Table 2. Summary of Defects Observed in Mice Targeted in Genes Encoding Neurotrophin Receptors Targeted Receptor Phenotype Sensory Activity nociception propioception PNS Defects sup. Cervical ganglion trigeminal ganglion nodose/petrosal ganglion vestibular ganglion dorsal root ganglia la afferents

p75

TrkA

TrkB

TrkC

partial normal

very low normal

normal normal

normal impaired

normal normal ND^ ND smaller ND

5%^ 30% normal ND 30% normal

normal 40% 10%^ ND 70% normal

75% ND ND ND 80% lost

Notes: ^approximate percentage of remaining neurons. "^D, not determined. '^Erickson, J.T. and Katz, D.T., personal communication.

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receptors are required for the outgrowth of these cholinergic fibers or for maintaining their cholinergic phenotype remains to be determined. B. TrkB Defective Mice

The trkB targeted mice carry a deletion in their tyrosine kinase sequences that prevents expression of the TrkB tyrosine kinase receptor but not of the noncatalytic isoforms (Klein et al, 1993). These mice develop to birth, however, most of them die within the first postnatal week, most likely due to their lack of feeding. Indeed, the first symptomatic difference between normal and homozygous trkB mice can be observed approximately 12 hours after birth, a time when most of the mutant animals do not have signs of milk in their stomachs. No gross lesions, including cleft lip or palate, were observed in the heads of these animals that may explain their inability to take nourishment (Klein et al., 1993). The TrkB-defective mice display significant neuronal cell loss in several ganglia of the peripheral nervous system (PNS) including the DRGs, the trigeminal ganglion and particularly in the nodose-petrosal complex (Klein et al., 1993; J.T. Erickson and D.M. Katz, personal communication) (Table 2). Neurons of the nodose-petrosal complex relay visceral sensory information from cardiovascular, respiratory, and gastrointestinal systems to the CNS. Therefore, it is possible that the significant loss of these neurons accounts for the early deaths of the TrkB kinase-defective mice. Interestingly, BDNF mutant mice have slightly longer life spans, and many animals survive 2-4 weeks (Jones et al., 1994; Emfors et al, 1994a). These mice display defective movement coordination and balance with head bobbing and spinning followed by long periods of inactivity. This defect is likely to be due to atrophy and loss of vestibular ganglion neurons which results in defective innervation of the inner ear. No such defects were observed in the TrkB mutant mice because they do not survive long enough to undergo maturation of their vestibular neurons. Interestingly, BDNF mutant mice do not display defects in their motor neurons (Jones et al., 1994; Emfors et al., 1994a). As indicated above, the TrkB tyrosine kinase is also a receptor for NT-4. Whether double "knockout" BDNF and NT-4 mice display the same phenotype as the TrkB-defective animals remains to be determined. Likewise, it is not known whether mice also lacking expression of the TrkB noncatalytic isoforms may display additional neuronal defects. Most of the structures known to express TrkB tyrosine kinase receptors, such as the cerebral cortex, the pyramidal cell layer of the hippocampus, and the thalamus appear at least morphologically normal (Klein et al., 1990). It is possible that some defects may be found in these structures following more detailed analysis. Alternatively, CNS neurons may require TrkB signaling postnatally, at times beyond the life span of these mutant mice. However, it is also possible that TrkB-expressing neurons may survive in the absence of this signaling receptor thanks to compensatory mechanisms, perhaps provided by the highly related TrkC receptors which are coexpressed in most of these structures.

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Disruption of the tyrosine kinase sequences of the trkC gene also results in severe sensory defects, but of a more distinct nature than those observed in TrkA- and TrkB-defective mice (Klein et al, 1994). Mice lacking TrkC kinase receptors are defective in propioception, the sensory activity responsible for localizing the limbs in space (Table 2). As a consequence, these targeted mice display abnormal movements of an athetotic nature resulting in highly abnormal limb postures. This sensory defect is due to the complete absence of la muscle afferents, the projections derived from large propioceptive DRG neurons that connect primary endings of muscle spindles in the periphery to motor pools in the ventral region of the spinal cord (Table 2). TrkC mutant mice have limited life spans most likely due to additional neuronal defects. However, a few TrkC-defective mice have survived for over six months. Mice, defective for the gene encoding its cognate ligand NT-3, display virtually identical sensory defects (Emfors et al., 1994b; Farinas et al., 1994). However, these mice display a more severe phenotype, and most animals do not survive beyond a few weeks. The reason for these differences is not known. It is possible that the TrkC-defective mice express noncatalytic TrkC receptor isoforms which may mediate some NT-3 activities. Alternatively, NT-3 may interact with TrkAand TrkB receptors, thus, partially compensating for the absence of TrkC tyrosine kinase activity in these mutant mice.

VI. THE ROLE OF THE p75 RECEPTOR IN VIVO The most relevant information regarding the function of the p75 receptor in neurotrophin activity has been provided by analyzing mutant mice carrying a targeted p75 gene (Lee et al., 1992, 1994a). These mice display sensory and sympathetic defects, thus, demonstrating that the p75 receptor is required for proper neuronal development (Table 2). Interestingly, these mice do not display defects characteristic of mice lacking TrkB (or BDNF) and TrkC (or NT-3) tyrosine kinase receptors. These observations indicate that p75, in spite of serving as a receptor for BDNF and NT-3 in vitro, may not be involved in mediating their biological activities in vivo (Figure 1). Alternatively, the absence of p75 might be compensated for by other molecules, such as the TrkB and TrkC noncatalytic receptors. The observed defects in the p75 null mice appear to be limited to NGF-dependent neurons, mainly sensory (nociceptive) and sympathetic neurons (Lee et al., 1992, 1994a). These defects however, are much more limited than those observed in either TrkA or NGF null mice (Table 2). For instance, mice lacking TrkA receptors (or NGF) lose most (>95%) of their sympathetic neurons within their first postnatal week. In contrast, the p75 null mice display normal sympathetic ganglia and normal innervation patterns to all their targets with the exception of the pineal and the sweat glands (Lee et al., 1992, 1994a). These observations raise the possibility that TrkA signaling may require p75 receptors only in a subset of NGF-dependent neurons. However, a more plausible

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interpretation of these results is that p75 plays a role in recruiting circulating NGF molecules rather than in NGF signaling (Barbacid, 1993a). Considering the rapid dissociation kinetics of NGF from p75, it is possible that the abundant expression of these receptors in neurons or in adjacent glial cells increases the local concentration of diffusible NGF. If so, the lack of p75 receptors will have only phenotypic consequences in those neurons for which the availability of NGF is limiting. In support of this hypothesis, p75-defective sensory and sympathetic neurons survive well in culture in the presence of NGF, but they need two to four times more NGF to achieve the same response levels as wild-type neurons (Davies et al., 1993; Lee et al., 1994b). Crossing p75 null mice with transgenic strains overexpressing NGF should provide the experimental tools to test this hypothesis. In summary, the advent of gene targeting has provided new means to study the function of the two classes of neurotrophin receptors in vivo and to define precisely the physiological role of each of these molecules, at least during development. Some of the results obtained (i.e., sympathetic and sensory defects in TrkA null mice) were expected based on earlier studies using immunological approaches to neutralize NGF activity. Others, such as the exquisite specificity of the NT-3/TrkC pathway in propioception or the limited defects displayed by the p75 targeted mice, were unexpected. Another important lesson learnedfi-omthese targeted mice is the differential role that neurotrophins play in the peripheral versus the central nervous system. In the PNS, ablation of Trk receptor genes (or those encoding their cognate neurotrophins) results in massive neuronal cell death. However, the CNS neurons of these mutant mice appear, for the most part, unaffected in spite of widespread neurotrophin and receptor expression. These observations illustrate the complex mechanisms involved in the development and survival of the nervous system and predict the existence of additional neurotrophic factors that await discovery. REFERENCES Anton, E. S., Weskamp, G., Reichardt, L. R, & Matthew, W. D. (1994). Nerve growth factor and its low-aflfinity receptor promote Schwann cell migration. Proc. Natl. Acad. Sci. USA 91,2795-2799. Barbacid, M. (1993a). Nerve growth factor: A tale of two receptors. Oncogene 8, 2033-2042. Barbacid, M. (1993b). The Trk family of neurotrophin receptors: Molecular characterization and oncogenic activation in human tumors. In: Molecular Genetics of Nervous System Tumors (A.J. Levine and H.H. Schmidek, eds.), John Wiley & Son, Inc., New York, Chapter 9, pp. 123—135. Barbacid, M. (1994). The Trk family of neurotrophin receptors. J. Neurobiol. 25, 1386-1403. Barde, Y—A. (1994). Neurotrophic factors: An evolutionary perspective. J. Neurobiol. 25, 1329-1333. Barker, P. A., Lomen-Hoerth, C , Gensch, E. M., Meakin, S. O., Glass, D. J., & Shooter, E. M. (1993). Tissue-specific alternative splicing generates two isoforms of the trkA receptor. J. Biol. Chem. 268, 15150-15157. Barker, P. A., & Shooter, E. M. (1994). Disruption of NGF binding to the low affinity neurotrophin receptor p75LNTR reduces NGF binding to TrkA on PC12 cells. Neuron 13, 203-215. Barrett, G. L., & Bartlett, P. F. (1994). The p75 nerve growth factor receptor mediates survival or death depending on the stage of sensory neuron development. Proc. Natl. Acad. Sci. USA 91, 6501-^505.

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