Biomembranes Signal Transduction Across Membranes
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Biomembranes Edited by Meir Shinitz...
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Biomembranes Signal Transduction Across Membranes
Balaban Publishers
3
4b
VCH
Biomembranes Edited by Meir Shinitzky Volume 1:Physical Aspects, 1993 Volume 2: Structural and Functional Aspects, 1994 Volume 3: Signal Transduction Across Membranes, 1995 See page 326 for further information.
0 VCH Verlagsgesellschaft rnbH, D-69451 Weinheim (Federal Republic of Germany), 1995
Distribution: VCH, P. 0. Box 101161, D-69451 Weinheim (Federal Republic of Germany) Switzerland: VCH, P. 0. Box, CH-4020 Basel (Switzerland) United Kingdom and Ireland: VCH (UK) Ltd., 8 Wellington Court, Cambridge CB1 1HZ (England) USA and Canada: VCH, 220 East 23rd Street, New York, NY 100104606 (USA) Japan: VCH, Eikow Building, 10-9 Hongo 1-chome, Bunkyo-ku, Tokyo 113 (Japan) ISBN 3-527-30023-6 (VCH, Weinheirn)
Biomembranes Signal Transduction Across Membranes Edited bv Meir Shihitzky
Balaban Publishers
3
4b
VCH
Weinheim . New York Basel . Cambridge . Tokyo
Editor: Prof. Dr. Meir Shinitzky Department of Membrane Research and Biophysics The Weizmann Institute of Science Rehovot 76120 Israel
This book was carefully produced. Nevertheless, authors, editor and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.
Published jointly by VCH Verlagsgesellschaft mbH, Weinheim (Federal Republic of Germany) VCH Publishers Inc., New York, NY (USA) Editorial Director: Miriam Balaban Production Manager: Dip1.-Wirt.-Ing. (FH) H.-J. Schmitt
Library of Congress Card No.: applied for
British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library
Die Deutsche Bibliothek - CIP-Einheitsaufnahme Biomembranes / ed. by Meir Shinitzky. - Weinheim ; New York ; Base1 ; Cambridge ; Tokyo : VCH : Brooklyn, NY : Balaban Publ. NE: Shinitzky, Meir [Hrsg.]
Vol. 3. Signal transduction across membranes. ISBN 3-527-30023-6
- 1995
0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim (Federal Republic of Germany), 1995
Printed on acid-free and chlorine-free paper All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form -by photoprinting, microfilm, or any other means -nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such are not to be considered unprotected by law. Printing: Strauss Offsetdruck GmbH, D-69509 Morlenbach Bookbinding: J. Schaffer, D-67269 Griinstadt Printed in the Federal Republic of Germany.
Preface
One of the major physiological aspects of biological membranes is signal transduction and processing. Volume 3 of this series is dedicated almost entirely to mechanisms associated with signal transduction. The unique framework of physical properties presented in Volumes 1 and 2 provides an essential ground for the understanding of such mechanisms and the three volumes therefore form a comprehensive series on membrane function. Rehovot, October 1994
Meir Shinitzky, Editor
Contents
Chapter 1
General Mechanistic Patterns of Signal Transduction Across . . . . . . . . . . . . . . . . . . . . . . . Membranes Marcel Spaargaren, Siegfried W de Laat and Johannes Boonstra
Chapter 2
Receptors for Neurotransmitters . . . . . . . . . . . . . . . E A n n e Stephenson and Philip G. Strange
Chapter 3
1
61
. . . . . . . . . . . . . . .
95
G Proteins in Signal Transduction . . . . . . . . . . . . . . . Lutz Birnbaumer and Marie1 Birnbaumer
153
Receptors to Peptide Hormones Sandra Incerpi and Paolo Luly
Chapter 4
Chapter 5
Membrane-Associated Protein Kinases and Phosphatases . . . . . David S. Lester
Chapter 6
Phospholipases in Signal Transduction . . . . . . . . . . . . . Daniela Corda, Marco Falasca, Maria di Girolamo and Tiziana Cacciamani Index
. . . . . . . . . . . .
. . . . .
253
283
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Biomembranes Edited by Meir Shinitzky Copyright 0 VCH VerlagsgesellschaflrnbH, 1995
CHAPTER 1
General Mechanistic Patterns of Signal Transduction Across Membranes MARCEL SPAARGARENl, SIEGFRIED W. DE LAAT2 and JOHANNES BOONSTRA3 'Onyx Pharmaceuticals, 3031 Research Drive, Bldg. A, Richmond, CA 94806, USA 2Hubrecht Luboratoty, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands 3Departrnent of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands
Contents 2 Abbreviations 4 Introduction 6 Receptors 8 Signal transduction 14 Cell Surface Receptors 15 Catalytic receptors 19 G-protein-coupled receptors 23 Receptors without catalytic activity or G-protein coupling 26 Signal Transduction Mechanisms 26 GTP binding/GTPase proteins 26 G-proteins 30 Ras proteins 33 Adenylate cyclase, cyclic nucleotides and phosphodiesterases 34 Phospholipases and phospholipid-derived second messengers 34 Phospholipase A, 37 Phospholipase C 39 Phospholipase D
M. Spaargaren et al.
2
39 Kinases 40 Protein kinase C 41 CAMP-dependent protein kinase 43 RAF-1 kinase 44 Mitogen-activated protein kinase 45 Signal Transduction in Development and Cancer 45 Signal transduction and development 49 Signal transduction and cancer 55 Acknowledgements 55 References
Abbreviations AA ACTH AP BDNF CAM-kinase CRE CREB CSF DG EGF EPO FGF FN FSH GAP GC G-CSF GH GM-CSF GNRP G-protein HGF I IFN IGF I1 IP3
- arachi donic acid
- adrenocorticotropic hormone - activator protein
- brain-derived neurotrophic factor
- Ca2+/calmodulin-dependent protein kinase CAMP responsive element CRE binding protein colony stimulating factor diacylglycerol epidermal growth factor erythropoietin fibroblast growth factor fibronectin follicle-stimulating hormone GTPase activating protein guanylate cyclase granulocyte-CSF growth hormone granulocyte-macrophage-CSF guanine nucleotide-releasing protein guanine nucleotide-binding protein hepatocyte growth factor insulin interferon insulin-like growth factor inter leukin inositol 1,4,5-triphosphate
General Mechanistic Patterns
KGF LH LH-CG LIF LPL LT M AP-kinase NDF NGF NF-1 NT-3 PA PAF PC PDE PDGF PG PI PIP PIP, PKA PKC PL PLA, PLC PLD Pro PT R RSK SCF SF SH-2 domain TGF TNF TPA TRE TRH TSH TX
- kerati nocyte growth factor
- luteinizing hormone - choriogonadotropin
- lymphocyte inhibiting factor
- lysophospholipid - leukotriene -
-
-
-
-
mitogen-activated protein kinase neu differentiation factor nerve growth factor neurofibromatosis type-1 protein neurotrophin-3 phosphatidic acid platelet activating factor phosphatidylcholine phosphodiesterase platelet derived growth factor prostaglandin phosphatidylinositol phosphatidylinositol 4-phosphate phosphatidylinositol 4,5-bisphosphate CAMP-dependent protein kinase protein kinase C phospholipid phospholipase A, phospholipase C phospholipase D prolactin pertussis toxin receptor ribosomal S6 kinase stem cell factor steel factor src homology 2 domain transforming growth factor tumor necrosis factor 12-0-tetradecanoyl-phorbol-13-acetate TPA responsive element thyrotropin-releasing hormone thyrotropin stimulating hormone thromboxane
3
4
M . Spaargaren et al.
Introduction A multicellular organism is composed of a wide variety of different cell types, each of them specialized to fulfill its function. Optimal functioning of an organism is only possible if the individual cells that make up the different tissues and organs are able to communicate with one another in order to coordinate their growth, division, development, differentiation, and organization. In this contribution we will discuss the mechanisms of intercellular communication and the intracellular processes resulting in the cellular responses (i.e., signal transduction), limiting ourselves mainly to mammalian cells. In general, cells are able to communicate in three ways (Fig. 1): (1) by small molecules (e.g., ions or metabolites, smaller than & 1 kDa) that can pass gap junctions, which connect the cytoplasm of two cells; or by signalling molecules (e.g., hormones, GFs and neurotransmitters) that are either (2) cell-surface-localized, or (3) secreted. The first two mechanisms are only available to adjacent cells, whereas the latter can act over some distance. In this chapter we will focus on cell-to-cell signalling by secreted signalling molecules since this mechanism is most widely used. It should be mentioned, however, that some of the secreted signalling molecules are also able to exert their effect on neighboring cells in a membrane-anchored fashion. Cellular communication via secreted signalling molecules can be classified in three categories, based on the distance over which the signal acts (Fig. lc). The first category, denoted endocrine signalling, occurs if the distance between the signal producing cell and the target cell is relatively large, the signalling molecules (hormones) being secreted by endocrine cells, usually organized in specific glands, and transported by the bloodstream to the target cells. As a consequence of the dilution in blood, the concentration of the signalling molecules is relatively low (in the pM range). The second category, paracrine and autocrine signalling, occurs if the signal producing cell is in the immediate environment of the target cell (paracrine), or if the signal producing cell itself is the target cell (autocrine). Examples for these signalling molecules are growth factors that are able to regulate the proliferation and differentiation of a variety of cell types, mainly at an intermediate concentration (nM range). Autocrine signalling is usually confined to certain cancer cells, both producing and responding to growth factors, which causes unrestrained cell proliferation and tumor formation. The third category is employed by neurotransmitters, acting in the synaptic signalling of the
General Mechanistic Patterns
5
a
b Fig. 1. Cellular communication. a) The small diffusible signal molecules are directly transported to the target cell by means of gap junctions. b) Signalling by cell-surfacelocalized signalling molecules. The signalling molecule is exposed on, and associated with, the cell membrane of the signalling cell, and acts on the receptor of a directly contacted target cell. c) Signalling by secreted molecules: (1) Endocrine signalling. The signalling molecule (hormone) is secreted by the signalling cell and transported by the blood stream to the target cell. (2) Para-/autocrine signalling. The signalling molecule (growth factor) is secreted by the signalling cell in the direct cellular environment and either acts on neighboring target cells (paracrine) or the signalling cell itself acts as target cell (autocrine). (3) Signalling by neurotransmitters. The signalling molecule (neurotransmitter) is secieted in the synaptic cleft and acts on the target cell which is in direct contact with the signalling cell. S=signalling cell, T=target cell.
nervous system. Although this type of signalling occurs in a paracrine fashion, it is regarded as a different category because of the extremely short distance between the signal producing cell and the target cell, which are only separated by the synaptic cleft. As a consequence of this,
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the concentration of the signalling molecule is relatively high (pM range). All three systems have in common that the signalling molecule has to interact specifically with a particular protein of the target cell, the receptor. In general, the receptors for endocrine and paracrine signalling molecules bind with relatively high affinity (approx. 0.1 nM) and respond to the relatively low concentration of ligand (pM-nM) within a time scale of minutes, whereas the receptors for neurotransmitter bind with relatively low affinity (1-100 nM) and respond to the relatively high concentration (pM) of ligand with a rapid response of the receptor (ms-s). In the next paragraphs of this introduction, these receptors will be briefly introduced; more detailed information about the different receptor types are provided in the next section.
Receptors In the case of a hydrophobic signalling molecule (e.g., the steroid hormones like estradiol, testosterone, progesterone, cortisol, corticosterone, aldosterone, thyroid hormone, or retinoic acid), the ligand crosses the plasma membrane and interacts with a specific intracellular receptor, located in the cytoplasm or nucleus of the cell. In most cases the receptor for a hydrophobic molecule can act as a transcription factor (steroidlthyroid hormone receptor), directly regulating the transcription of target genes. However, in this chapter we will focus on the hydrophilic signalling molecules that act by binding to plasma membranelocalized receptors (Fig. 2). In the case of a hydrophilic signalling molecule (e.g., hormones like insulin; growth factors like EGF, PDGF, IGF; cytokines like IL; or neurotransmitters like epinephrine, dopamine, or acetylcholine), the interaction occurs by binding of the ligand to a specific cell surfacelocalized receptor (Fig. 2). The main types of receptors involved in this process are coupled to intracellular effector molecules by means of their intrinsic catalytic activity, such as receptor protein tyrosine kinases, receptor protein serinelthreonine kinases, or receptor protein tyrosine phosphatases (designated catalytic receptors), by means of a G-protein (designated G-protein-coupled receptors), or by none of the above (designated receptors without catalytic activity or G-protein coupling). Binding of the signalling molecule to the receptor initiates a signal transduction cascade in which the signal is transmitted from the plasma
General Mechanistic Patterns
7
Hyd roph o bic
Fig. 2. Signalling by hydrophilic and hydrophobic signalling molecules. The hydrophilic signalling molecule binds to a cell-surface-localized receptor, initiates the activation of several signal transducing molecules in the cytoplasm, and finally results in activation of transcription factors that cause enhanced expression of target genes in the nucleus. The hydrophobic signalling molecule is able to cross the cell membrane and bind to its receptor in the cytoplasm or nucleus, and the receptor itself is a transcription factor that enhances the expression of target genes in the nucleus.
membrane to the cytoplasm, or even to the nucleus, where the transcription of certain target genes can be regulated. The effect of a ligand on a target cell is determined by the diversity of receptors and signal transducing molecules that are present in that particular cell. Thus, particular signalling molecules may, for example, elicit growth promoting activities in one cell but growth inhibitory activities in another cell.
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Signal transduction After binding of the ligand (first messenger) to the receptor, the first step in the signal transduction cascade is the activation of effector molecules by the activated receptors. Activation of the effector is established by the intrinsic catalytic activity of the receptor or by the Gprotein coupled to the receptor. The effector molecules are localized at the cell membrane as integral membrane proteins, or are bound to the cell membrane by interactions with cell membrane phospholipids or membrane proteins, or are cytoplasmic. These effector molecules include cyclases (AC and GC), phospholipases (PLA,, PLC and PLD), ion channels (e.g., for Ca2+ and K'), protein kinases (tyrosine kinases and serine/threonine kinases), and other enzymes or regulatory proteins (e.g., GAPS). Most of these effector molecules are able to induce changes in the intracellular levels of so-called second messengers, small non-protein molecules which either act in the cytoplasm or within the plane of the cell membrane. Among these second messengers are cyclic nucleotides (CAMPand cGMP), DG, phosphoinositides, Ca2+, and AA and its metabolites (eicosanoids). These second messenger molecules are able to regulate the activity of a variety of transducing proteins, mainly cytoplasmic protein serinehhreonine kinases, or membrane-bound Ras proteins. Finally, the kinases are able to regulate the DNA binding activity, the transcriptional activity, or localization of a number of transcription factors in a protein synthesis-independent way, thereby regulating the expression of target genes (Fig. 3). To provide an idea about the diversity and functioning of these signal transduction pathways, some of the main pathways will be briefly introduced in the last paragraphs of this introduction, although they will be more thoroughly discussed in forthcoming sections of this chapter. One of the first molecules identified as a second messenger is cAMP (Fig. 4). By now it is clear that many G-protein-coupled receptors are able to enhance cellular cAMP levels by the G-protein-mediated activation of adenylate cyclase, which in turn is able to convert ATP into cAMP [ 11I]. The next step in this signal transduction pathway is the activation of a serinekhreonine kinase, the CAMP-dependent protein kinase PKA. Due to the activation of PKA by CAMP, a number of specific protein substrates are phosphorylated, including transcription factors. An important finding was the recognition of another signal transduction pathway, activated by a diversity of ligands acting on different types
General Mechanistic Patterns
9
extracellular
t E
cell membrane
cytoplasm
e-'
T.F.
1
nucleus Fig. 3. Generalized schematic representation of signal transduction. The first messenger or ligand (L) binds to and activates its cell-surface-localized receptor (R). The activated receptor activates several integral membrane, membrane-associated, or cytoplasmic effectors Q, which in turn generate second messengers (SM). These second messengers activate a transducing protein (TP) that is either membrane-associatedor cytoplasmic. The transducing proteins may sometimes also be directly activated by the receptor (thus acting as an effector) or by an effector. The transducing molecules directly or indirectly cause the activation of transcription factors (TF)in the cytoplasm or nucleus, thereby initiating the enhanced expression of target genes in the nucleus.
of receptors. The first step in this bifurcating pathway is the hydrolysis of a minor membrane phospholipid, PIP2, by the enzyme PLC (Fig. 5). The activation of PLC can be mediated by its direct phosphorylation on tyrosine residues by receptor tyrosine kinases, or by means of G-protein activation by the G-protein-coupled receptors. The products of PIP2 hydrolysis are two second messenger molecules: DG and IP,. DG operates within the cell membrane and acts as an activator of PKC [72,99], whereas IP, is released into the cytoplasm and raises the level of another second messenger, Ca2+, by inducing its release from intracellular stores [ll]. In addition, it is noteworthy that some other phosphoinositides may also act as second messengers. Besides the IP,mediated release of Ca2+ from intracellular stores, the level of this
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Fig. 4. The adenylate cyclase-initiated CAMP signal transduction pathway. Binding of a ligand to its receptor, mainly G-protein-coupled receptors, activates the integral membrane protein AC by means of the G-protein. AC converts cytoplasmic ATP into CAMP,which is able to activate PKA. PKA in turn is able to directly or indirectly modulate the transcriptional activity of transcription factors.
second messenger can also be enhanced by the influx of Ca2+ through Ca2+-channels which can be operated by changes in the voltage difference across the cell membrane or directly by receptors. Ca2+ is, in addition to other processes, involved in activation of the Ca2+ and Calmodulin-dependent CAM-kinase (a serinehhreonine kinase) [34,61, 1171.
A third signal transduction pathway is initiated by the activation of the enzyme PLA, (Fig. 6). The activation of PLA, can be evoked by many ligands acting on different types of receptors and can be caused by G-protein-mediated activation and by its phosphorylation, although it remains to be established whether the activating phosphorylation event is a direct phosphorylation by the receptor tyrosine kinases or by activated cytoplasmic tyrosine or serine/threonine kinases. PLA, is able
General Mechanistic Patterns
11
Fig. 5. The phospholipase C-initiated diacylglycerol-, inositol 3-phosphate-, and Ca2+signal transduction pathway. Binding of a ligand to its receptor, which either has intrinsic tyrosine kinase activity or is coupled to a G-protein, activates PLC. PLC catalyzes the hydrolysis of the membrane phospholipid PIP,, thereby generating the two second messengers IP, and DG. IP, is released into the cytoplasm where it can bind to a receptor on the rough endoplasmatic reticulum, thus causing the release of another second messenger Ca2+ from this intracellular store. Ca2+ is involved in regulating the activity of a large number of enzymes, among which is CAM-kinase. DG stays in the inner leaflet of the plasma membrane and mediates the activation of PKC. Both CAM-kinase and PKC are involved in the regulation of trancriptional activity of transcription factors.
to hydrolyze membrane phospholipids at the sn-2 position, thereby releasing the second messenger AA. AA has been reported to be involved in the activation of PKC and ion channel-opening. In turn, AA
M . Spaargaren et al.
12
PI/@ AA
+
T.F.
R
t Fig. 6. The phospholipaseA,-initiated arachidonicacid and eicosanoid-signaltransduction pathway. Ligand-induced receptor activation results in G-protein- or phosphorylationmediated activation of PLA,. PLA, catalyzes the hydrolysis of membrane phospholipids, thus causing the release of AA into the cytoplasm. AA itself is able to activate PKC, and AA can also be metabolized into eicosanoids (eicos.) such as the bioactive PGs, LTs, and TXs. AA activates PKC and, by an unknown mechanism, AA and the eicosanoids modulate the transcriptional activity of transcription factors.
can be metabolized, for example, into PGs, LTs, and TXs, which have also been reported to have pronounced effects on important signal transduction-relevant processes [98]. The transcription of a number of transcription factor encoding genes and their target genes has been demonstrated to be regulated by the production of AA and its metabolites.
General Mechanistic Patterns
13
+
Fig. 7. The Ras- and kinase-mediated signal transduction pathway. In particular the ligand-induced activation of receptor tyrosine kinases appears to be able to transduce signals without the involvement of second messengers. These signal transduction systems include the direct phosphorylation-induced activation of cytoplasmic tyrosine and serinehreonine kinases, and the activation of Ras by a yet unknown mechanism. The activated kinases and Ras in turn activate mainly serinekhreonine kinases that are able to regulate the activity of transcription factors.
Finally, it should be mentioned that beside the signal transduction pathway using different second messengers, it is clear by now that signal transduction can also be carried out by a cascade of kinases apparently without the interference of these second messengers (Fig. 7). This type of signal transduction is mainly evoked by receptors lacking G-protein coupling such as receptor tyrosine kinases or receptors for cytokines which may be linked to cytoplasmic tyrosine kinases of the Src-family. Furthermore, signal transduction by these types of receptors in many cases involves the Ras protein, a GTP-binding/GTPase protein, of which its exact upstream regulation or downstream effector action remains to be established. In the next sections we will focus mainly on three subjects: (1) the receptors that start the signal transduction cascades; (2) most of the major components of these cascades; and (3) the relevance of signal transduction in developmental biology and the occurrence of cancer.
M. Spaargaren et al.
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Cell Surface Receptors A wide variety of signalling molecules acts by binding to cell-surfacelocalized receptors. Among these signalling molecules are polypeptides such as hormones, growth factors, differentiation factors and cytokines, as well as neurotransmitters. After binding of these ligands to their receptors, which span the cell membrane, information has to be relayed from the exterior to the interior of the cell in order to elicit certain cellular responses. The mechanisms by which the cell-surface-localized receptors transduce these signals across the plasma membrane are diverse in nature and are reviewed here. Based on the mechanisms by which the cell-surface receptors transduce the signal across the plasma membrane, the receptors can be classified into functionally distinct classes (Fig. 8): (1) receptors with intrinsic catalytic activity, in general these receptors have an extracellular ligand binding domain, a short hydrophobic transmembrane stretch, and an intracellular part containing the catalytic domain consisting of either a tyrosine kinase, serinelthreonine kinase, or tyrosine phosphatase
‘1
SIGNAL
Fig. 8. Cell-surface receptorclasses. Cell-surface-receptorscan be classified in three main receptor classes. a) Receptors that contain intrinsic catalytic activity. This receptor class is dominated by the receptor tyrosine kinase, which mainly include receptors for hormones and growth factors. b) Receptors that are directly coupled to a G-protein. This receptor class includes receptors for neurotransmitters, neuropeptides and hormones. c) Receptors that do not exhibit intrinsic catalytic activity or G-protein coupling, but may initiate intracellular signals by means of an associated enzyme. This class of receptors is dominated by the receptors for cytokines.
General Mechairistic Patterns
15
domain; (2) receptors coupled to a G-protein; in general these receptors have an extracellular domain, seven hydrophobic transmembrane domains believed to make up the ligand binding pocket, and an intracelMar domain coupled to a G-protein; and (3) receptors without catalytic activity or G-protein coupling; in general these receptors are transmembrane proteins with an extracellular ligand binding domain, a single hydrophobic transmembrane domain, and an intracellular domain that may associate with a signal transducing molecule. Catalytic receptors
A large number of receptors contain intrinsic catalytic activity as exemplified by the large family of receptor protein tyrosine kinases. In addifin,ntn, the rpszptm t . y m i w kim.w.s,~ h rw~q~.n,cs m &.c+h..,y ~.~.t~i.~.si.c catalytic activity as well. For example, it has recently been shown that the type I1 receptor for TGFP and the Activin receptor have a serinelthreonine kinase domain. Furthermore, several receptor-like protein tyrosine phosphatases have been cloned for which no ligands have yet been identified. However, since not much is known about the regulation and signal transduction of receptor serinehhreonine kinases or tyrosine phosphatases, in this part we will focus on the receptor tyrosine kinases. Probably the most extensively studied type of cell-surface receptors are those containing intrinsic protein tyrosine kinase activity [ 1,26,27, 135,1451. These receptors are mainly involved in mediating the signalling by growth factors which can be secreted by the cell or act as membrane-anchored growth factors. Activation of the tyrosine kinase of these growth factors, which is essential for their mitogenic signalling, results in a cascade of biochemical and physiological responses in the cell, finally leading to either stimulation of DNA synthesis and cell division, or differentiation. A number of these receptor tyrosine kinases have been identified by virtue of their presence in transforming retroviruses or transforming potential and are encoded by proto-oncogenes [ 1,17,27,67]. Based on sequence and structural similarity, these receptors can be classified into several subclasses (Fig. 9). 1. The first subclass of receptors is characterized by the presence of two cysteine-rich regions in the extracellular domain. It is exemplified by the receptor for EGF and TGFa, i.e., EGF-Rlc-erbB1, and includes the receptor for NDF, i.e., neu and its human counterpart HER2 or cerbB2, and the HER3/c-erbB3 protooncogene products as well.
16 EGF-R
I
a
PDGF-R
II
M. Spaargaren et al. I-A
FGF-R
HGF-R
NGF-R
..... ..... ..... -
I
111
Iv
V
VI
Fig. 9. Receptor tyrosine kinase subclasses. The receptor tyrosine kinases can be classified according to some characteristic motifs. Each subclass is designated by a prototype receptor (see text for additional members of the subclasses). Besides their eventual subunit composition, the subclassification is mainly based on the presence or absence of an insert region in the kinase domain (black box), the number of conserved cysteine-rich domains (dotted box), and the number of immunoglobulin-like domains (loop).
2. The second subclass of receptors is characterized by an extracellular domain with five immunoglobulin-like loops and an insert region in the kinase domain. This subclass includes and CY and p receptors for PDGF, the CSF-1-Rlc-fmr, and SCF(SF or SLF)-Rlc-kit. 3. The third subclass of receptors is characterized by an extracellular domain with a cysteine-rich region and has a heterotetrameric a2p2 structure linked by disulfide bridges, the (II chain having the cysteinerich region and being involved in ligand binding and the p chain containing the transmembrane part and the tyrosine kinase domain. This subclass includes the I-R, IGF-1-R, and c-rosywhich is a receptor for an unknown ligand. 4. The fourth subclass of receptors is characterized by an extracellular domain with three immunoglobulin-like loops and an insert region in the kinase domain. This subclass includes the receptors for the different types of F;GFs (aFGF, bFGF, K-FGF, KGF, and FGF-5) (i.e., the FGF-R llc.flg, FGF-R2/c-bek, FGF-R3 , and FGF-R4).
General Mechanistic Patterns
17
5. The fifth subclass of receptors is characterized by an extracellular
domain with a cysteine-rich region and has a heterodimeric structure, the
a and fi subunits being linked by a disulfide bridge. It contains the HGF
(or Scatter factor)-Rlc-met, and c-sea which is a receptor for an unknown ligand. 6 . Receptors in the sixth subclass have neither a cysteine-rich region nor immunoglobulin-like loops in the extracellular domain. It is composed of different receptors for neurotrophins, i.e., the NGF-Rlc-trk for NGF, trkB for NT-3 and BDNF, and trkC for NT-3. 7. Receptors in the seventh subclass have a single cysteine-rich domain and include eph, elk, and eck, for which no ligands have been identified yet (not shown). 8. The final subclass only includes c-ref, a receptor for an unknown ligand (not shown). In addition to this classification, it can be mentioned that the I-R, eph, elk, and eck, as well as a novel receptor tyrosine kinase, c-ml, share an additional common feature, i.e., the presence of tandemly repeated FN type I11 modules in their extracellular domain. These repeats are implicated in cell adhesion. In their inactive state receptor tyrosine kinases are present at the cell membrane either as monomers or as loose dimers. Their ligands are either monomeric (e.g., EGF, TGFa) or dimeric modules (e.g., PDGF, CSF-1, NGF, HGF). It is now commonly accepted that the tyrosine kinase of the receptor is activated by ligand-induced dimerization of the monomeric forms (e.g., EGF-R, PDGF-R) or enhanced association between dimerized subunits (e.g., I-R) [ 125,1351. For example, binding of the monomeric EGF results in the dimerization of the monomeric EGF-Rs, thus activating the tyrosine kinase. On the other hand, PDGF occurs as three different dimeric isoforms, made up of disulfide-bonded homo- or heterodimers, composed of homologous A and B chains, i.e., AA, BB, or AB. Two distinct receptors have been identified, the a receptor binding all three PDGF isoforms, and the p receptor which only binds PDGF BB with high affinity. Binding of PDGF leads to the formation of homo- or heterodimers, depending on which PDGF isoform is the ligand, and consequent activation of the tyrosine kinase. The third variation on this theme occurs within the class of receptors having a heterotetrameric structure (i.e., I-R, IGF-R) in which binding of the monomeric ligand induces an allosteric interaction between the two ap subunits already dimerized by a disulfide bridge between the a subunits.
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Fig. 10. Binding of SH-2 domain-containingproteins to receptor tyrosine kinases. Proteins having one or more SH-2 domains are able to bind to autophosphorylationsitesof receptor tyrosine kinases. The main autophosphorylation sites of the EGF-R are situated at its carboxy-terminal tail, enabling the reported binding of PLCy1, PI-3-kinase, and GAP (left). The main autophosphorylationsites of the PDGF-R are located in the kinase insert region, enabling the reported association of PLCyl, PI-3-kinase, GAP and the Src-kinase (right).
The two main mechanisms by which these receptors transmit their information to the interior of the cell are via phosphorylation and protein associations. The earliest response upon binding of the ligand, dimerization, and the subsequent activation of the tyrosine kinase is receptor autophosphorylation. This autophosphorylation removes competitive substrate sites from the receptor itself, thus enabling the phosphorylation of substrates. Furthermore, autophosphorylation of the receptor enables the high affinity association to its phosphorylated sites and the subsequent phosphorylation of several effector proteins. These signalling molecules include PLC-71, rasGAP, PI-3-kinase (the 85 kDa subunit), and members of the Src-family of tyrosine kinases [27,103]. Very recently the protooncogene product Vav, encoding a potential transcrip-
General Mechanistic Patterm
19
tion factor [25,89,127] and showing homology to the family of GNRPs [2,19], was also shown to be bound and phosphorylated by a number of receptor tyrosine kinases. These proteins all have a Src homology-2 (SH-2) domain in common, which enables them to bind to specific phosphorylated tyrosine sites on the receptor, either present at the carboxyl terminal tail (e.g., EGF-R) or in the kinase insert region (e.g., PDGF-R, CSF-1-R) [75,103] (Fig. 10). All or a specific subset of these effector molecules were found to become associated to, and phosphorylated and/or activated by, the different receptor tyrosine kinases: e.g., the PLC-71, rasGAP, PI-3-kinase, Src kinase, and Vav associate with the activated and autophosphorylated PDGF-R; PLC-y 1, rasGAP, PI-3kinase, and Vav with the EGF-R, and PI-3-kinase with the CSF-1-R. In addition, it is noteworthy that some of the receptor tyrosine kinases bind and activate the Raf-1 serinekhreonine kinase as well; however, the Raf-1 kinase does not possess an SH-2 domain and is probably activated by serine phosphorylation. Furthermore, recent studies have established an important role for the GTP binding/GTPase protein Ras in the signal transduction by the receptor tyrosine kinases, as it was shown to mediate the growth factor-induced activation (e.g., EGF, NGF, FGF, PDGF and insulin) of Raf-1 kinase and MAP kinase and its substrates RSK [39,78, 134,1431 and the AP-1 transcription factor component c-jun [14,108, 1241. G-protein-coupled receptors
A large number of receptors for neurotransmitters, neuromodulators, hormones and growth factors do not contain intrinsic catalytic activity. In order to transmit their signal, these ligands activate receptors that are coupled to intracellular effector molecules by G-proteins 1151. The G-protein is active in the GTP-bound state and inactive in the GDPbound state. The activation of the G-protein is initiated by binding of a ligand to the G-protein-coupled receptor, whereas the deactivation of the G-protein is mediated by its intrinsic GTPase activity. The regulatory mechanism, property and identity of the different G-proteins and intracellular effectors involved in this type of signal transduction will be discussed further on in this chapter. In this part we summarize and discuss the large number of ligands and receptors that are known to be coupled to such G-proteins for the generation of intracellular responses. A large number of G-protein-coupled receptors have been cloned that mediate the cellular responses for a variety of signalling molecules (Table I) [83,115]. As exemplified in Table I, the number of signalling
M. Spaargaren et al.
20 TABLE I
G-protein-coupled receptors (list of most of the known ligands for cloned Gprotein-coupled receptors) ~~
Neurotransmitters
Hormones
Others
Sensory system
Acetylcholine Adenosine Dopamine Epinephrine Histamine Norepinephrine Octopamine Serotonin
Angiotensin Bombesin Bradykinin Endothelin FSH LH LH-CG Neurotensin Oxytocin PTH Tach ykinins (Substance K, Substance P and Neuromedin K) TRH TSH Vasopressin
Cannabinoids Chemoattractants (e.g., FMLP) IgE LTs
Olfaction Taste (not cloned) Vision
PAF PGs
Thrombin
molecules that are known to activate G-protein-coupled receptors is quite large; however, the number of receptors involved is even larger. Especially, the most prominent occurring ligands for these receptors, the neurotransmitters, have a large number of receptors available for their signalling. For example, at least seven types of a-adrenergic and three types of P-adrenergic, five types of muscarinic acetylcholine, five types of dopamine, and three types of serotonin receptors are known (Table 11). Due to the diversity of receptors and the difference in effector-coupling among the receptor subtypes (as a consequence of different (;-proteins being coupled to these receptors), one ligand can elicit a variety of responses depending on the receptor subtype present in a particular cell (Table 11). All these G-protein-coupled receptors share significant homology in the amino acid sequence, which reveals a similar secondary structure (Fig. 11) [40,62,74,115]. The typical G-protein-coupled receptor consists of a single polypeptide chain having an extracellular NH,terminal domain which is often glycosylated. Seven hydrophobic
21
General Mechanistic Patterns
TABLE II
Neurotransmitter receptors and their effectors (list of some neurotransmitters, their receptors and receptor subtypes and the response of effectors, to emphasize the diversity of receptors available for one ligand and as a consequence the variety of responses that can be elicited by this ligand) Ligand
Receptor
Epinephrine and norepinephrine
a-adrenergic-R: (yl: (yIA, aIB, a l C
(y2: %?A9 a2B) (y2C, (y2D
Effector PLC t , PLA, t , PLD t ACJ, Ca2+4,K+t?
0-adrenergic-R:
P,, 02, 03
A c t , Ca2+t
Acetylcholine
Muscarinic acetylcholine-R: Ml-type: Mi, M,, M5 M2-type: M2, M4
P L C t , K+J AC4,K't
Dopamine
Dopaminergic-R: D,-type: D,,D5 Dz-type: D,, D3, D4
AC t AC 4
Serotonin
Serotonin-R: ST- 1A ST- 1c ST-2
AC4,K't PLC t AC t , Ca2+ 4
domains of equal length are postulated to be the membrane spanning domains and are believed to make up the ligand-binding pocket (Fig. llb). These transmembrane domains are separated by three extracellular and three cytoplasmic loops. The first part of the intracellular carboxy-terminal domain is, in combination with the second and third cytoplasmic loop which also determine the G-protein selectivity, thought to be involved in the G-protein coupling. Furthermore, nearly all Gprotein-coirpled receptors have two conserved cysteines in the first and second extracellular loop, which are involved in proper ligand binding. Most of the G-protein-coupled receptors can be desensitized by means of phosphorylation of the intracellular domain by, for example, the P-adrenergic receptor kinase or PKA, thereby rendering these receptors refractory to further stimulation after the initial response [40,74]. Many effectors are regulated by G-protein-coupled receptors. Which type of effector is activated by a specific receptor is dependent on the
M . Spaargaren et al.
22
Extracellular
e
lntracellular
Fig. 11. Model of generalized G-protein-coupled receptors. a) A schematic diagram of the proposed topology of a generalized G-protein-coupled receptor. The polypeptide chain is shown as a string of circles each representing an amino acid. The proposed membrane spanning domains are numbered on top, and the intracellular domains are numbered on the inside with roman numerals. The carboxy-terminal part of the receptor is often attached to the cell membrane by one or two palmitoylated cysteines (/\/\/\), and the amino-terminalpart is often glycosylated on asparagine (branched structures). The second and third intracellular loops and the carboxy-terminalpart are believed to be involved in G-protein coupling. Some kinase substrate amino acids involved in desensitizationof the receptor are marked (-P). b) A representation of the tertiary structure of a generalized G-protein-coupled receptor. The proposed transmembrane a-helices are believed to make up the ligand-binding pocket (L) which is buried deeply within the membrane.
General Mechanistic Patterns
23
type of G-protein linked to the receptor, since these G-proteins have specificity for the different effector molecules. Among the effectors are phospholipases (PLA,, PLC), nucleotide cyclases (AC and GC), cGMP-PDE, and ion channels (Ca2+ and K + ) [15].
Receptors without catalytic activity or G-protein coupling Only very recently the cloning of receptors for a number of cytokines (also known as lymphokines or hemopoietic growth factors) led to the recognition of a new type of receptors, such as those for Ils, GM-CSF, G-CSF, Epo, Pro, GH, IFNs, and TNFs [3,9,95,146]. These receptors are transmembrane glycoproteins with an extracellular ligand binding domain, a single hydrophobic transmembrane domain, and an intracellular domain that does not contain any domain exhibiting possible intrinsic catalytic activity or G-protein coupling. Until now, little has been known about the mechanism by which these receptors regulate cell proliferation or differentiation, although their activation has been reported to lead to the induction of protein tyrosine phosphorylation as well as G-protein activation. The src-like tyrosine kinases may participate in the signal transduction by some of these receptors. The receptors of this family can be classified mainly according to differences in their extracellular domain (Fig. 12). 1. The tirst class of receptors, the superfamily of hemopoietin receptors, predominantly for lymphokines, includes 11-1- to II-7-R, GM-CSF-R, G-CSF-R, and Epo-R. This class of receptors is mainly characterized by the presence of a conserved group of four cysteine residues, a juxtamembrane conserved box including the sequence TrpSer-X-Trp-Ser ( W S X W S ) and a fibronectin 3 homologous motif, and the presence of immunoglobulin-like domains. Further subclassification of this class of receptors is possible based on the variations of these structures. The first subclass includes the 11-1-R, which has three immunoglobulin-like loops only. The second subclass includes the G-CSF-R and 11-6-R /3-chain (also known as gp130), all of which have the three characteristics once. The third subclass includes the II-3-, 11-5and GM-CSF-R &chains and c-mpl, all of which have a duplication of both the cysteine group and the conserved box but lack the immunoglobulin-like domain. The fourth and largest subclass consists of the 11-2-, 11-3-, 11-5, 11-6- and GM-CSF-R a-chains, the I1-4-R, 11-7-R and Epo-R, all of which lack the immunoglobulin-like domain (the Pro-R and GH-R also lack the WSXWS conserved sequence). In addition, it can be mentioned that the 11-2, 11-3, 11-4 and Epo receptors are related in their
M . Spaargaren et al.
24 IL-1-R
IL-6-RP G-CSF-R
ILJIIL-Y GM-CSF-RP
c-mpl LIF-R
I
IL-2-RP IL-3-RU 1L-4-R IL-5-RU IL-6-RU IL-7-R GM-CSF-Ra
Pro-R GH-R
INFdPR INFyR
TNFdP-I-R TNFdP-II-R
& :.:.:.:. ....... ,.....
i....
Fig. 12. Cytokine receptor subclasses. The receptors without catalytic activity or Gprotein-coupling(dominated by the cytokine receptors) can be classified according to some characteristic motifs. The subclassification is mainly based on the number of the conserved WSXWS motifs (black box), the number of cysteine motifs containing four conserved cysteine residues and one tryptophan residue (dotted box), the number of immunoglobulinlike domains (loops), the presence or absence of a contactin-like sequence (hatched box), the presence of a single or duplicated region containing two conserved cysteine motifs of two cysteine residues each (flat dotted box), or the presence of cysteine-rich domains (dotted squares). Represented are cytokine receptors (or receptor subunits) fir Us, CSFs and some others 0, IFNs 0 and TNFs 0 , and the homologous Pro and GH receptors
0.
cytoplasmic domains as well, and the 11-3-Ra, GM-CSF-Ra, and 11-5-Ra share a common 0-subunit for constitution of a high affinity receptor. 2. The second class of receptors includes the Pro-R and GH-R which both lack the WSXWS motif but do contain the cysteine motif. 3. The third class of receptors is characterized by a single, as for the IFNa-R, or duplicated, as for the IFNy-R, sequence containing two groups of two conserved cysteine residues. 4.The fourth class of receptors is characterized by four cysteine-rich repeating elements homologous to the low affinity NGF-R and consists of the two types of TNF-Rs as recently identified. Most of the high affinity receptors for the cytokines are composed of at least two distinct subunits, for example the 11-2-R, 11-3-R, I1-5-R,
General Mechanistic Patterns
25
11-6-R, and GM-CSF-R, which comprise at least two distinct ligand binding components, the a-and &chain [66,95]. Both these components are able to bind the ligand, but the highest affinity is obtained by both chains combined. The 0-chain is responsible for the intracellular signal transduction, including, in the case of 11-2-R, the interaction with lck, a member of the src-family of protein tyrosine kinases. The 11-3-R, GM-CSF-R, and 11-5-R share the same &subunit for constitution of a high affinity receptor (i.e., at least in humans). Recent studies have indicated that the 11-2-R may even be composed of three subunits. Interestingly, a single GH molecule is able to bind and dimerize two monomeric GH-Rs by two distinct binding sites of the GH molecule [381. Many cytokines have been demonstrated to induce protein tyrosine phosphotylation in their target cells. The fact that none of their receptors has a tyrosine kinase domain suggests the association of an independent tyrosine kinase with these receptors. Indeed, the IL-2-R 0-chain was shown to be associated with Lck, a member of the Src-family of tyrosine kinases, independent of its SH-2 domain [63]. Recently, the signalling pathway of the IFN a/@-Rwas shown to include another protein tyrosine kinase, tyk2, although a direct interaction between the receptor and this tyrosine kinase has not been established yet [ 1381. In addition, an IFN a-activated protein tyrosine kinase is able to directly phosphorylate and activate a SH-2/SH-3 domain-containing transcription factor [35,52, 1161. Furthermore, the GH-R was found to be associated with a yet unidentified tyrosine kinase. In addition, certain effects (e.g., GM-CSF) can be inhibited by the G-protein-blocking PT; however, whether this is due to a direct or indirect involvement of a G-protein in the response remains to be established. Interestingly, the ligand-induced activation of a large number of the hemopoietin receptors, i.e., some interleukin receptors and GM-CSF, results in the activation of the GTP binding/GTPase protein Ras, indicating the involvement of Ras in the signal transduction by the receptors [95,114]. Besides the cytokine receptors, several other receptors have been identified without catalytic activity or G-protein coupling. Examples are the adhesion receptors or integrins that are able to generate signals across the cell membrane by means of association with signal transducing molecules. Furthermore, several ion channels are known to directly bind and respond to ligands by opening or closing, the so-called ligandgated ion channels. For example, acetylcholine binds to the pentameric niqtinergic acetylcholine receptor which is a cation-selective channel,
M. Spaargaren et al.
26
the GABA and glycine receptors are both chloride-ion channels, and the glutamate receptor is permeable to Na’ and Ca2+ ions.
Signal Transduction Mechanisms In order to elicit the diversity of responses in the cell, the receptors use a number of different mechanisms to transduce the signals. By their intrinsic catalytic activity, coupled G-protein, or associated enzymes, the receptors activate one or a number of effectors like cyclases, phospholipases, kinases or other enzymes or regulatory proteins, and may thus eventually cause the subsequent release of so-called second messengers, such as cyclic nucleotides, calcium, DG, phosphoinositides, AA, and eicosanoids. Thus the external information can be translated into a repertoire of internal signals, finally resulting in the desired cellular response. Many of the signalling systems and their components are known by now, and some of the most prominent ones are described below.
G P binding/GPase proteins Proteins that bind and hydrolyze GTP are involved in a variety of cellular processes [22,23], and play an important role in the signal transduction by almost all receptor types, being involved in the regulation of the activity of a number of enzymes. In particular, two classes of this superfamily of proteins are implicated in signal transduction, i.e., the classical heterotrimeric G-proteins, which are essential for proper functioning of the G-protein-coupled receptors, and the 21 kDa “small” products of the rus protooncogenes, which act as transducing proteins in signal transduction by receptor tyrosine kinases and cytokine receptors
v11. G-proteins In some of the transmembrane signalling receptor systems, the detection of the external signal (i.e., ligand binding) and the generation of an intracellular response are properties of the same protein (e.g., that have intrinsic catalytic activity). Other receptors, however, make use of heterotrimeric G-proteins in order to couple the extracellular signal to the intracellular signal transducing elements [15,16,86,130]. The G-
General Mechariistic Patterns
27
protein cycles between an inactive GDP-bound and an active GTP-bound state. The activation is catalyzed by the receptor and the deactivation is established by the intrinsic GTPase activity of the G-protein. These Gproteins are heterotrimers consisting of an a , p, and y subunit. The a subunit is able to bind and hydrolyze GTP. After binding of GTP, the a subunit and the by dimer dissociate. The by complex and possibly the a subunit are bound to the plasma membrane by means of an isoprenoid and myristic acid lipid anchor, respectively. G-protein-coupled receptors that are activated by extracellular ligands or photons (in retinal cones and rods) associate with the membraneassociated G-protein in the basal, GDP-bound, state and promote the dissociation of GDP from G-proteins. As a consequence of the subsequent GTP binding to the a subunit, the aby trimeric complex dissociates from the receptor, and the GTP-bound a subunit dissociates from the by complex. The GTP-bound a subunit then activates the effector enzyme or ion channel, although in some cases the effector coupling can be mediated by the By complex [79], as recently reported for AC [48,129], Ca2+ channels [73], and myocardial K+ channels. The intrinsic GTPase activity of the a subunit hydrolyzes the bound GTP, thereby terminating the interaction with the effector. The GDP-bound a subunit then reassociates with the By complex, after which the GDPbound aPy trimeric complex is available for activation by another receptor (Fig. 13). The effectors that are regulated by these G-proteins are very diverse [ 15,1301. They include enzymes such as AC, PLC, PLA,, cGMP-PDE, and ion channels for K', Ca2+, and Na'. The a subunit distinguishes the different G-proteins and defines the receptor and effector specificity of the G-protein. Before the different a subunits were cloned, a classification of the G-proteins was made based on their sensitivity to cholera and pertussis toxin as well as their effector function. The G-proteins sensitive to cholera toxin and able to activate AC were named G,; those PT sensitive and able to inhibit AC were named Gi, and the ones sensitive to both toxins and involved in the coupling of light-activated rhodopsin to a cGMP-PDE in retinal cones and rod outer segments were named G, (or transducin). At present approximately 18 different mammalian G-protein a subunits have been cloned [71,123] that can be classified into four subclasses based on amino acid similarity (Table 111): (1) The G, class consisting of the a, (having four splicing variants) and which is involved in olfaction; (2) The Gi the highly homologous aOlf class consisting of the highly homologous ail,a2 and ai3,aOA and aOB,
M . Spaargaren et al.
28
GbP
GbP
GiP
4
Signal
Fig. 13. Functioning of G-proteins in signal transduction. G-protein mediated effector activation upon binding of a ligand to a G-protein-coupledreceptor. (1) Receptors coupled to a G-protein have higher affinity for the ligand and ligand-bound receptors have higher affinity for the G-protein. (2) Binding of the ligand results in a conformational change in the receptor, which causes the dissociation of GDP and the exchange of it for GTP on the G-protein, followed by the dissociation of the G-protein from the receptor and of the GTP-bound or-subunit from the fly complex. (3) The GTP-bound a-subunit binds to and activates an effector which results in cellular responses. (4) By the intrinsic GTPase activity GTP is hydrolyzed to GDP and the GDP-bound a subunit reassociates with the fly complex, after which the G-protein may couple to another receptor.
the atl and at2 (transducins) involved in vision by rods or cones, respectively, ayg(gustducin) involved in taste, and the a,; (3) the G, class consisting of the highly homologous a,, a l l and ( ~ 1 4 ,and the a 1 5 and a l 6 ; and (4)the G,, class consisting of a12and ~ ~ 1 3 . The functional role of the a subunits does not strictly correlate with their classification [71,123] (Table 111). The two members of the G, class of a subunits, the Gas and Gaolf, are both able to activate AC, thereby increasing cAMP levels in the cell. Members of the Gi class are all PT sensitive except for the Ga,, both the Gai and Gao subtypes function in ion channel regulation, the Gai in inhibition of AC thus lowering cAMP levels, Gao in stimulating PLC, Gatl and GaQ in activation of the retinal cGMP-PDE, whereas the function of Gag and
General Mechanistic Patterns
29
TABLE I11
G-protein subclasses and their effectors (the different G-proteins are classified based on amino acid homology of the a subunits) Class
a-subtype
Gs
ffs
ffolf
Gi
ffil
ffi2 ffi3 ffOA
ffOB fftl
fft2
(312
PT sensitivity
Effector
-
AC t , Ca2+ t AC t
-
+ + + + + + + +
ffg ffz
-
ffq ff11 ff14
-
ff15 ff16
-
ff12
-
ff13
-
AC.C,K+t, PLC t , PLA2 t Ca2+ 4 , K + t , PLA,? cGMP-PDE cGMP-PDE ? ? PLC-p1 t PLC-p1 t PLC t ? PLC t ? PLC t ? ? ?
GaZ in effector regulation remains to be established. The members of the G, class are insensitive to PT, and the Ga, and G a l l have recently been demonstrated to be involved in the stimulation of PLCpl [ 12,1311. It has been suggested that the other members of this class may be involved in activation of different PLC isotypes. Little is known about the function of the members of the GI, class, which are all PT insensitive. The number of cloned a subunits as well as the number of purified G-proteins is growing rapidly. A number of additional G-proteins which have been identified based on functional characteristics, such as those involved in the activation of the different phospholipases or the opening of different ion channels, await further identification and cloning.
30
M . Spaargaren et al.
Ras protein The ras protooncogene product, which is a member of a large family of small GTP-binding/GTPase proteins, is a low molecular weight, (21 kDa) GTP-binding protein, and is homologous with the a subunit of the heterotrimeric G-proteins [22,23,42,71]. It is implicated in the signalling by a variety of growth factor- and cytokine-activated receptors, acting in a membrane-bound fashion. The Ras protein binds GTP and catalyzes its hydrolysis to GDP (by intrinsic GTPase activity, which is relatively low compared to that of the G-proteins), the GTP-bound form being the biologically active state of the protein, whereas the GDPbound form is inactive. The activity of Ras can be regulated by GAPs, which enhance the intrinsic GTPase activity of Ras, thus decreasing the signalling activity of Ras, or by GNRPs that enhance the exchange of GDP for GTP, thus increasing the signalling activity of Ras (Fig. 14) [20,43]. At least two GAPs, the rasGAP and NF-1, are known to promote the hydrolysis of GTP on Ras, thereby downregulating the Ras activity [20,43,56]. The rasGAP is phosphorylated on tyrosine by, and associates to, a number of activated growth factor-R tyrosine kinases [27,135]. However, recent studies have demonstrated that these modifications do not represent a major control mechanism for the activity of rasGAP or Ras by growth factors [43]. Furthermore, rasGAP is known to associate with other tyrosine phosphoproteins by means of its SH-2 domains, which may be an important upstream stimulatory control mechanism or downstream effector mechanism for Ras. Two of these proteins have been cloned: p190, which itself resembles GTPases and GAPs and is also related to a transcriptional repressor [118]; and p62, which is homologous to a hnRNP protein and may thus be involved in mRNA processing [142]. The other GAP is encoded by the NF-1 gene; the recent cloning and sequencing of this gene revealed that the encoded protein is homologous to the catalytic domain of rasGAP and is also capable of stimulating the hydrolysis of GTP on Ras. The NF-1 gene has been implicated in the hereditary disease Von Recklinghausen neurofibromatosis as well as in some sporadic tumors and is a so-called tumor suppressor gene, causing enhanced Ras activity upon its loss of function [6,36,82]. In addition to their upstream regulatory function, these GAPs have also been suggested to be downstream effectors of Ras, thus both inactivating and mediating the signalling of the Ras protein [20,21,55].
General Mechanistic Patterns
I
Inactive
1
31
GDP
GDP
I/
V
Signals Fig. 14. Regulation of Ras activity. Ras is inactive in its GDP-bound state. By GNRPs the GDP dissociates from Ras and is exchanged for GTP. The GTP-bound Ras is biologically active resulting in cellular responses. The intrinsic GTPase activity of Ras can be activated by GAPS, thus causing the hydrolysis of GTP to GDP, resulting in GDPbound inactive Ras.
In addition, the activity of Ras proteins is regulated by the GNRPs [44,120], some of which display specificity for the different members of the family of Ras-related proteins, and are also known as guanine nucleotide-(or GDP-)exchange factors, guanine nucleotide-(or GDP-) releasing factors, or guanine nucleotide-(or GDP-)dissociation stimulators. These proteins, as their names indicate, are able to enhance the dissociation of GDP from Ras (or the Ras-like proteins), thereby increasing the amount of Ras in the GTP-bound state (GTP is present in higher concentrations than GDP), thus causing its activation. Little is known about the regulation of the activity of the GNRPs; however, very recently their activation has been reported upon NGF treatment of cells, thus causing the activation of Ras [go].
M . Spaargaren el al.
32
I
RESPONSE
1
Fig. 15. Functioning of Ras in signal transduction. The Ras protein was shown to be required for receptor tyrosine kinase signal transduction and is implicated in the PKC signal transduction pathway. As already indicated in Fig. 14, Ras activity can be regulated by GNRPs and GAPs. The mechanism by which receptor tyrosine kinases modulate the activity of these Ras-regulatory proteins remains to be established. The receptor-activated PLA, may have an upstream Ras-regulatory function, since in vitro GAP activity can be regulated by AA and eicosanoids. How Ras activation signals to its downstream effectors remains to be elucidated, although GAPS have been suggested to be involved in this process as well. Recent studies have demonstrated that Ras mediates the growth factorand PKC-induced activation of Raf kinase, MAP kinase, and its substrate RSK, thus playing a central role in signal transduction by receptor tyrosine kinases.
AA and eicosanoids have been reported in to be involved, at least in vitro, in the inhibition (by AA, PGI,, and lipoxygenase products) and stimulation (by PGF,,, PGA, P G h ) of GAPs, and also the stimulation (by AA) of a GTPase inhibiting protein [20,58,112] (Fig. 15). Since the Ras protein is implicated in growth factor receptor tyrosine kinasemediated mitogenic signalling [ 17,42,67], PLA, activation and the
General Mechanistic Patterns
33
subsequent formation of AA and AA-metabolites which are able to regulate the activity of GAPS, may provide an important link between the growth factor receptor- and Ras-mediated signal transduction pathways. A number of studies have demonstrated that ras is required for cell proliferation and cell transformation in response to growth factors and tyrosine kinase oncogene products, respective [ 17,42,67]. Very recently several studies have reported that ras mediates the PKCand growth factor receptor-induced modulation of MAP kinases, RSK and the r f kinase (Fig. 15) [39,78,134,143]. However, neither the mechanism by which the ras protein is activated by growth factor receptor tyrosine kinases (upstream) nor the mechanism by which it regulates growth factor-induced signal transduction and cell proliferation (downstream) have been elucidated. Furthermore, it is noteworthy that a Ras-related protein, i.e., rho, is involved in assembling the actin network of the cytoskeleton by the regulation of actin polymerization P71.
Adenylate and guunylate cyclases AC and GC are able to synthesize cyclic nucleotides, CAMP, and cGMP, respectively, in response to extracellular signals [45,92,147]. These cyclic nucleotides in turn are able to act by activating effector molecules such as the CAMP- and cGMP-dependent protein kinases, cyclic nucleotide-gated ion channels, and cGMP-regulated PDEs. On the other side of the coin, cyclic nucleotide PDEs are able to attenuate the signals evoked by these cyclic nucleotides. The enzyme AC is a main effector enzyme for many G-proteincoupled receptors (see Fig. 4) but can also be activated by several growth factor receptors containing tyrosine kinase activity, as well as by several cytokine receptors [45]. The enzyme can be modeled as an integral membrane protein, having two hydrophobic domains which each contain six membrane-spanning domains, and two hydrophilic cytoplasmic domains. Its activation results in the conversion of ATP into cAMP which in turn can play a role as a second messenger, for example in the activation of PKA [ 1321. PDEs are involved in the degradation of cAMP and cGMP, thereby attenuating the signals evoked by these second messengers. The PDEs can be mainly classified in five functionally distinct families based on their modification by the different cyclic nucleotides. Type I PDEs are stimulated by Ca2+/Calmodulin, types I1 and 111 are stimulated by
M. Spaargaren et al.
34
cGMP, type IV are specific for CAMP, whereas type V specifically hydrolyzes cGMP (this type of PDE includes the ones involved in vision transduction in the rods and cones).
Phospholipases and phospholipid-derived second messengers Besides their importance for defining the cell membrane, phospholipids are involved in the regulation of a number of different cellular processes as they can be converted into bioactive hormones and phospholipid-derived second messengers. Due to the activation of different phospholipases, a number of phospholipid-derived hormones and second messengers can be produced such as DG, phosphoinositides, AA, eicosanoids (e.g., PG, LTs, TXs), PA, LPLs, PAF, ceramides, and sphingosine. The main phospholipases involved in the production of these compounds are PLA,, PLC, and PLD (Fig. 16). Nearly all the products of the hydrolyzing action catalyzed by these phospholipases have been implicated in signal transduction [37,49]. The different phospholipases and their main reaction products - as far as they are involved in signal transduction - are discussed below.
Phospholipase A , Several ligands for receptor tyrosine kinases (e.g., the EGF-R, PDGF-R, I-R), G-protein-coupled receptors (e.g., a 1-adrenergic-R, Acetylchol ine muscarinic-R, ACTH-R, GRH-R, Vasopressine- la-R, Angiotensin 11-R, Thrombin-R, P,-Purinergic-R, IgE-R, Bradykinin-R, Bombesin-R, Histamine-R, Substance P-R, Rhodopsin, PAF-R, LTD4-R, and LTC4-R) and receptors without catalytic activity or Gprotein coupling (e.g., 11-1, TNF-R, IFNa-R) have been reported to enhance cellular PLA, activity. PLA, catalyzes the hydrolysis of membrane phospholipids at the sn-2 position, thus producing lysophospholipids and free fatty acids (Fig. 16), among which AA is the most prominent one. AA and its metabolites (e.g., eicosanoids such as PGs, LTs, and TXs) play an important role in a wide variety of biological processes like the inflammatory response, immune responses, and the regulation of cell proliferation and differentiation, acting both as interand intracellular messengers [68,98,137]. The production of AA can also be accomplished by the sequential activation of PLC and DG-lipase, although PLA, provides the most prominent source. Either the activation or the induced expression of PLA, can cause enhanced levels of AA.
35
General Mechanistic Patterm
c =o
I 0 I
c-
c zo 0
I
c-
PLA, C
I
0
Ic- PLC 0 PLD
-0-PPO
I
X Fig. 16. Action of phospholipases in generating phospholipid-derived second messenger molecules. Membrane phospholipids constitute an important source for the production of second messengers. Several phospholipases are involved in the production of second messengers from the phospholipids, acting by catalyzing the hydrolysis of the ester bonds as indicated. PLA, action results in the production of AA, PLC in DG and IP,, and PLD in PA. (X represents the different head groups, e.g., choline, ethanolamine, inositol, serine.)
The released AA can be further metabolized into eicosanoids, by cyclooxygenases to PGs and TXs, or by lipoxygenases to LTs and HETEs (Fig. 17). The produced eicosanoids may act either as intracellular second messengers or in a paracrine or autocrine fashion as extracellular first messengers as for many of these bioactive lipids, such as PGs and LTs, G-protein-coupled receptors have been identified. PLA, is ubiquitous in nature and can be detected in almost all cell types. Several different types of PLA, have been purified and cloned: extracellular pancreas and venom types as well as various cellular types, which all have a typical low molecular mass of 14-18 kDa. Recently,
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Fig. 17. Function of PLA2-generated molecules in signal transduction. Activation of PLA, either by a G-protein or by phosphorylation results in the hydrolysis of membrane phospholipids, thus producing AA and LPLs or, depending on the hydrolyzed phospholipid, Lyso-PAF (L-PAF). AA can be metabolized by cyclooxygenases, for example, into PGs and TXs, or by lipoxygenases into LTs and hydr(oper)oxy-e.icosa-tetraenoic acids (HETEs). These eicosanoids are potent intracellular second messengers, but may also act in a paracrine or autocrine fashion as extracellular first messengers. L-PAF is a precursor for yet another bioactive mediator PAF.
however, the purification and cloning of a class of higher molecular mass (85 kDa) cytosolic PLA2s has been reported. These high molecular mass cytosolic PLA2s are AA-selective, respond to physiological increases in intracellular Ca2+ and pH with enhanced activity, and translocate to the membrane in a Ca2+-dependent manner [3 1,1261. Interestingly, sequence analysis demonstrated the presence of a Ca2+dependent lipid-binding domain homologous to PKC. GAP, and PLC, as well as several potential kinase phosphorylation sites (for both serinekhreonine and tyrosine kinases). Several events have been implicated in activation of PLA,, most of them including the activation of G-
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proteins. In addition, PKC and the enhancement of intracellular pH or Ca2+ have been implicated in PLA, activation. Recent studies have established that the high molecular mass cytosolic PLA, is activated by several growth factors and that the activation is due to its phosphorylation [84]. Therefore, these cytosolic PLA,s are likely candidates for playing a role in signal transduction by the receptor tyrosine kinases. Interestingly, AA and/or the eicosanoids have been reported to activate PKC, activate different ion channels (for Ca2+ and K+), and cause the release of Ca2+ from intracellular stores. Furthermore, the activation of PLA, and the subsequent formation of the AA-metabolites were shown to be involved in growth factor-induced c-myc, c-fos, and jun B expression. In addition, in vitro they inhibit rasGAP and NF-1, and stimulate a GTPase inhibiting protein, all regulators of Ras activity [ 1121. Since evidence has been provided that the Ras protein is implicated in growth factor receptor tyrosine kinase-mediated mitogenic signalling, PLA, activation may thus provide an important link between the growth factor- and the ras-signal transduction pathways (see Fig. 14). In addition to AA as the released fatty acid, PLA,-catalyzed hydrolysis of phospholipids results in the formation of lysophospholipids. Lysophosphatidylcholine has been implicated in stimulation of the CAMP-PDE and in activation of PKC (41. Another potent phospholipid mediator, PAF, can be formed upon acetylation of an alkylethercontaining lysophosphatidylcholine. PAF in its turn can act as a hormone by binding to the PAF-R which is a G-protein-coupled receptor, thereby causing phospholipid turnover via PLA,, PLC, and PLD [ 107,1211.
Phospholipase C A wide variety of hormones, growth factors, cytokines, and neurotransmitters stimulate the hydrolysis of PI by means of PLC (Fig. 16). As a consequence of the action of PLC, the intracellular second messengers DG and inositol phosphates are generated. Many distinct types of PLC have been cloned and sequenced, which indicate the existence of four types of PLC, named PLCa, PLCP (includes the family members PLCPl and PLCP2), PLCy (includes PLCyl and PLCy2), and PLCX (includes PLCX1, PLCX2, and PLCX3), each type containing several distinct family members. All four types are able to hydrolyze PI, PIP, and PIP,. PLCP is activated by a G-protein, the activation being impaired by PKC-induced phosphorylation of PLCP on serine residues.
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PLCyl is activated by tyrosine phosphorylation and forms a physical complex with the cytoplasmic domain of several receptor tyrosine kinases [109]. This binding of PLCy appears to be mediated by the SH-2 domains. Indeed, only the subtype PLCy contains two of these SH-2 domains as well as an SH-3 domain, the latter probably being involved in cytoskeleton association. The receptor-mediated hydrolysis of PIP, by PLC leads to the generation of two second messenger molecules, IP, and DG (see Fig. 5) [lo]. IP, mobilizes Ca2+ from intracellular stores by binding to a specific receptor [ 111. DG, in concert with Ca2+, activates the phospholipid-dependent kinase PKC, which in its turn phosphorylates a multitude of proteins, thereby controlling a host of cellular processes [72,99]. In addition to IP,, I(1,3,4,5)P4 has also been reported to cause the release of Ca2+ from intracellular stores, and several other phosphoinositides may exert second messenger functions [88]. In addition to being a substrate for PLC, PIP and PIP2 may play an important role in regulating the assembly and dynamics of the cytoskeleton of the cell. These phosphoinositides have been reported to interact in vitro with several actin binding proteins, such as profilin and gelsolin, that are known to regulate the amount and size of actin filaments. The positive effect of these actin binding proteins on actin polymerization is specifically inhibited by these phospholipids as a consequence of their direct interaction with these proteins. Besides PIP,, PC can also be an important source for the production of bioactive lipids [13,46]. In addition to the production of DG from PIP,, the main source of DG is hydrolysis of PC by a distinct type of PLC (not yet cloned). Furthermore, PA, generated by the hydrolysis of PC by PLD, can subsequently be converted into DG. DG, on the other hand, can be converted to PA by DG kinase, an enzyme that has a Ca2+-dependent activity and may become membrane-associated via phosphorylation by PKC. PA, to complicate matters even more, may have second messenger potential itself. DG can also be used as a substrate for DG lipase, thus giving rise to the bioactive AA and eicosanoids, which in their turn can act as messengers as well. Furthermore, several growth factor receptor tyrosine kinases and oncogenic tyrosine kinases are able to activate PI-3 kinase which is, by means of a SH-2 domain, present in its 85 kDa subunit, associated with and tyrosine phosphorylated by these receptors. As a consequence of the PI-3 kinase activation using PI, PIP, and PIP, as a substrate, phosphatidylinositides are produced that are phosphorylated at the 3 position of
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the inositol ring. The possible function of these phospholipids in low abundance remains to be established.
Phospholipase D There is now substantial support for the implication of PLD in signal transduction [37,49]. Unfortunately, amino acid sequence information is not yet available for PLD. PLD mediates the hydrolysis of phospholipids, mainly PC, thereby producing free choline and PA (Fig. 16). The mechanisni by which PLD activation is linked to receptor activation is not clear, although the involvement of a G-protein, Ca2+ influx and mobilization, as well as PKC, have been reported, suggesting that multiple PLD isoforms may exist. PA is implicated as a second messenger itself, although in addition its conversion to DG by a phosphatidate phosphatase (PA-phosphohydrolase) increases the effective DG pool involved in regulation of PKC activity. With respect to the possible role of PA as a mitogen, it is important to note that some of the initial studies may have dealt with the effect of contaminating lyso-PA instead of effects of PA itself. Recent studies have indicated that lyso-PA indeed has the capability of mobilizing Ca2+ and inducing a mitogenic response [ 1361. In addition to the effect of AA and eicosanoids on GAPs involved in the regulation of ras activity, it is noteworthy to mention that GAPs as well as a GTPase inhibiting protein as both sensitive in v i m to PA, the first one being inhibited by and the latter activated by PA.
Kimes Important candidates as signal transducing target enzymes for protein tyrosine kinase receptors, G-protein-coupled receptors, and the receptors without catalytic activity or G-protein coupling, are kinases with tyrosine-, or serinekhreonine-specificprotein kinase activity [60] or with dual specificity [85,106]. The serine-threonine kinase PKC is an important mediator of signals elicited by receptors coupled to PLC, which can be found in all before-mentioned receptor classes, as it is activated by DG. Other examples are the serinekhreonine kinases belonging to the family of MAP kinases, RSK, Raf-1 kinase, cdc2 kinase, and casein kinase 11, which are all mainly involved in signal transduction by the growth factor receptor tyrosine kinases, and believed to be activated as a consequence of their phosphorylation (dephosphorylation in the case of cdc2 kinase), albeit not directly by the receptor
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et al.
tyrosine kinases. Another group of serinekhreonine kinases are, like PKC, second-messenger-activated kinases, such as PKA and CAMkinase, PKA being mainly involved in signal transduction by G-proteincoupled receptors that act on AC. Furthermore, it has recently been established that tyrosine kinases belonging to the src-family are involved in signal transduction by a number of cytokine receptors and also by some receptor tyrosine kinases. Below we discuss the properties and signal transduction relevance for some of these kinases in more detail.
Protein kinase C
PKC, a serinekhreonine kinase, is a major component of many signal transducing systems, being activated by many ligands including hormones, growth factors and neurotransmitters, operating by means of the different receptor subtypes [47,144]. PKC, initially identified as a Ca2+/ phospholipid-dependent protein kinase, is activated by binding of the second messenger DG which is released by the action of PLC (see Fig. 5). Several tumor promoting agents such as phorbol esters (e.g., TPA) mimic the effect of DG, thereby activating PKC. Furthermore, it has been demonstrated that AA and other unsaturated fatty acids are also able to activate PKC (see Fig. 6). PKC is thought to be in the inactive conformation with the aminoterminal pseudosubstrate region of the regulatory domain interacting with the carboxyl-terminal catalytic domain (autoinhibition). Changes within the pseudosubstrate would cause unfolding and decreased dependence on cofactors. Before activation, PKC has to be bound to the cell membrane. This membrane interaction is accomplished by PKC binding to phosphatidylserine and Ca2+, Ca2+-binding sites being generated at the interface between PKC and the membrane. After binding to the membrane PKC can bind DG, thus causing the reorientation of the regulatory domain which is essential for its activation [94]. Molecular cloning has revealed that PKC exists as a family of multiple isozymes having closely related structures [ 1001. Initially, four isozymes were isolated and cloned: PKCa, PI, PII, and y (called group A). Later additional clones were characterized which were designated PKC A, E , 5, v, and L (group B). These group B, PKCrelated enzymes lack the conserved region that is involved in Ca2+ binding. The different PKCs have been found at a number of sites in the cell like the membrane, cytosol, cytoskeleton, and the nucleus. Furthermore, the different PKC subspecies have subtly different properties with respect, for example, to substrate specificity.
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Activation of PKC is involved in the regulation of various ion channels, the Na /H +-exchanger, cell-signal 1i ng pathways involving Ca2+ and CAMP, and in the negative feedback control or down-regulation of various receptors coupled to the phosphoinositide cycle, such as growth factor receptors which are often substrates for PKC. One of the most predominant substrates of PKC is the myristoylated alanine-rich C kinase substrate (MARCKS), which is a filamentous F-actin crosslinking protein and may be a PKC-regulated cross bridge between actin and the plasma membrane. Furthermore, it is clear that PKC plays a crucial role in the regulation of gene expression [50,69,70]. It has been established that PKC can directly phosphorylate IKB which is the inhibitory polypeptide of transcription factor NFKB.As a consequence of the phosphorylation of IKB,it can no longer associate with cytosolic NFKB,and the free NFKB translocates to the nucleus where it binds to specific response elements in the regulatory regions of genes leading to their enhanced expression. Furthermore, the PKC signal transduction pathway can activate the DNA-binding activity of the transcription factor complex AP-1 (by an intermediate component, probably a phosphatase, causing the dephosphorylation of the AP-1 component c-jun) [24]. The transcriptional regulation of a number of target genes, for example those having a TRE in their promoter region (e.g., c-jun), is directly controlled by the PKC pathway (Fig. 18). +
CAMP-dependent protein kinase PKA is a serinehhreonine kinase involved mainly in the signal transduction by G-protein-coupled receptors (see Fig. 4). PKA is inactive in the absence of cAMP and consists of two regulatory and two catalytic subunits. At this moment four regulatory subunit genes (RIa, RIP, RIIa, and RIIP) and three catalytic subunit genes (Ca, Cp,and Cy) have been identified. These isoforms differ in their expression pattern and subcellular localization and constitute different biochemical properties. In analogy with PKC the regulatory subunits contain a pseudophosphorylation site and thereby occupy the substrate binding site of the catalytic subunit in the holoenzyme complex. Binding of two cAMP molecules to each of the two regulatory subunits causes the dissociation of the dinieric regulatory subunits and the two monomeric catalytic subunits. Thus the catalytic subunits become activated and thereby enable the kinase to phosphorylate its substrates.
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4
lpKCl
‘r’ phosphatase ?
“I
I
Fig. 18. Regulation of transcription factor activity by phosphorylation. The activity of a large number of transcription factors is regulated by phosphorylation. This phosphorylation may modulate the DNA binding, the transcriptional activity, or the cellular localization of these transcription factors (see text for more details), thereby controlling the transcription of target genes.
Perhaps the best studied function of PKA is its phosphorylation of several key regulatory enzymes of different metabolic pathways such as the phosphorylase kinase and glycogen synthetase both involved in glycogen metabolism [33]. Another important function of PKA is the negative feedback regulation of G-protein-coupled receptors, mainly adrenergic receptors. These receptors are coupled by means of the Gprotein to AC, thereby increasing cellular cAMP levels and causing the subsequent activation of PKA. PKA is able to phosphorylate these receptors at the cytoplasmic face resulting in uncoupling of these receptors from the G-protein. The result is desensitization of the receptor, which is characterized by a reduction in effector stimulation over time, despite the presence of the ligand. The transcriptional regulation of a number of genes has been reported to depend on the production of cAMP and the subsequent activation of PKA [50,69,70].One of the PKA substrates is a transcription factor that belongs to the family of cAMP response element binding proteins, CREB. Upon activation, the catalytic subunit of PKA translocates to the nucleus in order to be able to phosphorylate CREB. CREB, upon phosphorylation on serine which activates its transcriptional activity, is able to bind to the CRE present in the promoter regions of several target
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genes, thereby inducing their transcription. The Ca2+/Calmodulindependent protein kinases can also phosphorylate and activate the transcription factor CREB. Furthermore, PKA-induced phosphorylation enhances the DNA-binding activity of the transcription factor complex AP-1 (apparently by phosphorylation of an AP-1 inhibitor IP-1), and the transcriptional activity of the transcription factor NFKB(KKB,a complex forming protein able to regulate the localization of NFKB,is believed to be phosphorylated by means of an intermediary kinase (Fig. 18).
RAF-1 k i m e One of the molecules thought to be critical for the transmission and amplification of mitogenic signals from the cell surface to the nucleus is the protooncogene product Raf-1. Raf-1 is a serinekhreonine kinase that itself is phosphorylated and activated in response to several growth factors, cytokines, and hormones [81,96]. Phosphorylation of Raf-1 occurs predominantly on serine residues but also, albeit at low stoichiometry, on tyrosine residues. Thus phosphorylation of Raf-1, in the case of receptor tyrosine kinases probably by an intermediate serine/threonine kinase, is likely to regulate its activity. However, at least a portion of the phosphorylation appears to be a consequence rather than a cause of activation of Raf- 1 ki nase activity, reflecting either au tophosphoryiation or phosphorylation by Raf-1-activated kinases. Furthermore, the Raf-1 protein was found to be associated with some of the growth factor receptor tyrosine kinases. Given the fact that Raf-1 lacks any SH-2 domains, this association will probably be the consequence of an indirect interaction. The amino terminal domain of Raf-1 is important in the regulation of its activity, either functioning to keep the kinase activity repressed or to regulate the interaction with substrates. The deletion or mutation of the amino terminal domain gives rise to the oncogenic version of the Raf-1 protein kinase, one of the few oncogenic serinekhreonine kinases. The activation and phosphorylation of Raf-1 is stimulated after activation of several receptor tyrosine kinases and PKC. Furthermore, recent studies have demonstrated that Raf-1 acts downstream of another protooncogene, the Ras protein, which was shown to be the intermediate between tyrosine kinase receptors and Raf-1 (see Fig. 15) [143]. Not much is known about the substrates or activators of Raf-1; however, during the preparation of this chapter, Raf-1 was reported to activate
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and phosphorylate the MAP kinase kinase, which is an activator of MAP kinase [76].
Mitogen-activated protein kinase MAP kinases are a group of protein serinelthreonine kinases that are activated in response to many extracellular stimuli, including those that activate several growth factor receptor tyrosine kinases (I, NGF, EGF, PDGF, and FGF), activators of PKC, G-protein-coupled receptors (thrombin, bombesin, and bradykinin), and cytokine receptors [ 18,105, 1331. The MAP kinases are a family of kinases that are also known under other names such as ERKs (extracellular signal-regulated kinases), MBP kinases (myelin basic protein kinases), RSK kinases (ribosomal S6 protein kinase-kinases), MAP-2 kinases (microtubule-associated protein-2 kinases), or ERT kinases (EGF-R threonine kinases). At the moment several different MAP kinases have been identified, the best studied ones are designated p42 MAP kinase (ERK2) and p44 MAP kinase (EKK1). Both isoforms are differentially distributed inside the cell; the p42 MAP kinase co-localizes with the microtubule network and the p44 MAP kinase is cytoplasmic but translocates to the nucleus upon stimulation of the cells [29]. The mechanism of activation of these MAP kinases is quite unique as they are dependent on tyrosine phosphorylation for their activity, and they require threonine as well as tyrosine phosphorylation to become active. The exact mechanism of action of MAP kinase has been a matter of some controversy. A kinase cascade may be involved in which the upstream kinase is regulated by agonists and phosphorylates MAP kinase; an intramolecular autophosphorylation may occur in which a non-kinase cellular factor may regulate the kinase activity of MAP kinase, or intermolecular transphosphorylation may occur in which the MAP kinase is activated allosterically. Indeed, the MAP kinases have been demonstrated to be capable of intramolecular autophosphorylation on tyrosine and threonine residues, albeit at very low levels [105]. However, recent studies have reported the purification of a MAP kinase activator that was able to induce in vitro threonine and tyrosine phosphorylation of both a kinase-negative mutant and a wild-type MAP kinase, the latter also being activated, thus establishing this factor as a MAP kinase kinase [77,97]. Interestingly, several recent studies have established an intermediate regulatory role for the protooncogene ras in the growth factor- and PKD-induced phosphorylation and activation of
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MAP kinase [39,78,134,143] (see Fig. 15), whereas during the preparation of this chapter, it was shown that another kinase downstream of Ras, the Raf-1 kinase, is able to phosphorylate and activate the MAP kinase kinase [76]. Once activated, MAP kinases can phosphorylate microtubulineassociated protein-2 (MAP-2), the EGF-R, and both phosphorylate and activate RSK and, at least in vitro, Raf-1 kinase, and the transcription factor protooncogenes c-jun and c-myc. In turn, besides the ribosomal S6 protein, RSK phosphorylates several transcription factors such as the serum response factor (SRF) and c-fos in vitro on sites that are also found to be phosphorylated in vivo [50,69,70] (Fig. 18).
Signal Transduction in Development and Cancer The optimal coordination of the above-described processes is vital, for example, in proper embryonic development (i.e., the development of the fertilized egg into the organism as a whole), whereas the deregulation of these processes is the cause of a number of diseases, among which cancer is the most prominent. Moreover, the research of both developmental biology and cancer have provided new insights in general signal transduction mechanisms. Therefore, in the following section these two fields of investigation are discussed in the context of the signal transduction mechanisms described above.
Signal trarzsduction and development The communication between cells and the above-mentioned signal transduction pathways appear to play important roles in the determination of cell fate in the early embryo, in the differentiation of cells during embryogenesis, and the differentiation of cells in the full-grown organism. Especially with respect to the signal transduction pathways initiated by growth factors, mainly mediated by the receptor tyrosine kinases, a growing body of evidence indicates their involvement in developmental processes ranging from the fruitfly Drosophila melanogaster and the nematode worm Caenorhabditis elegans to mammals [5]. Drosophila and C. elegans are powerful tools for developmental geneticists since the patterns of cell division and cell differentiations are known completely for C. elegans and partially for Drosophila. Below we highlight some of these developmental processes.
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The cell-fate specification of the R7 photoreceptor neuronal cell in the Drosophilu eye depends on a signal from a neighboring R8 cell [ 110, 1131 (Fig. 19a). Mutational analysis has revealed a number of genes involved in the normal development of the R7 cell. One of these genes from the K7 cell is sevenless (sev), which encodes a receptor tyrosine kinase of which the cytoplasmic domain most closely resembles the EGF-R. The Sev protein was found to be activated by a membraneanchored ligand, encoded by the bride of sevenless (boss) gene, present in the R8 cell. When mutations that decrease or increase the signalling by Sev were screened, several components of its signal transduction pathway were revealed. The involved loci are Rasl encoding a protein homologous to the GTP bindinglGTPase protein Ras, son of sevenless (sos) encoding a protein that is homologous to the yeast S. cerevisiue CDC25 protein, which is an activator of guanine nucleotide exchange by Ras proteins, and the Gap1 locus encoding a putative rasGAP. The lossof-function mutations of Rasl and sos result in a decrease of signalling by the Sev receptor tyrosine kinase [122], whereas such a mutation of Gap1 and a gain-of-function mutation in Rm1 mimics activation of Sev [51,53]. Furthermore, sevenmaker (sem) encodes a SH-2LSH-3 domain containing protein, and seven in absentia (sina) encodes a nuclear factor; they have been implicated in the signal transduction by the Sev receptor tyrosine kinase, and thus in Drosophilu eye development. Another receptor tyrosine kinase (PDGF-R-like, with a kinase insert domain), encoded by the torso gene, is involved in the control of Drosophila head and tail formation (anterior-posterior polarity), as determined by both loss- and gain-of-function mutations. Some of the genes involved in the Sev pathway, like rasl and sos, have also been implicated in the torso pathway. In addition, this pathway involves the polehole gene, which encodes a Raf serinehhreonine kinase. Another gene encoding the Drosophilu EGF-R homologue (DER) is involved in a number of developmental processes [ 1191. The DER loss-of-function mutants torpedo and faint little bull F b ) , and the gain-of-function mutation ellipse can give rise to different phenotypes, flb being lethal; torpedo being involved in dorso-ventral axis formation; and ellipse in pattern formation, cell division, and eye development. Finally, the breathless gene, encoding a Drosophilu GFG-R homologue (DFGF-R 1), is involved in proper trachea development. The signal transduction components involved in the development of vulva1 precursor cells in C. eleguns have also been extensively studied by mutational analysis [128] (Fig. 19b). These studies revealed the
General Mechanistic Patterns
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boss
I i n-3
+
LIGAND
t RECEPTOR TYROSINE KINASE
SHUSH3
PROTEIN
li
t
GNRP
t
GAP
e-
RAS
+
+
-
I
--“I-
4
?
Drosophila
RAF
C. elegans vulva development
I
Fig. 19. Sigual transduction pathways involved in Drosophila eye and C. elegans vulva development. T h e product of the genes, identified by genetic loss- or gain-of-function mutation analysis, involved in the signal transduction of either Drosophila eye development (a) or C’. elegans vulvadevelopment @) are represented. In the middle their function is indicated based on homology to their mammalian counterparts (see text for more details).
importance of the let-23 gene, encoding a receptor tyrosine kinase with similarity to the EGF-R, in proper vulva1 development. Its loss-offunction mutation resulted in a vulvaless phenotype, whereas its gain-offunction mutation resulted in a multivulval phenotype. The ligand for this receptor is believed to be a transmembrane growth factor precursor (EGF-like), encoded by the lin-3 gene, present in an adjacent anchor cell [65]. The loss-of-function mutation of the let-23 gene could be overcome
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by a gain-of-function mutation in another gene, let-60, whereas a lossof-function mutation of let-60 resulted in a vulvaless phenotype [7,59]. The let-# gene encodes a Ras protein. A new connection in this pathway was made by the discovery of the sem-5 gene, encoding for an SH-2/SH-3 domains-containing protein homologous to the protooncogene c-crk [32,104]. A loss-of-function mutation in the SH-2 domain of sem-5 prevented the development of the vulva [91]. Because c-crk binds to autophosphorylated receptor tyrosine kinases, sem-5 might be the direct mediator of signals from let-23 to let-60, by binding to let-23 and activating let-# by a yet unknown mechanism. Furthermore, the lin-45 gene is required for vulva1 differentiation by activated let-60. lin-45 encodes a protein homologous to the Raf serinekhreonine kinase apparently acting downstream of the Ras protein encoded by let-60. Many receptor tyrosine kinases and ligands are known to be expressed during mammalian embryogenesis, and besides their role in proliferation in the fully grown organisms, they also appear to have their effect in differentiation. For example, dominant mutations in the white spotting (W) and Steel (Sl) loci on the mouse both result in defects during embryological development and in adult life [139]. Both mutations affect pigmentation, germ cell systems and blood-forming systems, resulting in lack of hair pigmentation, sterility, macrocytic anemia, and mast cell deficiency, indicating improper melanogenesis, gametogenesis, and hematopoiesis. It turned out that the W gene encodes the receptor tyrosine kinase protooncogene product Kit [28,54], and the SZ gene encodes the ligand of Kit, known as the steel factor (SF) or stem cell factor (SCF) [141]. For proper mammalian development, SF in its membrane-associated form appears to be required [41]. The phenotypes found in mice carrying different mutations in the W locus are due to loss-of-function mutations that either reduce Kit tyrosine kinase activity or make Kit defective in receptor autophosphorylation, thereby preventing its normal signal transduction. Other receptor tyrosine kinases implicated in development are those belonging to the family of trk protooncogenes. Receptors in this family, including Trk, TrkB, and TrkC, are activated by the neurotrophic factors NGF, BDNF, and NT-3. Trk binds NGF only, TrkB binds both BDNF and NT-R, and TrkC binds NT-3 [102,103]. The members of the Trk family are exclusively expressed in tissues of neural origin (each member having its own specificity), and the Trk receptor tyrosine kinases and their ligands appear to function in the development of the central and peripheral nervous systems controlling the survival of
General Mechanistic Pattertu
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specific neurons. Originally, p75, a protein without tyrosine kinase activity, was shown to bind all the neurotrophic factors (therefore it was called NGF-R), however, all with relatively low affinity and without cellular responses. It is now believed that p75 forms a heterodimer with the different members of the Trk family to constitute a high-affinity receptor for the different ligands, and thus is able to participate in neuronal differentiation [64]. Finally, it should be mentioned that several growth factors, cytokines, and “differentiation” factors, such as TGFP and LIF, regulate the differentiation of different multipotent stem cells in the fully grown adult organism. An excellent example is provided by the cytokines (or hematopoietic growth factors) that act not only in the blastocyst on the pluripotent embryonic stem cells - thus directing the differentiation of these cells into hematopoietic precursor cells - but also in the adult organism on the multipotent hematopoietic stem cells, thus directing the differentiation of these cells into the whole orchestra of mature hematopoietic cells.
Signal transduction and cancer It has been clear for many decades that the disease called “cancer” is in some way related to defects in the normal mechanisms that control cell differentiation and proliferation. It is only in the last decade, however, that the precise molecular mechanisms involved in some forms of uncontrolled cell proliferation have begun to be elucidated. These new insights have renewed hopes that the underlying features of the cancerous process can be understood. The balance between protooncogenes, having a positive effect on cell proliferation [ 17,27,67], and tumor suppressor genes (or antioncogenes), having a negative effect on cell proliferation [90,140], is believed to play an important role in the proliferation of normal functioning cells. Any defects leading to the constitutive activation of one of the signal transduction components, especially those involved in signal transduction by growth factors as described in the previous sections of this chapter, may result in uncontrolled cell growth and consequently tumor formation. The mutated genes involved in this process are called either oncogenes, which are derived from their normal cellular counterparts the protooncogenes - by gain-of-function mutations [ 17,27,67], or tumor suppressor genes, which are inactivated as a consequence of lossof-function mutations [go, 1401. Below we focus on the oncogenes since
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more knowledge of their action in signal transduction is available as compared to the recently discovered tumor-suppressor genes. Oncogenes are defined by their ability to confer a transformed phenotype on cells in tissue culture and make them tumorigenic in vivo, and have been identified through their occurrence in the genome of acutely transforming R N A and DNA tumor viruses [17]. These oncogenes arise from normal genes, called protooncogenes, encoding for normal cellular counterparts like growth factors, their receptors and signal transducing molecules. In fact, a large number of signal transduction components have been identified by virtue of the presence of their encoding genes in transforming viruses. The viral integration, amplication, chromosomal translocation, or mutation of their encoding genes may give rise to cancer, as these oncogenes may thus constitutively activate or derange the mitogenic pathway of the growth factors [ 1,17, 27,671. Therefore, the elucidation of the mechanism of action of these growth factors has emerged as one of the fundamental problems in cancer research and may prove to be a crucial prerequisite for understanding the causes underlying the unrestrained proliferation of cancer cells. The oncogene products can be classified according to their function (Fig. 20):
1. Growth factors - Several oncogenes encode proteins that are secreted from the cell and are, or act like, growth factors. Examples of these oncogenes are sis which encodes a protein highly similar to the PDGF-B chain, int-2 (FGF-3), hst (K-FGF), and FGF-5, which all encode an FGF related protein, and Wnt-l (or int-1). The oncogenic potential of these proteins is due to overexpression of their encoding genes, thus causing the autocrine overstimulation of their cell surfaceor intracellular-localizedreceptors. This may be accomplished either by the adoption of the encoding gene in the retrovirus or by integration of the provirus in the encoding gene. 2. Receptor tyrosine kinases - A large number of oncogenes encode mutant forms of growth factor receptor tyrosine kinases. Examples of the protooncogenes are erbB which encodes the EGF-R; neu (or erbB2 or HER2) which encodes the receptor for N D F ; m which encodes the CSF-1-R; met which encodes the HGF-R; trk which encodes the NGF-R; trkB which encodes a receptor for BDNF and NT-3; trkC which encodes a receptor for NT-3; kit which encodes the receptor for SLF;flg and bek which encode different FGF-Rs; and eph, elk, eck, ret, sea, and ros-1, all which encode receptors for unknown ligands.
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General Mechanistic Parrerris I
I EXTRACELLULAR
Fl SH2 I
o
SH,
o
NROSINE KINASES
yes
SER/THR KINASES
GTP BINDING1 GTPaser
ASSOCIATED OR MEMBRANE-
blk
vav 7
akt
hck
c
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MOPLASMIC
k17
TRANSCRIPTION FACTORS
myc lyl-1
vav 1
for
jun
gli-1
etbA eVi-1
pbx
Hoa 2 4
re1 kl-3
myb els ski
0 0 0 NUCLEAR
ma1
cbl
Most of the oncogenic versions encode for a truncated or mutated receptor or are the result of amplification and overexpression of the protooncogene. For example, the oncogenic counterpart of the EGF-R is v-erbB which lacks the ligand binding domain, having a constitutively activated tyrosine kinase as a consequence, whereas the oncogenic potential of neu can be released by a point mutation in the transmembrane part or truncations in both the cytoplasmic and the extracellular domains, all resulting in a constitutively activated tyrosine kinase. Among growth factor-Rs, the most frequently implicated in human cancer have been members of the EGF-R family. The EGF-R gene is often amplified or overexpressed in squamous cell carcinomas, astrocytoma, and glioblastomas. The EGF-R homologous receptor, the neu/ c-erbB2IH ER2 protooncogene, is frequently overexpressed in adenocarcinomas of several tissues (e.g., breast, ovary, and stomach). This overexpression is correlated with the prognosis of the cancer. 3. Receptors without tyrosine kinase activity - A small number of protooncogenes encode receptors without catalytic activity such as mpl, which encodes a member of the hematopoietic growth factor receptor superfamily having a WSXWS box and conserved cysteines and a tandemly duplicated extracellular domain as described for the IL-3, LIF, and MG-CSF p chain, and mas, which encodes the seven membrane spanning G-protein-coupled angiotensin-R.
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The overexpression of mas, but also of serotonin or acetylcholine receptors, causes cell transformation and thus has oncogenic potential. mpl is probably oncogenic due to truncation of the receptor. 4. GTP bindinglGTPase proteins - A number of protooncogenes belong to the family of GTP binding GTPases, which encode either the small monomeric Ras proteins or the cy subunit of the heterotrimeric Gproteins. Examples are the members of the Ras family H-rm, K-ras, and N-rm, which are highly homologous and are named after the Harvey and Kirsten rat sarcoma viruses and neuroblastoma from which they were originally identified. Furthermore, some of the protooncogenes from this class encode for cy subunits of G-proteins such as gsp, which encodes an cy, subunit or gip, which encodes an cyi subunit. In addition, it is noteworthy that the oncogene dbl encodes a protein that specifically catalyzes the dissociation of GDP from rho, a ras-like GTPbinding protein, thus qualifying it as a GNRP. Since the vuv oncogene and the bcr gene share pronounced homology with dbl, these proteins may also function as exchange factors for Ras-like proteins. The most potent transforming oncogenes, and the most frequently detected oncogenes in tumors, are those of the ras family. Oncogenic mutations of ras are found in 30-50% of lung and colon carcinomas, in 90-95% of pancreas carcinomas, and in 30% of all human cancer [17]. The ras oncogene differs from its normal cellular counterpart by one or more point mutations. These mutations either reduce the rate of GTP hydrolysis on ras by blocking the stimulation of its GTPase activity by GAPS or increase the rate of nucleotide exchange [43]. Both mutations result in the accumulation of the ras protein in the GTP-bound active state. The gsp oncogene is found in some pituitary tumors in which its defect results in the constitutive stimulation of adenylate cyclase. 5. SH-2/SH-3 domain-containing proteins without catalytic activity - A large number of signal transduction relevant proteins such as members of the Src family of tyrosine kinases, GAP, PLC, and the p85 subunit of the PI-3-kinase contain the so-called SH-2 and/or SH-3 domains. The SH-2 domains in these proteins appear to be involved in the association of these proteins with autophosphorylated receptors, whereas the SH-3 domain appears to be involved in association with these proteins with the cytoskeleton. Besides these proteins which all display enzymatic activity, some protooncogene products have been found to encode for SH-2EH-3 domain-containing proteins which lack any catalytic activity. Examples of these protooncogenes are crk having one SH2 and one SH3 domain; nck, having one SH2 and three SH-3 domains; and vav, having one SH-2 and two SH-3 domains.
General Mechartistic Patterns
53
The oncogenic v-crk was shown to be complexed to several tyrosinephosphorylated proteins, and it has been proposed that v-crk induces transformation either by protecting the receptor tyrosine kinases from dephosphorylation by phosphatases or by enhancing the interaction of these receptors with their substrates. The recently identified vav protooncogene encodes for an intriguing protein that is a substrate for receptor tyrosine kinases, having sequence motifs commonly found in transcription factors such as a helix-loop-helix and leucine zipper-like domain similar to these motifs in the myc and max proteins, and a cysteine-rich segment proposed to represent a zinc finger [25,89,127]; see, however, [ 191. Furthermore, as mentioned before, homology has been found between the vav protooncogene and the protooncogene dbl, which encodes an exchange factor for Ras-like proteins [2,19]. The deletion of the helix-loop-helix motif causes the oncogenic activation of vav. 6. Membrane-associated tyrosine kinases - In addition to the receptor tyrosine kinases, a number of protooncogenes encode for nonreceptor tyrosine kinases, which are mainly membrane-associated but are also found in the cytoplasm or even in the cell nucleus. These nonreceptor protein tyrosine kinases include the structurally related members of the Src-family, i.e., the oncogenes src, yes, f g r , and the potential oncogenesfyn, blk, lck, lyn, and hck. Furthermore, the distinct tyrosine kinaseshs (or fes) and abl. The mutationally activated oncogenes deliver a continuous rather than a ligand-regulated signal. For example, in the oncogenic v-src a tyrosine is lacking which in c-src is phosphorylated in order to down-regulate the tyrosine kinase activity; thus this mutation results in enhanced kinase activity. The abl protooncogene can be converted into an oncogenic form by chromosome translocation, and it is thus fused with the bcr gene as found in certain forms of leukemia (chronic myeloid and acute lymphocytic leukemias). In the resulting fusion protein, it is thought that a phosphoserine residue from bcr interacts with a SH-2 domain in abl to activate its tyrosine kinase activity. Furthermore, deletion of the SH-3 region of the c-abl protooncogene activates the proteins transforming capacity. This SH-3 domain binds to a protein which is similar to bcr and a rho-specific GAP [30]. 7. Cytoplasmic serinekhreonine kinases - Besides the kinases having tyrosine specificity, a number of protooncogenes encode cytoplasmic protein serinekhreonine kinases. This group of protooncogenes includes raf (mil), pim-I, mos, cot, and akt. The ruf kinases include three
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members, ruf-1, A-ruf, and B-ruf, which are highly homologous and all three have oncogenic potential. The oncogenic forms of raf have all lost their regulatory N-terminal sequences, leading to the permanent activation of the kinase. Interestingly, the retroviral oncogene v-akt, which is most closely related to PKC, has a SH-2 like domain which may couple this serinelthreonine kinase to autophosphorylated receptor tyrosine kinases [8]. 8. Transcription factors - Protooncogenes in the final group encode nuclear proteins of which many have been shown to be transcription factors. Among these protooncogenes are the members of the myc family (myc, N-niyc, and L-myc), lyl-1, and ski, which contain a helix-loophelix motif in their DNA-binding domain; fos andjun, which both have a leucine zipper and basic DNA-binding domain and are members of the AP-1 transcription factor family; evi-I, gli-I, and erbA, which encodes the nuclear thyroid hormone receptor, all three of which have a zinc finger motif in their DNA-binding domain; and furthermore, pbx and Hox2.4, both of which encode homeobox genes, re1 which shares homology with the p50 subunit of the NF-KBcomplex, bcl-3 which can inhibit the p50 subunit of the transcription factor NF-KB, and the tranand cbl. scription factors myb, ets, ski, mf, The oncogenic properties of v-fos and v-jun are thought to involve fusion of the protooncogenes to a strong retroviral promoter, resulting in overexpression of their encoded proteins, and as a consequence, the aberrant transcriptional activation of their target genes. Furthermore, some mutations are known to involve the negative regulatory serine phosphorylation sites. The v-erbA oncogene encodes a thyroid receptor without thyroid binding but with an intact DNA-binding domain, thereby dominantly suppressing the transcription of inducible target genes, thus blocking differentiation. The oncogenic activity of v-re1 is probably due to its ability to act as a dominant negative mutant as a consequence of its truncation, thereby suppressing the transcriptional regulation of target genes by the normal NFKB-re1 complex. It is clear that further studies on the mechanisms of signal transduction by these (proto-)oncogenes may help new insight to be gained into the regulation of proliferation and differentiation of both normal as well as tumor cells, which seems likely to lead eventually to novel means of prevention, diagnosis, and treatment of cancer.
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Acknowledgements We thank Dr. Libert H.K. Defize, Dr. Paul J. Coffer, and Dr. Arie J. Verkleij for the helpful conversations and their critical reading of the manuscript; and the Department of Image Processing and Design (Biology), University of Utrecht, for the computer generation of the figures.
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Biomembranes Edited by Meir Shinitzky Copyright 0 VCH VerlagsgesellschaflrnbH, 1995
CHAPTER 2
Receptors for Neurotransmitters F. ANNE STEPHENSON'
and PHILIP G. STRANGE^
'Department of Pharmaceutical Chemistry, School of Pharmacy, 29-39 Brunswick Square, London, WClN lAX, UK 2Biological Laboratory, The University, Canterbury, Kent, CZ2 7NJ, UK
Contents 62 62 63 64
70 74 78
91 91
Abbreviations Introduction Ligand-Gated Ion Channel Receptors Nicotinic Acetylcholine Receptors 64 Peripheral nicotinic acetylcholine receptors 68 Neuronal nicotinic acetylcholine receptors 69 Serotonin, receptors 70 Adenosine triphosphate receptors GABA, and Glycine Receptors Glutamate Receptors G-Protein Linked Receptors 83 General structural features of G-protein linked receptors 86 Functional domains of G-protein linked receptors 90 Regulation of G-protein linked receptor activity Concluding Remarks References
F.A. Stephenson and P. G. Strange
62
Abbreviations CGS 21680
- (2-p-carboxyethyl)phenylamino-5‘-N-carboxamido-
CP 55940
- [ la,2P(R)5a]-(-)-5-(1,1-dimethylheptyl)-2-[5-hy-
DAMGO DPCPX
- Tyr-Dala-Gly-[NMePhe]-NH(CH2),0H - 1,3-dipropyl-9-cyclopentylxanthine
DPDPE GR113808
adenosine
droxy-2-(3-hydroxypropyl)-cyclohexylphenol]
- [DPen2, DPen’]enkephalin
- [ 1-[2-[(methylsulphonyl) amino]ethyl]4-piperidinyl] methyl-1-methyl-1H indole-3 carboxylate
GTI
- 5-hydroxytryptamino-5-0-carboxymethylglycyltyro-
ICI 11855
sinamide - erythro-DL-l-(7-methylindan-4-yloxy)-3-isopropylam inobutane-2-01
L365260
- 3R( +)-N-(2,3 dihydro- 1-methyl-2-oxo-5-phenyl- 1H-
NECA
1,4 benzodiazepine-3-yl)-Nf-3methylphenyl urea - 5’-N-ethylcarboxamidoadenosine
8-OH DPAT - 8-hydroxy-2-(di-n-propylamino)tetralin
SCH 23390
U 69593 YM091512
- 7chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-lH-3-
benzazepine-7-01 - 5 (Y ,7 a, - (-)- N -met h y I - N - [7 - ( 1- p y ro 11i d in y 1)- 1oxaspiro (4,5) dec-8-y 11benzene acetam i de - cis-N-(l-benzyl-2-methylpyrollidinyl-3-y1)-5-~hloro2-methoxy-4-methylamino benzamide
Introduction The interaction of a neurotransmitter with its receptor is a critical event in the normal function of a synapse. Neurotransmitter receptors are also key sites of action for many drugs used to treat brain disorders [45]. Therefore, understanding the nature and function of neurotransmitter receptors is of importance from both basic and clinical science perspectives.
Receptors for Neurotransmitters
63
From biochemical studies it became apparent that neurotransmitter receptors are integral membrane proteins with binding sites for the neurotransmitter; these binding sites are available extracellularly (synaptically), and the receptor also has the ability to signal to the inside of the cell. Biochemical, pharmacological and physiological studies also showed that on the basis of the speed and nature of the response produced, receptors for neurotransmitters could be divided into two broad classes: receptors associated with rapid changes in the permeability of the membrane to certain ions and receptors that lead to changes in certain second messenger molecules with the participation of guanine nucleotide (GTP) binding proteins (G-proteins). This broad division has been given a structural basis from the application of gene cloning techniques, and this chapter aims to summarize the present state of knowledge on these two families of receptors, the ligand-gated ion channel receptors (ionotropic receptors) and the G-protein linked receptors (metabotropic receptors). The information provided below on these two families of receptors is not exhaustive, rather the chapter outlines general principles. Some general references are given, however, that enable the reader to obtain more specific information.
Ligand-Gated Ion Channel Receptors For the ligand-gated ion channel receptors, the interaction of the neurotransmitter with the respective neurotransmitter receptor results within milliseconds in the opening of either a cation- or anion-selective integral ion channel. Depending on the selectivity of the channel, this results in depolarization or hyperpolarization of the recipient cell. Prolonged exposure to the neurotransmitter leads to a waning of the conductance changes, i.e., desensitization. Overall, there are fewer neurotransmitters that gate ion channels compared to those that activate G-protein coupled receptors; however, the number of receptor molecules may actually be more in number because of their heteromeric nature. The neurotransmitters that gate ion channels are, for excitatory responses, acetylcholine, glutamate, serotonin and adenosine triphosphate (ATP) and for inhibitory responses, y-aminobutyric acid (GABA) and glycine. With the exception of the ATP receptor for which at this time there are no primary structures available, all the ligand-gated ion channel neurotransmitter receptor proteins share the same overall predicted domain structure. At the sequence level, with the exception of the glutamate receptor
64
F.A. Stephenson and P. G. Strange
subfamily, they all share amino acid sequence similarity. The primary structures for all ion channel receptors, where known, are highly conserved across vertebrate species. The best characterized of the ligand-gated ion channels is the peripheral nicotinic acetylcholine receptor (nAChR). Therefore, in this section this protein will be discussed in detail. The other members of this group will then be discussed with respect to this prototypic receptor.
Nicotinic Acetylcholine Receptors Peripheral nicotinic acetylcholine receptors Peripheral nAChRs are found in vertebrates at the neuromuscular junction. They are characterized pharmacologically by their activation by the neurotransmitter, acetylcholine, and the classic agonist, nicotine. They are antagonized by d-tubocurarine and the a neurotoxins, in particular a-bungarotoxin isolated from the banded krait snake, Bungam multicinctus. nAChRs are cation-selectivechannelsand conduct Na+, K + and Ca2+ ions. The peripheral nAChRs are so well characterized because it was discovered that the electric organs of electric fishes such as the electric ray, Torpedo californica, and the electric eel, Electrophorus electricus, contain high concentrations of homogeneous nAChRs with the correct peripheral-type pharmacology. This abundant source of receptor together with the early availability of the high affinity ligand [1251] a-bungarotoxin facilitated peripheral nAChR purification, the cloning of nAChR genes and the study of their structure to, currently, a resolution of 9 A using electron image analysis. Peripheral nAChRs are heterologous, acidic membrane glycoproteins. They are composed of five polypeptide chains assembled as a pentameric complex a2&G. The high affinity acetylcholine binding site is localized to the a subunit, thus there are two binding sites per receptor oligomer. The nAChR was the first neurotransmitter receptor for which the genes were cloned due, in part, to serendipity, as described above and additionally, to the pioneering work of Numa and colleagues in Japan. The deduced primary structures of the nAChR subunits share structural features. Thus, the N-terminal sequence of each polypeptide is preceded by a short, mainly hydrophobic, sequence of 24 amino acids. This is characteristic of the signal peptide sequence which determines membrane insertion. The exact molecular weights of the Torpedo californica sub-
Receptors for Neurotransmitters
65
units are 50,116 (347 residues, CY subunit), 53,681 (469 residues, p subunit), 56,279 (489 residues, y subunit) and 57,565 (501 residues, 6 subunit). There is a high degree of homology in primary structure between the subunits, with an average of 40% amino acid sequence identity between all four chains. There is a closer homology between the CY and p polypeptides and between the y and 6 polypeptides, suggesting that the subunits originate from a common ancestral gene with a first branching for the CY and p chains and a second branching for the y and 6. Hydrophobicity analysis of the respective primary structures predicts that each subunit is polytopic. It is now generally agreed that there are four transmembrane segments - M1, M2, M3 and M4, respectively - each of at least 20 hydrophobic amino acids. The N-terminus of the protein is predicted to be extracellular. It is at least 200 amino acids in length and is hydrophilic in nature with consensus sequences for N-glycosylation. All the subunits contain, within this region, the cys-cys loop domain which is of unknown function but is characteristic of the ligand-gated ion channel superfamily. It has the conserved motif, Cys-X-X-X-X-X-X-hydrophobic-Pro-hydrophobic-AspX-X-X-Cys. The CY subunit is characterized by two adjacent cysteine residues, 192 and 193, which contribute to the high affinity acetylcholine binding site. Other amino acids within the CY subunit implicated in the high affinity acetylcholine binding siteare Tyr 93, Trp 149 and Tyr 190, thus suggesting a three-point attachment within this subunit class. The ion channel is thought to be formed by the transmembrane spanning regions with each of the subunit M2 domains forming the inner lining of the channel. The M2 amino acid sequence contains amino acids with hydrophilic side chains requisite for ion transport and, indeed, sitedirected mutagenesis of these residues showed decreases in ion channel conductances. The non-competitive antagonist of the nAChR, chlorpromazine, specifically labels residues Ser 262 of the 6 subunit M2 and both Ser 254 and Leu 257 of the 0 subunit M2. Cation selectivity of the channel is determined by the negatively charged groups, particularly between the M2 and M3 domains. Consensus amino acid sequences for phosphorylation by either protein kinase A, protein kinase C or protein tyrosine kinase are found within the cytoplasmic loops of the different respective nAChR subunits (see [49] for more detailed discussions). The salient features of the nAChR polypeptides are shown in Fig. 1. A more detailed discussion of peripheral nAChR structure can be found in [ 101 and [47].
F. A. Stephenson and P. G. Strange
66
a
192 C
Fig. 1 . Schematic representationof nAChR. a: Transmembraneorganizationof an nAChR o! subunit. Pertinent features are the large N-terminal extracellular domain, the four transmembrane regions 1-4, the hydrophilic cytoplasmic loop between transmembrane regions M3 and M4 and the Cys-Cys loop motif characteristic of all members of the ligandgated ion channel superfamily. T Sites of N-glycosylation; 0 Distribution of negative charges at the mouth of the channel. C-C 192,193 are the adjacent cysteines unique to all a subunits and thought to be involved in agonist binding; S 262 (6) and S 254 L 257 (both 0) are the residues in M2 labelled irreversibly by the channel blocker chlorpromazine. b: Receptor as viewed perpendicular to the plane of the membrane. Each of the segments represents a subunit. Reprinted from [42], with permission.
Receptors f o r Neurotransmitters
67
Fig. 2. Schematic representation to scale of the nAChR based on the three-dimensional structure determined by helical reconstruction from cryo-images of Torpedo californicu post-synaptic membranes. The figure shows a section of the nAChR through the plane of the membrane. Depicted are four of the five subunits. A part of the a-helical segment M2 from each subunit lines the narrow pore spanning the hydrophobic part of the membrane bilayer. Rings of negatively charged groups near or at the end of M2 are probably located adjacent to the regions where the pore widens and the channel is formed. Note that the majority of the protein is extracellular. Reprinted in part from [51], with permission.
The three-dimensional structure of the peripheral nAChR has been studied using electron image analysis of polymerized receptor molecules rapidly frozen and observed at low temperatures (i.e., nAChR cryoimages). Thus, as viewed perpendicular to the plane of the membrane, the nAChR appears as an 80 A diameter rosette with a central pit and five peaks of electron density symmetrically arranged around a central pore (Fig. 2). Each of these peaks is thought to be a subunit and the ordering around the rosette is apay6 in a clockwise direction. Viewed within the plane of the membrane, the nAChR appears as a cylinder of length 110 A which spans the bilayer but protrudes mostly on the extracellular surface and thus into the synaptic cleft. This view of the nAChR also shows that the molecule is virtually at right angles to the membrane. It was suggested that all the membrane spanning regions are CY helices, but more recent evidence is consistent with only the M2 as an a helix surrounded by a ring of continuous (3 sheet contributed by M1, M3 and M4 [51]. It is proposed that when the channel opens in the presence of neurotransmitter, the M2 transmembrane spanning helices tilt such that the hydrophobic amino acids within this region move from the central pore (i.e., closed state) into the space at the interface between the subunits thus creating the open conducting state.
68
F.A. Stephenson and P. G. Strange
Neuronal nicotinic acetylcholine receptors Neuronal nAChRs are found in the central nervous system and in autonomic ganglia. They are also cation-selective channels conducting Na', K + and Ca2+ ions, but they differ from their peripheral counterparts in both pharmacology and structure. Notably, the majority of neuronal nAChRs are insensitive to antagonism by a-bungarotoxin and thus are termed a-bungarotoxin-insensitive receptors whereas those still antagonized by the neurotoxin are a-bungarotoxin-sensitive,or alternatively, a-bungarotoxin binding proteins. The compound methyllycaconi tine also selectively antagonizes the a-bungarotoxin-sensi tive receptors, and interestingly, this compound has low affinity for peripheral receptors. Some responses are selectively antagonized by the neuronal neurotoxin k toxin, a minor component of Bungarus multicinctus venom. Additionally, neuronal nAChRs are generally more Ca2+-permeable than their peripheral counterparts [37]. With regard to their structure, only two types of neuronal nAChR subunits have been described, the a and the 0 subunits. The peripheral nAChR subunits have been designated a1 and 01; the neuronal genes identified to date are only of these two subunit types, but in contrast to peripheral nAChRs, multiple related genes have been identified (i.e., a2-a8 and 02-04). The nomenclature follows similarities in primary structures between subunits; a subunits all contain the adjacent cysteine residues identified in the peripheral nAChRs as being important for neurotransmitter binding, whereas 0subunits do not contain these amino acids, and they are referred to as non-a or structural subunits. The neuronal nAChRs are pentameric and the deduced primary structures share the same features and domain structures as the peripheral receptors, i.e., the predicted four transmembrane spanning domains and the cys-cys loop in the extracellular region. The subunit complements of the neuronal receptors are not known, but from expression studies it is predicted that generally they require the presence of an a and a 0 subunit in unknown ratios for correct receptor assembly and function. Both in situ hybridization and immunopurification studies propose that single neuronal nAChRs can be heterogeneous with respect to, at least, their a subunit complements. The a7 subunit is unique in that it will efficiently form homo-oligomers that are antagonized by a-bungarotoxin. Characterization of the other a0 cloned neuronal nAChRs has not so far revealed such clearly demarcated pharmacology, a1though graded differences in the potency of different nicotinic drugs and rates of
Receptors for Neurotrarrsmitters
69
desensitization have been reported. Selected properties of nAChR polypeptides are summarized in Table I. TABLE I
Characteristics of nAChR polypeptides Subunit
Classification
Characteristic features
a1
Peripheral
High affinity acetylcholine binding subunit
Coexpression of a and 0required for a bgtinsensitive channel activity;
Neuronal
a6 a7
Forms a bgt-sensitive homo-oligomers
a8
ARD
Invertebrate neuronal
01
Peripheral Neuronal
6
Coexpression of alflly6 required for channel activity
Y
Peripheral Peripheral
&
Peripheral
1
All termed structural subunits
(a1)2plyS (embryonic muscle/electric organ) (a1)~lsS (adult muscle)
Note: In this and subsequent tables, characteristics of each type of ligand-gated ion channel are summarized. More detailed information is found in the text, accompanying figures and the reference list.
Serotonin, receptors Serotonin, (5HT3) receptors are ligand-gated cation channels with permeability to Na' and K'. They have high affinity for the 5HT,-
70
F.A. Stephenson and P. G. Strange
selective antagonist compounds such as Ondansetron which is now used clinically for the treatment of cytotoxic drug-evoked emesis following anti-cancer chemotherapy. In the brain 5HT3 receptors are expressed at very low abundance; however, they are found at relatively high concentrations in several neuronal cell lines which is unusual for ligand-gated ion channels. Indeed, the 5HT3 receptor has been purified by affinity chromatography from NCB20 cells. It was shown that it has a size of 250,000 daltons and it is heteromeric consisting of two subunits with Mrs 54,000 and 38,000 [29]. More recently a cDNA encoding a 5HT3 receptor subunit was cloned by functional expression using the NCB20 cell line as the source of mRNA. The cDNA identified encoded a polypeptide with a deduced Mr 50,000 with all the features common to the ligand-gated ion channel superfamily [28]. Antibodies raised against the deduced primary sequence have shown that the 54,000 subunit is the protein product encoded by the cDNA. The cDNA when expressed in COS-1 cells formed 5HT-gated channel responses similar to those found in the NCB20 cell line [28]. Related 5HT3 receptor subunits have not been reported.
Adenosine triphosphate receptors Adenosine triphosphate (ATP) has recently been shown to function as a fast-acting neurotransmitter mediating excitatory responses in the peripheral and the central nervous systems (reviewed in [3]). ATP-gated channels are permeable to Na', K + and Ca2', and they are classified pharmacologically as the P,, purinoceptor by the relative potencies of different ATP analogues. The ATP receptor of the rat vas deferens has a sedimentation coefficient of 12.1 S which is a similar value to that found for other ligand-gated ion channels. The P2x purinoceptor ligand, [3H]a,P-methylene ATP, specifically photoaffinity labels a M, 62,000 molecular weight species which has thus been identified as the ATPbinding subunit [6]. It is predicted that the ATP receptors form heteromeric receptors which share structural similarities with nAChR and other members of the ligand-gated ion channel superfamily.
GABA, and Glycine Receptors GABA, and strychnine-sensitive glycine receptors are both ligandgated chloride channels that mediate inhibitory responses. Multiple genes
71
Receptors for Neurotransmitters
encoding these two types of receptor have been cloned, and analysis of the respective deduced primary structures has revealed that they share motif and amino acid sequence similarity with nAChR subunits. But, in contrast to the nAChR subunits which have an enrichment of negative charges that are involved in cation binding at the mouth of the channel, GABAA and glycine receptor subunits that coassemble to form anion channels have positively charged amino acids in the homologous positions. The overall amino acid sequence similarity between GABAA, glycine and nAChR polypeptides is 15% whereas between GABA, and glycine receptor subunits, the similarity rises to 50%. Summaries of the known respective subunits are given in Tables I1 and 111.
-
-
TABLE II
Characteristics of GABA, receptor polypeptides Subunit type
Comments
CYl a2
Type I BZ pharmacology in a1Plyl combinations Type I1 BZ pharmacology in a2Ply2 combinations Type I1 BZ pharmacology in ( ~ 3 0 1combinations ~2
(113
CY4
a5 016 Pl 02
03
P4 Yl Y2 Y3
a40272 receptors have high affinity for Ro 15 4513 only Type I1 BZ pharmacology and reduced Zolpidem affinity a6P2y2 receptors have high affinity for Ro 15 45 13 only
Splice variant found in human Splice variant 04' found in chick crl02yl combinations bind BZ agonists only This subunit confers a complete spectrum of BZ activities with ~ $ 3combinations; confers Zn2+insensitivity; splice variants 72s and y2L found This subunit confers a complete spectrum of BZ activities with a0 combinations
6
Enriched in cerebellar granule cells
P
Identified in the retina; forms robust homo-oligomers that are bicuculline-insensitive
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F.A. Stephenson and P. G. Strange
TABLE III
Characteristics of glycine receptor polypeptides Subunit type
“‘1
Comments ~
~~~
High affinity glycine binding subunit
Splice variant a l L
“2
Forms strychnine-sensitive glycine gated homo-oligomers
Developmental1y-regulated splice variant. Neonatal form is strychnineinsensitive
P
Structural subunit
Confers picrotoxin insensitivity on expressed (“0) receptors
Like the neuronal nAChR, only two subunit types, (I! and 0,have been identified for the glycine receptor, and within each subunit class multiple isoforms have been described. The (I! subunit contains the high affinity strychnine and glycine binding site. Mutagenesis studies have suggested that agonist binding to the (I! subunit occurs within the same domain as for high affinity acetylcholine binding to the nAChR. So far the only characteristic of the 0 subunit is that, when expressed in combination with an (I! subunit, it renders the channel insensitive to blockade by the classical GABA, receptor non-competitive antagonist, picrotoxin. The 0subunit may have a structural role analogous to that of the 0subunit of neuronal nAChRs. For the GABA, receptor the situation is more complex, with the identification of five distinct subunit classes (I!,0,y, 6 and p and their respective isoforms. The (I! subunits are specifically photoaffinity labelled with the anxiolytic drugs, the benzodiazepines, which are allosteric regulators of GABAergic function (see below). Subunit classification for both receptors is based on amino acid sequence identity with at least 75% identity between isoforms of the same subunit class which falls to the level of 35 % in pair-wise comparisons between different subunit types. Within subunit types, the divergent regions between isoforms are found
73
Receptors for Neurotransmitters
I
His 101 Required for 62 agonisl binding
313 319 Y2L insert
Splice variant in p 3 Gly 201
Required foi Type I BZ seleclivily
Fig. 3. Diagrammatic representation and interrelationship of important domains of mammalian GABA, receptor polypeptides. The numbered amino acid sequence of the bovine GABAA receptor subunit is shown, highlighting significant residues and regions within this and other GABAA receptor polypeptides. Arrowhead, consensus sequences for N-glycosylation; Ml-M4, transmembrane domains; Cys-Cys, conserved extracellular motif common to all members of the ligand-gated ion channel superfamily except the ionotropic glutamate receptors.
1
NH24
v
220
J
138c-c152
41 83 tr2Alu28
421
%8? 7%
M1
M2
Determlnanta of hlgh affinity strychnine binding
M3
325 n1L
misubunit
M4
h
333
Fig. 4. Diagrammatic representation and interrelationship of important domains of mammalian glycine receptor polypeptides. The numbered amino acid sequence of the rat glycine a1 subunit is shown, highlighting significant residues and regions within this and other glycine receptor polypeptides. Arrowhead, consensus sequences for N-glycosylation; Ml-M4, transmembrane domains; Cys-Cys, conserved extracellular motif common to all members of the ligand-gated ion channel superfamily except the ionotropic glutamate receptors.
in the predicted cytoplasmic loop domains and at the extreme N- and C-termini. Chemical cross-linking studies showed that the quaternary structure of the glycine receptor is a pentamer. The quaternary structures
74
F.A. Stephenson and P.G.Strange
of the GABAA receptors have not been determined, but it is generally assumed that, like other members of the ligand-gated ion channel superfamily, they are pentameric. For both receptors the in vivo subunit complements of the pentameric structures are not known, but it is possible to summarize findings related to this question. Each subunit mRNA has its own unique pattern of temporal and spatial expression. For the glycine receptors, an a subunit expressed alone can form glycine-gated chloride ion channels in contrast to single p subunit expression which suggests the existence of homomeric a subunit-containing and a0 subunit-containing heteromeric receptors (in unknown ratios and with the possibility in both cases of a subunit heterogeneity). For the GABAA receptors, although each subunit can form a homomeric agonist-gated chloride channel, only the p subunit forms channels with high efficiency, thus suggesting that only this subunit class may occur as a homo-oligomer in vivo. In expression studies the presence of an a, and y subunit is required for a GABA-gated response with full benzodiazepine pharmacology. The a variant receptors in a p y 2 combinations display different benzodiazepine subtype pharmacologies. Thus native receptors are thought to consist predominantly of, for example, alp2/3y2. (@2/3and y2 are specified because they are the most abundant mRNAs of their respective subunit classes.) Immunoaffinity purification studies isolating native receptor subpopulations from mammalian brain using isoform-specific antibodies support this. Additionally they have shown the presence of two a subunits per receptor oligomer (other subunit ratios are not determined). They have further shown the presence of different cx subunits within a single receptor, thereby increasing GABA, receptor structural diversity. Characteristic features of GABAA and glycine receptor subunits are given in Figs. 3 and 4. More detailed discussion of their properties can be found in Olsen and Tobin [33], Burt and Kamatchi [8] and Stephenson [42] for GABAA receptors and Betz [5] for glycine receptors.
Glutamate Receptors Glutamate receptors contain cation channels that conduct Na+ and K + , and some subtypes are also Ca2+-permeable. Until recently, the ionotropic glutamate receptors were the major class of ligand-gated ion channel for which there was no structural information. This changed, however, in 1989 with the cloning by functional expression of the first cDNA to encode a glutamate receptor subunit, GluR1, which is one of
Receptors for Neurotransmitters
75
TABLE IV Characteristics of ionotropic glutamate receptor polypeptides Subunit type
Pharmacology
Comments
GluRl (A)* GIuR2 (B)' GluR3 (C)' GluR4 (D)*
High affinity for AMPA
Flip and flop splice variants exist for each. GluR2 is RNA edited 100% in TM2. Cation selectivity is dominated by GluR2, e.g., GluRl/GluR2= impermeable GluRl =Ca2+ permeable
GluR5 (01)' GluR6 (02)' GluR7 (03)'
High affinity for kainate; forms homooligomers gated by glutamate
RNA edited in TM2 RNA edited in TM1 and TM2 Forms robust homo-oligomers
High affinity for kainate. Will not form homomeric glutamategated channels
Expression studies suggest heteromeric receptors formed with GluR5-7
NMD A-selective
Seven splice variants found; High affinity MK8Ol binding subunit
NMDARl (rl)* NMDAR2A (&I)* NMDAR2B NMDAR2C NMDAR2D
*Letters in brackets show alternative nomenclature. AMPA=or-amino-3-hydroxy-Smethyl-4-isoxazolepropionate. NMDA =N-methyl-D-aspartate.
the non-NMDA receptor pharmacological subclasses [22]. The identification of related genes followed, and then in 1991, again via cloning by functional expression, the first cDNA encoding an NMDA glutamate receptor subunit was identified [30]. Amino acid sequence similarity between the non-NMDA (e.g., GluR1) and NMDA receptor subunits is of the order of 11% identity and of 36% similarity, and they can be considered as two subfamilies (see below). Thus, in current nomenclature based upon pharmacological specificity, the non-NMDA receptors comprise the AMPA and the kainate receptors whereas, so far the NMDA receptors stand alone. Unexpectedly, although all the glutamate receptor gene products identified share the same domain organization as the members of the ligand-gated ion channel superfamily described above, at the level of amino acid sequence there is little similarity
F.A. Stephenson and P. G. Strange
76
889
1
NH2 {
j
j
I
GluRl
Flip/ Flop exons 7421\793 R586'in 2 CJ in1,3,4 890
GIuR5
935 KA1
NH2
1
920
1 ,4b>
h
191 Insertion
NMDARl Deletion
Important for Ca2+ permeability
1
NHZi
v
V V
V
V
111
I
1445
I
NMDARPA
N Important for ~ g 2 block +
Fig. 5. Diagrammatic representation and interrelationship of important domains of ionotropic glutamate receptors. The numbered amino acid sequence of the rat subunit for each subtype of glutamate receptor subunit is shown, highlighting significant residues and regions within the sequence depicted and related subunits. Arrowheads, consensus sequences for N-glycosylation; the positions of the predicted transmembrane spanning regions M1-M4.
between them even within the predicted transmembrane spanning regions. The predicted subunit sizes of all the glutamate receptor subunits are significantly larger than for other ligand-gated ion channels and vary from 99,000 up to 160,000 daltons (see Table IV and Fig. 5). Further,
77
Receptors for Neurotransmitters
in comparing divergent amino acid sequence regions between isoforms of one subunit type, again differences are found. In general, for the ligand-gated ion channels described above, it is the predicted N-terminal region where the primary structures are conserved, whereas for the glutamate receptors, the converse is found with the putative intracellular domain being highly conserved. Despite the difference in primary structures with nAChRs, glutamate receptors are generally considered to be heteromeric pentamers. For the non-NMDA receptors this has been suggested for native proteins by chemical crosslinking between receptor subunits in synaptic membranes [55].Coexpression of different combinations of non-NMDA receptor subunits and the two types of NMDA receptor subunits further supports their heteromeric nature. The ionotropic glutamate receptors have the potential to yield the most extensive structural diversity within the family of ligand-gated ion channel receptors. This is not only because of the number of subunit types that may coassemble but also because extensive alternative splicing of their genes occurs, and uniquely, that RNA editing of some of their genes has been found (summarized in [41]). For example, GluR1-4 can exist in two forms denoted “flip” and “flop. These arise by alternative exon usage of a segment of 38 amino acids in the intracellular loop region. Both forms are differentially expressed throughout development as shown by in situ hydridization, and they confer differential functional properties on cloned receptors. The first example of RNA editing of a brain protein was described for the GluR2 gene. In the predicted M2 region of this gene, there is a base change which, when translated, results in the transition from glutamine to arginine (Q+R). This has a profound effect on the permeability properties of the channel. Thus the presence of GluR2 dominates the selectivity of the channel in coexpression studies. The edited form of GluR2, i.e., R-containing, is Ca2+ impermeable whereas an R-Q point-mutated or unedited GIuR2 is Ca2+ permeable. In practice, the GluR2 has always been found in the edited state. RNA editing also occurs at this site for GluR5 and GluR6, but here both edited and unedited forms are found for endogenous mRNAs. Recently RNA editing at two points in M1 was described for GIuR6. For the NMDA receptor, so far two subunit types have been described. These are the NMDARl subunit, of which there is only one isoform - but it can exist in seven alternative splice forms - and the NMDAR2 subunits A-D. The level of amino acid sequence similarity between these subunit types is low and only 15% . Detailed discussions of ionotropic glutamate receptors can be found in [32,41].
-
78
F.A. Stephenson and P. G. Strange
G-Protein Linked Receptors Members of this large family of receptors were originally recognized from their relatively slow effect on second messenger systems, e.g., adenylyl cyclase. Subsequently, it was realized that for these kinds of receptors the signalling systems consist of a receptor protein, a guanine nucleotide binding protein (G-protein) and an effector molecule (e.g., adenylyl cyclase). In the past few years with the isolation of some of these species and the application of gene cloning techniques to these G-protein linked receptor systems, the amount of knowledge about these systems has greatly multiplied. It is now clear that G-protein linked receptors constitute a large family of related gene products, and this part of the chapter will concentrate on the structures and properties of these receptor proteins. Much of the discussion will concentrate on the &-adrenergic receptor. This is not only a typical G-protein linked receptor but also was one of the first of this class of receptor to be purified [4] and cloned and an amino acid sequence obtained [ 121. It has therefore been extensively analyzed, and it can be viewed as a prototype for other members of this group of receptors (see [24,34] for recent reviews). Where there are differences in detail, these will be noted between the &-adrenergic and other G-protein linked receptors. The G-proteins themselvesare heterotrimeric proteins consisting of cup and y subunits (see also Chapter 00). There are at least 17 different cy subunits known [39] and four and four y species have been recognized [26]. The effectors that are regulated by these receptors are adenylyl cyclase, phospholipase C, K + channels and Ca2+ channels. Multiple subtypes of adenylyl cyclase and phospholipase C are now known [11,25]. Some specificity has been recognized in the interaction of particular receptor subtypes with G-protein and effector isoforms, but it must be clear that these signalling systems afford very considerable complexity. Table V summarizes some of the properties of the G-protein linked receptors with amino acid sequences determined by gene cloning. In Table V the receptor species are grouped according to their ligands; and for each receptor the size of the protein portion of the receptor subtype, its signalling system, and radioligands suitable for ligand-binding assays are given.
79
Receptors for Neurotrarisniitters
TABLE V Principal G-protein linked receptors for neurotransmitters In this table the principal families of G-protein linked receptors for neurotransmitters and their subtypes are given based on the Receptor Nomenclature Supplement, 1993, Trends in Pharmacological Sciences [54]. Other G-protein linked receptors have been identified and the amino acid sequence determined for substances that are not neurotransmitters. These are not shown here but can be found in [54]. For each neurotransmitter receptor family the receptor subtypes and the size of the corresponding protein are given where this is known. The number of amino acids refers to the human sequence unless otherwise stated. The principal effector pathways and one radioligand suitable for use in ligand binding assays are also given. For more details refer to [54]. CAMPrefers to effects on adenylyl cyclase, IP,/DAG refers to effects on phospholipase C.
Receptor family
Adenosine
Receptor subtype
A1
Amino acids
326
409
328
Radioligands
[3HJDPCPX
[3H]CGS21680
[3H]NECA
Principal effectors
CAMP .1. K+ channel t Ca2+channel .1.
CAMP t
CAMP t
A20
Adrenergic ~~
~
alA
alB
alc
alD
-
515 (rat)
466 (bovine)
560 (rat)
[3H]prazosi~i (non-selective) IP?/DAG
IPq/DAG
ZP,/DAG
-
Adrenergic
01
P2
P3
477
413
402
[3Hlbisoprolol
[3H]IC1118551
[ 125]iodocyanopindolo1
CAMP t Ca2+ channel t
CAMP t
CAMP t
F.A. Stephenson and P. G. Strange
80 Bombesin BBI
BB2
390
384
[ ‘251]-[Tyr4]bombesin
[12’Y]
IP,/DAG
IPSIDAG
[DTyr6]bombesin6-13 methylester
Bradykinin
Cannabinoid
-
Bl
B2
-
364
472
[3HlBK, - 8
[I2’I] [Tyr8]BK
[3H]CP55940
-
IPJDAG
CAMP I-
Cholecystokinin CCK,
CCKB
444 (rat)
447
[3H]devazepide
[3HjL365260
IP3/DAG
IP3/DAG
Dopamine Dl
D2
D3
D4
D5
446
443
400
387
477
I3HI-
l3HI-
i3~1-
i3~1-
I3HI-
SCH22390
YM091512
YM091512
YM091.512
SCH23390
CAMP t
CAMP IK+ channel t Ca2+ channel I-
-
CAMP I-
CAMP t
GABA
Glutamate (metabotropic)
GABA,
mGluRl
mGluR2 mGluR3
mGlu%
mGluR5
mGluR,
1199 (rat)
872 (rat)
912 (rat)
1171 (rat)
871 (rat)
IP,/DAG
CAMPI- CAMP& CAMPI- IP3IDAG
CAMP IK+ channel t Ca2+ channel I-
879 (rat)
-
Receptors for Neurotransmitters
81
Histamine HI
H2
H3
491 (bovine)
359
-
[3H]mepyramine
[3H]tiotidine
[3H]N-crmethylhistamine
IP,/DAG
cAMP t
-
5-h ydrox ytryptamine ~WIA
5m1 B
~HTID
~HTIE
~HTIF
42 1
386 (rat)
377
365
367 (mouse)
[3H]8-OHDPAT
[1251]GTI
[1251]GTI
[3H]5hydroxytryptamine
[3H]LSD
cAMP 4
cAMP C
cAMP C
CAMP 4
CAMP C
K + channel t
5-h ydrox ytryptamine ~ H T ~ A
~ W B
47 1
479
[3H]ketanserin IP,/DAG
~HT~c 458
5m4
[3H]5-hydroxytryptamine
[3H]mesulergine
[3H]GRl13808
IP,/DAG
IP3/DAG
cAMP t
-
Muscarinic acetylcholine Ml
M2
M3
M4
M5
460
466
590
479
532
[3~pirenzepine
-
-
-
-
cAMP 4 K + channel t
IP3/DAG
CAMPC
IP3/DAG
[3H] (-)-Nmethylscopolamine (non-selective) IP3/DAG
F.A. Stephenson and P. G. Strange
82
Neurotensin
Neuropeptide Y
-
Yl
y2
384
-
[1251]NPY
[ 1251]NPY
cAMP .C
424 (rat)
cAMP C Ca2+ channel C
IP,/DAG cAMP C
Opioid
Cr
6
K
-
372 (mouse)
-
[3H]DAMG0
[3H]DPDPE
[3H]U69593
cAMP C K + channel t
CAMP 4 K+ channel t
Ca2+ channel 4
Somatostatin SSI
ss2
ss3
ss4
391
369
418
383 (rat)
K+ channel t
CAMP C
CAMP .1.
[1251]-[Tyr'']SS (non-selective)
-
Ca2+ channel C
Tachykinin NKl
NK2
NK3
407
398
468
[3H]BH [Sar9,Met(02)'']SP
[3H3NKA
[3H]senktide
IP,/DAG
IP,/DAG
IP,/DAG
Receptors for Neurotransnritters
83
Vasopressidox ytocin '1,
Vl B
v2
OT
394 (rat)
-
371
388
-
[3H]d[Val4]AVP
[ 251]d(C€€& [Tyr (Me)2, Thr4, Om*, Tyr9NH2]OT
IP3/DAG
CAMP t
IP,/DAG
IP,/DAG
'
Vasoactive intestinal polypeptide VIP
GRF
PACAP
Secretin
359 (rat)
-
-
449 (rat)
"2SI]VIP
[ 251] GRF
['251]PACAP
[12sI]secretin
cAMP t
cAMP t
CAMP t
CAMP?
General structural features of G-protein linked receptors When the methods of gene cloning were applied to these receptors, several features became apparent. First, although subtypes of receptors for particular neurotransmitters had been described using pharmacological and biochemical approaches, the extent of the subtypes uncovered by gene cloning was much greater. Generally, in this family of receptors diversity is achieved by multiple genes coding for distinct receptor subtypes, but there are also examples of diversity based on alternative splicing of a common gene, e.g., D2(short)/D2(long) dopamine receptors [18], mGluRlalP receptors [32]. Second, for all G-protein linked receptors, the amino acid sequence, when subjected to hydropathy analysis, was found to contain seven stretches of hydrophobic amino acids long enough to form transmembrane a-helices. It is assumed, therefore, that the structures of these receptors are composed of seven transmembrane spanning a-helical segments linked via loops of protein outside the membrane with an extracellular amino terminus and intracellular carboxyterminus [9] (Fig. 6 ) . There is some experimental support from biochemical [ 141 and immunological studies [53]for this layout of the sequence with respect to the membrane.
I
F.A. Stephenson and P. G. Strange
84
r-----NH2
out ---Membrane
----
in
-------
COOH
Fig. 6. A two-dimensional model for the generalized structure of a G-protein linked receptor. The model shows the disposition of the amino acid sequence with respect to the membrane. Receptors differ in the size of the extracellular amino terminal section, the third intracellular loop and the carboxy terminal region; see the text for details of this variation which is indicated in the figure by broken lines.
When the amino acid sequences of receptor subtypes for one neurotransmitter are compared, considerable homology is seen (see, for example, [l]), and this homology is highest in the putative membrane spanning regions, e.g., for the dopamine receptors, D2 and D, dopamine receptors are 75 % homologous [40] and the D4dopamine receptor is 28 % homologous to D,, 41 % homologous to D2 and 39% homologous to D, [52]. There is also considerable homology between receptors for similar neurotransmitters, e.g., within the transmembrane regions the D, dopamine receptor is 48 % homologous with the a2 adrenergic receptor, 41 % homologous with the 5HT,, serotonin receptor, 27% with the MI muscarinic receptor, 26% with the NK, tachykinin receptor and 32% homologous with the TRH receptor [7,46]. For some G-protein linked receptors, however, the homology with other family members is negligible, and it seems that within the G-protein linked receptor superfamily subfamilies can be recognized as follows: Group 1. These receptors are 300-400 amino acids long with a relatively small amino-terminal extracellular region and bind small ligands, e.g., CY and p adrenergic, dopamine, muscarinic acetylcholine, opiate, tachykinin and hypothalamic releasing hormone receptors. The
Receptors for Neurotrarisniitters
85
ligands for these receptors are thought to bind in a cavity formed by the transmembrane regions (see below). As indicated above, homologies between receptors in this class tend to be greater for related ligands. It has been proposed on the basis of homology comparisons that receptors for cationic amines and for peptides may comprise separate structural subgroups [23], and evidence cited below indicates that these subgroups may be reflected in the structure of the ligand-binding sites. There has also been some interest in comparing the length of internal loops of these receptors. The G-proteins must interact with the internal face of these receptors so it might be possible to discern differences in the internal loops of different receptors related to interaction with different G-proteins. From early studies on cationic amine receptors, it seemed that those interacting with G, had relatively shorter third intracellular loops and longer C-terminal tails, and the converse was true for those receptors that coupled to GJG, [9]. With the description of other receptor sequences, this correlation seems less clear. Receptors for peptide neurotransmitters have rather short third intracellular loops, but the functional relevance of this characteristic is unclear. Group 2. The G-protein linked receptors for the neurotransmitter glutamic acid (metabotropic glutamate receptors) are 900-1200 amino acids in length and show negligible homology with the Group 1 receptors [32]. A motif of seven transmembrane spanning regions is seen together with a very large extracellular amino terminal segment ( 600 amino acids) and a large carboxyterminal segment (- 350 amino acids). It is not known at present which part of the receptor sequence functions to bind the glutamate, but there are weak homologies in the extracellular amino terminal segment with the GluR1 subtype of the glutamate receptor (ion channel linked, Table IV), so this could contain the ligand-binding domain. Group 3. Receptors in this group are 400-600 amino acids long with a large amino terminal section. Ligands for these receptors are mostly not neurotransmitters but are mentioned here for completeness. They include: calcitonin, PTH, secretin and vasoactive intestinal polypeptide. Within the group there are significant homologies between receptors, but these receptors do not show amino acid sequence homologies with other groups of G-protein linked receptors. Group 4.This is a group of receptors for glycoprotein hormones (LH/CG, FSH, TSH) included here for comparison. These receptors are about 800 amino acids long with a large extracellular amino terminal
-
86
F.A. Stephenson and P. G. Strange
domain responsible for recognition of the hormone. The transmembrane spanning regions of Group 4 and Group 1 receptors show significant homology. Sites for post-translational modification of G-protein linked receptors have been identified. Within the extracellular amino terminal section of many sequences, potential N-glycosylation sites are found, and in some receptors a further site is found in the second extracellular loop. For the P2-adrenergic receptor evidence has been presented that two oligosaccharide chains are attached to the amino terminal extracellular portion of the receptor [43]. The oligosaccharides add about 16 kDa to the protein molecular weight of 65 kDa. The oligosaccharides are probably important in targeting the receptor to membranes during its biosynthesis; they do not directly alter the binding of ligands to receptors but may affect receptor/G-protein interaction. Sites for phosphorylation by protein kinases (A and C) have been identified in the third intracellular loop and C-terminal tail of the b2adrenergic receptor [48]. Phosphorylation is thought to be important in receptor desensitization and regulation (see below). For the P2-adrenergic receptor a cysteine residue has been identified in the C-terminal tail to which a palmitoyl group is added. The interaction of this palmitoyl group with the membrane may form a fourth intracellular loop. Similar cysteines have been recognized in the sequence of other G-protein linked receptors, so palmitoylation may be a more general phenomenon. For the P2-adrenergic receptor palmitoylation may have an important role in the ability of the receptor to couple to G-proteins, and the level of palmitoylation may be dependent on agonist stimulation [31].
Functional domains of G-protein linked receptors The two-dimensional model of a G-protein linked receptor in Fig. 6 is not very illuminating with regard to the true three-dimensional structure of these receptors. There is very little firm experimental information on the three-dimensional structures, and ideas in this area have been based on presumed analogies with the protein bacteriorhodopsin. This bacterial protein is not G-protein linked, but it does contain seven transmembrane spanning regions and is assumed to be related to rhodopsin, the G-protein linked light harvesting protein of the eye, which is in turn thought to be related to the G-protein linked receptors [15]. A highresolution three-dimensional structure of bacteriorhodopsin is available
Receptors for Neurotransmitters
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[20], and it has been assumed that the G-protein linked receptors are assembled in a similar manner. Thus the seven a-helical regions are likely to be bundled together to form a cavity and the folded structure secured by a disulphide bond between the first and second extracellular loops. This cavity then forms the binding site for ligands for Group 1 receptors (Fig. 7). There is some experimental evidence from fluorescence quenching studies that the ligand binding site of the &adrenergic receptor is within the membrane [50],and mutagenesis studies of several Group 1 receptors for cationic amines locate the ligand-binding region of the receptor about a third of the way in from the external membrane surface [44]. This may not be the case for Group 1 receptors for peptide ligands (see below). For G-protein linked receptors of Group 2 and 4, the ligand-binding sites may be located in the extracellular portions. Attempts have been made to generate three-dimensional models of Group 1 G-protein linked receptors (see, e.g., [21,27]). These are based on the three-dimensional structure of bacteriorhodopsin on to which is overlaid the sequence of suitable G-protein linked receptors. The models generated allow sensible predictions to be made about groups that may be involved in receptor-ligand interaction and allow images of the receptors to be generated. The validity of using bacteriorhodopsin as a model is unclear, and a recent study of the structure of rhodopsin [38] and theoretical analyses of G-protein linked receptors [2] suggest that the packing of the helices in bacteriorhodopsin and rhodopsin or the G-protein linked receptors may be subtly different (Fig. 8). Further information on the structure of the ligand-binding sites of these kinds of receptors may be obtained by identifying amino acid residues within the putative transmembrane a-helical regions that are highly conserved and which would have important functional roles. Mutagenesis studies can then be performed to probe their importance. Such consideration highlights: 1. An aspartic acid residue about two-thirds of the way down helix 11. This is conserved in many Group 1 receptors and seems to be important for maintaining the conformation of the receptor and also conferring Na+ sensitivity on ligand-binding for certain receptors. 2. An aspartic acid residue about a third of the way down helix 111. This is conserved in Group 1 receptors for cationic amines and is thought to provide the counter ion for the cationic amine. 3. A cluster of serine and cysteine residues (three in total) about a third of the way down helix V in receptors that bind catecholamines.
F.A. Stephenson and P.G. Strange
88
Fig. 7. The relative disposition of the transmembrane a-helices (shown as cylinders) in bacteriorhodopsin. The diagram is taken from [20] and is reproducedwith permission. The protein bacteriorhodopsin is thought to be related structurally to G-protein linked proteins (rhodopsin and the receptors) and so may provide a guide to the structure of the G-protein linked proteins as discussed in the text.
0
Fig. 8. Arrangements of the seven a-helices in bacteriorhodopsin and rhodopsin. The diagram (taken from [38] with permission) shows the arrangements of the a-helices seen as contours ill the projection density map of bacteriorhodopsin(1efi) and rhodopsin (right). The slightly different relative arrangements of the a-helices can clearly be seen.
Receptors for Neurotransmitters
89
These may be important in hydrogen bond interactions with the catechol hydroxyl groups. The importance of some of these residues has been investigated for P,-adrenergic, a,-adrenergic and D, and D, dopamine receptors and supports the role of these residues as hydrogen bond donors (see, e.g., [13,36]). What is not clear at present is whether pairs of these residues can function equally well or whether there is a favoured pair. Further careful mutagenesis studies will be required. 4. A series of hydrophobic aromatic residues (Phe, Trp) found in helices V and VI that provide stacking interactions with the catechol ring for catecholamine ligands and provide an hydrophobic pocket around the ligand-aspartate (see 2 above) ion pair [13,21]. These are just some of the interactions that have been highlighted between ligands and G-protein linked receptors, and they locate the ligand-binding site for catecholamine ligands about a third of the way in from the membrane, as indicated above. For the receptors that bind catecholamines, it is likely that quite different interactions will be involved for the non-catecholamine ligands at these receptors. Similarly, at receptors for peptides different interactions again may be involved [35], and preliminary mutagenesis studies suggest that the ligand-binding site for Group 1 peptide receptors may be near the external surface of the receptors and may involve parts of the external loops [ 16,171. The site of interaction of the receptor with the G-protein has also been extensively investigated, mainly for the P2-adrenergic receptor. Mutagenesis and deletion studies have shown that the second and third intracellular loops and the C-terminal tail contribute to form the interaction site of the receptor and G-protein. It is presumed that there is some specificity in this interaction with a particular receptor subtype interacting preferentially with a particular G-protein subtype and thus influencing a particular effector system. The amino terminal part of the third intracellular loop (12 amino acids approximately) and part of the second intracellular loop appear to determine the specificity of coupling. For example, exchange of a 12 amino acid stretch of the amino terminal segment of the third intracellular loop of the M, muscarinic acetylcholine receptor with the equivalent domain of the P-adrenergic receptor altered G-protein coupling specificity [56]. The carboxyterminal region of the third intracellular loop and the carboxyterminal tail are thought to be important in maximizing the efficiency of receptor/G-protein interaction [24]. Interaction between receptor and G-protein may depend upon amphipathic a-helical regions in the third intracellular loop of the receptor [34].
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F. A. Stephenson and P. G. Strange
Regulation of G-protein linked receptor activity The activity of G-protein linked receptors can be regulated. In the short term this represents desensitization after a challenge with an agonist; in the longer term this can represent down-regulation of receptors through loss of receptors from the surface of a cell. Again this has been most studied at the molecular level for the P2-adrenergic receptor, but it is assumed that common mechanisms apply for other G-protein linked receptors. Desensitization of the P2-adrenergic receptor, that is loss of the agonist response, occurs after agonist challenge for seconds to minutes. Desensitization seems to depend on phosphorylation of the receptor. For low concentrations of agonist phosphorylation by CAMP-dependent protein kinase in the third intracellular loop is important. This phosphorylation uncouples receptor and G-protein and provides a mechanism for the receptor generated CAMP signal to feed back and regulate receptor responsiveness. For high doses of agonist a second mechanism is important. The receptor occupied by agonist is susceptible to phosphorylation by a specific 0-adrenergic receptor kinase (PARK) which phosphorylates the agonist-receptor complex in its carboxyterminal tail. This phosphorylation does not interfere directly with receptor/(;-protein interaction, but an additional protein, P-arrestin, interactswith the PARK-phosphorylated receptor and disrupts receptor/G-protein interaction. Down regulation of P2-adrenergic receptors occurs after much longer (> 1 h) exposures to agonist. The initial phase of this response is a loss of receptors from the cell surface due to an increased rate of removal. This phase may be mediated by clathrin coated vesicles which then interact with the endosomal systems of the cell. The later phase (4-24 h) of down regulation is due to a reduction in the biosynthesis of new receptors and is predominantly caused by more rapid degradation of p2adrenergic receptor mRNA [ 191. Another phenomenon that has been observed which may be related to the processes of desensitizationldown regulation is sequestration. This is the rapid (mins) agonist-induced sequestration of receptor in a vesicular compartment associated with the plasma membrane. Although such sequestration could be involved in the desensitization or down regulation processes, recent evidence suggests that it is a separate phenomenon associated with receptor resensitization by dephosphorylation [57]. It seems that there may be a continuous cycling of receptors into this sequestered compartment where dephosphorylation can occur. The
Receptors for Neurotransmitters
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mechanisms leading to agonist induced sequestration are at present unclear.
Concluding Remarks It should be clear from this chapter that knowledge on neurotransmitter receptors has increased greatly in the recent past. Earlier biochemical and pharmacological studies combined with more recent molecular biological approaches for the characterizationof the molecular properties of these receptors has underlined their division into two broad families. Within each of these, subfamilies of receptors have been identified and their properties outlined above. These kinds of structural analyses of receptors have provided some insights into their respective functions. However, the understanding of the functional significance of the potential diversity of highly conserved receptors of one subtype particularly within the ionotropic receptor subclass is in its infancy. Further studies will provide increasingly detailed analyses of the structures of the receptors which will enable these more challenging questions to be addressed.
References 1 2 3 4 5 6 7 8 9 10
11 12 13
Attwood, T.K., E.E. Eliopoulos and J.B.C. Findlay, (1991), Gene 98:153-159. Baldwin, J.M., (1993), EMBO J 12:1693-1703. Bean, B.P., (1992), Trends in Pharmacol. Sci. 13:87-90. Benovic, J.L., R.G.L. Shorr, M.G.Caronand R.J. Lekowitz, (1984), Biochemistry 2 3 ~ 4 10-45 5 18. Betz, H., (1992), Quart. Rev. Biophys. 25:381-394. Bo, X., J. Simon, G. Burnstockand E.A. Barnard, (1992), J. Biol. Chem. 267: 1758117587. Bunzow, I.R., H.H.M. Van Tol, D.K. Grandy, P. Albert, J. Salon,M. Christie, C.A. Machida, K.A. Neve and 0. Civelli, (1988), Nature 336:783-787. Burt, D.R. and G.L. Kamatchi, (1991), FASEB J 5:2916-2923. Caron, M.G. and R.J. Lefkowitz, (1988), J. Biol. Chem. 263:4993-4996. Changeux, J.-P., J.-L. Galzi, A. Devillers-Thiery and D. Bertrand, (1992), Quart. Rev. Biophysics 25:395-432. Cockcroft.,S. and G.M.H. Thomas, (1992), Biochem. J. 288:l-14. Dixon, R. A.F., B.K. Kobilka, C.J. Strader, Benovic, J.L. Dohlman, H.G., T. Frielle, Bolanowski, M.A. Bennett, C.D., F. Rands, R.E. Diehl, R.A. Mumbord, E.E. Slater, I.S.Sigal, M.G. Caron, R.J. Lefkowitz and C.E. Strader, (1986), Nature 321:75-79. Dixon, R.A.F, I.S.Sigal and C.D. Strader, (1988), Cold Spring Harbor Symposia on Quantitative Biology, LIII 487-497.
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14 Dohlman, H.G., M. Bouvier, J. Benovic, M.G. Caron and R.J. Lefkowitz, (1989, J. Biol. Chem. 262:14282-14288. 15 Findlay, J.B.C. and D.J.C. Pappin, (1986), Biochem. J. 238:625-642. 16 Fong, T.M., M.A. Casciere, H. Yu, A. Bansal, C. Swain and C.D. Strader, (1993), Nature 362:350-353. 17 Gether, U., T.E. Johansen, R.M. Snider, J.A. Lowe, S. Nakanishi and T.W. Schwartz, (1993), Nature 362:345-348. 18 Giros, B., P. Sokoloff, M.P. Martres, J.F. Riou, L.J. Emorine and J.C. Schwartz, (1989), Nature 347:923-926. 19 Hadcock, J.R. and C.C. Malbon, (1993), J. Neurochem. 6O:l-9. 20 Henderson, R., J.M. Baldwin, T.A. Cesca, F. Zemlen, E. Beckmann and K.H. Downing, (1990), J. Mol. Biol. 213:899-929. 21 Hibert, M.F., S. Trumpp-Kallmeyer, A. Bruinvels and J. Hoflack, (1992), Mol. Pharmacol. 40:8-15. 22 Hollmann, M., A. O’Shea-Greenfield, S.W.Rogers and S. Heinemann, (1989), Nature 342~643-648. 23 Kieffer, B.L., K. Before, C. Gaviereux-Ruffand C. Hirth, (1992), Proc. Natl. Acad. Sci. USA 89:12048-12052. 24 Kobilka, B., (1992), Ann. Rev. Neurosci. 15:87-114. 25 Krupinski, J., T.C. Lehman, C.D. Frankenfield, J.C. Zwaagstra and P.A. Watson, (1992), J. Biol. Chem. 267:24858-24862. 26 Lefkowitz, R.J., (1992), Nature 358:372. 27 Livingstone, C.D., P.G. Strange and L.H. Naylor, (1992), Biochem. J. 287:277-282. 28 Maricq, A.V., A.S. Peterson, A.J. Brake, R.M. MyersandD. Julius, (1991), Science 254 ~432-437. 29 McKernan, R.M., N.P. Gillard, K. Quirk, C.O. Kneen, G.I. Stevenson, C.J. Swain and C.I. Ragan, (1990), J. Biol. Chem. 265:13572-13577. 30 Moriyoshi, K., M. Masu, T. Ishii, R. Shigemoto, N. Mizuno and S. Nakanishi, (1991), Nature 354:31-37. 31 Mouillac, B., M. Caron, H. Bonin, M. Dennis and M. Bouvier, (1992), J. Biol. Chem. 267:21733-21737. 32 S. Nakanishi, (1992), Science 258597-603. 33 Olsen, R.W. and A.J. Tobin, (1990), FASEB J 4:1469-1480. 34 Ostrowski, J., M.A. Kjelsberg, M.G. Caron and R.J. Lefkowitz, (1992), AM. Rev. Pharmacol. Toxicol. 32:167-183. 35 Perlman, J.H.., D.R. Nussenzweig, R. Osman and M.C. Gershengorn, (1992), J. Biol. Chem. 267:24413-24417. 36 Pollock, N.J., A.M. Manelli, C.W. Hutchins, M.E. Steffey, R.G. Mackenzie and D.E. Franck, (1992), J. Biol. Chem. 267:17780-17786. 37 Role, L.W., (1992), Current Opinion in Neurobiol. 2:254-262. 38 Schertier, G.F.X., C. Villa and R. Henderson, (1993), Nature 362:770-772. 39 Simon, M.I., M.P. Strathmann and N. Gautam, (1991), Science 252:802-808. 40 Sokoloff, P., B. Giros, M.P. Martres, M.L. Bouthenet and J.C. Schwartz, (1990), Nature 347: 146- 151. 41 Sommer, B. and Seeburg, P.H., (1992), Trends in Pharmacol. Sci. 13:291-296. 42 Stephenson, F.A., (1993), In: Neurotransmitter Receptors, F. Hucho (Ed.), Elsevier, Amsterdam, in press. 43 Stiles, G.L., J.L. Benovic, M.G. Caron and R.J. Lefkowitz, (1984), I. Biol. Chem. 259:8655-8663.
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44 Strange, P.G., (1991), Curr. Op. Biotech. 2:269-273. 45 Strange, P.G., (1992), Brain Biochemistry and Brain Disorders. Oxford University Press, Oxford. 46 Straub, R.E., G.C. Frech, R.H. Joho and M.C. Gershengorn, (1990), Proc. Natl. Acad. Sci. USA 87:9514-9518. 47 Stroud, R.M., M.T. McCarthy and M. Schuster, (1990), Biochemistry 29:1100911023. 48 Summers, R.J. and L.R. McMartin, (1993), 1. Neurochem. 6O:lO-23. 49 Swope, S.L., S.J. Moss, C.D. Blackstone and R.L. Huganir, (1992), FASEB J. 6 :2514-2523. 50 Tota, M.R., M.R. Candelore, R.A.F. Dixon and C.D. Strader, (1991), Trends Pharmacol. Sci. 12:4-6. 51 Unwin, N., (1993), Neuron 10 (Suppl):31-41. 52 Van Tol, H.H.M., J.R. Bumow, H.C. Guan, R.K. Sunahara, P. Seeman, H.B. Niznik and 0. Civelli, (1991), Nature 350:610-614. 53 Wang, H., L. Lipfert, C.C. Malbon and S. Bahouth, (1989), J. Biol. Chem. 264: 14424-1443 1 . 54 Watson, S.P. and D. Girdlestone, (1993), Receptor Nomenclature Suppl., Trends in Pharmacological Sciences Suppl:1-43. 55 Wenthold,R.J., N. Yokotani, K. Doi and K. Wada, (1991), I. Biol. Chem. 267501-
507.
56 Wong, S.K.F., E.M. ParkerandE.M. Ross, (1990),1. Biol. Chem. 265:6129-6224. 57 Yu, S.S., R.J. Lefkowitzand W.P. Hausdorff, (1993), 1. Biol. Chem. 268:337-341.
Biomembranes Edited by Meir Shinitzky Copyright 0 VCH VerlagsgesellschaflrnbH, 1995
CHAPTER
3
Receptors to Peptide Hormones S. INCERPI a d P. LULY Department of Biology, University of Rome “Tor Vergata, ” Rome, Italy
Contents 96 96 104 106 112
139 141 146
Introduction The Peptide Hormones Physical Parameters of Hormone-Receptor Interactions Kinetics of Hormone-Receptor interactions Mechanism of Peptide Hormone Action 112 The “second messenger” hypothesis 114 Hormone receptors and adenylate cyclase 115 Guanine nucleotide-binding regulatory proteins 119 The “dual messenger” hypothesis 122 Metabolism of inositol phospholipids 123 Inositol-l,4,5-trisphosphateand diacylglycerol as messengers 124 Guanine nucleotide regulatory proteins and phospholipid hydrolysis 125 Glycosyl-phosphatidylinositolhypothesis 128 Other phospholipids as possible sources of second messengers 129 Receptor phosphorylation 136 Internalization of hormone-receptor complexes 138 Cellular sensitivity to hormones: up and down regulation Evolution of Peptide Hormones and their Receptors Clinical Relevance of Hormone-Receptor Interactions References
96
S. hcerpi and P. Luly
Introduction Hormones are elaborated by a variety of tissues and distributed to all cells by the circulatory system (Fig. 1). However, only those cells containing binding proteins of high affinity for the hormone (i.e., the receptors) have the ability to respond. So the first concept that comes out is: the presence of the receptor (on the plasma membrane or inside the cell) makes a cell competent for that specific hormone. Endocrinology borrowed the concept of receptor from pharmacology , as pharmacology paved the way with descriptions of functional drugprotein interactions [3,132]. The term was adopted and used loosely, since for many years only correlative information was available to link receptor interactions directly to hormonal responses [84,100,106,128]. Only in recent years, with the isolation of pure receptors and the cloning of specific genes for hormone-induced proteins, has the functional role of receptors been unequivocally demonstrated [55,58,124]. Hormones are a group of substances that belong to different chemical classes but can be functionally divided on the basis of their mechanism of action: steroid and phenolic hormones freely permeate the cellular plasma membrane and interact with specific receptors in the cytoplasm and nucleus of target cells [4,107]; in contrast, polypeptide hormones and neurotransmitters first bind to specific receptor sites located on the plasma membrane of target cells [28,80,122].
The peptide hormones An updated compilation of the most important polypeptide hormones of vertebrates is reported in Table I. Some of them are “classic” hormones; others are, in a sense, not “true” hormones but factors (such as NGF, EGF, ANF, etc.), produced not by a typical endocrine tissue but by other tissues such as the heart, the nervous tissue, submaxillary glands, etc. [104]. Polypeptide hormones vary greatly in molecular size from a tripeptide to multi-subunit proteins (> 30 m a ) ; some of them are glycopeptides. The determination of the primary structure of bovine insulin by Ryle et al. [125] provided the first known molecular sequence of a hormone: a real cornerstone of both endocrinology and protein chemistry. Since then the primary structures of most of the known hormones have been established and a few have been extensively investigated. For example, X-ray crystallographic structures are available for several forms of insulin [ 161. Furthermore, analogues of many of the smaller hormones have been synthesized.
Receptors to Peptide Hormones
m
I Ca2+
97
receotor for secretion-
\
second essenger
(from outside or inside cell 1
E N D O C R I N E HORMONEPRODUCING CELL
endocrine
endocrine hormone secretion by exocytosis
t
capillary fenestration
&
m general blood circulation
1
rece tor for polypeptide e n d c r i n e hormone
c
second messenger
t Fig. 1. A classic scheme of hormone production in an endocrine tissue, release in the circulatory system, and effect on a distant target.
Hormones have been isolated from endocrine tissues by following the bioactivity associated with that tissue through a process of extraction and fractionation. Initially it was believed that the primary structure of insulin contained all the information needed for the formation of the secondary and tertiary structures. The failure of reduced insulin to regain bioactivity upon mild oxidation with air meant, however, that there had to be a larger precursor form of insulin. With the introduction of the radioimmunoassay technique, larger inactive forms of many polypeptide hormones were detected in human plasma. Finally, the techniques of gene expression provided incontrovertible evidence that some hormones are produced as larger, inactive precursors called “prohormones.”
Adenohypophy sis Posterior pituitary Adenohypophysis
Adenohypophysis
Adenohypophy sis Adenohypophy sis
Adenohypophysis (pars intermedia) Adenohypophysis
Adenohypophysis
Adrenocorticotropic hormone (ACT@
Arg-Vasopressin ( A W or ADH)
Follicle-stimulating hormone (FSH)
Growth hormone (GH)
Lipotropin (LPH) Luteinizing hormone (LH)
Melanocyte-stimulating hormone WH) Prolactin (PRL)
Thyroid-stimulating hormone (TSH)
Pituitary
Source
Peptide hormones: sources and effects
TABLE I
~~
Analgesic actions in CNS
Stimulates the secretion of thyroid hormone in thyroid gland follicles
Synthesis of milk constituents in mammary gland; stimulates testosterone production; mammary gland secretory cell differentiation
CNS functions, skin-darkening reaction
Fat mobilization, originates opioid peptides stimulates Leydig cells development and testosterone production (in male); stimulates corpus luteum and .progesterone production (in female) .
Modulates somatic cell growth: needs somatomedins, hyperglycemia, hepatic steroid metabolism, bone sulfation reactions
Stimulates development of ovarian follicle and secretion of estrogen; stimulates seminal tubules and spermatogenesis
Increases water reabsorption in kidney
Stimulates production of cortisol and dehydroepiandrosterone in adrenal cortex
Effects
(VIP)
Stimulates gallbladder contraction and bile flow; increases secretion of pancreatic enzymes
Stimulates gastric acid secretion and gastrin release CCK-like effects
Frog skin Frog skin
Bombesin
Caerulein
Skin
Inhibits gastric secretion; stimulates insulin secretion
Neurotransmitter in peripheral autonomic nervous system; relaxes smooth muscles; stimulates water and bicarbonate (ions) secretion in pancreas and gut (small and large intestine)
Duodenum
GI tract, nervous system
Gastric inhibitory peptide (GIP)
Vasoactive intestinal peptide
Increases secretion of gastric acid and pepsin; stimulates bile flow; many other effects
G cells in midpyloric glands in
Gastrin stomach antrum
Stimulates pancreatic acinar cells to release bicarbonate and water which elevate pH of duodenum
Duodenum when pH of content is less than 4.5
Secretin
Kidney
Cholecystokinin (CCK) (pancreozy min)
Analgesic actions in CNS
Adenohypophysis (pars intermedia) and CNS cells Acts on bone marrow to induce terminal differentiation, initiation of hemoglobin synthesis
Effects
Source
Erythropoietin
&endorphin
TABLE I, continued
Hypothalamus, CNS, milk, gonadal cells containing receptors for GRH Hypothalamus, spinal cord, extrahypothalamic brain, pancreas, stomach, intestine
Gonadotropic-releasing hormone (GRH)
Growth hormone release-inhibiting hormone (somatostatin)
Growth hormone-releasing factor (GRF)
Hypothalamus
Corticotropic-releasing factor (CRF)
.Releases GH from anterior pituitary
2.
3.
R
3
9
8
Inhibits release of GH and TSH from anterior pituitary, modulates pancreative hormones
Releases FSH and LH from anterior pituitary
Releases ACTH and &endorphin from anterior pituitary
Stimulates proliferation of cells of ectodermal and mesodermal origin with serum
Epidermal growth factor (EGF) (urogastrone)
Hypothalamus
Acts on development and maintenance of sympathetic neurons
Nerve growth factor (NGF)
Submaxillary gland
Acts like PRL and GH
Effects
Human placental lactogen (hPL)
Source
LH-like functions; maintains progesterone production during pregnancy
continued
Human chorionic gonadotropin ( h w
Placenta
TABLE I,
Increases smooth muscle contraction in GI tract; possible pain transmitter
Hypothalamus, CNS, intestine
Substance P (SP)
Vasodilator, lowers blood pressure
Plasma, gut, other tissues Human leukocytes
Bradykinin
Osteoclast-activating factor (OAF)
Stimulates in vitro bone resorption
Stimulates synthesis and release of aldosterone in the zona glomerulosa cells of adrenal cortex
Blood, lungs, brain
Angiotensin II
Blood
Atrial natriuretic factor (ANF) (atriopeptin)
Decreases blood pressure; stimulates renal sodium excretion; increases GFR and urine volume
Possible neurotransmitter; effects on gut at pharmacological doses
Hypothalamus, intestinal mucosa
Neurotensin
Atria
Regulates reproductive glands
Hypothalamus, pineal gland
Arg-Vasotocin
Heart
Releases TSH and PRL from anterior pituitary
Hypothalamus, extrahypothalamic brain, spinal cord and brain stem
Thyrotropic releasing hormone CTRH)
Releases PRL from anterior pituitary
Prolactin releasing hormone (PRH)
Effects Releases MSH from anterior pituitary, probably not in humans
Source
Melanotropin release-inhibiting factor (MIF)
TABLE I, continued
g
-$
b
6
a-
3
5 c,
Peripheral cells of pancreatic islets
Pancreatic polypeptide (PP)
Seminiferous tubule, ovary
Inhibin
Parafollicular C cells of thyroid gland
Calcitonin (CT)
Thyroid
Liver, muscle, kidney
Somatomedins (insulin-like growth factors I and 11, IGF)
Liver
Fetal Sertoli cells
Antimullerian hormone
Testes
Relaxin Corpus luteum
A-cells
Glucagon
Ovaries
B-cells
Source
~
Insulin
Pancreas
TABLE I. continued
Lowers serum calcium
Cartilage sulfation, somatic cell growth; insulin-like effects
Inhibition of FSH secretion from anterior pituitary
Mediates involution of the mullerian ducts
Increases during gestation (possible inhibitor of myometrial contractions)
Shows several effects on gut at pharmacological doses
Glycogenolysis in liver
Hypoglycemic effect; pleiotypic stimulator of metabolism
Effects
*F
h
8
h
HLA antigen (see somatomedins/IGF)
Colony-stimulating factor; macrophage growth factor
Miscellaneous
Stimulates conversion of white blood cell precursors to granulocyte and mononuclear phagocytes
Stimulates differentiation into immunocompetent T cells of the precursor cells; stimulates phagocytes
Thymopoietin I and I1 (a-thymosin)
Stimulates bone resorption and phosphate excretion by the kidney
Effect
Stimulates adenylate cyclase in thymus and spleen cells
Kidney, lung, spleen
Parathyroid glands
Source
Thymic humoral factor (THF)
Thymus
Parathyroid hormone (PTH)
Parathyroid
TABLE I, continued
8
03
8
%.
Yl
2 3
43
3
S. Incerpi and
104
P. Luly
Physical Parameters of Hormone-Receptor Interaction Hormone-receptor interaction is a simple bimolecular reaction that at equilibrium obeys the law of mass action and can be described as follows:
ka kd
[w,
where [R] and [HR] are, respectively, the concentrations of free hormone, of free receptor binding sites, and of the hormone-receptor complex; ka is the association rate-constant, kd is the dissociation rate-constant; Ka is the association constant (expressed as l/mole or M - ') and Kd is the dissociation constant (expressed as moles/l or M). There are several mathematical methods to estimate the interaction of small ligands with macromolecules [13,34,87,115-117,1291. These methods involve several relevant assumptions: (1) the hormone is in a homogeneous form; (2) labelled and unlabelled hormone behave identically; (3) the hormone and the receptor are univalent (i.e., one hormone molecule binds to one binding site); (4) the hormone-receptor interaction is a simple bimolecular reversible reaction; (5) full equilibrium is achieved; and (6) bound and free hormone can be separated without perturbing the equilibrium. Other important concepts to be kept in mind when using a receptorbinding assay are the following: High specific activity of the ligand is important to allow a reliable measurement of radioactivity at low concentrations, since in most binding assays high-affinity binding sites become saturated in the nanomolar concentration range and only a small fraction of the added radioligand is bound; The specific activity of labelled stock solutions should be checked before each assay to avoid changes due to decomposition, evaporation of solvent or decay of the labelled ligand. The isotope most commonly used to label ligands is tritium. Tritium-labelled molecules have high specific activity ( > 20-
Receptors to Peptide Hormones
105
50 Ci/mmol) as well as a relatively long radiochemical half-life ( > 12 y). When a very high specific activity is required, 1251-or 1311-labelled compounds can be used ( > 2000 Wmmol). A drawback in the use of this labelling is a short half-life (60 to 8 days) and also a possible biological hazard for the users of these compounds [71]. The binding of the radiolabelled hormone to a plasma membrane preparation or to a cell population must fulfill the criteria of saturability and reversibility. A basic assumption for a binding assay is that a tissue contains a finite number of receptor sites which are saturated in the presence of increasing concentrations of the labelled hormone. The binding of the ligand can be considered as the sum of at least two components: (1) a “specific” binding to saturable sites, and (2) a “nonspecific” unsaturable binding. The “nonspecific” components include low affinity binding sites and adsorption to the employed labware (e.g., tubes, filters, etc.). In a physiological concentration range, the binding of a peptidic ligand occurs to specific sites in a reversible and saturable manner, whereas nonspecific binding can be estimated in the presence of a large excess of
Ligand
Concentration
b
Fig. 2. Specific binding is obtained by subtractingnonspecificbinding from total binding.
S. lncerpi and P. Luly
106
unlabelled hormone. The saturation assay is the basis for the estimation of binding parameters: (a) the equilibrium dissociation constant (Kd);and (b) the number of binding sites (Bmax).Practically, the saturation assay is performed at steady-state conditions in tubes in which the same amount of tissue preparation (or of whole cells or plasma membranes) is incubated with increasing concentrations of the radioactive ligand in the presence (nonspecific binding) or absence (total binding) of an excess of unlabelled hormone. The specific binding can be calculated by subtracting nonspecific binding from total binding (Fig. 2). This is the simplest and most expensive way to measure specific binding. The most convenient and most widely used method is to employ small amounts of labelled ligand (around the physiological concentration range) as a tracer together with increasing amounts of unlabelled ligand up to the desired concentration. The amount of bound hormone can be determined in this case by multiplying the fraction of labelled ligand specifically bound by the total hormone concentration.
Kinetics of Hormone-Receptor Interactions The analysis of the kinetics of hormone-receptor interactions is based on the Law of Mass Action and on the Michaelis-Menten equation for enzyme-substrate interactions. The study of ligand-receptor interactions, as well as of the mathematical equations to derive the estimation of kinetic parameters, is beyond the scope of this chapter. We will just focus on the application of the basic principles for the estimation of binding parameters.
Scatchard plot In most binding studies the equilibrium binding affinity constant (Kd) and the maximum number of specific binding sites (BmJ are estimated from the Scatchard plot [ 1291of the saturation isotherm obtained by using the Scatchard equation for the analysis of binding data:
B -_ F
Bm,x
-
Kd
(4)
which reptesents the equation of a straight line. In fact, measuring the specifically bound ligand (B) and knowing the concentration of free
Receptors to Peptide Hormones
107
Fig. 3. A typical Scatchard plot of binding data indicating a single class of binding sites. B, bound hormone; F, free hormone.
ligand (F)in the incubation medium at equilibrium, the ratio of bound vs. free ligand (BIF) is plotted against the amount of bound ligand (B), and a best fit can be obtained by linear regression (Fig. 3). The equilibrium binding constant (Kd) is easily calculated from the plot as the negative reciprocal of the slope, and the maximum number of binding sites (BmaX) is given by the intercept of the line with the x axis (Fig. 3). If the hormone binds to a single set of non-interacting sites, the Scatchard plot yields a straight line; but when more types of binding sites are present, as in the case of insulin, the Scatchard plot gives a curved line with an upward concavity (Fig. 4). This behavior can be interpreted in two ways: (1) two different classes of binding sites are present, i.e., a high affinity and low capacity together with a low affinity and high capacity, with no mutual interaction; (2) a unique class of binding sites is present, but its affinity changes according to the amount of hormone bound, from an “empty” conformation (high affinity) to a “filled” conformation (low affinity). A non-linear Scatchard plot, in the case of binding to two sets of independent, non-interacting sites, can be resolved into two components
S. Incetpi and P. Luly
108
INSULIN BOUND
(MAO-~)
P
G R O W T H HORMONE BOUND
(M/10'"
Fig. 4. Examples of linear (lower curve) and non-linear (upper curve) Scatchard plots of insulin binding to human lymphocytes.
Receptors to Peptide Hormones
Y
0
I
109
I
I
30
I
I
I
60
Minutes of dissociation
Fig. 5. Effect of unlabelled hormone on the dissociation of labelled insulin bound to rat liver membranes. Upper curve reports data from a “dilution only” experiment, whereas the lower curve reports data from a dilution experiment in the presence of cold hormone. In both cases liver membranes were equilibrated with labelled insulin and then transferred to medium containing fresh buffer with or without cold hormone. Clearly a saturating concentration of cold hormone speeds up the dissociation of labelled insulin, pointing to the existence of site-site interactions between sites filled with unlabelled insulin and sites filled with labelled insulin.
either by a simple graphical analysis, as described by Rosenthal [101, 1201 or by computer programs now readily available [ 1021. An important alternative, which must be considered when a curvilinear Scatchard plot is obtained, is that negative cooperativity may be present. In this case, as we reported before, the receptor sites do not show a fixed affinity, but the affinity decreases as receptor occupancy increases (and vice versa). The analysis of steady-state data alone does not allow us to differentiate between the “multiple sites” model and the “cooperative model.”
S. lncerpi and
110
P. Luly
1 .o .8
.6 .4
.2
n .O m ml
ij
ILL ol
-
-2 -.4
-.6
-.8 -1.0
Fig. 6 . A typical Hill plot of binding data.
For a correct evaluation of the best model to fit our data, the method of choice is based on the kinetics of ligand dissociating from its receptor [35](Fig. 5); but alternatively one could also apply the Hill plot [64], first applied to the dissociation curve of oxyhemoglobin and described by the equation
v
=
100
Kx"
where y is the saturation percentage of hemoglobin with O,, x is the tension of 0, in the solution, K is the equilibrium constant and n is a number > 1. The Hill plot, in the case of peptide hormones, can be obtained from saturation binding data by plotting log BI(B,,-B) against log [L],where L is the free hormone, B is the amount of bound ligand and B,,, is the maximum number of binding sites estimated by the Scatchard analysis. The slope of the line fitted by linear regression is
Receptors to Peptide Hormorres
111
Total sites
I
I
z.
Fig. 7. Evaluation of the average affinity At a given point on the curve, i is the level of bound hormone and Bi corresponds to a (B/F)i (bound/free) value. The value of g for that occupancy is -gi, or (B/F)i/(Ro-Bi).
denoted as the Hill coefficient (n)where n > 1 indicates positive cooperativity, n=O indicates no cooperativity, O < n < 1 indicates negative cooperativity [26] (Fig. 6). The equilibrium constant (Kd) can be estimated from the Hill plot as the abscissa value for log [B/(B,,,-B)] =0, i.e., the intercept on the x axis is obtained for B=(B,,,/2); this Kd denotes an “average” affinity constant. Interaction among sites cannot be evaluated by Scatchard analysis, and the application of the Hill plot has also been sometimes considered limited, providing only a slope factor indicating the presence of cooperativity for n # 1. Cooperativity can also be evaluated by the graphic analysis elaborated by De Meyts and Roth [36]: from this
S. incerpi and P. Luly
112
analysis a new parameter can be calculated, i.e., the average affinity of the receptor sites 5 calculated as
A plot of 5 vs. logy=log(B/Ro) gives y=O when all sites are empty, and 7 = 1 when all sites are occupied. So can be considered as an average affinity for each level of receptor occupancy (Fig. 7). The meeting point between the average affinity profile and the Hill plot is that the high affinity (K,) (empty conformation) and the low affinity (KJ (filled conformation) can be determined also from the abscissa intercept of the asymptotes of the Hill plot [64]. Mechanism of Peptide Hormone Action
The “second messenger” hypothesis In 1957-1958 Sutherland and Rall made the pivotal experiment that after a short time led to the discovery of cyclic AMP [143]. When epinephrine was added to liver slices, there was a fast, large and reproducible appearance of glucose in the incubation medium due to glycogen breakdown. However, the 100,OOOxg supernatant of rat liver homogenate in the presence of epinephrine did not induce the same effect: the production of free glucose. Nevertheless, the addition of the 600Xg fraction to the same supernatant gave the physiological response. These findings indicated that the stimulating effect of epinephrine on glycogenolysis was dependent on “something” that was present in the whole cell. They already knew that the glycogenolytic effect of epinephrine in liver was due to the activation of the limiting reaction from glycogen to glucose, i.e. the phosphorylase activity, according to the following: glycogen
phosphorylase
i,
glucose- 1-phosphate
It was very easy at that point to assume that epinephrine activated the phosphorylase pathway and that the presence of the fraction sedimenting at 6OOxg was necessary. Sutherland and Rall observed that the 600Xg fraction, in the presence of ATP and magnesium, produced a heat-stable molecule that could convert the inactive phosphorylase b to the active phosphorylase a. The activator was identified by Sutherland and Rall with
Receptors to Peptide Hormones
I13
the help of Lipkin, an expert chemist in nucleotide metabolism [ 1431. It was found, in fact, that a nucleotide coming from a barium hydrochloride-digest of ATP was the activator, and this molecule was called cyclic adenosine monophosphate (cyclic AMP). It was found later that the cyclic AMP concentration in tissues reflects a balance between two opposing reactions: (1) formation from ATP, and (2) inactivation through conversion to 5’-AMP. The next series of studies was devoted to the enzymes involved in these two opposing reactions: adenylate cyclase and phosphodiesterase. Rodbell and coworkers [118] at the National Institutes of Health showed that the GTP molecule is essential for the transduction mechanism generating cyclic AMP. Before the information flows through the membrane, two events must take place: on the cell surface an external signal must interact with a receptor and, at the same time inside the cell, a GTP molecule must bind to its own G-protein. This process has been studied in great detail by Gilman and coworkers [53,121] (Fig. 8). Adenosine-3’,5’-cyclic monophosphate (cyclic AMP) is now recognized as a ubiquitous regulatory molecule, controlling many different metabolic processes in both prokaryotic and eukaryotic cells. In animal cells it acts as an intracellular “second messenger” in the transduction of
GTP
Cyclic AMP
Fig. 8. Rodhell’s classic model of the organization of hormone-stimulated adenylate cyclase. The receptor R is hypothesized as an integral membrane protein having two different domains, one that binds the hormone and the other one linked to the nucleotidebinding regulatorysubunit N . The GTP-binding subunit Nactivates the catalyticcomponent C.
S. Incerpi and P. Luly
I14
information brought by hormones and circulating factors. A survey of the hormones acting on the modulation of intracellular cyclic AMP levels is reported in Table 11. TABLE I1
Hormones affecting adenylate cyclase and cyclic AMP levels Inhibition
Stimulation
Insulin a2-adrenergic agonists Muscarinic cholinergic agonists Somatostatin Some prostaglandins (e.g., PGE,) Some opiate peptides
Glucagon Vasopressin Parathormone Calcitonin Melanocyte stimulating hormone 0-adrenergic agonists Some prostaglandins (e.g., PGE,) Hypothalamic releasing hormones Anterior pituitary hormones (FSH, ACTH, TSH, LH, hCG)
The synthesis of cyclic AMP is catalyzed by the hormone-sensitive adenylate cyclase system [ATP pyrophosphate lyase (cyclizing) E.C.4.6.1.11. Hormone-sensitive adenylate cyclase activity is found in almost all animal cells (some erythrocytes and cultured cells are exceptions) and can be stimulated by many hormones, biogenic amines, proteins, prostaglandins and local factors. There are also drugs that inhibit the cyclase; among these are opiates, a-adrenergic amines and adenosine. The hormone-sensitive adenylate cyclase system is composed of at least three proteins [ 1211: (1) a catalytic protein, relatively inactive; (2) a guanine nucleotide-binding protein that mediates the action of different regulatory ligands; and (3) one or more hormone receptors.
Hormone receptors and adenylate cyclase Receptors for polypeptide hormones are individual proteins embedded in the lipid bilayer, distinct from adenylate cyclase. A direct demonstration that adenylate cyclase and hormone receptors are distinct proteins came from the cell fusion experiments by Orly and Schramm [131]. The
Receptors to Peptide Hormones
115
Sendai virus was used to fuse Friend erythroleukemia cells (which have adenylate cyclase but lack 0-adrenergic receptors) to turkey erythrocytes treated with N-ethylmaleimideto deactivate the adenylate cyclase. Plasma membranes from the resultant erythrocyte-Friend cell heterokaryons displayed catecholamine-sensitive adenylate cyclase activity. Cell fusion and membrane preparation were performed in the presence of cycloheximide to prevent de novo protein synthesis, so that the source of stimulation was the interaction of Friend cells with the erythrocyte P-adrenergic receptor [ 1313. The response of the heterokaryon membrane was very rapid (about 5 min). These experiments unequivocally demonstrate the “mobile receptor” hypothesis according to which hormone receptors and adenylate cyclase are discrete molecules, relatively free to diffuse and interact in the plasma membrane lipid bilayer. The ligand-binding properties of a wide variety of hormone receptors acting through adenylate cyclase have been studied in the plasma membrane of target cells using radioactively labelled ligands; a number of receptors has also been characterized after detergent solubilization [ 1,39, 42,43,149,152]. As to the adenylate cyclase molecule, little is known except that it is a 150 kDa integral membrane protein modulated by G-proteins, but also affected by calcium ions and calmodulin. The paucity of information is due to the difficulty of preparation of purified catalytic subunits. The catalytic subunit is known to have a binding site for ATP or preferably for a divalent cation-ATP complex.
Guanine nucleotide-binding regulatory proteins Specific receptors for certain ligands, the guanine nucleotide coupling proteins, and the catalytic subunit of adenylate cyclase are known to be distinct proteins under separate genetic control [14]. Much more is known about the guanine nucleotide-binding regulatory proteins (also named G- or N-proteins) than about the catalytic subunit because of their greater stability and ease of preparation and radioactive labelling (Fig. 8). Several different preparations of G-proteins, resolved from the catalytic subunits, are now available at different degrees of purity. The first data on the composition of the nucleotide regulatory proteins came from the to label work of Pfeuffer [ 1111 who used [32P]-GTP-y-azidoanilide 2000 Dalton band pigeon erythrocyte membranes. The fractionation of a on dodecyl sulphate polyacrylamide gels was consistent with its involvement with adenylate cyclase. Further studies were performed using
116
S. Incerpi and P. Luly
Fig. 9. Lipid involvement in membraneassociation of G-proteins. The G-protein or-subunit has a myristate attached by amide linkage to the amino terminal glycine residue, whereas the G-protein y-subunit has a geranylgeranyl moiety attached by a thioether linkage to the carboxy-terminal cysteins residue; the latter is also carboxymethylated. The interrelationships among the three subunits are not yet defined. The coupled membrane receptor is an integral membrane protein with seven membrane-spanningdomains. The carboxy-terminal end of the receptor protein is thought to be partly linked to the membrane by palmitoylation through a thioesther bond to a cysteine.
cholera toxin [76]. Strong evidence [99] suggests that the crucial step in the stimulationof adenylate cyclase activity by toxin is the ADP-ribosylation of the protein involved in the activity modulation by guanine nucleotides. ADP-ribosylation of the protein is time and temperature dependent and requires GTP and a cytosolic protein - features strikingly similar to those observed for the activation of adenylate cyclase. The number of G-proteins reported has increased in recent years [14], and several types of G-proteins have been distinguished: four Gs (s =stimulatory) [62,105]; three Gi (i =inhibitory) [37,78]; one Go (o=other) [6,78]; one G z/x [51,97]; and two classes of sensory G proteins, one involved in vision transduction process (transducin) [60,90] and another one involved in olfactive perception [78].
Receptors to Peptide Hormones
117
TABLE I11
Peptide horinones known to act through a G-protein system Membrane function affected
Effect
AC PLC AC AC AC K-channel Ca-channel PLA2 PLC Ca-channel PLC
Stimulation Stimulation Inhibition Stimulation Inhibition Opening Closing Stimulation Stimulation Opening Stimulation
PLC PLD PLA, AC PLC AC
Stimulation Stimulation Stimulation Inhibition Stimulation Stimulation
AC PLA2 Ca-channel AC K-channel Ca-channel AC PLC AC AC AC
Stimulation Stimulation Opening Inhibition Closing Closing Stimulation Stimulation Stimulation Stimulation Stimulation
Hypothalamic Corticotropic releasing hormone Thyrotropin releasing hormone Growth hormone releasing hormone Somatostatin
Gonadotropin releasing hormone
Ox ytocin
Vasopressin V-la (vasopressor, glycogenolytic)
V-lb (pituitary) V-2 (antidiuretic)
Pituitary Adrenocorticotropin (ACTH)
Opioid
Luteinizing hormone (LH) Follicle stimulating hormone (FSH) Thyrotropin (TSH) Melanocyte stimulating hormone (MSH)
118
S. Incerpi and P. Luly
TABLE ID. continued Membrane function affected
Effect
AC PLC Ca-pump AC AC PLC AC AC PLC AC PLC AC AC Ca-channel PLC PLD PLA2
Stimulation Stimulation Inhibition Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation Inhibition Stimulation Stimulation Stimulation Stimulation
Other hormones Glucagon
Secretin Cholecystokinin (CCK) Chorionic gonadotropin Parathyroid hormone (PTH) Vasoactive intestinal peptide (VIP) Calcitonin Angiotensin I1
AC, adenylate cyclase; PLC, phospholipase C; PLD, phospholipase D; PLA,, phospholipase A,.
G-proteins are heterotrimers composed of subunits a,/3 and y. They are considered a subset of a larger family of GTP binding proteins, including factors controlling protein synthesis (Elongation Factor Tu = EF-Tu) and a group of small GTP binding proteins with a MW of 2025 kDa, among which are the “ras” gene products [53]. G-proteins are localized at the inner surface of the plasma membrane where they function as signal transducers, coupling receptors to effector proteins (Fig. 9). Before the discovery that GTP is an essential activator of adenylate cyclase and that hormones enhance the action of nucleotides, the current thought was that hormone receptors interact directly with the catalytic unit and that fluoride ion, a non-physiologic activator, directly affects the
Receptors to Peptide Hormones
I19
catalytic unit. G-proteins are currently considered to be universal couplers between membrane receptors (R) of extracellular signals and membrane effectors (E) of various intracellular messengers. Practically all membrane transduction processes (hormonal, neuronal, sensorial, immunological, chemotactic, growth andlor differentiation factors) involve a G protein and its coupling to an effector [23]. G-proteins, in fact, have been found to control the activity not only of adenylate cyclase and cGMP phosphodiesterases (PDE) but also of phosphatidylinositol 4,5-bisphosphate (PIP2)-specificphospholipase C (PLC) [ 15,191. A list of peptide hormones acting through G-proteins is reported in Table I11 ~41.
m e “dm1 messenger ’’ hypothesis The second major transduction system of peptide hormones is the polyphosphoinositide (PPI) pathway. Phosphatidylinositol is a typical phospholipid component of the plasma membrane, and it covers less than 10%of total cell phospholipids, being located mainly in the inner plasma membrane leaflet. Phosphatidylinositol is easily phosphorylated at positions 4 and 5 to become PPI which, in turn, is hydrolyzed by hormone-dependent phospholipase C. Two second messengers are produced: a phosphorylated head-group inositol 1,4,5-trisphosphate (IP,) and diacylglycerol (DAG). The IP, molecule raises the cellular levels of calcium, whereas the DAG molecule stimulates a calcium-activated phospholipid-dependent protein kinase (protein kinase C or PKC) . The DAG molecule can be hydrolyzed by a DAG lipase to release a fatty acid, which in most cases is arachidonic acid, a precursor of eicosanoids. The overall relevance of phosphoinositide hydrolysis in cell regulation by hormones and other ligands has been recognized quite recently, but agonist-stimulated phospholipid turnover was first described in 1953 by Hokin and Hokin [67] whose pivotal discovery of acetylcholine-stimulated incorporation of [32P]-phosphateinto pancreatic phosphoinositides [68] shed some light on a major transduction mechanism of muscarinic agonists and provided the background for the understanding of hormone actions. Examples of receptors coupled to phosphoinositide metabolism are the following: muscariniccholinergic, al-adrenergic, serotoninergic, dopaminergic, H -histaminergic, peptidergic (e. g ., cholecystokininpancreozymin or CCK-PKZ, thyroid stimulating hormone or TSH, thyrotropin-releasing hormone, corticotropin-releasing hormone, angiotensin 11, V1-vasopressin, bradykinin, substance P).
S. Incerpi and P. Luly
120
A general scheme (Fig. 10) relating receptor-mediated phospholipid turnover to intracellular signal transduction was proposed in 1975 by Michell [98], who suggested that accelerated phospholipid turnover is a common feature of calcium-dependent hormones being increased, phosphatidylinositol (PI) hydrolysis the cause rather than an effect of increased cytosolic calcium concentration during hormone action. The original candidates for calcium mobilization during ligand-induced phospholipid turnover were intermediates formed during PI hydrolysis such as phosphatidic acid and inositol 1,2 cyclic phosphate [ll]. However, other polyphosphoinositides, phosphatidylinositol4monophosphate Hormone 1 Hormone 2 secreted
-
Cell membrane
Exocytos i s
Hormone 2
phosphorylat ions
t o be secreted
Biologics I activity
Cytoplasm
Endoplasrnic
reticulum
Fig. 10. Hormone action on phospholipid metabolism in the plasma membrane. Hormone binding to the receptor induces the activation of the hydrolysis of PIP, to form IP3 and DAG, two putative second messengers acting, respectively, on intracellularcalcium levels and protein phosphorylation. IP, is degraded by dephosphorylationto IP and subsequently recycled to PI. A secondary effect of hormone 1 could be the release, mediated by a calcium-modulated exocytosis, of hormone 2. For abbreviations, see legend to Fig. 11.
Receptors to Peptide Hormones
121
(PIP) and particularly phosphatidylinositol-4,5-bisphosphate(PIP,) were also found to undergo rapid turnover during hormone action and PIP, has been identified as the major signal-generating precursor, which is degraded to produce the calcium mobilizing messenger inositol-l,4,5trisphosphate (IP,) during agonist occupancy of receptors for calciumdependent hormones [30] and many other ligands responsible for cell activation and growth (Table IV).
TABLE IV Signals and receptors coupled to the phosphoinositide transduction system
Growth factors Insulin, EGF, NGF, PDGF Vasopressin, bradykinin, bombesin, thrombin Auxin (plants), aggregation factor (sponges) Angiogenin
Inhibitory agents Dopamine Prostacyclin AMP, adenosine cYz-agonists
Hormones and transmitters Serotonin (5HT,), histamine (HI), octopamine, glutamate Vasopressin (V,), oxytocin, angiotensin 11, cholecystokinin, bradykinin Glucagon Muscarinic cholinergic agonists (MI) Adrenergic agonists (a,) Gonadotropin-releasing hormone, thyrotropin-releasing hormone Substance P, neurotensin, VIP, bombesin, substance K f-Met-Leu-Phe, platelet activating factor (PAF), thrombin, leukotriene B, ATPIADP
Other signals Glucose Ouabain Photons Antigen, interferon, phytoemagglutinin Spermatozoa
S. incerpi and P. Luly
122
Metabolism of inositol phospholipids Inositol-containing phospholipids, i.e., PI and its phosphorylated derivatives PIP and PIP,, are more abundant in nervous tissue and were first found and characterized in the brain [63]. The biosynthetic pathway involves the combination of myoinositol with cytidine diphosphodiacylglycerol (CDP-DAG) by enzymes located on the cytosolic surface of the endoplasmic reticulum [7]. Myoinositol is formed from D-glucose via glucose-6-phosphate, so it can be both a dietary constituent and an endogenous metabolic product. The PI produced is then phosphorylated to PIP and PIP,. The inositol phospholipids are enriched in arachidonic acid at position 2 , possibly via a phospholipase A2-mediated cycle of deacylation-reacylation, and they represent a major source of arachidonic acid [57]. The hydrolysis of inositol phospholipids is performed by phospholipase C (PLC) which exists in membrane-bound and cytosolic forms and catalyzes the breakdown of phosphoinositides localized in the inner leaflet of the plasma membrane [69]. Phosphoinositides are hydrolyzed by PLP-C to DAG and several inositol phosphates, among which IP, is the most abundant (Fig. 11). The biosynthetic pathway and the hydrolysis are shown in Fig. 10. Multiple forms of the enzyme have been found in several tissues [ 1261 whose activity and substrate specificity are influenced by calcium ions. PIP, is the preferential substrate for the hormoneactivated enzyme at a calcium concentration of 100-200 nM [12].
DAG
C D P-DAG
I
i
PIP
I PIP, Fig. 1 1 . Pathway of phospholipase C-mediated hydrolysis of phosphatidylinosital-4’,5’bisphosphate (PIP,). DAG, diacylglycerol; PA, phosphatidic acid; CDP, cytidine diphosphate; PI, phosphatidylinositol; PIP, phosphatidylinositol monophosphate; IP,, inositol-l‘,4’,5’-trisphosphate.
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Inositol-I ,4,5-trisphosphate and diacylglycerol as messengers A number of studies on permeabilized cells and cell fractions have shown that IP, releases calcium ions from an ATP-dependent nonmitochondria1 pool that seems to be located within the endoplasmic reticulum (ER) and close to the plasma membrane [12]. IP, mobilizes calcium by binding to specific intracellular receptors that promote the opening of calcium channels in vesicular storage sites associated with ER. These receptors are widely distributed in target tissues (e.g., brain) of calcium mobilizing hormones. Where does calcium come from? Due to the steep gradient across the plasma membrane, the calcium was initially thought to come from the extracellular fluid, but intracellular calcium also rose when extracellular calcium was removed from the medium. The analysis of calcium content of cellular organelles showed that the primary source was the endoplasmic reticulum; it has been shown [151] that the source is a large group of vesicles associated with the endoplasmic reticulum and the plasma membrane called calciosomes, containing a calsequestrin-likeprotein that binds calcium ions. Apparently mitochondria are not involved in the rise of calcium levels. The other primary product of phospholipase C action on phosphoinositides is DAG (Fig. 11). The formation of this supposed mediator is very rapid, and it has been shown to occur following the stimulation of phosphoinositide breakdown in platelets [74], pancreas [38], mast cells [73], 3T3 cells [61] and liver [145]. DAG positively modulates the activity of protein kinase C (PKC), a family of calcium/phospholipiddependent enzymes, by influencing its affinity for calcium and phosphatidylserine [ 1031. The DAG released during phospholipid hydrolysis, via activation of calcium/phospholipid-dependentenzymes, is responsible for the regulation of a number of cellular functions including growth, differentiation, gene expression, secretion and neurotransmission. Tumor-promoting phorbol esters, such as phorbol-12-myristate-13acetate (PMA), exert their biological activity as analogs of DAG through the binding to the regulatory region of PKC [ 1031. The PKC thus functions as an intracellular receptor for phorbol esters. PKC is considered to be involved in many receptor-mediated cell responses to hormone action and secretion, but most of its functions are based on the ability of phorbol esters and synthetic DAG to mimic responses to hormones and other ligands. Direct evidence for the role of PKC in hormone action is difficult to obtain and it has not been reported so far.
S. Incerpi and P. Luly
I24
Guanine nucleotide regulatory proteins and phospholipid hydrolysis The modulation of adenylate cyclase activity is due to the interaction with GTP-binding regulatory proteins, either stirnulatory (Gs or Ns) or inhibitory (Gi or Ni); similarly the activation of phospholipase C is due
rM-
L
/
Choline
/
n
\
ACYL- COA
/
\
C3P
r;noiine
Fig. 12. Phosphatidylcholine (PC) cycles for the production of second messengers diacylglycerol (DAG) and eicosanoids. DAG can be generated directly via phospholipase C (PLC) or by the action of phospholipaseD (PLD) to give phosphatidic acid (PA) which is cleaved to DAG by PA phosphohydrolase(PAP). The hydrolysis of phosphatidylcholine may also be catalyzed by phospholipaseA2 with the production of lyso-PC and arachidonic acid, a precursor of eicosanoids. Lyso-PC may be re-esterified to PC or catabolized to glycerophosphocholine (GPC) which can be degraded to glycerol-3-phosphate (G3P) and choline. G3P can be converted to DAG through PA synthesis.
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125
to a guanine regulatory protein called Gp. This protein, not yet characterized, is similar to Gi in that it is sensitive to pertussis toxin but is also similar to Gs in that it is stimulated by fluoride ions. It seems that many receptors for neurotransmitters, peptide hormones, growth factors and prostanoids are coupled to a G-protein (see [14] for review) (Table 111). It is likely that both stimulatory and inhibitory G-proteins are involved in the regulation of the plasma membrane phospholipases responsible for the generation of intracellular signals derived from phospholipids. The complexity of this potential regulatory system is emphasized by recent data on the involvement of phospholipids other than phosphatidylinositol, i.e., phosphatidylcholine as a precursor of DAG in certain stimulated cells [46]. Moreover, the activation of other phospholipases, such as phospholipase A, and phospholipase D, has been considered to be involved in the generation of other signal molecules such as arachidonic acid and phosphatidic acid or phosphocholine [14,47] (Fig. 12). The family of intracellular messengers is increasing day by day, thus stressing the importance of the phosphoinositide hydrolysis system as well as of the cyclic AMP generating signals in the control of cell functioning by hormones and transmitters.
Glycogd-phosphutidylinositol hypothesis The involvement of phospholipid hydrolysis has also been considered a possible transduction mechanism for insulin. In particular, in recent years the attention of the authors has focused on a particular type of phosphatidylinositol: glycosylphosphatidylinositol (GPI) [94]. This phospholipid has been found to serve as an anchor on the cell plasma membrane for certain membrane proteins [95,96,127]. Many glycoproteins are anchored to the cell surface by hydrophobic interactions of the protein itself with the lipid bilayer. The most known mechanism of anchoring involves one or more stretches of hydrophobic amino acids spanning the membrane and resulting in an attachment to the plasma membrane [94]. The alternate and less known mechanism of anchoring involves the covalent linkage of the C-terminal amino acid of the protein to a glycosylated form of phosphatidylinositol termed glycosilphosphatidylinositol (GPI). This mechanism has been found in a variety of cell types, and an updated list is provided in Table V. In Fig. 13 the structure of the GPI protein anchor is reported. The C-terminal amino acid is linked by an amide bond to ethanolamine, connected, in turn, to an oligosaccharide of variable structure. The terminal monosaccharide of
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TABLE V
Proteins with a glycos yl-phosphatidylinositol anchor
Mammalian antigens Thy-1 RT-6 Qa Ly-6 Carcinoembryonic antigen Blast-1 CD14 Non-specific cross-reacting antigen Protozoal antigens SSp-4 Trypanosoma Variant surface glycoprotein Trypanosoma Surface protein Paramecium 195-kDa antigen Plasmodium Cell adhesion LFA-3 Neural cell adhesion molecule Heparin sulfate proteoglycan Contact site A Dictyostelium PH-20 guinea pig sperm
Hydrolytic enzymes Alkaline phosphatase 5 ‘-nucleotidase Acetylcholinesterase Alkaline phosphodiesterase I Trehalase Lipoprotein lipase Aminopeptidase P Renal dipeptidase p63 protease Leishmania Merozoite protease Plasmodium Miscellaneous Homologous restriction factor GP-2 Scrapie prion protein Decay-accelerating factor Folate receptor Elongation factor EF-1 alpha Tegument protein Schistosoma Antigen 117 Dictyostelium 125-kDa glycoprotein Saccharomyces 130-kDa hepatoma glycoprotein 34-kDa placental growth factor Oligodendrocyte-myelin protein FcIII receptor
the glycan is non-N-acetylated glucosamine, linked at the C-1 position to the C-6 hydroxyl of the inositol ring. The structure of the GPI moiety has been determined from studiesusing bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from Staphylococcus aureus, B. subtilis or B. thuringiensis [94]. It has been hypothesized, but not definitely proved, that the putative long-searched insulin messenger could be a substance containing inositol, derived from glycosyl phosphatidylinositol hydrolysis. PI-PLC added to liver plasma membranes was found to reproduce the effects of insulin by facilitating the generation of the “enzyme modulator. ” The insulin-
Receptors to Peptide Horniotres
I
bH
*
.-
127
c
Membrane
Fig. 13. Structure of a glycosyl-phosphatidylinositol protein anchor. The C-terminal amino acid is linked through an amide bond to ethanolamine, which is, in turn, linked through a phosphodiesterbond to an oligosaccharideof variablecornposition and structure. The terminal monosaccharide of this glycan is a non-acetylated glucosamine which is linked at position C-1 to the C-6 hydroxyl of inositol.
sensitive glycosyl-PI appears to have a great similarity to the glycosyl-PI protein anchor. The insulin-sensitive glycosyl-PI, however, apparently lacks two of the features found in the protein anchor, ethanolamine and aminoacids (i.e., it appears to have a smaller size with respect to similar molecules bound to proteins). Also the topological distribution of the insulin-sensitive glycolipid in the plasma membrane is under debate; some studies have suggested a cytoplasmic orientation [94], others have suggested an intracellular location for the lipid. Thus the topological question remains open until definitive evidence is available. The observation that insulin stimulates the hydrolysis of glycosyl-PI led to the search for a specific phospholipase C; this enzyme was isolated from liver plasma membranes and appears to be calcium-independentand specific for glycosyl-PI.
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The next question arising is how the regulation of glycosyl-PI hydrolysis is coupled to the activity of the insulin receptor; surely this is the most difficult step to establish. Studies with anti-receptor antibodies, or site-directed mutagenesis, indicated that the tyrosine-kinase activity of the receptor is necessary for the expression of all the biological activities of insulin [ 1191. Cells transfected with mutant insulin receptors lacking tyrosine-kinase activity do not show glycosyl-PI hydrolysis in response to insulin, whereas cells transfected with wild-type receptors respond normally [94]. We can infer that the activation of the glycosyl-PI-specific phospholipase C by the receptor might occur as a consequence of a tyrosine-kinase activation, leading to changes in the state of phosphorylation of the enzyme; the coupling factor could be a specific GTP-binding protein able to activate the phospholipase C. The involvement of a G-protein in insulin action has been suggested since the pertussis toxin and antibodies to the GTP-binding ras p21 protein can block certain actions of the hormone. Also the literature on a G-protein involvement in insulin action is very controversial and contradictory [70], but we can suggest recent useful reviews on this “hot” topic [70,83].
Other phospholipids as possible sources of second messengers In the last few years alternative pathways of phospholipid turnover related to hormonal signal transmission have been reported [48]. Another phospholipid, phosphatidylcholine (PC), has been found to be an important source of diacylglycerol (DAG). DAG can be generated via phospholipase C (PLC), as indicated before, or via phospholipase D (PLD) to yield phosphatidic acid (PA) which is cleaved to DAG by PA-phosphohydrolase (PAP) (Fig. 12). The activationof PC, found almost exclusively in eukaryotic cell membranes, is due to a PC-specific PLC in response to hormones such as vasopressin, PDGF, bombesin and insulin [46,48,49,110]. The hydrolysis of PC would be mediated, also in this case, by a G-protein, Gp [46,48,110]. A variety of hormones (insulin, insulin-like growth factors I and 11, epidermal growth factor, vasopressin, thyrotropin-releasing hormone) stimulate PC synthesis [46,48,49,1lo]. In fact, the DAG that comes from PC hydrolysis can also act as a substrate for PC synthesis. We can, therefore, infer the existence of a PC-cycle (Fig. 12). The presence of a PC-cycle gives some advantages when compared to a transduction mechanism based on a PI-cycle. First of all PC is much more abundant than PI; thus its hydrolysis may offer more copious precursors. Further-
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5
Recep or
r
G - P otein
+
PIP2 PLipase C
pip2 V D A G -
+
Prot Kin C
P C PLipaseC PC PLipase D Fig. 14. Mechanism of phosphatidylcholine-phospholipase(PC PLipase) activation involving protein kinase C. The scheme indicates that PC phospholipase activation is started by the productionof DAG from PIP, following theactivation of PIP, phospholipase C via a receptor-mediated G-protein mechanism. DAG activates protein kinase C either directly or indirectly. DAG is produced directly from PC via PC phospholipasec or indirectly via PC phospholipaseD with the subsequent activationof PA phosphohydrolase. For abbreviations, see legend to Fig. 11.
more, the resynthesis of PC from DAG is much simpler and less expensive in metabolic terms in comparison with the resynthesis of PIP, (Figs. 11 and 12). As an additional advantage, PC hydrolysis appears to be more selective, since it gives rise to DAG without any effect on cytoplasmic calcium; DAG, in turn, might preferentially address the hormonal signal to the activation of protein kinase C (PKC). The activation of PKC via PC hydrolysis is likely to be more prolonged than that due to PIP, breakdown, and the PC hydrolysis could be maintained by positive feedback from DAG derived from PC or via PA (Fig. 14). It has been proposed that PC breakdown can be involved in long-term mechanisms (growth, differentiation, neuronal plasticity, alterations in receptor functions) requiring a prolonged activation of PKC [46].
Receptor phosphory lation As indicated in The Concise Oxford Dictionary, “desensitization” expresses a reduction of the sensitiveness, or in biochemical language, the trend to a reduction of the biological response over time, despite the continuous presence of the stimulus (i.e., hormone or agonist). Receptor phosphorylation is a mechanism first known to lead to the desensitization of the receptor itself. Desensitization can be homologous, when a substance desensitizes the cell to a second exposure to the same agent, or
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heterologous, when other transduction systems are affected by the same
agent [ 1351. The best known example of heterologous desensitization is the one involving the 6-adrenergic receptor. When adenylatecyclase is stimulated by hormones, intracellular levels of cyclic AMP rise, protein kinase A is activated and the 0-adrenergic receptor becomes phosphorylated. The phosphorylation occurs exclusively on serine residues [ 136,140,1211. Protein kinase C (PKC) can also phosphorylate the P-adrenergic receptor; in fact, treatment of intact cells with phorbol esters which activate PKC also causes receptor phosphorylation and desensitization [82,137]. Homologous desensitization of the 0-adrenergic receptor also appears to involve receptor phosphorylation [ 1381. Benovic and coworkers [9] have identified a cyclic AMP-independent kinase that seems to be involved in homologous desensitization and phosphorylation of the P-adrenergic receptor. This enzyme, called 0-adrenergic receptor kinase (or P-ARK) is mainly cytosolic. Phosphorylation and functional uncoupling of the receptors are also the events leading to internalization of the receptors within the cells. These aspects of the regulation of 6-adrenergic receptor function closely resemble the mechanism regulating the “light” receptor rhodopsin [88]. In fact, the structure and organization into the plasma membrane of the 0-adrenergic receptor and rhodopsin are very similar (Fig. 15). Other receptors acting through G-proteins, including peptide hormones, show agonist-induced desensitization and, in some cases, downregulation. These are receptors for catecholamines (al-adrenergic), acetylcholine (muscarinic cholinergic), insulin, vasopressin and angiotensin. Phorbol esters lead to desensitization of al -adrenergic receptorpromoted PI turnover [27] and also to the phosphorylation of the al-adrenergic receptor [89]. al-agonists also give rise to al-adrenergic receptor desensitization and phosphorylation. The muscarinic cholinergic receptor has properties similar to those of the al-adrenergic receptor, and it is homologous to the P-adrenergic receptor [93]. A general pathway for the P-adrenergic receptor stimulation could be as shown in Diagram 1. Other receptors, particularly those for growthfactors including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor I (IGF-I) and insulin are themselves proteintyrosine kinases. In this case the phosphorylation not only leads to desensitization and/or internalization but also can be one of the very first steps leading to the transduction system.
Receptors to Peptide Hormories
.
. . . .. .
HOOC
131
NH2
C*.’.‘.‘ ., . , . . . . . I
Fig. 15. Scheme of the mammalian 0-adrenergic receptor and rhodopsin as they might be organized in the plasma membrane. For both proteins the seven spanning domains are shown in the plasma membrane. The amino- and carboxy-terminus together with the target sites of protein kinase C on the 0-adrenergic receptor are also indicated.
The P-subunit (90 kDa) of the insulin receptor contains a sequence of 23 lipophilic amino acids, on which depends the insertion of the receptor complex into the plasma membrane bilayer. It is interesting to observe that the same sequence provides the anchoring to the plasma membrane for other receptor proteins, i.e., the EGF and the LDL (low-density lipoproteins) receptors, including the “src”-family of protein tyrosine
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P. Luly
P-adrenergic receptor
agon i st
d activation of adenylate cyclase
L translocation of the 0-adrenergic receptor kinase from the cytosol to the plasma membrane
.1
phosphorylation of the 0-adrenergic receptor
-1 reduction of the interaction receptor G-proteins
-1
internalization of the receptors and recycling Diagram 1.
kinases (Figs. 16 and 17). When insulin binds to the a-subunit of the receptor, this interaction elicits the kinase activity of the @-subunit,which can be very fast (1 min) (211; therefore, the phosphorylation of the receptor was included among the fast responses of the hormone (see Table VI), and its biochemical aspects have been investigated (Table VII). Different patterns of phosphorylation have been observed using intact cells or cell-free systems. In intact cells, phosphorylation is one of the fastest events following insulin stimulation mainly involving serine and threonine, and partly tyrosine residues [75,153], whereas in cell-free systems the phosphorylation of the @-subunitaffects mainly tyrosine residues [29]. Phosphorylation of the @-subunitof the receptor increases its ability to phosphorylate exogenous substrates, and the receptor kinase becomes insulin-insensitive [86]; insulin sensitivity is restored by dephosphorylation. Insulin receptors seem to be under a double control through the phosphorylation of the @-subunits.Autophosphorylation of the P-subunit leads to enhanced tyrosine kinase activity toward exogenous substrates, and this represents one of the very first steps of the insulin transduction mechanism. On the other hand, the phosphorylation of the @-subuniton serine and threonine residues results in decreased receptor autophosphorylation and tyrosine kinase activity, leading perhaps to desensitization. It is conceivable that receptor kinase might mediate the metabolic effects of
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N"2
0
Ex tracellular Ligand binding Domain
Transmembrane Domain
Extracellulac Ligand binding Domain
I
I
I
I
Transmembrane Domain
Cytoplasmic
Cytoplasmic Tyrosine kinase Domain
Domain
COOH
Fig. 16. Schemes of insulin and epidermal growth factor receptors. Regions rich in cysteine residues are shown as open boxes, transmembrane domains as black boxes, and the tyrosine domains as stippled boxes. Left,epidermal growth factor; right, insulin. TABLE VI
Sequence of insulin effects
Seconds Binding to the receptor Activation of receptor protein (tyrosine) kinase Receptor autophosphorylation
Seconds to minutes Stimulation of plasma membrane-bound transport systems (hexose, ionic pumps) Hormone-mediated receptor internalization Changes in gene transcription Alterations of intracellular enzyme activities
Hours
Synthesis of proteins, lipids, nucleic acids Up and down regulation of the receptor Cell Growth
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I'AXXXXA
A Epidermal g r o w t h
factor
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I
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-
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@ I Y&.-.............. .:.:.:.x..j i.x...:I.,t.:.*.:z.<
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Fig. 17. Receptors showing tyrosine kinase activity. These receptors have a single membrane-spanning domain (shown in black); the amino- [N](extracellular) and the carboxy-terminus [C] (intrace1lular)are also indicated. The clustered cysteine residues are shown as cross-hatched bars, whereas the scattered residues are not shown. The tyrosine kinase domain contains both a catalytic site (stippled) and an ATP-binding site (hatched). Phosphorylated serine and threonine residues are shown as circles, whereas the phosphorylated tyrosine residues are shown as boxes. The length of the receptor is proportional to the number of amino acids, ranging between 972 (F) and 1367 (B).
insulin, sirice severe defects of receptor phosphorylation are found in patients with extreme insulin resistance [56,59] and in streptozotocininduced diabetes in rats [79]. In addition, agents that mimic insulin, such as vanadate and trypsin, stimulate the insulin receptor kinase. Therefore it is reasonable to think that receptor phosphorylation could have a physiological role in mediating insulin action (Table VIII). As to the EGF receptor, its activation leads to enhanced intracellular protein phosphorylation on tyrosine residues [72,113]. The phosphorylation pattern is similar to that reported for the insulin receptor in intact cells and cell-free systems [31]. The binding of EGF to its receptor rapidly induces receptor phosphorylation on tyrosine residues, and
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TABLE VII Major features of insulin receptor kinase
Intrinsic to the receptor ATP-binding site on the receptor 0-subunit Phosphorylation of highly purified receptors Co-purification of insulin binding and insulin-stimulated kinase activity
Modulators Insulin ATP (as phosphate donor) Mg++,Mn++
Substrates Receptors (autophosphorylation) Endogenous and exogenous subtrates
Phosphorylated amino acids in receptor Tyrosine and serine (in intact cells) Mainly tyrosine in cell-free systems
TABLE VIII Evidence for a role of insulin receptor kinase in insulin action
Impaired insulin receptor kinase activity in insulin-resistant states Streptozotocin-diabetic rats Cultured melanoma cells Syndrome of insulin resistance and Acunthosis nigricuns type A Obese mice after gold thioglucose treatment
Increased insulin receptor phosphorylation induced by insulinomimetic agents Concanavalin A Wheat germ agglutinin Trypsin Vanadate
serine and threonine residues are also phosphorylated; the residue threonine-654 on the cytoplasmic domain seems to be the major target of this phosphorylation [32]. EGF receptor phosphorylation can also be catalyzed by protein kinase C . The phosphorylation of the EGF receptor
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and P. Luly
by PKC could have the following effects: (1) inhibition of tyrosine kinase activity [40]; (2) decreased binding activity of the EGF receptor [33]; (3) internalization of the receptor [50].
Internalization of hormone-receptor complexes Several hormones undergo internalization of the hormone-receptor complexes, and the significance of this phenomenon is not fully understood. Hormones and growth factors known to be internalized are: insulin, EGF, TSH, IGF,, IGF, and NGF. The purposes of the internalization could be: (1) a means to degrade the ligand after having expressed its action at the cell surface; (2) a means to degrade the receptor; and (3) a means to degrade the ligand and/or the receptor in the cell interior in order to give a possible second messenger and/or a signal for a physiological response to the hormone. Internalization is an endocytotic process: hormones bind to receptors forming coated pits, which are indented vesicles in the plasma membrane containing hormone-receptor complexes (Fig. 18). Plasma membrane receptors are associated through their cytoplasmic tails with multimeric complexes of the protein clathrin to form coated pits [ 1081. Clathrin has a molecular weight of 180 kDa [109]; in the coat there are also other proteins of lower molecular weight, such as a pair of polypeptides of 3335 kDa (called clathrin light chains), a group of proteins of 100-1 10 kDa and a 50 kDa phosphoprotein. In vitro, and perhaps also in vivo, these proteins self-assemble into a structure containing three heavy and three light chains. This structure has been termed triskelion because of its “three-legged” appearance [147] (Fig. 18). After the binding of the ligand, the regions of the coated pits rich in receptors begin to invaginate. The coated pits pinch off from the membrane to form an internalized coated vesicle. The clathrin coat depolymerizes to leave an uncoated vesicle (endosome or receptosome). The vesicles fuse with an uncoupling vesicle of receptor and ligand with an acidic pH (about pH 5); the low pH favors the dissociation of the ligand from the receptor. The receptors are usually recycled to the plasma membrane, whereas the hormone molecules are usually degraded into the lysosomes. A small percentage of internalized receptors is also degraded. Intracellular receptors for polypeptide hormones have also been identified, but their physiological significance is not known [54]. These receptors appear to be mostly associated with intracellular membranes
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5.
Normal
Receptosome
P
sma
Membrane
-
\
Uncoupling Vesicle
\
Fig. 18. Endocytosis and recycling of hormone-receptor complexes. Internalization includes [ 11 hormone binding to membrane receptors; invagination [2] of coated pits; endocytosis [3] of clathrin-coated vesicles; depolymerization[4] of clathrin leaving clathrin triskelions and receptosomes; coupling of receptosomes [S]with uncoupling vesicles (low pH) followed by the dissociation of the hormone-receptor complex; recycling [6] of receptors to the cell surface; and hormone degradation and release of metabolites [7].
such as the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum [54].It has been shown [54] that some of the rapid effects of polypeptide hormones do not need internalization of the hormonereceptor complex to be triggered. Endocytosis seems to be a more essential step for the long-term effects of hormones because the hormonereceptor complexes can directly interact with intracellular and/or nuclear structures [54].
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Cellular stmitivity to hormones: up and down regulation The internalization of the hormone-receptor complex leads to a decrease in the number of receptors on the cell plasma membrane. Therefore, the receptor number is often regulated by the hormonal level of blood plasma. This phenomenon is known as down-regulation. For instance, insulin receptor-complexes are continually internalized leading to a decrease in the number of receptors on the plasma membrane. Thus we could guess that in the cell the number of membrane receptors is not fixed but can be modulated by the concentration and time of exposure to hormones and their agonists. Down-regulation by internalization or shedding of receptors has been shown for a variety of plasma membrane receptors when they are exposed to an excess of ligand for periods of time. Insulin is one of the hormones for which the number of receptors plays a critical role directly affecting the clinical state. In insulin resistance patients have circulating insulin levels two to three times higher than normal. Therefore, obese patients often have hyperinsulinemia and a reduction of insulin sensitivity due to a decrease of the number of TABLE IX
Insulin receptor status when insulin sensitivity is altered State
Receptor concentration
Receptor affr nity
Normal
Normal Normal
Insulin resistance Leprechaunism Uremia Glucocorticoid excess Lipoatrophic diabetes Growth hormone excess Type A syndrome Type B syndrome Obesity Hyperinsulinemic diabetes
LOW
Normal/low High Normal LOW Normal Normal
Insulin sensitivity Exercise Growth hormone deficiency Glucocorticoid deficiency
Normal High Normal
High High High
LOW
Normal Normal/low LOW
Low/normal Normal/high/low LOW
LOW
Receptors to Peptide Hornioties
139
insulin receptors in target and non-target cells; but in the case of severe insulin resistance, patients require a 5- to 50-fold increase of endogenous and exogenous insulin to have a normal response. Patients with non-insulin- dependent diabetes have decreased insulin sensitivity, which means high levels of hormone and a reduction in the number of receptors. Less common seems to be the opposite phenomenon, i.e., the increase of receptor number called up-regulation. The up-regulation of insulin receptors has been reported in the Swarm rat chondrosarcoma chondrocyte due to insulin [142]. The increase in 1251-insulin binding was reversible and dependent on the concentration of insulin in the culture medium [142]. Patients with insulinomas have shown a decrease in receptor concentration when circulating insulin increases. In addition, there is an increase of receptor affinity, with a concomitant increase of insulin binding; newborns also show high levels of plasmatic insulin with hypoglycemia and high receptor number [ 1231. Cases of altered insulin sensitivity are reported in Table IX.
Evolution of Peptide Hormones and Their Receptors It is quite interesting to note that hormone molecules have no function by themselves, but the function is usually determined by the quality of the receptor, not to mention the numbers, affinity and features that determine the biological function of the hormones (down-regulation, internalization, desensitization): all these factors depend mainly on the receptor. Therefore, a change in a hormone molecule gives rise to a change in the receptor. The evolution of receptors is bound to the evolution of hormones and vice versa, so it is better to consider this issue as an evolution of the endocrine system. Taking all these premises into account, we can say that the “speed” of evolution is greater for hormone molecules than for receptors, the latter being faster than the evolution of transduction systems: speed of evolution hormones > receptors > transduction systems From an evolutive point of view peptide hormones can be divided into three families [ 181: 1. Cholecystokinin (CCK) - Gastrin family. CCK-like peptides are found throughout the Arthropoda and Vertebrate phyla, but gastrin is found only in reptiles, birds and mammals. The CCK-receptor is the
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same in all vertebrates, whereas the gastrin receptor is first detected in birds and mammals. In reptiles gastrin acts through the CCK-receptor.
2. Growth hormone (GH) - Prolactin (PRL) family. The main members of this family, GH and PRL, are separated all through the vertebrates, as are their receptors. There is no cross-reaction between the two hormones, with the exception of primate GH, which can be both a potent lactogen and a growth factor. Particularly interesting appears to be prolactin. This hormone is important for the production of milk, a function typical of mammals. In all other vertebrates, PRL, which is very ancient, has different functions: (a) Osmoregulation, particularly in fishes and amphibians - this is a pivotal function for those fishes which move from fresh to salt water; PRL modulates ion and water permeability in skin; (b) Larval growth in amphibians and reptiles - this function is important at very early stages, before GH starts to be effective; (c) Reproductive functions in higher vertebrates - it has been shown that PRL induces brooding behavior in birds, has a gonadotropic function in rodents and, as already reported, induces milk production in mammals. 3. Insulin family - Several peptides belong to this family that probably arose by gene duplication [17]. Insulin-like growth factors I and I1 are the most closely related, and nerve growth factor (NGF) is also considered to be a member of this family. These peptides basically affect two functions: growth and metabolism. Every peptide has a major function, e.g., insulin is a metabolic hormone, but can also act as a growth factor. The relative affinities of insulin family receptors toward the other members are shown in Table X. TABLE X Relative affinities for each receptor of the insulin family toward family members Receptor
Relative affinities
Insulin IGF I IGF I1 Relaxin NGF
Insulin > proinsulin > IGF I1 > IGF I S- Relaxin ( z 0) IGF I > IGF I1 > insulin E proinsulin IGF I1 2 IGF I S insulin E proinsulin Relaxin > NGF > proinsulin > IGF 4 insulin ( g0) Only NGF binds
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Clinical Relevance of Hormone-Receptor Interactions Alterations of hormone receptors leading to clinical disorders were divided many years ago in the simplest way: (1) too much or (2) too little of a hormone. If the patient had too little, it was enough just to administer the hormone or an agonist; if the patient had too much, the prescription was either to remove the gland or to suppress the hormone synthesis. The scheme below gives an indication of prefixes used to indicate “too much” or “too little.”
State Below normal Normal Above normal
Prefix HYPOEuHyper-
At present, progress in molecular biology and recombinant DNA technology has shed light on a number of endocrinopathies. Endocrinopathies involving peptide hormones can be due to: (1) alterations of hormone concentration; (2) alterations of peptide hormone receptors; (3) presence of anti-hormone and anti hormone-receptor antibodies; (4) alterations of transduction systems; and (5) alterations at the post-receptor level. 1. Alterations of hormonal concentration - The simplest condition for a hypoendocrinopathy takes place when the gene for the hormone is missing. One example is offered by the GH deficiency type IA [112]; there are two genes for human GH, and both of them are clustered along a 48 kb stretch of DNA. Human growth hormone-N (hGH-N) is expressed in the hypophysis, whereas hGH-V is expressed in the placenta [52]. In GH deficiency type IA, hGH-N is deleted [ 1121; hGH-V remains but it is not functional in the hypophysis. Therefore, patients are short and tend to hypoglycemia. Hypoendocrinopathy may also result from a defective hormone molecule. These types of alterations have been reported for insulin and antidiuretic hormone. Insulin deficiency is known to induce diabetes mellitus and mutant insulins have also been characterized [ 1441; among these [Leu B25]-insulinand [Ser B24]-insulin have been identified in diabetic patients. These residues are in the normotype represented by phenylalanine and are found in the receptor binding domain of insulin. Besides these types of mutations, there are processing defects [114]. Insulin is synthesized as a single peptide, with a chain C as a bridge between A and B subunits. In familial hyperproinsulinemia, the B-C
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junction is cleaved normally but the A-C is not; therefore, the C peptide is not cleaved. As a consequence of this partial cleavage of proinsulin (the resulting molecule has 60%of biological activity with respect to normal insulin), the pancreas compensates by secreting more hormone in order to keep high serum levels. Other processing defects have been studied in rodents. Brattleboro rats have a form of hereditary diabetes insipidus, resulting from a deficiency of antidiuretic hormone (ADH) and its neurophysin. Both molecules are cleaved from a single polyprotein. The gene sequence in these animals shows a deleted base; as a consequence, although ADH is normal, its neurophysin is abnormal and cannot bind ADH [130]. 2. Alteration of peptide hormone receptors - Several insulin receptor mutants have also been found in patients with severe insulin-resistant diabetes mellitus. After the discovery of insulin and its use to treat diabetes, it was found that this syndrome could be divided into two types: (a) insulin-dependent diabetes mellitus (IDDM) and (b) non-insulindependent diabetes mellitus (NIDDM) [65]. IDDM was treated by the administration of insulin, whereas NIDDM was found to be insulin-insensitive; in the latter case the level of the pancreatic hormone was normal or even above the normal average. Molecular biology has shed light on at least some of these cases of insulin resistance, leading to the identification of mutations of the insulin receptor that impair its function [8]. It has been found that the insulin receptor in patients with severe insulin resistance (e.g. leprechaunism, Rabson-Mendenhall syndrome, severe insulin resistance type A with acanthosis nigricans) is altered in its structure. The cloning of the insulin-receptor cDNA [44,146] and of the insulin-receptor gene [ 133,1341 has led to the finding that these patients have mutations in one or both alleles of the insulin-receptor gene. The insulin receptor mutations can be divided into six classes depending on which step is affected (Fig. 19) [8]: Class 1: synthesis, when the receptor protein is hardly detectable or very low levels of mRNA are present; Class 2: delayed transport to the plasma membrane; Class 3: defective insulin binding; Class 4: defective internalization of the insulin-receptor complex; Class 5: altered recycling of the receptor to the plasma membrane; Class 6: altered tyrosine kinase activity and intracellular signaling. Mutations in each of these classes have been described so far, except for class 4. However, because mutations leading to defective internalization of the low-density lipoprotein (LDL) receptor have been described [66], we can also expect similar mutations for the insulin receptor. A GH
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Fig. 19. Different classes of mutations affecting the structure and function of the insulin receptor causing severe insulin resistance. Classes of mutations are: [ 11 synthesis; [2] transport to the plasma membrane; [3] insulin-binding; [4] internalization; [5] recycling; [6] insulin signalling.
receptor defect is the likely possibility in Laron dwarfism [45]. Laron dwarfism is an autosomal recessive disorder where patients have short stature but have elevated GH levels. The hormone is biologically active, and thus a defect at receptor level seems to be a likely hypothesis; in fact, these patients also lack a plasma GH-binding protein [5,30] which is the cleaved amino terminus of the GH receptor [91].
3. Presence of anti-hormone and anti-hormone receptor antibodies Another group of endocrinopathies is known as “autoimmune diseases. ” These are characterized by the inability of an organism to recognize certain endogenous molecules as “self”; therefore, they are responded to as antigens giving rise to autoantibodies. These antigens can be either hormones (anti-hormone antibodies) or hormone receptors (anti-receptor antibodies). The first case is less common; high levels of anti-insulin antibodies that characterize the resistance to exogenous insulin have been observed occasionally in insulin-treated IDDM patients [ 1541. When these antigens are hormone receptors, several possibilities arise. In fact, anti-receptor antibodies have been implicated in the pathogenesis of three diseases: Graves’ Disease, with antibodies to the thyroid-stimulating hormone receptor [20]; Myasthenia gravis, with antibodies to the acetylcholine receptor [ 1501, the treatise of which is beyond the scope of this review; and Acanthosis nigricans Type B, with antibodies to the
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GRAVES DISEASE
NORMAL
1r’
PITUITA R THYROID STI M UL AT1NG HORMONE (TSH)
THYROID ST IMULATI NG
T4
THYROID
Fig. 20. Interaction between tyrotropin (TSH) and the thyroid gland in normal subjects and in Graves’ disease. T, and T,, thyroid hormones.
insulin receptor and severe insulin resistance [81]. In all cases antibodies compete with the hormone or the transmitter for the binding to the receptor on the cell plasma membrane. In Graves’ disease antibodies against the thyroid-stimulating hormone (TSH)-receptor block the binding of TSH to thyroid cells and also interact with the hormone receptor, mimicking the action of TSH itself; therefore, cyclic AMP production is increased as from the normal physiological stimulus. As a consequence, there is an abnormal production and secretion of thyroid hormone, giving rise in these patients to severe symptoms of hyperthyroidism (Fig. 20). Acanthosis nigricans is characterized by severe insulin-resistance to both endogenous and exogenous insulin. The tissue resistance gives rise to a compensatory hyperinsulinemia with basal and insulin-stimulated levels 10-100 times greater than normal (144). In some cases, the hyperinsulinemia is sufficient to overcome the insulin resistance, in others it is not and diabetes results (144). 4. Alterations of transduction systems - Pseudohypoparathyroidism, described in 1942 by Albright and coworkers [2], was the first hereditary hormone resistance syndrome to be observed. It was found to be an x-linked - or autosomal - dominant disorder characterized by hypoparathyroidism with neuromuscular irritability, hypocalcemia, hyperphosphatemia and elevated levels of parathyroid hormone (PTH), often associated with other minor abnormalities such as short stature, obesity, short neck and mental retardation [81]. Patients with pseudohypoparathyroidism type I show renal parathyroid hormone-resistance, with an
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145
TABLE XI
Major sites of expression and putative functions of glucose transporters Designation
Type
Major sites of expression
Functions
GLUT 1
Erythrocyte
Placenta, brain, kidney, colon
Basal uptake
GLUT 2
Liver
Liver, 0-cell, absorptive epithelial cells of kidney and small intestine
Bidirectional transport by hepatocyte, 0-cell glucose sensor, efflux of absorbed glucose across the basolateral membrane of epithelial cells of kidney and intestine
GLUT 3
Brain
Many tissues in humans (brain, placenta, kidney and others)
Basal uptake
GLUT 4
Muscle/fat
Skeletal and cardiac muscle, brown and white adipose tissue
Insulin-stimulated uptake
GLUT 5
Small intestine
Small intestine (jejunum)
Dietary absorption
impaired response of urinary cyclic AMP to parathyroid hormone administration [25], indicating a defect of the parathyroid hormone receptor-adenylate cyclase system and a decrease of the adenylate cyclase affinity for ATP [41]. However, PTH levels and hormone receptors appear to be normal; the adenylate cyclase is also normal since it can be stimulated by forskolin. However, fluoride and non-hydrolyzable analogs of GTP (e.g., GTP-y-S) do not activate the adenylate cyclase; fluoride and GTP act through the a-subunit of G,. It has been found that these patients have low levels of a-subunit mRNA [22,92]; therefore, it seems that the disease involves a defect in the gene for the a-subunit of G,. This raises a question: why does G,, which should be sensitive to all cyclic AMP-dependent hormones, affect only PTH action? In fact, this defect is not limited to PTH, but the deficiency is found in all tissues [148]and
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involves all cyclic AMP-dependent hormones; but the effects are subclinical and are not so severe as to give clear symptoms.
5. Alterations at the post-receptorial level - A good example of defects at the post-receptor level is alterations of the insulin-sensitive glucose transport system in NIDDM patients, known to be a primary cause of insulin resistance. NIDDM patients show decreased glucose transport activity and a decreased number of glucose transporters [8]. Also in this case molecular biology has made a great contribution to the understanding of glucose transport proteins and their regulation in normal and disease conditions. cDNA cloning studies have shown a family of structurally related glucose transporters, encoded by different genes. A number has been given to each transporter (Table XI); the insulinsensitive is called GLUT-4, and the 0-cell glucose sensor is GLUT-2 [8]. The isolation and characterization of cDNA encoding for the glucose transporters in mammalian cells have provided new cDNA probes and specific antibodies for the study of glucose transport and its modulation by hormones. These studies have shown that insulin resistance in adipocytes and impaired insulin secretion by the pancreatic 0-cells may be due to decreased expression of GLUT-4 and GLUT-2, respectively [ 10,24, 77,1391. Studies on the regulation of glucose transport in adipocytes suggest a model where insulin affects both the translocation of GLUT-4 from intracellular stores to the plasma membrane and the expression of GLUT-4 [8]. The hormonal effects on the two processes have different time-courses: the translocation would be fast, within minutes, whereas the effect on the mRNA for the GLUT-4 would be slow, within hours [ 1391 (see also Table VI).
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137 Sibley, D.R., J.R. Peters, P. Nambi, M.G. Caron and R.J. Lekowitz, (1984), J. Biol. Chem. 259:9742-9749. 138 Sibley, D.R., R.H. Strasser, M.G. Caron and R.J. Lefkowitz, (1985), J. Biol. Chem. 260:3883-3886. 139 Sivitz, W.I., S.L. Desautel, T. Kayano, G.I. Bell and J.E. Pessin, (1989), Nature 340:72-74. 140 Stadel, J.M., R. Rebar, R.G.L. Schorr, P. Nambi and S.T. Crooke, (1986), Biochemistry 25:3719-3724. 141 Stadel, J.M., P. Nainbi, R.G.L. Schorr, D.F. Sawyer, M.G. Caron and R.J. Lefkowitz, (1983), Proc. Natl. Acad. Sci. USA 80:34173-34177. 142 Stevens, R.L., K.F. Austen and S.P. Nissley, (1983), J. Biol. Chem. 258:29402944. 143 Sutherland, E.W., (1972), Science 177:401-408. 144 Tager, H.S., (1984), Diabetes 33:693-699. 145 Thoinas, A.P., J.S. Marks, K.E. Coll and J.R. Williamson, (1983), J. Biol. Chein. 2585716-5725. 146 Ullrich, A., J.R. Bell, E.Y. Chen, R. Herrera, L.M. Petruzzelli, T.J. Dull, A. Gray, L. Coussens, Y. Liao, M. Tsubokawa, A. Mason, P.H. Seeburg, C. Grunfeld, O.M. Rosen and I. Ramachandran, (1985), Nature 313:756-761. 147 Ungewickell, E. and D. Branton, (1981), Nature 289:420-422. 148 Van Dop, C. and H.R. Bourne, (1983), Ann. Rev. Med. 34:259-266. 149 Vauquelin, G., P. Geynet, J. Hanoune and A.D. Strosberg, (1973, Proc. Natl. Acad. Sci. USA 74:3710-3714. 150 Vincent, A., (1980), Physiol. Rev. 60:756-824. 151 Volpe, P., K.H. Krause, S. Hashimoto, F. Zorzato, T. Pozzan, J. Meldolesi and D.P. Lew, (1988), Proc. Natl. Acad. Sci. USA 85:1091-1095. 152 Welton, A.F., P.M. Lad, A.C. Newby, H. Yamamura, S. Nicosia and M. Rodbell, (1977), J. Biol. Chem. 2525947-5950. 153 White, M.F., S. Takayarna and C.R. Kahn, (1985), J. Biol. Chem. 260:94709478. 154 Yalow, R.S. andS.A. Berson, (1961), Am. J. Med. 31:882-891.
Biomembranes Edited by Meir Shinitzky Copyright 0 VCH VerlagsgesellschaflrnbH, 1995
CHAPTER 4
G Proteins in Signal Transduction LUTZ BIRNBAUMER
and MARIEL BIRNBAUMER
Department of Anesthesiology, UCLASchool of Medicine, Los Angeles, CA90024, USA
Contents 154
Abbreviations
155
Introduction
160
The G Proteins 162 The regulatory cycle - Mechanism of action of a receptor 168 fly-Dimers as potential activators of a-subunits by transphosphorylation 169 Molecular diversity of G protein subunits 177 Lipid modification of G protein subunits - Membrane attachment and function in signaling 176 Myristoylation of a-subunits 177 Palmitoylation of a-subunits 177 Polyisoprenylation of y-subunits 178 Patterns of G protein subunit expression 179 The a subunit: structure and function 181 a-Subunits as proto-oncogenes 182 The three-dimensional structure of the a-subunit
L. Birnbaumer and M . Birnbaumer
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186
205
217 221 222 23 1
G Protein Coupled Receptors 190 Subfamilies 198 Naturally occurring receptor mutations 198 Rhodopsin mutations 199 VP2-Vasopressin receptor mutations 199 The melanocyte stimulating hormone (MSH) receptor as a genetic model 203 G protein activation by non-heptahelical receptors Molecular Diversity and Regulation of Adenylyl Cyclases and Type-CP Phosphatidylinositol-SpecificPhospholipases 205 Molecular diversity of &-subunits - Multiple effectors for a single G protein 207 Signaling through 0-y dimers 207 Adenylyl cyclases 21 1 Inhibition of adenylyl cyclase 212 Regulation by phosphorylation 212 Phospholipase C 215 Subunit concentrations that cause half maximal effects Role of GAP activity of the effector 216 Specificity of receptors for G proteins Conclusion Acknowledgements References Appendices
Abbreviations AC DAG IP3 PLC a x , or Gxcr
-
adenylyl cyclase diacylglycerol inositol 1,4,5-trisphosphate phospholipase C
- a-subunit of G,, with a composition of aXPr
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Introduction The primary structure of the components involved in G protein mediated signal transduction are now well known. They include a large family of transmembrane receptors, a large family of heterotrimeric (spy) G proteins activated by GTP under the influence of receptors, and a series of molecularly unrelated effectors that are regulated by G protein a,G protein By dimer, or both G protein a and Py dimers (Table I). The list of extracellular compounds depending on G proteins for their signalling includes hormones, neurotransmitters, and auto- and paracrine factors, and illustrates the central role of G protein mediated signal transduction in cell regulation and body homeostasis. The cellular functions regulated by the activated G proteins mainly include adenylyl cyclase (AC) and the type CP phospholipase (PLCP), which are the enzymes responsible for the production of the classical second messengers, CAMP- by AC - and inositol 1,4,5 tris phosphate (IP3) and diacylglycerol (DAG) - by PLCP. Other functions that are expressed in some but not all cells are also targets of G protein regulation. These include visual phosphodiesterase (PDE), Ca2+ channels and a variety of K+ channels; and there may be more (Table 11). Specificity and selectivity parameters define which G protein is activated by which receptor, and which effector is activated by which G protein. These specificity and selectivity parameters are stringent enough to avoid undesired crosstalk between distinct G protein regulated pathways, for example AC vs PLC, but they are not absolute. Due to this, it is possible that under extreme conditions the activation of one G protein mediated signalling pathway, say activation of AC, may be accompanied by that of another (e.g., PLC). This gives rise to responses that may vary in interesting ways, depending on receptor density and receptor occupancy. Here we will summarize the most relevant aspects of the structural and functional features of the “receptor+G protein+effector” axis (Fig. 1). We shall begin with the G proteins which transduce the conformational change of the receptor induced by agonist into a regulated effector function. This will be followed by a discussion of structural features of the receptors. The article will finish with a short description of molecular diversity within the two main effector functions, ACs and PLCPs and their responses to G protein subunits. An overview of the historical development of this field was published and will not be reiterated here [l]. For a previous, complementary review see [2].
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TABLE I Molecular elements of the G protein transmembrane signal transduction path
A. >200 Receptors 1. For neurotransmitters and autacoids a. Biogenic amines catecholamines adrenaline noradrenaline dopamine serotonin (5HT) histamine melatonin b. Non-biogenic amines y-amino butyric acid (GABA) glutamate acetylcholine cannabinoid: anandamide purines adenosine ATP 2. For opioid peptides &endorphin leu- and met-enkephalins dynorphin neo-dynorphin 3. For pituitary hormones adrenocorticotropin (ACTH) glycoprotein hormones luteinizing hormone (LH) thyrotropin (TSH) follicle stimulating hormone FSH) melanocyte stimulating hormones (a-, 0- and y-MSH) vasopressin oxytocin 4. For pituitary release factors thyrotropin release hormone (Tl2I-J) corticotropin release factor (CRF) somatotropin release factor (GRF) gonadotropin release hormone (GnRH) pituitary adenylate cyclase activating peptide (PACAP) somatostatin (SST, SRIF')
5. Forkinins tachykinins or neurokinins Substance P Substance K Neurokinin B bradykinin angiotensin II endothelin 6. For gastrointestinal peptides
cholecystokinidpancreozymin (CCK) gastrin secretin glucagon glucagon-like peptide 1 glucose-dependent insulino-tropic peptide (GIP) gastrin releasing peptide (GRP) peptide YY (PYY)
7. For neuropeptides neuropeptide YY (NPY) vasoactive intestinal peptide (VIP) peptide histidine-methionine-wide (PHM) 8. For endocrine and paracrine
hormones parathyroid hormone calcitonin calcitonin-gene related product amylin pancreatic polypeptide pancreastastin galanin
9. For chemokines and chemotactic agents interleukin 8 formyl-peptides C5a C3a platelet activating factor thrombin
G proteins in Signal Transduction TABLE I,
157
continued
10.For arachidonic acid metabolites prostaglandins and prostacyclin thromboxanes leukotrienes 11.For sensory inputs light: opsins (dim, red, green, blue) odors taste (sweet, bitter) 12.For divalent cations Ca2+
3. Ion channels K channels inward rectifier - 1 gene ATP-sensitive - 1 gene Ca2+-activated- > 1 gene Ca channels cardiac - C type skeletal muscle - S type presynaptic - types: N,L.?others 4. Others
plasma membrane Ca-pump ?glucose transporter ?Na/H exchanger
B. G Proteins: Combinations of a and By
D. Examples of Molecular Diversity of Cloned Receptors
1. a-subunits
1. Catecholaminereceptors 3 0-adrenergic @l,D2,@3) 3 al-adrenergic (1A, 1B,1C) 3 a2-adrenergic (C 10,C2,C4) 5 dopaminergic (Dl-D5)
a-S
wolf
a-q a-1 1 a-14
01-15/16 a-12 a-13 a-01 (woA) a-02 (a-oB) a-il a-i2 a-i3 a-Z
a-t(rod) - transducin a-t(cone) a-gust - gustducin 2.
By dimers
D l through 05 y l through y7
C. Response Elements (Eftectors) 1. Adenylyl cyclases - 8 genes 2. Phospholipases (PLCP - 4 genes)
2. Serotonin receptors > 10: 5 type SHT-lA, -lB (also 1D@, -lD (also lDa), -1E, -lF; 3 type 2: 5HT-2A (former 2) -2B (former 2F) -2C (former 1C) other: 5HT-4,5HT-5,5HT-7 3. Acetylcholine 5 muscarinic (Ml-M5) 4. Peptide hormones
5 somatostatin 3 vasopressin (W-la, VP-lb, VP-2) 3 opioid: p , b , ~ 3 MSH 3 NPYIPPY 011, Y2) 2vIP 2 endothelin (ET-a, ET-b) 2 angiotensin 11(type I and II) 2 bradykinin (BK1 and BK2)
5. Odorant
>40
8 types (I-VI ...). All stimulated by G p , except 40 kDa sperm AC; Some stimulated by Ca2+/CaM (e.g., types I and 111). Two or more are inhibited by G p s (e.g., type V, VI and 11) Effect of Goy may be: mhibition (type I); stimulation conditional on G,a-stimulation (type 11, IV); no effect (e.g., type 111, v , VI) Gta (aT)interacts with and relieves the inhibitory action of ypDE on activity of a/3 complex
A. Adenylyl cyclases (ACs) transmembrane,
B. cGMP-phosphodiesterase (cG-PDE) peripheral membrane protein: aPy2
Several subtypes (?), e.g., 40-pS cardiac muscarink K + channel; 50-pS GH cell acetylcholine and somatostatin-stimulated inward rectifier. Stimulated in heart by three G p s , not by G,a; one or more may be stimulated by fly and/or arachidonic acid metabolite(s)
> 6 subtypes: type S : skeletal muscle, L-current (inh. by DHP). Type C: sensitivity to: DHPs, w-CTx-GVIA, w-CTxheart/smooth muscle/neurons/endocrine cells, L-current (inh. by DHP); MVIIC, w-Aga-IVA; transmembrane, 24 TMs Type B: neurons, N-current (inh. by w-CTx-IVA); Type D: neuron/ in al;alternative splicing of al; endocrine cells, L-current (mh. by DHP); Type A: neurons, Q-current (inh. by w-CTx-MVIIC); Type E: neurons, R- (residua1)current
E. Ca2+ channels - Voltage-gated; variable
transmembrane protein, structure: GIRKI: 2 TMs
D. Inwardly rectifying K + channel(s)
C. PIP2-specific phospholipase Cs (PLCDs) 4 isoforms: /31,/32,&rP4. PLCP,: stimulated by a of G,, GI1, G,, and (less) peripheral membrane protein, single subunit G16; expressed ubiquitously. PLCP2: stimulated by by and (less) by G16a, enzyme, part of family of PIP2-specific PLCs: as well as by high concentrations of other Gq-type as; expressed predominantly in blood-borne cells. PLC&: sensitive to both Py and some P,YJ G,-type as. PLCP4: a vs fly sensitivity not known
12 TMs
Comments Molecular diversity-types, subtypes
Effector class (structural/functional characteristics)
Examples of mammalian effector systems regulated by G proteins
TABLE 11
8
3
R
9 3Q-
%
k
3
3
Q
3 Q-
$
P
Type P: P-current (ihn. by w-Aga-IVA; underlying a1still unknown). Stimulation by G p : proven for type S (skeletal muscle) and type C (cardiac). Inhibition by Gola and GO2amediated pathways seen in whole cell recordings, direct regulation by G a not yet proven in a cell-free system; L- and N-type Ca" channels are regulated in this way. Stimulation by either a Gia or a G$y triggered pathway, e.g., PTX-sensitive stimulation of Ca2+ currents in Y 1 adrenal cells by angiotensin I1 and in GH3 cells by TRH
E., continued
Several subtypes based on electrophysiology. One type stimulated by G,a: proven for pig coronary artery and rat uterine smooth muscle channels in lipid bilayers
G . Ca'+-dependent K+ channels
(charybdotoxin sensitive, transmembrane proteins, 6TMs; cloned mSlo &a channel: 1200 aa; purified tracheal channel: 62 kDa a and 31 kDa 0;subunit structure: aJA?
Several subtypes by pharmacology and affinity labelling of SU receptor site. Stimulated by three G p s , not by G,a or G,a in inside out-membrane patches
F. ATP-sensitive Kf channel(s) (inhibited by sulfonylureas (SUs), transmembrane protein, structure/subunit composition unknown)
six a1genes known: a l A , alB, a l C , a l D , alE, a 1 S ; /3 isoforms (f11-/34); general subunit structure (a2G)a,/3[ 571; subtype defined by type of al,presence of y proven only in sk. muscle
Comments Molecular diversity - types/subtypes
Effector class (structural/functional characteristics)
TABLE 11, continued
3
8. 0
E
2 2R
Y
G3 i%
GI
-. 3
2
a s.
Q
'a
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Hormone
Fig. 1. The transduction of the signal emitted by the hormone occupied receptor by G protein involves the activation of the G protein by GTP and concomitant dissociation of the protein into two signalling molecules, a-GTP and &, of which each regulates diverse effector functions.
The G Proteins Three separate aspects of heterotrimeric G proteins are important: 1. The existence of a basic regulatory cycle involving a GTPasedependent unidirectional subunit dissociation-reassociationcycle in which hormone receptor drives the activation by GTP and the consequential dissociation into an active GTPoa complex plus a 0-y dimer; and the GTPase activity, which, by converting a GTP into a GDP, causes the deactivationof the a-subunit with concomitant acquisition of high affinity for /3r dimer (Fig. 2); 2. The existence of a rather large degree of molecular diversity among all three of the G protein subunits, of which the different a-subunits can be grouped into structurally and functionally related subgroups; and 3. The fact that both as and 07 dimers are signalling molecules (i.e., have the potential of regulating effector functions), and that single as and 07s are able each to regulate more than a single effector function. The regulatory cycle insures that G protein mediated processes are rapidly reversible and dependent on a second-by-second basis on agonistdriven receptor activity. This cycle is relatively slow and appears to involve separation of the activated form of the G protein from the receptor so that during the lifetime of an activated G protein, a single receptor has the ability of activating several G protein molecules. Thus, occupancy of one receptor may lead to activation of not one but several
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r""' f
I
GTP
. I
\
11,-
/
Fig. 2. Basic regulatory cycles of a G protein involving both GTP-induced activation and subunit dissociation and GTPase-dependent inactivation and subunit association.
effector molecules. G protein mediated signalling not only involves the transduction of the input signal, receptor occupancy by ligand, into modulation of effector activity altering the intracellular level of a second messenger, but also the amplification of the input signal whereby occupation of a single receptor may activate several G proteins; and each G protein due to the generation of both an Q signal and a Py signal may modulate not one but two effector molecules. Molecular diversity of G protein subunits provides both for a certain degree of redundancy, most clearly shown in a recent knockout experiment, and for the potential ability of a given cell to alter its primary or secondary response patterns by switching or up- or down regulating selected isoforms of as, P s , and ys. The potential ability of multiple actions of a single G protein in turn provides for a potentially large variation in the response pattern that may be elicited in a given cell by activation of a given G protein. The particular response pattern elicited depends on the type of G proteins and the type of effector functions that the cell is expressing.
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The regulatory cycle - Mechanism of action of a receptor The experiment shown in Fig. 3A illustrates the effect of receptor to accelerate the activation of G, by guanine nucleotide. The experiment in Fig. 3B illustrates what appears to be the molecular basis for this effect of the receptor, i.e., the reduction in the requirement of Mg2+ for G, activation by guanine nucleotide from supraphysiologic (app. Km ca. 1520 mM) to well below that of normal prevailing intracellular levels (app. Km ca. 10 pM). It is clear from these two experiments that receptors
20
40
Time
60
(min)
80
100
Fig. 3A. Key characteristics of receptor mediated stimulation of G protein activation and reconstitution of receptor induced activation of G protein with purified proteins. Receptor accelerates activation of G, by a non-hydrolyzablenucleotide(GTPyS) (from Abramowitz et al. [172]).
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accelerate activation by guanine nucleotide. This is due to catalysis of nucleotide exchange, GTP or GTPyS for GDP as is illustrated in Fig. 3C with a reconstituted system where the only protein components are receptor and G protein placed into phospholipid vesicles. The experiment Liver membrane Gs and Glucagon Receptor (AC inactivated with NEM) 1
z
E
G T P r j + Glucagon
.x
~
U
c
$ 2 $
10
0 0
20
Mg2+
30
40
50
(mM)
Fig. 3B. Key characteristics of receptor mediated stimulation of G protein activation and reconstitution of receptor induced activation of G protein with purified proteins. Receptor reduces the concentration of Mg2+ required for activation of G protein by GTP or a GTP analog (adapted from Iyengar and Birnbauemr [ 173I).
Gs reconstituted with BAR
i "0
5
10 Time (min)
15
Fig. 3C. Key characteristics of receptor mediated stimulation of G protein activation and reconstitution of receptor induced activation of G protein with purified proteins. Basal and agonist-stimulated GTPyS binding to G, in phospholipid vesicles (adapted from Cerione et al. [174]).
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in Fig. 3B illustrates that, at high enough Mg2+, activation of G, by guanine nucleotide is quasi-independent of a receptor and that the primary effect of receptor is to reduce, by a factor of close to 1000, the concentration at which M$+ causes nucleotide exchange. The mechanism by which this increase in affinity of G protein for M$+ occurs is not well understood, but it, rather than the more commonly considered nucleotide exchange reaction, may be the underlying mechanism of receptor mediated activation of a G protein. Activation of a purified G protein by a non-hydrolyzable GTP analogue (GMP-P(NH)P or GTPyS) leads, as it does in membranes, to its persistent, quasi-irreversible activation. Hydrodynamic analysis of the G protein before and after activation showed that activation is associated not only with stable binding of the guanine nucleotide but also with a decrease in molecular weight due to a dissociation reaction of the type:
In the presence of GTP, the reversal reaction involves GTP hydrolysis and reassociation of a with 07 to give the starting aGDP.0y complex according to the reaction sequence
Taken together these reactions allow for the postulation of the minimal regulatory cycle of the type shown in Fig. 2. Several individual rate constants that make up this cycle have been assessed with native (e.g., [3], reviewed in [4]) and recombinant (e.g., [5,6] G proteins. The rate at which an individual a-subunit hydrolyzes GTP to GDP has been estimated by various means at about 3-5 min-'. A purified heterotrimeric G protein exhibits steady state GTPase activities that are limited by the rate of exit of GDP from the catalytic site at about 0.03 min-'. This slow rate is due both to a slow intrinsic exit rate from a,at about 0.2-0.4 min-', and to an inhibition of GDP dissociation by the & complex, for which the a *GDP complex has high .affinity, to 0.01-0.03 min-'. At the prevailing intracellular concentration of Mg2+ of ca. 0.5 mM, spontaneous reactivation of a G protein is essentially nil. This insures a minimum of "noise" in the absence of an activating ligand. Stabilization of binding of GTPyS to &free a , steady-state GTP hydrolysis by a 07-free a,and a jump in intrinsic tryptophan fluorescence
G proteins in Signal Transduction
165
of GTP- or GTPyS-liganded cr depend on M 2 ' with an apparent K,,, of ca. 10 nM. Dissociation of GDP from a,on the other hand is independent of M 2 + , proceeding at about 1/10 the intrinsic rate (kcat)of GTP hydrolysis, and is inhibited by about ten-fold by 07.This inhibition is relieved and dissociation is stimulated by high mM Mg2+ (apparent K,,, ca. 10 mM). Concomitant with stimulation of GDP release from cr& trimer, M g + also stimulates steady-state GTP hydrolysis. These data have been used to construct the scheme of Fig. 4. In contrast to regulation of GTP hydrolysis by a G protein cr-subunit and of its nucleotide binding by /3y and M$+, the mechanism by which a receptor modulates this process is less well understood. Interpretations
Mg? Pi
koff = 0.3/rnin (no effect of Mg)
I
aGTP
I
koff = 0.3/rnin ~
koff values refer to nucleotide; kcat. catalytic rate of GTP hydrolysis; 4 , h g h affinity form of receptor.
Fig. 4. Summary of kinetic events occurring during a G subunit dissociationheassociation cycle in the absence and presence of a hormone receptor complex. For details see text.
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as to the role of receptors vary somewhat depending on the point of view. Most models describe receptors as simple exchange factors and very few include M?' as a participating regulator or attempt to locate the site to which Mg2' binds in the process of G protein activation. Yet one of the first effects of receptor stimulation to be described was a reduction of the requirement for M$+ (e.g., [7]; also Fig. 3B). The experiments on the effect of M?' on GDP release and steady-state hydrolysis of GTP by and suggest it as a site of trimeric G protein point to an active role of action of M?'. Indeed, while 07 inhibits GDP dissociation from a , Mg2' Py acts as a guanine nucleotide release stimulator (GDS), promoting not only GDP release but also the binding of GTP. Liganded receptors could act by simply reducing the K,,, for M 2 ' at 07 so that nucleotide exchange is but the stimulation of the exchanger function of the Mg2+*py complex. In support of this, to interact with as, liganded receptors (HR complexes) require not only that a be present, in the context of an a07 complex [8], but also M2'. Interaction of G protein with receptors can be assessed not only by studying changes in nucleotide binding and hydrolysis but also, because of a reciprocal effect of G protein on receptor, by determining the agonist affinity state of the receptor (Fig. 5). The formation of this complex may require an agonist and can generally be detected because it stabilizes the receptor in a conformation that often, but not always, has higher affinity for the agonist (Rh) than what is observed in the absence of the G protein (R,)[9-131. In support of the assumption that for receptors such as the 0adrenergic receptor the high affinity form of the receptor represents G R, purified G protein-free receptor shows the same low affinity for the agonist as seen after saturation of membranes with guanine nucleotide and acquires high affinity for an agonist when reconstituted with purified G protein (e.g., Fig. 5). Since either a guanine nucleoside diphosphate (GDP or GDPPS) or a guanine nucleoside triphosphate (GTP or one of its non-hydrolyzable analogs) promotes low affinity binding (e.g., [9]; Fig. 5B), high affinity binding can be taken largely as the measure of the receptor complexed with nucleotide-free heterotrimeric G protein (HR G), poised for association with either GDP or GTP, thus allowing a "free" exchange to occur. In one case, GDP, the complex relaxes without activation of G. In the other case, GTP, the complex advances along a not totally understood path, one version of which is shown in Fig. 4,to new forms of HR G. Because of a conformational change in a induced by 0
G proteins in Signal Transduction A.
9 9 cyc- Membrane (NoGs)
S49 wt Membrane
9
B.
167
8
7
6
5
9
0
(-)lsoproterenol (-log M)
Tulkey ErythrocyteMembrane
98765 4 M) (-)lsoproterenol (-log
4
c.
7
6
5
I
1 . 4
.
J-AR reconstituted with Gs
1 3
(-)lsoproterenol (-log M)
Fig. 5. Guanine nucleotide and Mg2+ regulation of affinity of receptor for agonist reflects the interaction of G proteins with receptor. A: Regulation of receptor affinity for agonist is dependent on G,, as seen from a comparison of agonist displacement curves obtained with G,-containing (left) and G,-deficient (right) S49 cell membranes and on Mg2+ in the medium. B. The high affinity form of the receptor (obtained in the presence of Mg2+) represents the complex of agonist-occupied receptor and the guanine nucleotidefree G proteins. C. Reconstitution of regulation of agonist affinity to purified receptor by purified G , protein. In the absence of G, the inhibition of antagonist ('251-CYP) binding by an agonist only reveals low affinity binding (not shown). (Adapted from Birnbaumer et al. [175] and Cerione et al. [174].
MgGTP binding, the a-0-yinteraction is reduced. This promotes dissociation of the HR G(Mg) GTP(Mg) complex into activated a GTP Mg plus HR 0-y Mg. Possible fates of the HR * 0-y complex are illustrated in Fig. 6. One path leads to the formation of the 0-y dimer plus the HR complex; the other lets H dissociate before @y does, leading to the formation of an 9
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Fig. 6. Model of receptor G protein interactions within the context of a G protein regulatory cycle showing various possible fates of the HR 0-y complex. Although only free a-GTP and free 0-y are assumed to be competent to regulate an effector, this has not been tested. Note that >P may form without or with formation of free P-y and that HR P-y is a postulated substrate of G protein coupled receptor kinases.
R fly complex. There is no information as to the role of M?' in determining the type path followed, but whether or not Py forms appears to be important from the signalling point of view because there are G protein
signalling pathways that are mediated by the dimers rather than the a subunits. a-subunits activate adenylyl cyclases (for molecular diversity see below), a type of Ca2+-dependent K + channel, K + channels of the inwardly rectifying and ATP-sensitive type, skeletal and cardiac voltagedependent Ca2+ channels, visual phosphodiesterase, various isoforms of PLCP, etc. ,By-dimers, on the other hand, stimulate certain forms of PLCP as well as certain types of adenylyl cyclase, and possibly also the inwardly rectifying K+ channel and one of the Ca2+-activated K+ channels. While in the case of a-subunits it appears to be clear that it is the activated free a-subunit that is responsible for receptor signalling, it has not been shown, for example, whether only free fly dimers or whether fly HR and Py R complexes can also modulate effector hnctions. fly-dimers as potential activators of a-subunits by transphosphorylation A surprising and not as yet much =cognized finding is that fly dimers can activate the G,-adenylyl cyclase system by a mechanism involving a-
G proteins in Signal Transduction
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subunit activation. This depends on the method of preparation. Activation occurs if fly dimers are prepared from heterotrimeric G proteins by activation with GTPyS but not if prepared using GMP-P(NH)P as the it dissociating agent. By the use of 35S-labeledGTPyS and [T-~~P]GTP, was now shown that the P-subunit can become phosphorylated and that the phosphorylated Py can transfer its phosphate to membranes under conditions that lead to activation of adenylyl cyclase or onto GDP but not ADP, GMP, or GDPPS. The overall reaction is that of a nucleotide diphosphate kinase and the acceptor is very likely a,-bound GDP. Phosphorylation of fly requires micromolar concentrations of Mg2+. Chemical properties of the bound phosphate are such that the most likely acceptor amino acid is histidine, and treatment of Py with a histidinemodifying reagent inhibits itsphosphorylation [ 14,151. This then presents an alternate possibility for receptors to catalyze G protein activation:
It is clear that many if not all of the arguments made above for how receptors promote activation of a G protein are applicable here as well. The reaction might proceed slowly only in the absence of receptors; receptors could accelerate the reaction by lowering the requirement for Mg2+, etc. One might be tempted to suggest that fly-catalyzed activation of a-GDP to a-GTP by direct transfer of phosphate may be the preferred mode of activation of G proteins. Yet the fact that adenylyl cyclase, and hence G,, is also activated with the non-phosphorylating analogue GMPP(NH)P, and that this reaction is stimulated by a receptor would indicate that phosphorylation of bound GDP cannot be the whole story. The question of reagent purity (i.e., whether GMP-P(NH)P preparations are really free of GTP) becomes of paramount importance for correctly interpreting experiments with fly dimers.
Molecular diversity of G protein subunits Purified unactivated G proteins have molecular weights in the range of 100,000, being composed of three subunits a,P , and y, in decreasing order of size. Biochemical, chemical, and eventually molecular cloning of mRNAs showed that each of the subunits exhibits molecular diversity. This diversity is large for as and ys and moderate for f l s . All are subject
L. Birnbaumer and M . Birnbaumer
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to post-translational modifications, notably lipidations. Without being transmembrane in character, G proteins nevertheless localize to membranes within the cell, being found not only in plasma membranes where they are clearly engaged in signal transduction but also in Golgi and endosome membranes where their role(s) is(are) not yet clear. The a-subunits constitute both the most numerous and the most complex set. At 354 to 380 amino acids, they are similar but on the average only 47% homologous (the least: 36%, aolfvs. a12;the most: 88%, auqvs. a l l or ailvs. ai3;the most frequent homology among them: 40-45%) (Fig. 7; Appendix I). There are 16 non-allelic mammalian a-subunit genes known (Table 111). Four are sense related: two for vision (rod and cone transor gustducin). ducins), one for olfaction (aoE),and one for taste (agust The remainder are a, (four splice variants of uncertain functional difference), three ais, a, (two splice variants), az,aq,a 11, a12,' ~ 1 3 ~, ~ 1 and a15 (mouse)/a16 (human). An amino acid sequence alignment of G proteins a-subunits and a phylogenetic tree that relates their evolutionPhylogeneticTree of G Protein Subunits
a Subunits
x (354-395 aa)
p Subunits
p3cp4
asca16 60%
Gs
(340 aa) 22010
3 16
Family
12 .- i 1.
13
Subunits
(68-75 aa) "(1 C p 65%
t-cone'
Fig. 7. Phylogenetic trees of G protein a, p, and y-subunits. Calculations were performed using the algorithm proposed by Hein [176].Adapted from Birnbaumer [96].
4 ,
G proteins in Signal Transduction
171
TABLE III
Chromosomal location and main sites of expression of G protein cr subunits Chromosomal location" a-subunit
Human
ff-S
20q 13 2 (18q21-ter) 18
ff-olf
Mouse
Main sites of expression Ubiquitous, except mature sperm cells Olfaction, also: p cell, liver, lung, testis
19p13
14 10 14 10
Ubiquitous Ubiquitous Epithelial cells In cells of hemopoietic lineage
ff-12 a-13
(7P) (17q2)
5 11
Ubiquitous Ubiquitous
a-0
16
8
CNS, peripheral nerve cells, endocrine glands, not in skeletal muscle
a-t-rod ff-t-cone ff-gust
3p2 1 lp13 [7q21?]
9
Retinal rod cells Retinal cone cells, also in @-cell Certain taste receptor cells
a-i 1 a-i3 a-i2
7q2 1 lp13 3p21
5 3
Semi-ubiquitous Semi-ubiquitous Ubiquitous
aa
224 11
10
ff-q ff-11 a-14 a-15/16
19p13
3
9
Platelets, other
'an is linked to (downstream of) %; cyi3 is linked to %; cy4 is linked to q4; all is linked to als,16; cy, has introdexonstructure that suggests that it is an expressed pseudogene that arose from reverse transcription of an ancestral aigene; exons 7 and 8 coding for aO2 (also aoB)are upstream of exons 7', 8', and 9' that code for aOl(also cyoA). (For further details, see [ 1461.)
ary relations is shown in Table IV, Fig. 7, and Appendix I. In addition, it is known that there are five non-allelic p genes (4 cDNA sequences published), and seven non-allelic y genes (5 cDNAs published) (Fig. 7; Table V; Appendices I1 and 111). Combinatorial analysis shows the possibility of hundreds of distinct G proteins. However, not all these genes are expressed in any single cell, so that the actual cellular G protein complexity is somewhat less staggering, but still impressive.
L. Birnbaumer and M . Birnbaumer
172
TABLE IV Sequence alignment of the major classes of mammalian G protein a-subunits (palmtoyl)
I .
47 49 40 (mry)NGCTL--------------SAEERAALERSKAI6XNLKEDCISARKDLLLLC 40 (mry)MGAGA--------------SAEEK----HSR-LKEDAEKOAR~LLL~ 36 ( m r y ) M C C R Q - - - - - - - - - - - - - - S S E ~ ~ S R R I D ~ L R S E S Q R Q R R E I K L L L f f40 i MTLESIHACCL--------------SEFAKEARRINDEIWHVRRDKRDAR8ELKLLLLC 46 MSGVVRTLSRCLLPAEAGAREGAARDAEREARRRSRDIDALLARERRAVRRLVKILLLG 62 HARSLlWRCCPW----- - - -- -CLTEDEKAAARVDQEINRILLEQKKQDRCELKLLLLG 49 h-+ NccLGNS------KT-ECQREEKAQREANKKIWQLQKDK
NCCLCN~S-----KTA~'JDEKERREANKKIKKQLQKERLAYKATHRLLLLG (mry)NCCTL-- - - - - - - -- -SAEDKAAVERSWIDRNLREDGEKAAKEVKLLLLG
-
-
-
....
-
hum-gs rat-golf hum-gi3 mus-go1 hov-gtrod rat-gz mus-gq mus-912 hum-gl6
hum-gs A C E S G K I ; T I Y K Q M R I I , H V N G F N C E C C E E D P P L S N S ~ ~ ~ E ~ ~ Q D I K N N L K U I E T I V ~ M S N L V P117 ~~ rat-golf A C E S G K S T I V K Q H R I L ~ C F N P E - - - - - - - - - - - - - - - E K K Q K I L D I ~ A L Y T I I S ~ S T I I P104 W A G E S G K S T I V K Q N K I I H E D C Y S E D - - - - - - - - - - - - - - - E C K Q Y ~ S ~ I Q S I I A I I ~ C ~ K95 I D F hum-gi3 A G E S G K S T I V K Q N K I I H E ~ F S C E - - - - - - - - - - - - - - - D V K Q Y K P ~ S ~ I Q S L A A I 95 ~ ~ L ~ Emus-go1 Y bov-gtrod A G E S C K S T I V K Q N K I I H Q f f i Y S L E - - - - - - - - - - - - - - - - E C L E F I A I I Y G ~ L Q S I ~ I ~ L N I Q91 Y rat-gz TSNSGKSTIVKQMKIIHSCCMLE----------------ACKEYKPLIIYNAIDSLTRII~~LRIDF 95 mus-gq n ; E S G K S T F I K Q N R I I H C S C Y S D E - - - - - - - - - - - - - - - D K I I C F T K L W Q N I ~ A n Q ~ I ~ ~ K 101 IPY mus-g12 A C E S G K S T F L K Q N R I I H G R E F D Q K - - - - - - - - - - - - - - - A L L E F ~ I F D N I L K C S R V L V D ~ K117 ~I~ P G E S G K S T F I K Q N R I I H G A G Y S E E - - - - - - - - - - - - - - - ~ G F ~ L W Q N I N S M ~ I ~ ~ L104 Q I P F hum-916
..,.. ... . .
ELANPE--NQFHVDYILSVMNVPDFDFPPEFYEIWLWEDffi~CY~SNEYQLIDCAQYFLDKIDVI 185 PLAI~PE--lIQFRSDYIKSIAPITDFEYSQEFFDHVKKLWDD~~CFER~NEYQLIDCAQYFLERIDSV 172 CEAhRA--DDARQLNLACSAE-ffi~PELACVIKIIL~VQACFSRSREYQLNDSASYYLNDLDRI162 C . ~ K F P K - - ~ ~ K H V C ~ W S R H F . O T E P F S A E L L S ~ H R L W C D S G I O E C F N R S R E Y O ~ D S A K Y Y L D S L 163 DRI is8 163 167 QHSWEKHGHFLHAFWKAGLPVEPATFPLWPATFQLWPALSAL~SGIR~FS~EFQLGE~YFLDNLDRI187 S R P E S K - - H - - H A S L V n S Q D P Y K V I T F E K I I Y ~ Q W L W R I170
KYEHNK--A--HAQLVREMVEKVSAFENP~AIKSLWNDP ..\I.
~. . ..
hum-gs rat-golE hum-gi3 mus-go1 bov-g trod rat-gz mus-yq mus-gl2 hum-g16
hum-gs K Q R O W P S D Q D L L R C R V L T S G I F E T K F Q V D K V N F H N F D V U ) Q R D ~ I Q C F N D ~ A I I F W A S S S Y N N255 rat-golf SLMYTPTDPDLLRCRVLTSGIFETRFQVDKVNFHMFDVGCQRDERRKWIPEMD\PPAIIWAACSSYNH 242 hum-gi3 S Q S N Y I P T Q Q D V L R T R V K ? T G I V ~ F T F K O L Y F W F D V G C Q R S E ~ I H C F E C ~ A I I F C V A L S D Y D L 232 C A C D Y O P T W D I L R T R V K ~ I V ~ H F T F ~ L H F R L F D V ~ R S ~ I H C F D Y T A I I F ~ A L233 S G Y D P mus-go1 Y T P G ~ P T ~ D V L R S R V K ~ G I I ~ F S F K D L N F R H F D V G C Q R S E R K X W l H C F f f i ~ C I I F I A A L S 228 A Y D M bov-gtrod PIAADYIPTVEDILRSRO~cIVENKFTFKELTFKMmVGCORSERKKWlHCFEIIFCVELSAYDL 233 rat-a2 237 mus-iq ~LNYFPSKQDILLARKATKCIVEHDNIKKIPF)(HWVGCQRSQRQ)(WFPF~ITSILFINSSBEYDQ251 mus-912 T E E C W P T A Q D V L R S R H P I T C I N E Y C F S V Q ~ L R I W V G C Q K S ~ I H C F ~ I ~ I Y L A S L S E240 YDQ hum-gl6
A~~~VLPTPQDVLRVRVPITCIIEYPFDLQSVIFRHMVGCPRSERRKWIHCF~SIMFLVALSEYDQ
. . .
I...
.
.....
VIREDNOTNRLOULNLFKSI~RWLRTISVILFLNKODLLAEKVLACKSKIEDYFPEFARYT-TPEDA324 hum-as V m E D N i r R L r ; ~ S L D L F E I ~ R ~ SRITILFLNK6DHLAEKVLAGKSYI I DYFPEYANYT-VPEDA 3 11 rat -iolf V L A E D E ~ R n H E S H K L F D S I C " K W F T E T S I I L F L N ~ L F E E K I - - ~ S P L T I C Y P E Y T C S N - ~299 EU hum-gi3 VLHEDETMRHHESLWLFDSICNNKFFIDTSIILFLNKKDLFCW(I--KKSPLTICFPEY~SFI-TYWA 300 mus-go1 VLVDDEVNRHHESLHLFNSICNHRYFA~SIVLFLNKKDVFSW(I--KKllllLSICFPDYNGPN-~WA 295 hv-gtrod KLYEDNQTSRHAESLRLFDSIC~FINTSLILFLNKKDLLAEKI--RRIPLTICFPEYK~N-~EU300 rat-gz V L V E S D N E N R H E E S K A L F R T I I ~ P W F Q N S S V I L F L N K K D L L E E K I - - M Y S ~ L ~ Y F P ~ D G ~305 ~ A Q ~ mus-yq VLNEDRR~RLVESHNIFETIVNIJKLFFNLTSIILFLNWDLLVEKV--KSVSIKKHFPDFKCDPHRLWV 325 nus-gl2 C L E ~ Q W R H ~ L A L F C T I L E L P W F K S T S V I L F L N ~ I L E E K I - - P T S H ~ ~ F P S F ~ P308 K Q D A ~hum-916
..
* .
. ....* .
T P E P C W P R V T R A K Y F I R D E F L R I S T A S G D - - - - - - - - - - - G ~ Y C Y P H ~ A ~ E N I ~ V ~ D C R 383 DII TPDACEDPK\P17W(FFIRDLFLRISTATGD-----------CKHYCYPH~A~ENIRRV~DCRDII370 A A - - - - - - - - - - - - - Y I Q C Q F E D L N ~ - - - - - - - - - - - - ~ -TDTIUWQFVFDAVTDVI -I~~A 343 A A - - - - - - - - - - - - - Y I Q ? P F E S ~ R S - P N - - - - - - - - - - - K E - - I Y C H ~ A ~ I Q W F D A V T D343 II G N - - - - - - - - - - - - - Y I K V Q F L E L N M ~ V - - - - - - - - - - - K E - - I Y S H ~ A ~ ~ N F D A V T339 DII A V - - - - - - - - - - - - - Y I Q R Q F E D L N ~ K ~ - - - - - - - - - - - K E - - I Y S H ~ A ~ S N I Q F V F D A V T D344 VI RE-------------FILKHNDLEIPDSD------------KI--IYSH~A~ENIRNFM-I 348 Q R - - - - - - - - - - - - - Y L V P F D R K R R N 8 S - - - - - - - - - - - - K P - - L F H H P I T A I I I R W F ~ ~368 I K R - - - - - - - - - - - - - F I L D M Y T R M Y T C C V D C P E G S K K C A R S R R - - L F S H ~ A T ~ N I ~ F363 ~~SV QRHHLRQYELL QRHHLKQYELL IKNNLKECCLY IANNLRCCCLY IKENLKDCCLF IQNNLKYIGLC LQLNLKEYNLV LQENLKDINLQ LARYLDEINtL
394 381 354 354 350 355 359 379 374
hum-gs rat-golf hum-ai3 mus-go1 bov-gtrod rat-gz mus-gq mus-g12 hum-gl6
t+t
I).
.
hum-gs rat-golf
hum-gi3
mus-go1 bov-gtrod rat-yz mus-gq mus-glf hum-g16
Note: there are 16 non-allelic mamnalian genes known: four are sense related: two transducins. one olfaction and one taste ( ~ s t d u c i n ) . the remainder are a, (four splice variants). three a l ' s , a,, (two splice variants), ~ 2 . %. alx ail, al,. a14. and aI5. aI6 is the human om0 o w e of mouse a15.
*, absolutely conserved amino acid in all mammalian a-subunits (including aoz,ail, q-,,,,,a l l , 0113, a14,and agWt (gustducin). 0 , Cys conserved in all a s . cysteine conserved in all except a I 2and a13.h- , amino acids thought to contact fly dimer. Cys108 is unique to ao and reactive to NEM and does not participate in crosslinking to p [ 147,1481.
+
+,
G proteins in Signal Transduction
173
TABLE V 41 40
40
40 40
40 40 75 85 84 84 84 84 84 84 121 127 126 126 126 126 126 126 163
iF
STE4
172 171 171 17 1 17 1 171 171 209
4 STE4
214 213 313 211 213 213 213 257
jf
If
266 265 265 265 265 265 265 298
1 STE4
300
299 299 299 299 299 299 378
4 STE4
BF
341 340
340 340
340 340
STE4
-, amino acid identical to those in 01; -, gap; C, all cysteins are highlighted; 0 , cysteines conserved in all mammalian and the yeast /3 (STE4); , cysteines conserved in all mammalian 0s but absent in STE4. Cys-25 of D l can be crosslinked to Cys-36 or Cys-37 of y1 [149]; *, conserved in all; *, conserved in mammalian sequences only; under- and over-lined bolded sequences in STE4 are important for activation of the STE20 kinase. Box in a: consensus sequence of WE-40 motif. The five complete (A-B) and two partial (B) WD-40 of G protein @subunits are highlighted.
+
I74
L. Birnbaumer and M. Birnbaumer
Phylogenetic analysis subdivides a-subunits into three major families: as, aq and ai(Fig. 7; Appendix I). a, and aOlf are the lone members of the as family. The aq family has two main branches: brunch I contains aq,a l l ,and a relatively closely related ~ ~ 1 branch 4; 2 contains a12 and a 1 3(homology between branches: 43%). The aifamily is the most complex containing the three a ; ~ao, , the three remaining sense transducers atl (rod), atC(cone) and agust, and a,. Except for a, all of the members of this a-subunit family are PTX substrates, and all of the known PTX substrates are in this family. From a structural view, the 0-subunits (each of 340 animo acids) are the least variant of the three G protein subunits, being between 7 8 4 6 % homologous. They belong to the type of proteins with WD-40 motifs highlighted in the inset of Table V). WD-40 motifs divide the 0s into eight blocks, of which the first has a hypervariable region (@25-40) and the remaining seven have either a complete WD-40 motif (blocks 2 , 4 , 5 , 6 , and 8) or only the second (B) half of the motif (blocks 3 and 7). Homologues have been found in nonmammalian organisms as well as in yeast where the @-subunit is known as STE4. STE4 is co-linear with animal 0s in its full extent, except for the presence in STE4 of a 35-amino acid N-terminal extension, four inserts, and a 4-amino acid C-terminal extension. The C-terminal end of the last WD-40 motif is “imperfect” in that it either lacks the last amino acid (mammalian 0s) or has a non-consensus amino acid, S , in place of D or N (STE4). Amino acid identity within the co-linear portions of STE4 and mammalian p1 is 37%; the four mammalian @sshown are around 75% identical (Table V). The WD-40 motif is intriguing in that it has been found in a variety of apparently unrelated proteins (Table VI). Like the phosphotyrosinebinding SH2 domains and the “PPPVPPR”-like motif recognizing SH3 domains of non-receptor tyrosine kinases and the GRB2hem5 adaptor protein, the WD-40 motif may encode a protein-protein interacting function for which the partner has yet to be identified. Another possibility is that they contribute to a metal binding site, such as appears to be required for the @-subunit function to stimulate nucleotide exchange. The y-subunits, 68-75 amino acids long, are only 28-43 % homologous and constitute a set of the subunits that is as heterogeneous as asubunits (Fig. 7; Table V; Appendix 111). The interaction of @swith ys to form dimers may involve a coil-coil interaction with the participation of a large proportion of y and the
G proteins in Signal Transduction
175
TABLE VI
WD-40 motif and its occurrence
A. The consensus motif A LXGHXXXIXX@X~
F
L
V
B
--- ODSGGXDXMDXIWDS' TAA N
WD-40 Motif
S
C LFN VY
4, hydrophobic; 6 , n o t charged
B. Gene products with WD-40 motifs
References
Subunits of heterotrimeric G proteins Component of the year nuclear cytoskeleton Product in the chicken MHC-locus Enhancer of split, neurogenic gene in Drosophila Stable component of yeats U4/U6 small nuclear ribonucleoproteinparticle (snRNP) AER2rrUP1 Transcriptional repressor in yeast Negative regulator of the RAS-CAMPpathway in MSIl Yeast COR Coronin, component of the actidmyosin complex of Dictyostelium discoideum Clbp Chlamydomonas protein of unknown function PWPl Yeast protein with periodic tryptophan residues MAKll Apparently a membrane associated protein required for maintenance of killer MI double-stranded RNA AAc3 Deduced product of a developmentally regulated transcript in D. discoideum with long ACC (Q) repeats CDC20 Yeast gene product required for several microtubule dependent processes at several stages of cell cycle COP1 Arabidopsis regulatory gene that represses photomor photogenesis in the dark dTAFE80 Subunit of Drosophila TFIID, a component of the RNA polymerase I1 transcriptional apparatus LIS 1 Miller-Dieker lissencephaly (smooth brain) gene product
cf. cf. cf. cf. cf.
GP
CDC4 12.3 WPI) PRP4
[150] [150] [150] [150] [150]
cf. [l50] cf. [150] cf. [150] cf. [150] cf. [150] cf. [150] cf. [150]
[1511 [1521 [I531
[W
176
L. Birnbaumer and M. Birnbaumer
N-terminus of 0[ 161. Given the rather large sequence variability of y and the fact that the different Ps show sequence variability in their N-termini, it is thus not surprising that not all the 0s combine with all of the ys. For example, upon expression in cells or in vitro, 02 did not dimerize with y l , and 03 did not dimerize with either y l or y2 [17-191. An anti-sense signalling interference assay showed pairing of /33 with y4 [20,21]. The co-expression of y, Pl(1-129) and Pl(130-340) leads to formation of a stable Py dimer [16]. Pl(1-129) encompasses the N-terminal variable domain, the first (complete) WD-40 motif and the first incomplete WD40 motif (Table V), and /31(130-340) encompasses the remainder of the &subunit, including at its N-terminus a sequence found in yeast STE4 to be important for interaction with its effector, the protein kinase STE20. This suggests the existence of at least two independently folding structural domains in 0.
Lipid modijication of Gprotein subunits - membrane attachment and function in signalling Myristoylation of a-subunits Some G protein a-subunits have been shown to be myristoylated at the and N-termini. These include the two ats, the three ais, the two aos, aYgust az, which are the a-subunits that have the MGXXXS consensus myristoylation signal. Since amino termini of all subunits of purified G proteins are blocked, it stands to reason that non-myristoylated a subunits are nevertheless modified at their amino termini. For transducin it has been shown that the myristic acid at position 2 may be replaced by other lipids. Mutations of a-subunits that prevent myristoylation (e.g., G2A) prevent their localization to the membrane [22,23]. In one in vitro reconstitution assay that measures inhibition of adenylyl cyclase by recombinant G p , only the myristoylated form of Gia was found to be active [24; Codina and Birnbaumer, unpublished]. Myristoylated as exhibit a markedly enhanced affinity for fly dimers [25]. It remains to be seen therefore whether membrane localization of as is driven by high affinity for Py dimers or whether the myristic acid contributes to membrane localization by serving as a lipophilic anchor. It is likely that both factors contribute to the membrane localization of Gas.
G proteins iri Signal Transduction
177
Palmitoylation of a -subunits In contrast to myristoylation, which affects only few a-subunits, most if not all as appear to be palmitoylated at Cys-3 or another of the cysteines located near the N-terminus [26-281. This post-translational modification appears to contribute further to the membrane localization of Gas as shown for a, and aq.Two cysteines (Cys-9 and Cys-10) are palmitoylated in aq,and the mutation of both to Ser (aqC9S,C10S) not only delocalizes aqbut also interferes with its capacity to be activated by a receptor or to activate phosphoinositide breakdown, even if it is activatedby the Arg-183 to Cys mutation (R183C) (see below). Receptormediated stimulation of phosphoinositide turnover by aqC9S,C10S can be restored by attachment of the myristoylated N-terminus of at, at(1-9), to non-palmitoylated aq(16-rest). For as,removal of the palmitoylation site by the aSC3S mutation produces loss of membrane localization accompanied by loss of receptor-mediated activation of adenylyl cyclase, but only in a minor impairment in its action if it is constitutively activated by an R+C mutation. In contrast to the result with the aJaqchimera, the myristoylated, non-palmitoylated at(1-9)aS(17-rest) chimera restores membrane localization but confers an intrinsic activity not present in the wild type as without restoring receptor-mediated activation of adenylyl cyclase [28]. Also, in contrast to myristoylation which is permanent, palmitoylation is a reversible modification that varies with the metabolic or regulatory state of cells. It may thus be that cells contain “active” and “inactive” pools of G proteins that are regulated by palmitoylation. Changes in palmitoylation of G,a after stimulation of cells through the G , pathway have been observed [29]. Polyisoprenylation of y-subunits Like a-subunits, @- and y-subunits are also blocked at their N-termini with an as yet unknown blocking group [Codina and Birnbaumer, unpublished]. However, no specific post-translational lipidations have thus far been described for @s.y-subunits, on the other hand, have at their Ctermini a CAAX consensus polyisoprenylation signal and are polyisoprenylated [30-321. Full processing of ys involves both the polyisoprenylation at the Cys at -4 followed by cleavage of the three C-terminal amino
178
L. Birnbaumer and M . Birnbaumer
acids and carboxymethylation of the polyisoprenylated cysteine. y 1, which is expressed almost exclusively in the retinal cells (y-transducin) is farnesylated (C15); the remainder of the known ys are geranylgeranylated (C20). While polyisoprenylation is not necessary for association with Ps, it was found to be essential for interaction with adenylyl cyclase [17] and to increase affinity for a-subunits. Thus, both myristoylation and polyisoprenylation contribute to the high affinity interaction between inactive GDP-liganded a and Py dimers.
Patterns of G protein subunit expression Of the genes listed above, as,a2, and a l lappear to be expressed in all cells. Most cells also express auqand either ail or ai3(functional homologs of a l l and a2, respectively), and one or both of the a12and a13genes. Thus, all cells express eight “ubiquitous” a-subunits. Expression of ao, ~ ~ 1 4~ ,~ 1 and 5 , a, is not ubiquitous but also not and agust. Their prodexclusive to single cell types, as appear to be aYts ucts are found in groups of cells or tissues that often, but not always, have common embryonic origin. aolf,which was originally thought to be expressed exclusively in cells of the olfactory neuroepithelium, has a somewhat wider but nevertheless still restricted expression, being found also in basal ganglia of the central nervous system, in pancreatic islets, testis, lung, and liver. aolfco-migrates with the short form(s) of as as shown by immunoblotting and interpretation of effects of receptors, originally attributed by default to activation of a single G,, may have to be reconsidered because of the presence of two “G,” with differing efficiencies in both receptor coupling and effector activation. aos (ao1and aO2) are preferentially expressed in cells derived from the neural crest and in endocrine cells (pituitary, pancreatic fl cell) as well as in other selected cell types such as cardiac myocytes. a14 is expressed primarily in stromal and epithelial cells, and a I 5is expressed in many but not all cells with hematopoietic lineage. a, is found primarily in neurons and platelets and also in small quantities in red blood cells and other cell types. It follows that out of the repertoire of 16 a-subunit genes, a standard cell expresses between 9 and 10. On a comparative basis, it is difficult to properly measure the relative amounts of the different a-subunits in a cell. The difficulty arises from the large difference in sensitivity of the antibodies available for quantifi-
G proteins in Signal Transduction
179
cation (mostly anti-peptide) and from the heterologous nature of the standards (recombinant proteins) that are used to quantify the measurement of the proteins (SDS-denatured membrane associated proteins). There is, however, a consensus, derived not only from immunoblotting but also from the yields with which individual proteins are purified, as to which are more abundant and which are less so. Thus, levels of as,now the sum of a, plus aOlpin non-olfactory cells are on the low side, which for the sake of this discussion can be set at a value of 1.O. In contrast, aolf is a major protein in cells of the olfactory neuroepithelium where it accumulates in cilia to very high levels (to ca. 500 times standard a, levels), and where it appears to act to transduce the input of any one of over 40 olfactory receptors. Like a,, the levels of expression of aq and a l lare also low, differing from those of asby at most a factor of two. , a I 2and ( ~ 1 3 .In contrast, the The same appears to apply to ~ 1 4 (Y15/16, levels of expression of ai2 appear to be on the average at least five times those of a,, ail,and ai3(of which at least one is co-expressed with aDat about half the abundance of a2)are found. In neutrophils, and possibly also in other white blood cells, ayi2 and ai3are higher than in other cells, probably 10-20 times those of a,. In neurons, a, may be 50-100 times more abundant than as,contributing to up to 1% of total membrane protein. Highest of all appears to be transducin (atr! in rod cell discs, where it accumulates to 5% of total membrane protein. Although the tissue distribution of /3 and y has been less well studied, it is clear that most cells express at least two /3 and two y genes and more likely three of each. Together with the standard a-subunit repertoire, this makes for 40-90 distinct G proteins engaged in transducing receptor signals into modulated effector activities if, as one assumes, all asubunits have the ability to combine with any combination of Pr dimers.
The a-subunit: structure and function The a-subunits have been the object of extensive study over the last years. Secondary and tertiary structures have been inferred based on (1) sequence homology with other regulatory GTPases that had been crystallized, notably the bacterial elongation factor EF-TU and p21-ras (Ras); (2) the effect on activity of directed mutagenesis of selected amino acids and N-terminal truncations; and (3) blockade or mimicry of G protein regulation by receptors with receptor peptides. These studies have led to
L. Birnbaumer and M . Birnbaumer
180 G1 + GZ-5: GTP Bmding and
Intera-3onwth Receptors (e.g.. adrsnerglc. rnuscarinic, vasclpessm)
GTP Hydrolysis
Snterachon Hnth Efector Systems
(low GTP affinty:
high affinrty for Mg. R-,H
wrtla'v aarve)
]
R - X gsp, gf&?
Fig. 8. Summary of assignments of functional domains to structural domains of asubunits. A typical a-subunit is represented in linearized form. Amino acid sequences of interest are highlighted. These include: 1. the identity (id) box conserved in a,,ai's, ao, q s , and aOK; and the deviations found in az,a q / a l l and , a I 2 / a l (termini 3 of id are either Arg (R) or Lys (K)); 2. an Arg (R) ADP-ribosylated by CTX; a Gln (Q which when mutated leads to loss of GTPase activity and spontaneous activation by GTP and a Gly (G) residue which when mutated impedes activation by guanine nucleotides; 3 . a BamHI restriction site used in the construction of an ai/a, chimera that retained a, function; 4. conserved sequences involved in GTP binding (black); 5. the carboxyl termini of various a-subunits, with the location of the Cys (C) ADP-ribosylated by PTX and an Arg (R) at position -6, which when mutated uncouples the G protein from a receptor (the carboxyl terminal amino acid of the known PTX substrates is either Tyr (Y) or Phe 0). A truncated version of ai3,starting with Met18, has reduced affinity for 07 dimers. Several as are myristoylated at the N-terminus; most are palmitoylated on cysteine(s) near the N-terminus.
a general picture, such as shown in Fig. 8, in which regions of the achain involved in GTP-binding and hydrolysis, in recognition of a receptor, in interaction with an effector, and in interaction with the dimer had been identified (reviewed in [33]. The receptor interaction with C-termini of a-subunits was deduced primarily from the findings that pertussis toxin (PTX) uncouples receptors from G proteins (reviewed by [34]) and acts by ADP-ribosylating the cysteine at -4from the C-terminus [35], and that the uncoupled phenotype
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of the UNC allele of S49 lymphoma cells was due to the mutation of the arginine at -6 from the C-terminus to proline [36]. A recent switch in receptor specificity due to the switching of merely three amino acids in the C-terminus [37] and the finding that a synthetic 20 amino acid polypeptide encoding transducin 309-328 mimics transducin in stabilizing photoactivated rhodopsin in its MetaII state [38], support the notion that the C-terminal section of a-subunits is involved in receptor-G protein interaction. The N-terminal end (first 21 amino acids), on the other hand, participates in association with py-subunits. This interaction involves various aspects: lipidation of the y-subunit of py and at least two subdomains of the N-terminus, one close to the N-terminus proper and another at about 20 amino acids from it [39-41; references in [40]). Mutational analysis of a,showed that determinants that define effector interaction, in this case adenylyl cyclase, are in the last third of the molecule [42]. Consistent with this, 01, peptides derived from regions around 50 amino acids from the C-terminus can stimulate adenylyl cyclase, and a similar peptide from at interacts with y p D E and leads to stimulation of the catalytic activity of aPpDE[43]. Activation by GTP - experimentally by GTPyS - causes a fundamental conformational change which, for the a-subunits of heterotrimeric G proteins, involves a hinge region that is part of the so-called G3 or switch-I1 region of Ras and contains the DVGGQ motif. Specifically a Gly (226 in a,)is necessary for activation by GTP. Its replacement in a, with Ala to give a, G226A (also H21a or reverse-UNC; Fig. 8) results in inhibition of activation by GTPyS, without blocking the binding of GTPyS or the guanine nucleotide-induced regulation of the affinity of p2adrenergic receptor for an agonist of the type shown in Fig. 5. a-subunits as proto-oncogenes
Two activating mutations merit comment at this point. One, Q+L, is a mutation in which the conserved glutamine of the DVGGQ motif in a, (Q227) is changed to leucine. In Ras this mutation is oncogenic and inhibits its GTPase activity. In a,it also inhibits GTPase causing persistent activation in the absence of receptor stimulation. This mutation was found in pituitary and thyroid adenomas and given the oncogenic name gsp [44]. The other is a mutation in which the arginine of the RVXT
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motif, a, R201 and a, R179, is changed to either histidine, cysteine or serine, R-.H/C/S. In a,and transducin a this arginine is the site of ADPribosylation by cholera toxin, a modification that inactivates the GTPase activity of these a-subunits, causing their receptor-independent activation by GTP [45-481. Like Q+L mutations, R+C, R+H, and R+S of a,were found in growth hormone secreting tumors and thyroid adenomas, and also in patients suffering from McCune-Albrights Syndrome, a mosaic endocrinopathy; R+H and R+C mutations of ayi2 were found in adrenal cortical carcinomas, and ovarian granulosa and theca cell tumors. Expression of mutationally activated ai2, referred to as gip2, in Rat-1 cells leads to MAP kinase activation and induces their transformation to an oncogenic state [49,50]. G, and G, are not the only G protein a-subunits with oncogenic potential, for also the Q+L forms of aq,ao,and a12induce transformed states [51-531. Even though it would thus appear that each of the major classes of a-subunits may be potential oncogenes, this concept has recently been challenged by the demonstration that mutationally activated a,,rather than potentiating Ras-induced transformation, actually suppresses it [54], and colonic epithelium of an aQ-knockout mouse tends to develop adenocarcinomas [55].
l%ethree-dimensional structure of the a-subunit The 2.2 A crystal structure of transducin a (atemd) has been solved by Noel et al. [56] (Fig. 9). Though it is missing the first 25 amino acids, it represents a landmark in that for the first time it allows a comparison between the real structure of a GTP-liganded G protein a-subunit and the inferred ras/EF-TU based structure. By and large there have been no major surprises. The GTP-binding domain of at is indeed similar to that of Ras and EF-TU. What the crystal provided is the positon of sequences that represent the major difference in the amino acid sequence betwen Ras and at. One of these differences, a 120-amino acid insert plus connecting linkers (Fig. 10) and lies between the GAGES G1 and the DVQQ G2 motifs. It constitutes a helical domain (Fig. 9) and is responsible for major functional differences between the small and the large GTPases. The crystal shows that this helical domain forms a lid over the GTP-binding site of the GTPase domain, and that its linker at the Cterminal end (linker 2) contains R174, the cognate of a, R201 and a,
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n N
I
n
Fig. 9. Ribbon model of the three-dimensional structure of transducin or derived from its crystal structure. Thin lines, sequences homologous to those found in Ras and EF-TV; heavy lines, N-terminus, and 1-1 through 1-4 sequences unique to or-subunits; R between orF and p2, or, Arg-174; Q between 03 and or2, Q of DVGGQ motif. Adapted from Noel et al. [56].
R179. The lid structure of the helical domain explains the much slower nucleotide exchange rates of G protein a-subunits compared to other regulatory GTPases. The arginine, which has no parallel in other type of GTPases, turned out to be at the center of the GTP-hydrolysis mechanism, together with a lysine next to the G1 motif (K42) and a general base that catalyzes the nucleophilic attack required for the separation of the y-phosphate. Fig. 10presentscomparativesequence alignments illustratingstructure fbnction relationships as they are currently understood. In terms of effector regulation, the picture that may be emerging is that loops connecting a-helices to a-helices and to 0-pleats may constitute the
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52 63 63 62
56
121 hum-as
mus-m
133 HVNGFNCEELUTKVQDIKNNLKEAIETIV~SNLVPPVE~PENQFRMYILSVMNVPDFDFPPEFYE 133 -GS-YSD-D-RGFTKLWO-IFT-MQN.IIR--m-KI-YKYEH_-KAHAQLVREVDVE-VSA-ENPYVD 130 126
<---- 1-2 ----> Switch I1 203 -p3--> <;-a2---> <-p4--> <----a3-----> 4 5 I H C F ~ C I I F T ~ S A Y D ~ L V E D D ~ H E S L H L F N S I C N H R Y F A261 ~SIV bov-art F R M F D V G G Q-R S E R K K W____ P eeeee eeeeeeeeeeeeeeeeeeeeeeeeeee 343NLKDCGL349 273 273 270 266
-
bov-atr
314
xl-as 343 hum-as L F L N K Q D L ~ ~ C K S K I E D ~ P E F ~ ~ P E M T P E P C E D P R ~ ~ I R D E F L R I S T A343 SG~RHY mus-aq _ _ _ _ _ K---E--I-MFHLV-----YEGPQRDAQ-AR--LKM-VDLN-PDS--KI 323 mus-ai2 _ _ _ _ _ K--FE--T-TQ-SLTIC---YWANKYDE-AS Y-QSK-EDLNKRKDT-KE 319 <86-> <-------as-------> bov-atr IYSHMTCATDTQNVKFVFDAVTDIIIKENLKCCGLF 350 ETITET PTX rrrrrrrrrrrrrr rrrr&rr 2 12EGVT215 *c5*
x1-m _ _ _ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - - - -379 ---------hum-as CYPHFTCAVDTENIRRVFNIXRDITQRMHLRQYELL 379 mus-aq I - S - - - - - T - - - - - - F - - A - T - L Q L N - K E - N - V 359 nus-ai2 I-T-----T--K-VQF--DAVl-V-IKNN-KEG-F355
Fig. 10. Delineation of amino acid sequences of a-subunits involved in GTP binding and hydrolysis in interaction with receptor and in interaction with effector as deduced from analysis of the crystal structure of bovine human q,from analysis of a, mutations and from effects of synthetic a-subunit peptides.
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effector-regulatory domains. Substantiation of this concept - which will surely be tested in many laboratories - would imply that effector regulation involves not one but several independent points of contact.
Fig. 10, continued. Features of crystal structure. Heterotrimeric G protein as are formed of two distinct domains: a GTPase domain and a ca. 115 amino acid helical domain, connected by two linkers L1 and L2. The alignment analyzes the primary a-subunit sequence in terms of homology to the sequence of the smaller p21 Ha-ras. Since Gas are longer, this gives rise to inserts 1 0-1) through 4 (I-4), of which 1-1 constitutes a structurally separate helical domain. a-Helices and &pleats of sequences homologous to those of Ras are numbered sequentially a1-a5 and fll-fl6. a-Helices arising from, or due to, the inserts are denoted as aA-aG. Amino acids contacting the guanine base, ribose, and phosphates are subscripted with g, s, and p, respectively. Amino acids involved in coordinating the Mg are subscripted with m. GTP hydrolysis is proposed to involve a general base attack of the y-phosphoryl group by Glu203, and both Arg174 and Lys42. Sequences subscripted eee are implicated in the interaction with an effector on the basis of mimicry of PDE activation (bolded amino acids) or simple binding to PDEy [56,177]). Sequences subscripted rrr are implicated in a contacting receptor (rhodopsin) on the basis of the ability of a peptide comprising the 11 C-terminal amino acids to compete with a& transducin binding to rhodopsin and to stabilize the active Meta-II state of rhodopsin [38]. In agreement with this ADP-ribosylation of Cys at -4 from C termini of G protein asubunits impedes receptor-mediated activation. The C-terminus makes van der Waals contacts with the a2/04 loop, which is part of a conformational GDPIGTP switch (Switch I1 region). Trp in the switch Wa2 region served to monitor Mg-induced conformational changes in a,-GTP complexes upon hydrolysis to GDP [3,178,179]. bbb, sequences that are known to be required for interaction with fly dimers (e.g., Graf et al. [40] and references therein). Alignment with sequences of other a-subunits and the structure function inferences obtainedfiom mutations and chimeras. The sequences of Xenopus laevis 4 , murine aq, and murine.ai2are compared to that human og (splice variant: short, with Ser). -, amino acid identity, -, gap. Amino acids in bold in hum-a, have been shown to confer a, activity to chimeras. Those comprising regions I-III plus the extended sequence that includes region IV confer a,activity to an ai[ 1-212/a,[220-341]/ai[3 18-35] [42]. Amino acids in a, superscripted by in regions I-IV are essential for a, activity of a n a i [ l 212/a,[220-379] chimera. Amino acids in hum-a, subscripted in region V by are necessary for a, activity of an XI-a,.xl-og does not activate mammalian adenylyl cyclase but a xl-a,[ 1-35]/hum-c$[35-l57]/xl-a8[ 158-3791 does [Antonelli et al., unpublished]. a,[320-341] stimulates adenylyl cyclase (Hamm, pers. comm.). *G1*-*G5*are amino acid sequences that had been inferred from EF-TU and Ras structures to be involved in binding and hydrolyzing GTP. Actual points of contact are shown below the sequence of
+
%*
+
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L. Birnbaumer and M. Birnbaumer
G Protein Coupled Receptors
Receptors that act through G proteins are encoded in a large, structurally closely related superfamily of molecules that is referred to variously as Gprotein coupled receptors, which relates to their mode of action, or as seven transmembrane spanning or hepathelical receptors, which relates to their common structure. The arrangement of these seven transmembrane segments (TMs) in relation to each other has been inferred from structural homology of rhodopsin to bacteriorhodospin, for which it has been possible to obtain a three-dimensional structure by electron microscopy with a resolution between 3.5 and 7 8, (Fig. 11A). TMs appear perpendicular to the plane of the membrane enclosing a central space that is thought to form the ligand binding pocket (Fig. 11B) [57,58]. Fig. 11C shows an idealized view from the bottom or intracellular side of a G protein coupled receptor, based on the transmembrane arrangements observed for bacteriorhodopsin and alignments of amino acid sequences of bacteriorhodopsin and G protein coupled receptors. Fig. 12 shows a scheme in which the receptor has been flattened to simplify description of its primary features. The following features are apparent: seven transmembrane domains, an extracellular N-terminal domain, three intra- and three extra-cellular loops, and an intracellular Cterminal tail. Most, but not all, have at about 13 amino acids from the end of TM-VII a cysteine that is palmitoylated, providing for what is assumed to be another membrane anchor and a fourth intracellular loop. In some cases there is no C-terminal cysteine; in others there is a pair of vicinal cysteines (e.g., rhodopsin, a type-2 vasopressin receptor). While removal by mutagenesis of the palmitoylation site may not affect function (rhodopsin, a2-adrenergic receptor), it impairs function in others. For example, a 62-adrenergic receptor mutant (Cys-341 to Gly) is hyperphosphorylated and largely uncoupled from the GJadenylyl cyclase system. Most, but also not all, have a cysteine in both the first and second extracellular loops, which in rhodopsin and by inference in other receptors may form a disulfide bridge that may contribute to stabilization of the seven transmembrane structure. A variable number of variably located Nglycosylation sites has been found in the N-terminal extension or in one of the extracellular loops. While not required for function once assembled, for some receptors removal of the glycosylation sites impairs their proper assembly and/or membrane insertion resulting in defective cell surface expression. Little is known about the folding and membrane insertion process except for the fact that a receptor can be constructed
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C Fig. 11. Basis for the three-dimensional structure assumed for G protein coupled receptors. Top left: Electron density map of the bacteriorhodopsin in purple membrane of Halobacterium halobium showing three molecules, each with seven transmembrane regions, grouped around a three-fold axis. The probable boundary of one of them is indicated by the broken lines. Top right: Model of a single bacteriorhodopsin molecule in the purple membrane (7 resolution) showing seven closely packed a-helical segments that span the plasma membrane in a roughly perpendicular fashion (from Henderson and Unwin [57]). Bottom: Model of 'view" fron the cell's inside of typical a G protein coupled receptor based on the bacteriorhodopsin model and multiple amino acid sequence alignments. Grey, extracellular; black, intracellular.
A
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Fig. 12. Structural elements of a G-proteins coupled receptor and their hnctional import.
I and 11: The tertiary structure and relative positioning of the transmembrane segments depends not only on connecting extra- and intracellular loops but also on interactions among transmembrane segments. Co-expression of an N-terminal half (N-terminus plus TMs I-V plus part of the third intracellular loop) and a C-terminal half lead to an active receptor [ 1801; proteolytic digestion of purified receptor protein leads to a limiting digest formed of all seven TMs [181]; mutations that interfere with TM-UTM-VII interaction prevent receptor folding [182]. A. Small agonists (retinal, catecholamines)interact with the intramembrane region of the receptor to trigger a response. Larger agonists are also assumed to interact with the transmembrane domain to activate the receptor, but their overall binding involves more than this domain. This has been proven for glycoprotein hormones and is likely also to apply to intermediate size peptides such as vasopressin, kinins, angiotensin, etc. The Nterminal domain of the LH/CG receptor binds hCG with the same affinity as the entire receptor [183]. B. Sites on transmembrane regions VI and VII define specificities for antagonists in biogenic amine receptors. A chimera that is P2AR for TMs I-V and a2AR for TMs VI-W (plus C-tail) activates adenylyl cyclase @2 effect) and is blocked by yohimbine (a2AR blocker [180]; a single amino acid change in TM-VII of the 5HTIBchanges affinity for an antagonist (pindolol) by three orders of magnitude [184]. Antagonist binding may involve more than the B domain (non-peptide NK1 antagonist interacts with epitopes that are on top of both TM-VI and the TM-VII, even though neither epitope is required for NK1 (Substance P) binding [185]. C. N-terminal segment of third intracellular loop may be involved in defining specificity of G protein interaction. Replacement of 17 amino acids of the N-terminal end of intracellular loop 3 of the PLC-activating M3 muscarinic receptor, with the cognate 16 amino acid !stretch of the adenylyl cyclase inhibiting M2 muscarinic receptor resulted
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from two independently folding domains, one encompassing an Nterminal half (N-terminus to the third intracellular loop) and one Cterminal half (from the third intracellular loop to the end). Once assembled, intra-transmembrane domain forces and possibly disulfide bridges maintain the tertiary structure, so that it is possible to fragment the molecule by proteolytic digestion without loss of function.
Fig. 12, continued. in loss of PLC stimulation and acquisition of adenylyl cyclase inhibitory activity to a level that was 25% of control [186]. D. Ser-Thr rich regions in either or both the third intracellular loop and the C-terminal tail are substrates for G-protein coupled receptor kinases (GRKs). These sites are exposed upon activation of receptor by an agonist and initiate a desensitization cascade. Except for rhodopsin kinase which is anchored to the membrane through a C-terminal polyisoprene and phosphorylates light-activated rhodopsin without requiring other proteins, the other GRKs appear to phosphorylate HRs only when presented in the context of an HR 07 complex [187-191; reviewed in 1921. E.Potential protein:protein interaction site for receptor: Gor interaction. Point mutations in the C-terminal end of the third intracellular loop may lead to constitutive (agonist independent) activation of receptors as shown through artificial mutations for the 0 1 ~ [73], or2- [75], and p2- [74] adrenergic receptors and found in two natural mutations of the TSH receptor [159]. Peptides derived from this region can activate purified G proteins in virru [71,72,90]. F. Addition of GTP to membranes lowers affinity of receptor for glucagon [ 9 ] , catecholamines [ 10,111, and carbachol [ 12,131 but increases the affinity for prostaglandin in the case of platelet prostaglandin receptors [193], and has no effect on the vasopressinV2R interaction (Birnbaumer, unpublished). In the case of the cloned EP3-prostaglandin receptor, which mediates inhibition of adenylyl cyclase in adipose tissue, there are two splice variants that structurally differ in their C-terminus. For one variant, GTP addition lowers agonist affinity; for the other it increases it. The C-terminal tail may thus contribute to interaction with G protein [194]. G . A “DRY” motif (consensus D/e-R-Y/f/h/c) of which the R is invariant) is found in all 200 (or thereabout) G protein coupled receptors, including yeast STE2. Exceptions: receptors for the glucagon-related peptides (glucagon, GLP- 1, GIP, secretin, VIP (type 1 and 2), GRF, PACAP), CRF, PTH, and calcitonin, which constitute a subfamily of heptahelical G protein-coupled receptors. The DRY motif appears to be important for coupling to G protein. Mutation of its R in the VP2 vasopressin receptor results in loss of coupling to G, without changes in agonist binding or loss sequestration in response to hormone binding [ 160; Birnbaumer, unpublished]. Other: N-termini tend to be glycosylated. Extracellular loops 1 and 2 may linked by disulfide bridges [195], and C-terminal tails tend to exhibit at 13-15 amino acids from T M - W one or two vicinal palmitoylated cysteines. Removal of these cysteines impaired function in the 0-adrenergic receptor [ 1961, but had no functional effect on rhodopsin [ 1951 or the a2-adrenergic receptor [ 1971.
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Most G protein-coupled receptors have been cloned by now, giving a wealth of information about conserved and divergent parts of the receptors. While the transmembrane core, extracellular loops, and first and second intracellular loops are relatively similar in size forming a similarly sized family of proteins of ca. 350 amino acids, their N-termini, third intracellular loop, and C-termini vary very much in length. The longest N-termini appear to be the ca. 350 amino acid long glycoprotein hormone binding N-termini of the receptors for LH, FSH, and TSH. Hormone specificity of these receptors lies in their N-termini. Depending on the length of the N-termini, they may have at their N-termini a signal sequence (LH, FSHITSH receptors; glucagon, PTH) or not (neurotransmitter receptors, rhodopsin, a-thrombin receptor).
Subfamilies Analysis of sequence alignments has defined the existence of at least three structurally distinct subfamilies. The first subfamily has as its structural signature a DRY-motifand encompasses the vast majority of the > 200 G protein coupled receptors. They have about 12 highly conserved amino acids in their TMs - 3-4 in TM-11, 2 in TM-VI and 2-3 in TMVII - and, at the interface between the membrane and the cytoplasm at the end of TM-I11 a so-called DRY consensus motif (Asp-Arg-Tyr), of which the R is absolutely conserved in all receptors of this subfamily. The D is usually D, but can also be E (rhodopsin) and the Y, while present in about 100 members, varies in the others, the most frequently found being F, H, and C. An amino acid sequence alignment of a selected few of the receptors belonging to the class with the DRY motif can be found in Appendix IV. DRY-motif containing receptors do not show a G protein preference or preference for a neurotransmitter (small ligand) vs. a peptide hormone (large ligand). To the second subfamily belong the receptors for the glucagon-related peptides and for CRF, PTH, and calcitonin. Receptors of this class share little meaningful sequence homology with the DRY-motif containing G protein coupled receptors. They all stimulate adenylyl cyclase (i.e., activate GJ. Due to the presence of conserved cysteines, extracellular loops 1 and 3 may be connected by a disulfide bridge. The alignment of their sequences is presented in Appendix V. It is of interest that six of the nine evolutionary related receptors bind the six also evolutionarily related
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glucagon-related peptide hormones (Appendix VI). Although this may represent an example of co-evolution of interacting elements, the evolutionary paths followed from ancestral genes to present are not obvious (compare results from applying Hein’s phylogenetic analysis to conserved portions of the two protein families in Appendix V and VI). The Ntermini of all the members of this family (ca. 120 amino acids in length), with the apparent exception of one (GIP), have at their N-termini a hydrophobic sequence encoding a signal peptide and are, like the transmembrane cores, evolutionarily related (Appendix V). To the third subfamily belong the four metabotropic glutamate receptors and the Ca2+ sensing receptor. Because of the small number of members, this family is not distinguished by any special characteristics other than having a very long extracellular N-terminal domain, in spite of the smallness of the ligands - Glu and Ca2+ - and having the general feature of (putatively) traversing the plasma membrane seven times, and being functionally able to activate a G protein. The following three major functions are encoded into the primary structure of a G protein coupled receptor: 1. a binding site that when occupied changes conformation in a manner that it can be sensed by a G protein; 2. the ability of forming a complex with specific G proteins, which alters in some but not all cases the affinity parameters of the receptoragonist interaction; 3. the susceptibility to be desensitized by a desensitization machinery that senses the receptor’s occupation by an agonist. For small ligands the binding site is fully located within the plane of the membrane involving residues of the transmembrane domains (Fig. 13). Receptor activation involves conformational changes that modify the relationship among the transmembrane domains thereby acquiring high affinity for one or more subtypes of G proteins. While for larger ligands the primary ligand binding function resides in their Ntermini and various aspects of the extracellular loops, it may be assumed on the basis of structural similarity of the transmembrane cores and certain aspects of the intracellular appendages that the “activating” function o f the ligand is still exerted at the level of the transmembrane aspect of the receptor. Two cases of special interest are the rhodopsin and a-thrombin receptors. Both are “pre-associated” with their ligand, and receptor
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Rhod opsin
P2-Adrenergic Receptor
Salt brdge: LYS-296- Glu-113
Fig. 13. Proposed intramembrane, inter-TM domain location of 11-cis-retinal, the lightsensitive ligand of rhodopsin (upper), and of norepinephrine, the agonist of the 0adrenergic receptor (lower). Left:The drawings depict rhodopsin with the protonated Shiff-base of 11-cis-retinal bonded to Lys-296 of TM-VII, as it is thought to be in the non-excited resting state, and all-trans-retinal bonded to the same Lys-296 but now unprotonated, with the proton delocalized, as it is thought to be in the fully activated Meta-II state. This form of rhodopsin has a relatively long life-span allowing it to sequentially catalyze activation of many transducin molecules before it is deactivated (after [ 198-2001. Although the natural photoreceptor has the chromophore covalently attached through the Shiff base, this is not an absolute requirement. [Lys296-.Gly]opsin binds the n-propylamine Shiff base of 11-cis-retinal and is activated by light [201]. Right: Mutational studies that monitored the effect of structural changes on ligand affinity suggest that binding of an agonist - a catecholamine - and activation of the padrenergic receptor involve Asp-113 of TM-111, acting as a counterion to the cationic amino group of the ligand, Ser-204 and -207 of TM-V, creating hydrogen bonds with the two -OH of the catechol and Phe-290 of TM-VI stabilizing the aromatic group of the catecholaminethrough r-a bonding of the aromatic rings (after [60]).Asn-3 12 in TM-VII has been shown to be intimately involved in conferring high affinity binding to antagonists of the pindolol type, not only in this but also in the type-lBIDP serotonin receptor [184].
activation is, in fact, the consequence of ligand activation. In the case of rhodopsin (Fig. 13), the ligand, 11-cis-retinal, is bound through a Schiff base to Lys-296 of TM-VII. The ligand is activated upon light absorption
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which induces photoisomerization to all-truns-retinal followed by deprotonation of the Schiff base. In the case of a-thrombin, the N-terminus is a substrate for thrombin containing a latent receptor ligand. This ligand is “exposed” by the proteolytic action of thrombin. This results in the removal of the first 41 amino acids of the receptor. The new N-terminus now constitutes a tethered ligand that activates the receptor by presumably curling onto itself and entering into a binding pocket formed by the TMs of the receptor [59]. Extensive mutational analysis of the 02adrenergic receptor has led to the identification of the major residues in the binding of an activating catecholamine [60; reviewed in 611. Screening of amino acid sequence alignments identified conserved aspartic acids in biogenic amine binding receptors. Using the adrenergic receptors as the main test subjects, these aspartates have been subject to analysis through removal by site-directed mutagenesis (reviewed in [60-621). These studies identified the 02-adrenergic receptor Asp-1 13 in TM-I1 and its cognate in other receptors as a counterion for the binding of the amino-group of catecholamines and acetylcholine. They also identified PZadrenoceptor Asp-79 (80 in the D2 dopamine receptor and 383 in the receptor for luteinizing hormone) as responsible for regulating affinity by sodium (decreased for agonist and increased for antagonists) with consequences on coupling to G proteins that varied from a non-detectable, LH receptor, to a selective for one vs another type of G protein, an a2-adrenergic receptor, to mere changes in agonist concentrations required for half maximal activation, a 02-adrenergic receptor (Table VII). Of these, possibly the most interesting is the finding that for the a2-adrenergic receptor the Asp-79 to Asn mutation led to a loss of K + channel regulation, an effect of Gi, with unaltered inhibition of adenylyl cyclaseand unaltered inhibition of Ca2’ currents [63]. Opposing regulation of antagonist and agonist binding was an early finding for opioid receptors before they were known to be G protein coupled receptors [64]. Although the sodium-induced state was then referred to as the antagonist state to differentiate it from an agonist state, the functional role of this regulation is still unclear. Mutations of the third conserved aspartate, which is the D of the DRY motif, uncouple the receptor while increasing agonist affinity (Table VII). The G protein coupled receptors have recognition elements for both the G protein a-subunit and the G protein fly dimer, as as will not interact with receptors in the absence of flys [8,65,67], but Pys interact with a receptor in the absence of an a-subunit [67,68]. Figs. 4 and 6
Site
S69L
E92K
L98P
l i loop
TM I1
l e loop
MSHR (mouse)
G246frshft R137H R113W
V2R (human)
3i loop DRY motif l e loop
D619G A6231
P23H F27L
K296E A292E V20G
TSHR (human) 3i loop 3i loop
N-term N-term
Opsin (human) TM VII TM VII N-term
H183frshfi
D578G
LHR (human) TM VI
2e loop
D60G
GRFR (mouse) N-term
ACTHR (human)
S 120R
R201stop S74I
Mutation
TM 111 3i loop TM I1
A. Naturally occurring
Receptor
Inactive; CNDI ( 4 5 allele) Inactive; CNDI ( 4 2 allele) Reduced affinity for ligand and reduced expression; CNDI ( 4 3 allele)
Constitutive activity; thyroid adenoma Constitutive activity; thyroid adenoma
Constitutive action; retinitis pigmentosa Constitutive activation; stationary night blindness Impedes processing and damages cell causing autosomal dominant retinitis pigmentosa Same as V20G Same as V20G
Constitutive activation, hyper-responsive to MSH Phenotype: dominant extension of black: I?* (tobacco coat) Constitutive activation, unresponsive to MSH (somber coat) Phenotype: dominant extension of black: Constitutive activation. Phenotype: dominant extension of black: Po (somber); inactive (frame shift) Phenotype: nonextension of black: e (yellow coat)
Constitutive activation, partial and stimulable by LH; hereditary autosomal dominant male precocious puberty
lit/lit mouse: hypoplastic anterior pituitary; lack of GRF action
Inactive; hereditary familial glucocorticoid deficiency; autosomal recessive Inactive; hereditary familiar gllucocorticoid deficiency; autosomal recessive Inactive; familial glucocorticoid deficiency; autosomal recessive
Comments (receptor properties, phenotype, etc.)
Mutant forms of G protein coupled receptors
TABLE W
Refs.
R198E E298K
N-term N-term
El 134 (counterion)
TM 111
3i loop
TSH (human)
A623K D623E
TM I1
TM I1
LHR (rat)
D79A
TM I1
D383N
D80A D80E
D79N
D79N
TM I1
D2R
fY2-AR
Conserved Asp of transmembrane domain II:
TM VI
LHR (rat)
D578N
K296A
TM VII
1993
K296G
Opsin (human) TM VII
Expansion of natural mutations:
R796W
3i loop
continued
B. Made-made
CaSensingR (human)
TABLE W,
Loss of Na+-induced decrease in hormone binding
Loss of Na+-induced increase in antagonist binding and of Na+-induced decrease of agonist binding
Increase in Kd and Kact for Is0 (lox); antagonist binding unaffected; G, activated to 50% of control Normal low affinity agonist binding, no GTP sensitive high affinity binding, increased Kact to cause G, activation to ca. 15% of control
Unchanged ligand binding, but loss of regulation by Na. Signalling: inhibition of AC and Ca2+ currents unaltered, but stimulation of K+ currents (inward rectifier) severely impaired
No effect No effect
Unchanged activity; nature of mutation matters
Constitutive activation, suppressed by n-propylamine; Schiff base of 11-cis retinal giving light sensitive receptor Constitutive activation, suppressed by n-ethylamine; Schiff base of 11-cis retinal giving light sensitive receptor Constitutive activation, suppressed by 1I-cis-restinal; giving light sensitive receptor at pH 6.0 instead of pH 7.5
Fails to elicit response to Ca2+ in Xenopus oocytes after cRNA injection; cause not determined. Familial hypocalciuric hypercalcemia (FHH) (heterozygous) FHH; not functionally expressed FHH; homozygous: neonatal severe hyperparathyroidism (NSHPT); not functionally expressed
11641 1631
E
4
Q
Site
continued
Mutation
Comments (receptor properties, phenotype, etc.)
Quadruple: L266S, H269K and L272A
p2-m
D130N
p2-AR
Normal antagonist binding, increased high affinity agonist binding, altered TRP shift, no G , activation
Binds retinal but not transducin Binds retinal but does not activate Gt Loss of retinal binding Loss of retinal binding Binds retinal and activates transducin at 150%of control efficacy
Constitutive activation
Constitutive activation, graded (K is best); lower Kds for agonist
Constitutive activation, graded, lower Kd for agonist
Constitutive activation; lower Kds for agonist
[171]
Ref.
TM, transmembrane domain; i loop, intracellular loop; e loop, extracellular loop; N-term, N-terminal; frshft, frame shift; A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; K, Lys; L, Leu; M, Met; N, Asn; P, pro; Q, Gln;R, Arg; S , Ser; T , Tre; V, Val; W, Trp; Y, Tyr.
DRY motif
E134R, R135RE, R135Q, R135L R135W, E134Q
Opsin (human) ERY motif
Mutations in the DRY motif
3i loop
T348F,A,E,C or K
Triple: R228K, K290H and A293L A293 any AA
CY~~~,,-AR 3i loop
3i loop
3i loop
Cunstitutivr activations due to mutation of C-terminal end of third intrucellular b o p
Receptor
TABLE W,
2.
f?
8
Lu
G proteins in Signal Transduction
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depict our current thoughts as to the events that precede and follow interaction of a receptor with the G protein, and Fig. 5 depicts the effect that interaction of a G protein may have on the receptor’s affinity for an agonist . Efforts are being made to locate the regions of a receptor responsible for G protein activation, measured either as stimulation of GTPase, stimulation of GTPyS binding, or as activation of the G protein response pathway in membranes or intact cells. Attention has focused on the third intracellular loop. This loop is highly variable in length and composition which made it a candidate for conferring G protein specificity to a receptor (Appendix IV). Tests of this hypothesis have born interesting fruit. Exchange of a 17 amino acid sequence of the N-terminal end of the third intracellular loop of M3 and M2 muscarinic receptors substantially switched the receptor G protein specificity of these receptors [69]. Removal of a similarly located sequence from the PZadrenergic receptor uncoupled it from G protein interaction [70]. A 02-adrenergic peptide comprising N-terminal sequences required for function is able to stimulate GTP hydrolysis of G, [71]. Also the C-terminal end of intracellular loop 3 is important, for P2-adrenergic receptor peptides comprising this end of the loop are also able to stimulate GTP hydrolysis and GTPyS binding [71,72]. Through the use of peptides derived from receptor sequences , it has been suggested that other non-heptahelical receptors may also activate G proteins (see below). Similarities in amino acid composition of the C-terminal ends of the intracellular loops of the a l and PZadrenergic receptors led Lefkowitz and collaborators to explore chimeras in which the a1 receptor was made @,-like and vice versa. This led to identification of a l - 0-, and a,-adrenergic receptor mutants with constitutively enhanced agonist-independent activity [73-751. The potential role(s) of the third intracellular loop in the coupling process between receptor and G protein was also investigated by transient expression of the entire loop or N- and C-terminal 27-amino acid long portions of the loop [76]. Using as a template the alB-adrenergicreceptor, the loop or loop fragments had no effect by themselves on IP3 formation, as would be expected if one G protein that couples the alBreceptor to phosphoinositide hydrolysis were activated. In contrast, coexpression of the full alB-receptor and its complete third intracellular loop resulted in inhibition of the PLC activating effect of the receptor. This was partially mimicked by the N-terminal segment of the loop but
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L. Birnbaumer and M. Birnbaumer
not the C-terminal segment. Expression of the third intracellular loop of the D1,-dOpamine receptor had no effect on alB-receptor action but inhibited the effect of the D,,-receptor on PLC. The C X ~ ~ - I O O had P a small effect on M 1-muscarinic receptor stimulated PLC stimulation. Increasing the expression of full length receptors tended to overcome the inhibitory effect of the loop. These data are consistent with the idea that the loop acted by interfering with a protein-protein interaction and hence that the third intracellular loop may physically contact the G protein.
Naturally occurring receptor mutations Insight into structure-function relations have more recently also come from the study of genetic diseases that were uncovered to be due to mutations of G protein coupled receptors (summarized in Table VII). Two of these receptors, rhodopsin and the VP2-vasopressin receptor, have given a wealth of information because they give non-lethal phenotypes.
Rhodopsin mutations Mutations of the rhodopsin receptor cause retinitis pigmentosa, a group of dominant autosomal diseases that characterize themselves in that malfunction of rhodopsin leads to progressive retinal degeneration. In some cases this may occur because the mutant opsin is not properly processed causing damage to the protein processing and vesicle traffcking components of the rod cells [77]; in other cases, the reasons for the retinal degeneration are not clear. Of interest within the context of the present discussion is the opsin Lys-296 to Glu mutant. A study of this mutant opsin, expressed in COS cells, purified and then reconstituted with transducin, showed it to have constitutive activity, independent of light or retinal addition. An artificial mutation Glu-113 Gln, based on the fact that Glu-113, the opsin homolog to PZadrenergic receptor Asp-113, is the counter ion to the Lys-2961 retinal Schiff base, was also found to have constitutive activity in the absence of retinal. The intrinsic Iigans’ independent activity can be suppressed by addition of the 11-cis-retinal. This novel light-sensitive receptor differs from the natural in that it requires a lower pH for light sensitivity to aid in the deprotonation of the Schiff base [78]. Analysis of the man-made Lys-296 to Ala and Lys-296 to Gly mutants, which are
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also constitutively active, showed that activity could be suppressed by alkylamine Schiff bases of 11-cis-retinal and reactivated by light. This indicated that mechanistically the covalent attachment of retinal to opsin is a convenience designed by nature to maximize the light-capturing efficiency without being an essential part of the response mechanism [78].
VP2-Vasopressin receptor mutations A second large group of mutations has been identified in the type 2 vasopressin receptor gene. This receptor was cloned in 1991, located to the Xchromosome and soon proven to be the cause of many, if not all, cases of X-linked congenital nephrogenic diabetes insipidus [79-821. This disease manifests itself mainly through the inability of the kidney to respond to the antidiuretic effect of vasopressin (aka antidiuretic hormone or ADH), where patients cannot concentrate urine. Newborns become dehydrated, resulting in reduced growth, severe mental retardation, and - if untreated by the administration of water - death. Water is the only treatment required. Thirty-eight independent mutations of the VP2 gene have been identified at this time (Fig. 14). Eleven result in frame shifts with codon changes arid subsequent premature protein truncations; seven create a stop codon and also cause premature protein termination. All others are single amino acid changes of which ten occur in the predicted transmembrane domains, seven in the second extracellular domain, and two just prior to the beginning of TM-111. One Arg-137 to His is cytosolic just after TM-111 at the center of the DRY motif, which in this case is DRH. As mentioned and also summarized in Table VII, this mutation completely uncouples the receptor from G,, while other agonist-dependent functions (binding and receptor sequestration) remain unaltered. This identifies the DRY as important in G protein-receptor coupling. It is to be expected that further analysis of other amino acid changes will reveal new aspects of structure function relations in the VP2 receptor that might be extrapolated to other receptors as well.
The melanocyte stimulating hormone (MSH) receptor as a genetic model An interesting model that has potential for additional genetic analysis emerged liom the study of MSH receptor mutants. MSH regulates
. -
A
frameshift and truncation
Fig. 14. Physical map of W2-vasopressin receptor mutants found in patients suffering from congenital nephrogenic diabetes insipidus. From [81,82,160,161,203-2061.
0 single amino acid change
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melanin synthesis in melanocytes of hair follicle cells and skin. The MSH receptor is coupled to adenylyl cyclase through G, and its stimulation by the intermediary pituitary lobe hormone MSH (melanocyte stimulating hormone) elevates CAMP,which in turn regulates the transcription of the enzyme tyrosinase. Tyrosinase is the rate-limiting step in the synthesis of melanins, of which melanocytes make two kinds: phaeomelanin and eumelanin. Hair made from follicular cells synthesizing phaeomelanin is yellow/red in color; hair made from follicular cells synthesizing excess eumelanin over phaeomelanin is browdblack. At low tyrosinase activity the levels of dihydroxyphenylalanine (DOPA) made from tyrosine are low, and the default pathway is synthesis of DOPA-quinone followed by cysteinyl-DOPA which is incorporated into melanin to give phaeomelanin. IJpon excess production of DOPA, as results after maximal stimulation of the MSH/G, system, excess DOPA is converted to DOPAchrome, which when incorporated into melanin gives black eumelanin (summarized in Fig. 15). One of the MSH receptor genes, of which there are at least three, has been identified as the gene product of the extension locus. This is the gene that controls the degree to which dark color extends into the red/yellow background of hair [83; reviewed in 841. As diagrammed in Fig. 15C, hair color is controlled by a second genetic locus, the agouti locus. The gene product of agouti is a protein with the characteristics of a secreted polypeptide with signal sequence and processing signals. The agouti signal is made in paracrine fashion by cells that surround hair follicles, and although its mechanism of action has not been definitively established as yet, it is most likely a competitive inhibitor of MSH action on the MSH receptor. Inactivation of the extension locus is autosomal recessive, activation of the extension locus is dominant, inactivation of the agouti locus is recessive, and activation of the agouti locus can have variable effects depending on the strength of the normal extension locus. Three dominant murine extension mutations have been characterized as MSH receptor mutants, and the biochemical consequence was determined for two (Fig. 15D:i [83]. One, ESo0.”(sombre) causes the receptor to be constitutively activated, giving 50%the activity obtained with wild type receptor after MSH stimulation, but unresponsive to further stimulation by MSH; the second, Etob (tobacco), causes the receptor to have some constitutive activity and to be hyperstimulated by MSH, due to what appears to be increased efficacy. This mutation, Ser-69 to Leu, affects the first intracel-
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lk
L
CAMP
&
4
+
Tyrosinase DOPA
+
DOPA-Quinone
DOPA-Quinone
4
\r
d
Cysteinyl-DOPA
Cysteinyl-DOPA DOPA-Chrome
-&
I
+
Tyrosine Hlgh Tyrosinase DOPA
Tyrosine LOW
Phaeo-
Phaeo-
EuMelanin
Melanin
Melanin
C. MSKR (8HmdOn IoCUS)
V
CAMP
Yellow/Red
Phaeo-
Melanin
-9
MSH-R
EuMelanin
E+
BrownIBlack
wild typt
1
dominan recessiv
(E92K)
\
Etob (S69L)
Fig. 15. Summary of regulation of synthesis in melanocytes of phaeomelanin (A), of phaeomelanin plus eumelanin after MSH receptor stimulation (B), of the interactions of the extension (MSH receptor) and agouti loci (C), and of murine MSH receptor mutants responsible for the e, E",Em,3J, and I?"' alleles. For details see text and Robbins et al. ~331.
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Mar loop; the activating Es0>J3mutation, Glu-92 to Lys, is close to the end of TM-11. The extension locus could be a powerful model to search for receptor mutants using coat color as an initial selection marker. Parameters such as intrinsic activity (efficacy) of a receptor which confers at equal receptor abundance an enhanced or decreased capacity of the receptor to stimulate G, should be especially amenable to study in this way. Naturally occurring mutations that have been found in several other receptors are summarized in Table VII. It is interesting to note that several of them are of the activating type and that two of these, found in the TSH receptor, are due to change in amino acid composition of the C terminal end of the third intracellular loop, where Lefkowitz and collaborators had previously identified the potential for such functional activity. One, causing constitutive activation of the LH/hCG receptor, changes an Asp in TM-VI to Gly [85]. Although it has been speculated that activation may be the result of disruption of ion-pairing with a counter ion from another TM, the structural cause for activation must be another since mutating the same Asp to Asn instead of Gly did not activate the receptor 1861. Taken together, studies of naturally occurring receptor variants and mutants, as well as man-made mutants, indicate a participation of all the intracellular appendages in their interaction with G proteins.
G protein activation by non-heptahelical receptors In 1989, Nishimoto and collaborators demonstrated functional coupling of Man-6PIIGF-I1 receptor to G , in reconstituted phospholipid vesicles. GTPyS decreased the affinity of the receptor for IGF-I1 by a factor of close to 8 and IGF-I1 stimulated GTPyS binding in a PTXsensitive manner [87]. This last effect was mimicked by a Man-6P/IGF-I1 receptor peptide comprising a 14-amino acid sequence just inside the plasma membrane [88]. The peptide kinetically mimicked “classical” G protein coupled receptors in that it acted to reduce the concentration at which Mg2+ promotes nucleotide exchange [88]. Residue substitution studies suggested the signalling motif to be between 14 and 20 amino acids long, to have two basic residues at the N-terminal side, and to contain the C-terminal sub-motif B-B-X-B or B-B-X-X-B, where B is a basic amino acid and X is any amino acid (Fig. 16). Sequences with these
L. Birnbaumer and M . Birnbaumer
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Man-GPIIGF-II Receptor
0.9 IGF-II R Peptide
2410
R V G L R
G
E
K A R K
G GDP
Gi
2423
Control
0.3
7 a ~
~
2 GTP@ +
Mg2+ (-log M) Fig. 16. Sunimary of mode of action of the “Nishimoto couplone” of the cation independent mannose-6-phosphate/insulin-likegrowth Factor II (Man-6PIIGF-II) receptor as seen upon addition of the couplone peptide shown in the left panel is added to phospholipid vesicles reconstituted with G,. Note the effect of the couplone to left-shift the concentration of Mg2+ at which GDP/GTPyS exchange at Gi2is obtained.
general characteristics can be found in the second and third intracellular loops of several heptahelical receptors, including the C-terminal sequence of the human &-adrenergic receptor. A test of the P2-adrenergic receptor sequence was indeed found to stimulate GTPyS binding to G, [72]. Further studies identified a Gi/Go activating sequence in the C-terminal end of the third intracellular loop of the M4-muscarinic receptor [89] and a variant but very potent Gi/Go activating sequence, in the comparable position of the a2C10-adrenergic receptor, having at its C-terminus B-BX-X-F instead of B-B-X-X-B [go]. These rather tantalizing studies (reviewed in [91]) provide for a structural basis of the PTX-sensitivity of some of the effects of nonheptahelical receptors, such as those of the already mentioned Man6P/IGF-II receptor [87] and of the transforming activity of the type I1 TGFP receptor in NIH-3T3 cells [92]. This latter receptor has a “Nishimot0 couplone” in the middle of its cytosolic kinase domain. Also the Alzheimer P/A4-amyloid precursor protein (APP) has a Nishimoto motif, and, upon examination, APP was found to form a complex with Go, the most abundant neuronal G proteins, in a M$+-dependent and GTPyS-
G proteins in Signal Transduction
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sensitive manner. Mutations of the motif interfered with complex formation [93]. These type of studies are not only interesting in their own right but they also provide a focus for hrther studies on structural aspects of the mechanics by which heptahelical receptors activate a G protein. As a word of caution, it must be mentioned that the determination of both the actual structural pattern of a receptor that activates the G protein and its specificity will not be easy. Other substances mimic receptors, just as Nishimoto couplones do. These include mastoparan and polylysine [94,95].
Molecular Diversity and Regulation of Adenylyl Cyclases and TypeC/3 Phosphatidylinositol-Specific Phospholipases
Molecular diversity of a-subunits G protein
-
Multiple eflectors for a single
Activated a-subunits are regulators of effectors. The structural subdivision that emerged from the phylogenetic analysis of a-subunits (Fig. 17) correlates rather well with their known and suspected functional both subdivision. Activated forms of the closely related a, and aOlf stimulate adenylyl cyclases; those of aq,a11 ,~ ~ 1and 4 , (Y15/16stimulate PIspecific PLCs of the &type and the three q s , the type-1 a, splice variant and a, inhibit adenylyl cyclase. Among the three sensory PTX sensitive as - atr,atc,and agust - the mechanism of action of atr,activation of visual PDE through interaction with inhibitory PDEy subunits, is well which understood; that of atcis assumed to be similar, and that of agust, is involved in perception of sweet and bitter tastes, is still in need of confirmation at the biochemical level. The effector complements of a I 2 and ~ 1 3 a, sub-branch of the aqfamily, are still under debate and are the subject of intense research. A similar situation applies to a,s. While one is able to inhibit adenylyl cyclase in a reconstitution assay, the other is not. Both have been clearly shown to mediate inhibition of L- and N-type Ca2+ channels in mammalian cells and in mollusc synapses and to mediate of stimulation of PLC in Xenopus oocytes, but for neither has the effect been shown to be direct. These two a-subunits, each of 355 amino acids, are encoded by the same gene and differ in that the open reading frame of aOluses exons 7 and 8 that differ from exons the 7' and 8' used
L. Birnbaumer and M . Birnbaumer
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a Subunits
Functional Correlates$
ai I *
*, PTX substrate **, tentative $ Assignment of a function does not negate additional functions.
Fig. 17. Summary of known effects of G proteins on cellular effector functions (adapted from Birnbaumer [96]).
to build As a result, a,s differ in 26 of their last 106 amino acids in a region into which both receptor and effector specificities are encoded. It has been shown that a, and ais can regulate more than a single effector system. In addition to stimulating adenylyl cyclase, a, can stimulate Ca channels of the L-type and a subclass of smooth muscle channels). These are Ca2+-activatedvoltage-dependent K channels (ha functions that are expressed only in specific tissues and cell types so that responses to receptors that activate G , may vary from tissue to tissue. For example, Ca channel stimulation by asoccurs in tissues such as skeletal muscle and heart where it potentiates the stimulatory effect of the CAMPPKA system, but not in liver or endothelial cells that lack voltage-gated Ca channels; stimulation of the ha channel by a,occurs in tissues such as coronary smooth muscle, where it potentiates the relaxing effect of the CAMP-PKA system, but not in atrial cells of the heart that lack K,,
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channels. Likewise, ail, a2,and ai3,which inhibit adenylyl cyclases, can also activate at least two classes of K+ channels, the “muscarinic”type inwardly rectifying K + channel found primarily in cardiac atrial cells and in neuroendocrine cells, and the ATP-sensitive K + channel found in cardiac ventricle cells and pancreatic islet cells. These channels are also referred to as G-protein-gated K channels. The fact that single a-subunits may stimulate or inhibit more than one effector is relevant in terms of the ultimate elucidation of the roles that each G protein may play in a cell. The complexity of the responses of a cell to G protein stimulation is likely to be large.
Signaling through Py dimers While signaling through a-subunits is the most common mechanism by which receptors activateor inhibit effector hnctions [e.g., stimulation and inhibition of adenylyl cyclase by a, and ais, stimulation of ,&subtype of phospholipases C (PLCPs], G proteins also signal through their Py dimers. These responses require both a respectable concentration of G protein and expression of the adequate fly responsive effector system (reviewed in [96]).
Adenylyl cyclases Eight non-allelic adenylyl cyclase (AC) genes have been identified. Of these, full-length cDNAs of six and the partial sequence of the eighth have been published (Appendix VII). In contrast to membrane-associated G protein subunits, ACs are true transmembrane proteins. On the basis of Kyte-Doolittle plots, they are predicted to traverse the membrane 12 times, beginning with a cytosolic N-terminus and ending with a cytosolic C-terminus. Most intracellular loops are predicted to be short, but the fourth intracellular loop and the C-termini are quite long. N-termini are variable in length (e.g., short for type-I AC). This separates the first six transmembrane segments from the second six transmembrane segments. In addition, dot-matrix amino acid sequence comparisons reveal sequences in the first half of the molecules that are homologous to similarly placed sequences in the second half of the molecules, indicating an ancestral gene-duplication event. However, the lengths and the placements of these internal homology repeats vary from AC to AC, indicating
208
L. Birnbaumer and M . Birnbaumer 12 3
123456
A.
b.. pp
1
..........................
+.
..........................................
...........................................
B.
....
~~~~
...........................
45 6
G proteins in Signal Transduction
123456
C.
209
12 3
45 6
c ..... ............
Fig. 18. Kyte-Doolittle hydropathicity and similarity matrix analysis of type U and type V adenylyl cyclases. The presumed (6 +6) transmembrane regions are highlighted in the Kyte-Doolittle plots. Comparison of type I1 to type I1 (A) and type V to type V @) shows existence of two internal similarities in type I1 but only one in type V adenylyl cyclase. Comparison of type II to type V (C) shows extensive regions of similarity between the two molecules (central diagonal lines), of which the thickened portions encompass sequences that are similar among all six ACs. Non-colinear similarites between AC-II and AC-V (i.e., sequences of the first half of one molecule that are similar to sequences in the second half of the other molecule and vice versa) are highlighted in panels A and B.
that the duplication events occurred early in their evolution to the present state. Comparisons within the AC-I1 and the AC-V molecules and of ACI1 to AC-V, together with their respective Kyte-Doolittle plots, are shown in Fig. 18. Adenylyl cyclases are thus constituted of two similarly organized halves, each with six transmembrane domains or segments and a rather long C-terminus. Catalysis depends on the cooperative interaction of both halves and is carried out by the segments that are common to all ACs (reviewed in [97]).
L. Birnbaumer and M. Birnbaumer
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Fig. 19. Transmembrane model of adenylyl cyclases as originally proposed by Krupinski et al. [207] and supported by hydrophobicity and similarity analysis, and summary of main regulatory features (adapted from Iyengar [208]). ch (common homology), regions of adenylyl cyclases with conserved amino acids among all; ih (internal homology), regions where sequences in the first half are similar to sequences in the second half of the molecule.
-
-
IV
ubiquitous
I
olfactory epithelium, other (neuroendocrine cells, basal ganglia) brain
Ill
-
-V
ubiquitous, high in brain, hear, liver, kidney
'1 080-1144 aa; Vlll is incomplete; 191 completely conserved sites in AC 1 - VI Fig. 20. Phylogenetic tree derived from analysis of amino acid sequences comprising the C-terminal half of type I through VI plus type VII adenylyl cyclases and patterns of expression.
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Fig. 19 incorporates in cartoon form these structural features and summarizes the rather surprising differences in regulatory feature of the various adenylyl cyclase subtypes. As is the case for G protein a-subunits, a phylogenetic three of adenylyl cyclases I through VI and VIII (Fig. 20; Appendix VII), shows a good correlation between structural similarity and functional similarities. From a functional viewpoint, all ACs have in common that they are stimulated by the activated form of a, and by the diterpene drug forskolin, but they differ strikingly in their responses to Ca2+ and calcium/calmodulin (Ca/CaM). In addition, some but not all ACs are effectors for fly dimers. Type I and type I11 are stimulated by Ca/CaM. Types V and VI are inhibited by micromolar Ca2+. Type I is inhibited by flr dimers; in contrast, two others, type I1 and type IV, are stimulated by /3r dimers. While inhibition of type I is independent of other regulatory input@) (i.e., affects basal, a,-, forskolin- and Ca/CaM-stimulated activity), stimulation of type 11 and IV ACs only happens if the cyclase is simultaneously stimulated by a, (i.e., Pys act to potentiate an existing stimulatory input). The close phylogenetic relation of type VIII AC to type I AC, found on the basis of knowledge of about 60% of the molecule, is intriguing. It will be interesting to see whether, upon examination of the properties of the complete enzyme encoded in the type VIII gene, this relationship will hold up at the functional level by showing stimulation by Ca/CaM and inhibition by @ydimers. Studies are being carried out on the tissue distribution of the various adenylyl cyclases. The picture that is emerging is complex. Type I and VIII cyclases appear to be of primarily neuronal expression. Type I11 is very highly expressed in olfactory neuroepithelium. Like aOlpType I11 AC was originally thought to be restricted to this cell type but is expressed quite ubiquitously, as are all other types studied (V, VI, 11, and IV). It is worth mentioning that in one study in which expression of AC subtypes was tested for by analyzing RNA by RT-PCR, a single cell type was found to express five out of six ACs tested for.
Inhibition of adenylyl cyclase The first indications that guanine nucleotides are involved in inhibitory regulation of adenylyl cyclase were published in 1973 [98,99], and the
212
L. Birnbaumer and M. Birnbaumer
first isolations of the PTX-sensitive, putative Gi proteins were published purified in 1983 [ 100,1011. All indications were that adenylyl cyclases should be direct targets of ai-mediated inhibition of activity, as indicated by the presence of inhibitory regulation in a,-negative S49 cells [ 102, 1031 and the lack of an effect of cholera toxin, known to reduce affinity of a, for fly, on inhibitory regulation of adenylyl cyclase [104]. Nevertheless, the in vitro reconstitution of inhibition of adenylyl cyclase by aisubunits was not achieved until 1993 [105; Codina and Birnbaumer, unpublished] (Fig. 21). The inhibition is non-competitive with respect to activation of as,indicating that the ACs should have independent sites for interaction with a, and ai(Fig. 21A). Although not as marked as with Py dimers which act at the 100 through 1000 nM level, the concentrations of recombinant ais required for half-maximal inhibition (5-10 nM) are about 100-fold higher than required for half-maximal stimulation with recombinant a, under the same conditions (Fig. 21B). As seen with natural membranes (Fig. 21C) and in transient expression assays [ 106, 1071, the extent of inhibition varies with AC subtype. Inhibition appears to be 100%when operating on AC-VI but less so when operating on AC11. Moreover, AC-I1 inhibition by aiis suppressed by phorbol esters, presumably through PKC-mediated phosphorylation.
Regulation by phosphorylation Although studies on regulation of ACs by kinases are still in their infancy, the initial results that are emerging point to a complex and again type-specific response pattern. Type-I1 AC is stimulated by PKC, while Types V and VI appear to be inhibited by PKA.
Phospholipase C
Known to be a target of G protein regulation [ 108,1091, phosphoinositide (PI)-specific phospholipase C (PI-PLC) turned out to be a complex family of related proteins. The structures of PI-PLC’s were deduced from combined biochemical, genetic, and molecular biological studies ([ 110113; reviewed in [ 1141). PI-PLC’s are structurally subclassified into p, y, and 6, and for each of them there are at least three closely related subtypes, of which only the @-subclassis a target of G protein regulation. Four mammalian members of type /3 PLCs are known, as well as one
G proteins in Signal Transduction A.
0 Conkol
63.2nM [Q204L]mi3 24 nM [Q209L]m
nM[02124r& prnolcAMPlrninlrng
0.m
1.26
13
89
213 B.
q
12.6
533
ram
(nM)
Fig. 21. Characteristics of ai-mediated inhibition of adenylyl cyclases. A: Dependence on concentration of a;.In the experiment shown ca 5 nM a i 3 ( p L )caused 50% of maximal inhibition which in several experiments averaged 60%. Assays were in the presence of GTP. Similar data in terms of potency and extent of inhibition were obtained with wilt type as in the presence of GTPyS. B: Non-competitive nature of the inhibition by ai3 with respect to stimulation by a,. C: Extent of inhibition depends on the origin of membranespresumablydue to expression of different adenylyl cyclase subtypes. (Unpublished data from Codina and Birnbaumer).
Xenopus laevis homolog and two Drosophila melanogaster homologs, norpA and PLC 21 (Fig. 22; Appendix VIII). The amino acid alignment of 0,y, and 6 PLCs reveals two stretches of highly conserved sequences, X and Y, which presumably constitute the enzymatic core, and divergent N- and C-termini as well as diverging X-Y linkers. PLCy’s have the longest X-Y linkers, containing two src homology 2 (SH2) domains and one src homology 3 (SH3) domain. They participate in the intracellular transduction of signalling pathways that involve protein tyrosine kinase (PTK) activities. SH2 domains bind autophosphorylated PTKs and are phosphorylated by them. This results in PLCy activation, formation of diacylglycerol (DAG) and inositol
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PI-PLC X
PIP2
src Homology Domains
Type
+DAG
pi PI-PLC p2
PI-PLC
TP
PI-PLC p3 PI-PLC p4
None None None None
p 1 - p y~ ~ SH2 8 SH3 PI-PLC
6
None
Stimulated by
aq, all,a14 >> a16 >> a1 6 >> a1 1
Py >>> aq,a1 1 a andlor Py PTKs ?
Fig. 22. Summary of some of the structural and regulatory features of phosphoinositide specific C-type phospholipases. Based on Rhee and Choi [ 1141 and references listed in the text.
trisphosphate IP3, and the consequential activation of protein kinases of the C type (PKCs) and Ca2+ mobilization. In view of the fact that both PKC and Ca2+, alone or in combination with calmodulin (CaM) modulate adenylyl cyclases in a type-specific manner, PLCy activation may or may not be accompanied by CAMP changes. Type-6 PLCs have both short X-Y linkers and short N- and Ctermini. They lack SH2 and SH3 domains, and little is known at this point about factors or stimuli that regulate their activity. All four mammalian type-@PLCs are regulated by G protein subunits (summarized in Fig. 22). Their X-Y linker segments lack SH2 and SH3 domains and are short; in contrast, they have long C-termini when compared to y and 6 PLC C-termini. Mammalian type-0 PLCs are structurally more related to Drosophila's norpA PLC than y- and &type mammalian PLCs, and this was the first indication that 0-type PLCs were likely to be the G-protein-sensitive PLCs. NorpA (noreceptor potential & flies are blind due to disruption of the rhodopsin signalling pathway, which in insects is PLC- rather than PDE-dependent [ 1151. PLC-0 regulation by G proteins is being worked out in reconstitution studies [ 116-1281 (summarized in Fig. 22). The picture that is emerging from these studies is that PLCps show differential sensitivities to CY and by dimers. Studies have been reported with PLC-pl,-p2, and -03; PLC04 was only recently cloned, and little is known about its regulation by G protein subunits. The aq/allhave been ranked for effectiveness in stimulating PLC activity in one laboratory as P L C P l 2 PLCp3 % PLCp2, and the response to by as PLC03 > PLCp2 % PLCP 1, with PLCp4 being
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possibly unresponsive. For PLCP2 the rank order of responses appears to be a16>aq/al1. Studies of this type are ongoing, and ranking of reactivities may change depending on the type of assay (e.g., in vitro reconstitution vs. overexpression in COS or HEK-293 cells vs. mere analysis of responses of normal cells). Regardless of the exact final outcome, it is clear that, as is the case with ACs, the responses of cells to the activation of a given G protein by a receptor may vary in intensity and complexity depending on the complement of PLCs that is expressed in these cells. C-terminal truncation of PLCP results in loss of stimulation by the asubunit without loss of response to 0-y dimers [124,129; Schnabel and Gierschik, pers. comm.]. This indicates the existence of separate response domains for the two regulators.
Subunit concentrations that cause halfmaximal eflects- l%erole of GAP activity of the eflector As is the case for adenylyl cyclase regulation by P-y dimers, that of dimers also requires high levels of this protein, 100PLC by lo00 pM, as compared to the much lower concentrations required to obtain regulation with a-subunits (compare results in [ 119,1201 to those in [130]), raising the question of physiological relevance of the P-y regulation. Two arguments support a physiological role for 0-y dimers in PLC regulation: one relates to the cell type in which fly-regulationis proposed to be relevant - the neutrophil and the neutrophil-like HL60 cell; the other relates to the finding that PLCP is a GTPase activating protein (GAP) for a, [131]. In neutrophils and HL-60 cells PLC activationby the formyl-Met-LeuPhe (fMLP) receptor is blocked by PTX [ 132-1341 and hence not likely to be mediated by any of the PTX-insensitive G proteins of G, class that stimulateo-typePLCs [118,121,135,136]. WLPstimulatescholera toxin mediated ADP-ribosylation of the HL-60’s two main PTX substrates, ai2 and ai3,which supports the idea that in this cell Gis rather than the G, class of G protein(s) signal PLC activation [ 137,1381. Both the levels of G, proteins (> 100 pmol/mg protein) and the fMLP receptor density ( > 100,000/cell) are very high in HL-60 cells, making it plausible that Py dimers rather than as mediate PLC activation in this cell in spite of the high concentration required.
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The finding that PLCPl stimulates the intrinsic GTPase activity of aq indicates that reconstitutions with a-subunits that have been persistently activated with non-hydrolyzable GTP analogs are “artefactual” [ 1051, since the EC50 is a composite value that incorporates the affinity of the active form - a ratio of on and off rate constants and the rate at which an a-subunit inactivates due to GTP hydrolysis - so that the EC50 or Kact= ( k , f f + ~ p a s J / k o n .For GTPase-stimulating effectors, it follows that EC50 values for effector regulation - stimulatory or inhibitory by a-subunits activated by GTP are up-shifted with respect to EC50 values for as activated with non-hydrolyzable GTP analogs or AIF,- by a factor given by the b P a s e / k o nratio. This ratio has been reasoned to be in the order of 100 or more [105], thus bringing the effective concentration of an a-subunit needed to modulate PLC into the range of the concentrations required for stimulation of PLC by /3y dimers. Since Py dimers are shared by a-subunits to form G proteins, and apparently a-subunits do not discriminate in a major way between Py dimer subtypes, fly signalling is most the likely basis for cross-talk between AC and PLC signalling pathways (reviewed in [139]). Specij?city of receptors for G proteins
Analysis of electrophysiological responses to agonists in cells previously injected with antisense nucleotide and studies of cells transiently expressing receptors and G protein subunits indicate that receptors select a G protein not only on the basis of its a-subunit but also on the basis of its /3 and y subunit. The specificity rules emerging from these studies are by no means simple and easy to understand. For example, by measuring agonist-induced inhibition of voltage-activated Ca2+ currents in pituitary cells previously injected with subunit specific antisense oligonucleotides, it was shown that the pituitary cell somatostatin (SST) receptor interacts but not with any Go having sol, p3, or with a Go of composition aO2p1y3 y4 as its component, and that the M3-muscarinic receptor in these cells interacts selectively with a Go of composition aOlP3y4and not with a Go having either aO2,p2, or y3as its component [20,21,140]. Yet the subunit selectivity is not absolute, for both the M3 and the SST receptor inhibit adenylyl cyclase and inwardly activate rectitjing K + channels, which are effects that are mediated by Gi protein(s) [ 104,1411.
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Results obtained via transient expression of various types of receptors in COS cells in combination with G protein subunits are consonant with those obtained by suppression of G protein subunit synthesis. For example, expression of C5a receptors in COS cells has no effect on aqor phosphoinositide hydrolysis unless they are co-expressed with a , could not be substituted for a,,. Expression of the platelet activating factor (PAF) receptor, in contrast, mediates PTX-insensitive stimulation of the COS cell’s PLCp(s), and this occurs through G, and/or GI, [142]. Expression of Interleukin 8 (IL8) receptors in COS cells leads to stimula5 , (Y16 but not with aqor a l l ; tion of PLC if coexpressed with ~ ~ 1 ~4~, 1 or and this stimulation is PTX-insensitive. Expression of IL8 receptors with fly-sensitive PLCp2, on the other hand, leads to PTX-sensitive phosphoinositide hydrolysis, indicating interaction with ~ O I I - C Xor~ ~n ~ n - a ~ ~ / ~ ~ type G protein, that is of the Gi-type [143]. Thus, IL8 receptors interact with PTX-insensitive GI, and GI, and with PTX-sensitive Gi(s), but do not interact with G, or G , A very curious situation exists with a2-adrenergic receptors. At low density of expression, or at low agonist (adrenaline) concentration, they inhibit adenylyl cyclase activity via G,, and at high levels of expression, or high agonist concentrations, they stimulate adenylyl cyclase via G, [144], as well as PLC via Gq. Since the delineation of many of these interaction pathways involves over-expression of the interacting components, some of them may never occur under normal circumstances, and their acceptance in physiological terms may have to be reviewed. On the other hand, failure to establish an interaction under conditions of overexpression and proper controls is probably a very meaningful result. In vitro reconstitution assays point to a role for y in specifying with which heterotrimer a receptor interacts. This was shown for the interaction of rhodopsin with transducin reconstituted from a,-GDP and recombinant ply1, ply2, or ply3. It was found that only p l y l binds to lightactivated rhodopsin [ 1451.
,.
Conclusion The broad strokes of G protein-mediated signal transduction as well as many of the molecular players are now well known: hormone binds to receptor, receptor changes conformation - which constitutes the first
L. Birnbaumer and M . Birnbaumer
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1-
Growth Factors
Fig. 23. Signal transduction by G proteins incorporating possible locations of Mg2+ binding sites, the existence of Nishimoto couplones, and the paradox finding that expression of the third intracellular loop containing a Nishimoto couplone and bearing the site responsible for conferring constitutive activity of receptors, acts as an anticouplone.
response to agonist - and promotes activation of a G protein by GTP (Figs. 1 and 23). However, the details of these interactions are for the most part nebulous. One would like to know which aspect of a receptor interacts with which subunit of a G protein and then with which aspect of the G protein subunit. While it is clear that receptors “look” at the carboxyl-termini of a-subunits to decide with which G protein to interact, it is not known whether other important points of contact exist on as and which the points of contact on 0s and ys are. We have pointed out that fly, upon binding Mg2’, acquires the function of a nucleotide exchanger and raised the possibility that receptors may be acting merely by aiding in the activation of Py by Mg2’. Implicit, of course, is also that Pys have one or more specific site(s) for Mg2’, and we have raised the possibility that the WD-40 motif may be an ion-binding motif. A comparison of receptor sequences reveals three subfamilies - and possibly more if receptors from lower eukaryotic species are taken into consideration. Yet the three appear to operate functionally in the same
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way. For example, the shift in agonist binding affinity by GTP was discovered by studying the interaction of glucagon with its receptor, a non-DRY motif containing receptor; and the same shift in affinity for agonists is also found with receptors containing the DRY motif. Even though the motif is absent from two of the three receptor subfamilies, mutation of the R in some but not all DRY-type receptors results in complete loss of interaction with the G protein without an effect on agonist binding, except for the fact that only low-affinity binding is seen because of a lack of interaction with the G protein. For the glucagon or PTH receptor, one would like to know the location and identity of the functional equivalent of the DRY motif. Sequence alignments do not provide clarification in this regard (Appendix IVd). Receptors have what can be called “intrinsic activity” or efficacy. In its simplest terms this is the number of G protein molecules that it can activate in the presence of GTP per unit time. This then translates into the number of effector molecules affected per unit time by a single receptor and hence the x-fold stimulation measured with one receptor vs. another. Mutations are being found that affect intrinsic activity, some natural, others man-made. One would like to know the kinetic parameter or parameters responsible for altered intrinsic activity. Possibilities include rate of nucleotide exchange and rate of dissociation from one G protein and association to another and dissociation or not from Pr in the course of an a-subunit activation cycle. Intrinsic activity of DRY-type receptors has been markedly elevated by mutation, primarily at the C-terminal end of the third intracellular loop, but in other places as well (e.g., first intracellular loop, sixth transmembrane domain). One would like to know whether equivalent mutations can be made or will be found in receptors of the glucagon responsive family. An understanding of the role of the N-termini and Ctermini of the third intracellular loop in normal receptor functioning should be helpful, but data with respect to this are difficult to interpret. One would like to know why the expression in a cell of a segment of a receptor interferes with receptor-G protein coupling, but addition of a polypeptide derived from this segment promotes activation of a purified G protein by GTP (Fig. 23). Also, in terms of cell physiology there are important questions that need clarification. We now know that a receptor may signal to more than one effector using two distinct a s by interacting with two G proteins or
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by using the a and Py from the same G protein. Some of the effector systems have the capacity of responding to both the a! and the 07, showing synergistic effects. One would like to know the physiologic relevance of these findings. Such dually responsive effectors may act as signal integrators being modulated by co-stimulation of distinct a!-subunit pathways. One interesting aspect is that fly only operates at high concentration, much higher than that of as, not withstanding the GTPaseactivatingargument, One would like to have better measurements both of the potencies of a and 0y-subunits and of the GTPase-activating effect of the effectors. Convincing evidence has thus far been obtained only for the GAP activity of PLCp1. Do all PLCs have GAP activity? Do any of the adenylyl cyclases have GAP activity? The role that one may assign to Pys as signalling molecules depends on the levels of activated G protein. For circulating hormones, full receptor occupancy does not occur. It may thus be that at any time only a small proportion of the target G protein is activated, yielding submaximal amounts of as, and due to the higher concentration requirements, to ineffective amounts of Pys. On the other hand, for postsynaptic neurotransmitter receptors, full occupancy and high local receptor density is the rule. so that in this case one may expect signalling through both a! and 07. 07s also appear to be the signalling arm of receptors in nonneuronal cells such as neutrophils. Here it is clear that PLC (02) activation has to be via 07s because the effect of ligands such as fMLP and IL8 is blocked by PTX. Neutrophils contain no PTX substrate other than Gis and q s do not stimulate PLCs. One would like to know the subcellular distribution and relative concentrations of G proteins and effectors, not only in general, but especially in cases where 07-mediation is suspected. The concentration argument raised above may be irrelevant, for example, if receptor, G protein, and effector exist as a preassembled complex. One would like to know whether receptors exist as monomers, dimers, or oligomers. One would also like to know to what extent signalling through G proteins occurs within macromolecular complexes in which one could find several receptors (or the same or mixed type), a set of G proteins (also different kinds), and relevant effectors - a transducesome (pronounced transdusome). Signal transduction, cross-talk between signalling pathways, and signal integration would occur locally under conditions where the concentrations required in reconstituted systems have no relevance.
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Other questions relate to the effector systems. Some, like adenylyl cyclase and PLCP, are primary, expressed in all cells and shown unequivocally in reconstituted systems to be regulated by one or the other of the two signalling arms of G,, Gp, etc. Ion channels, on the other hand, are responses restricted to cells expressing the particular channel of interest. At the moment of this writing, G protein regulation of ion channels has not yet been accomplished by reconstitution from purified components, and in some cases laboratories cannot agree on the results. In several cases the channel in question is regulated not only by G protein but also by phosphorylation, making it difficult to establish whether the effect of G protein activation is due to a boruiide G protein-effector interaction or to the result of a feed back regulation via a second messenger activated protein kinase or via the activation of a phosphatase. One would therefore like to see purified channel proteins reconstituted with purified G proteins akin to what was done to demonstrate the interaction of receptor and G proteins, and this under conditions where the state of phosphorylation is known. Finally, one would like to know all of the biochemical functions of each of the G protein a-subunits and of each of the Py dimers. At the time of this writing, G,, and G,,, neither of which is PTX-sensitive, are still orphan. But it may be that all others are partially orphans because, while we know of one or two effectors, there may be more.
Acknowledgements We would like to thank Dr. Heidi Hamm for making the crystal structure of atavailable to us before publication. We also thank Dr. Juan Codina for providing us continued encouragement and constructive criticisms. This work was supported by NIH grants DK-19318 and HL45198 to LB and DK-41244 to MB. We also acknowledge Baylor College of Medicine’s Molecular Biology Computation Resource (MBCR) which made it easy for us to have access to the molecular biology computation software, Eugene and GCG Suite, that were needed to handle, analyze, and print the sequences presented in the appendices.
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231
G proteins in Signal Transduction
Appendix I Sequence alignment and phylogenetic tree of G protein a-subunits Asterisk, amino acid identity in all sequences compared. For this and all other appendices, phylogenetic relations were calculated according to J. Hein [176]. R and Q found to confer oncogenic potential to some a-subunits are highlighted, as are palmitoylated motif near the N-terminus that affects affinity for cysteines near the N-terminus. h -
+,
or.
a. Percent identity in alignment: 0
100
1
81 100
4 5
42 45
100
70 69 44 100
69 67 43
44 42 39 88 43 100 8 6 43 100 43 100
6
7 8 9 10 11 12 13 14 15 17
69 65
44 93
50 50 56 52 50 50 42 100
39 40 37 41 40 40 38 42 100
9
38 39 39 40 40 39 39 46 65
100
10
49 49 56 51 50 50 40 80
41 45
100
12
13
43
44 71 69 70 43 49 41 42 51 92 100
62 62
71 69 71 44 48 42 42 49
100
60 60
14
50 49 56 52 50 51 41 88
43 45 81 49 49 100
15
44 42 37 43 41 42 79 40 36 36 39 43 42 40 100
16
57 55 42
68 66
67 41 46 41 39 48 59 57 47 40
100
Nmber of completely conserved sites: 6 6
G Protein m-subunit
17
78 79 43 67
bov-gtcone bov-gtrod hum-g16 hum-gil hum-gi2 hum-gi3 hum-gs-long mus - g 11 mus-gl2
66 65
43 49 41 39 48 61 69 49 42 57
mus-gl3
mu s - g 14 m u s -go1 mus-go2 mus-gq ra t-go1f rat-gz rat-gust
100
b. Amino acid alignment: (palmitoylation) r-gz b-gtrod r-gust b-gtcone h-gi2 h-gil h-gi3 m-go1 m-go2 h-gs-1 r-golf
m-912
11-913
h-a16 m-gl4 m-aa m-911 -
1
r-qz
b-gtrod r-gust b-gtcone h-gi2 h-gil
h-gl3 m-go1 m-go2 h-gs-l r-golf 11-912 m-q13 !l-g16 m-gl4
m-aa m-iii
I-
fmrY)MGCRQ--------------SSEEKEAARRSRRIDRHLRSESQRQRREIKLLLLGTSN
43 39 43 43 43 43 43 43 (msrlMGCTL--------------SAEERAnLERSKAIEKNLKEDGISARKDVKLLLLGRGE 43 MGCLGNS------KT-EDQRNEEKAQREANKKIEKQLQKD~Q~~THRLLLLGAGE50 MGCLGNSS-----KTAEDQGVDEKERREANKKIEKQLQKERLA~THRLLL~AGE52 MSGWRTLSRCLLPAEAGARERRAGAARDAEREARRRSRDIDALLARER~VRRLVKILLLGAGE 65 M A D F L P S R S V L S V C F P G C V L - - - - - - - - - - - - - - T N G E A E E 58 MARSL?WRCCPWCL-----------TEDEKAAARVDOEINRILLEOKKODRGELKLLLLGPGE 52 ~MAGCCCL--------------SAEEKESORISAEIERHVRRDKKDARRELKLLL~TGE45 MTLESIMACCL--------------SEFN(EARRINDEIERlIVRRDKRDARRDKRD~RELKLLLLG~E 49 ~LESMMACCL--------------SD~ESKRINAEIEKQLRRDKRDARRELKLLLL 49 G~E ttllt h-+ (mry?)MGAGA--------------SREEK-----HSRELEKKLKEDAEKDAR~LLLLGAGE (myr?)GSGI--------------SSESKESAKRSKELEKKLQEDAERDAR~LLL~AGE fmyr?)MGAGA--------------SAEDKELAKRSKELEKKLQEAGE (myr)MGCTV--------------SAEDKAAAERSKMIDKNLREDGE~R~LLL~AGE fmyr)MGCTL--------------SAEDKAAVERSKMIDRNLREDGE~REVKLLL~AGE fmry)MGCTL--------------SAEDKAAVERSKMIDRNLREDAGE f m r y ) MGCTI: - - - -- - - - - - - -SAEERAALERSKAIEKNLKEDGISIVU(DVKLLLLGAGE
-
-
SGKSTIVKOMKIIHSGGFNLE---------------ACKEYKPLIIYNAIDSLTRII~LRIDFHNPD~ 101 97 SGKL;TIVK@MKIItlQDGYSLE-- - - - - - - - - - - - - - E C L E F I A I I Y G N T L Q S I L A I V W L N I Q Y G D S A R Q S C ; K S ' I ' I V K Q M K I I t I K W G Y S - K - - - - - - - - - - - - - - - Q C M E f K A W Y S N T L \ 2 S l ~ I V ~ L G I D ~ P100 RSR
~-
SGKSTIVK@MKIlHODGYSPE--------------ECLEYKAIIYGMlLOSILAIIRAMPTLGIDYAEVSCV 101 SGKSTIVKQMKI IIIEDGYSEE--- - - - - - - - -ECRQYRhWYSNTIQSIMRIVKAMC.NI~QIDFADPSRA 1 0 1 StiKSTlVK~MKIIHEAGYSEE---------------ECKQYKAWYSNTIQSIIAIIR/\MGRI.KIDFGDSARn 101 SGKSTIVKQMKIIIiEDGYSEL)---------~------ECKQYKVVWSNTIQSIlAIIRAMGRLKIDFGEAAKR 101 SGKSTIVKQMKIIIIEIX;F~;GE---------------DVKQYKPVWSNTIQSLRAlVRAMDTLGVEYGDKERK 101 SGKSTIVKQMKIIHEDC;F!:GE---- - - - - - - - - - - DVKQYKPVWSNTIQSLAAIVRAMDTLGVEYGDKERK 101 S G K S T I V K Q M R I L H V N G F N G E G G E E D P Q ~ R S N S ~ E K A T K V Q D I K ~ L K ~ I E T I V ~ S N I ~ V P ~ V1E2L3A N P E ~~
~
~
~
S ~ ; K S T I V K Q M R I L H V N G F N P E - - - - - - - - - - - - - - - E K K Q K I L D I K K ~ D ~ ~ I I S ~ S T I I P P 111 VPL~PE SC.KSTFLKQMRIIHGREFL)VK---------------ALI.EFRDTIFDNILKGSRVLVDAKDKLGIPWQHSENE 123 S(:KSTFLKQMRIIHGQDFDOR---------------AREEFRPTIYSMlIKCMRVLVDAREKLHIPWGDNKNQ 1 1 6 SGKSTFIKQMRIlliGAGYSEE---------------ERKGFRPLWQNIFVSMRAMIF~EKLQIPFSRPESK110 SGKSTSlKCMRIIllGSGYSCE--------------3iii(GFTKI,VYVNIF?'AMVAMIRAMDTLRIQYMCEQNK 103 S G K S T F I K O M R ~ I H G S G Y S D E - - - - - - - - - - - - - - - D K R G F T K L W O N I F T A M O A M I R A M ~ T L K I P Y K107 YEH~ SGKSTFIK~MRI IHGAGYSEE--- - - --- - - - - - - -DKRGFTKLWQNIFTAMQAMVRAMETLKILYKYEQNK 107
***..
tt*
*
L. Birnbaumer and M . Birnbaumer
232 K-uz b-itrod
- - Y D A V O L F A L T G P A E - - - - S K - G E I T P E L L G V M R R L W A D D
167
m-go1 m-go2 h-gq-1 r-golf m-gl2 m-g13 t1- g 1 6 m-gl4 m-gq
--TDSKMVCDWSRME----DT-EPFSAELLSAMMRLWGDSGIQECFNRSREYQWDSAKYYLDSLDRIGAGD
167
--UDARKLMIIMADTIE-----E-G~PKEMSDlIQRLWDSGIQACFDRASEYQLNDSAGYYLSDLERLVTPti 1 6 2 r-gust --EDQQLLLSMI\N~LE------VGDMTPQLAPIlKRLWGDPGlQACFEW\SEYQLNDSAAYYLNDLDRLTAPG1 6 5 b-gtcone - - D N G R Q L I I N L A D S I E - - - - - E - G l ~ P P E L V ~ I R K L W ~ V Q A C F D ~ E Y Q ~ D S A S Y Y L ~ Q L D R I 1'6~6A i ' D --DDARQLFALSCTAE-----EQGVLPDDLSGVIRRLWADHGVaACFGRSREYQLNDSAAYYLNDLERIA~SD 1 6 7 h-qi2 h-oi 1 - -DDAR~LFVLAGAAE-- - - -E-GFMTAELAGVIKRLWKDSGVOACFNRSREYOLNDSAAYYLNDLDRIA~PN1 6 6 h-913 - -DDAHQLFVLAGSAE-- - - -E-GVMTPELAGVIKRI.WRDGGVQA~FSRSREYQLNDSASYY~NDLDHISQSN 1 6 6
- - T D S Y ~ C D W S R M E - - - - U T - E P F S A E L L S ~ R L W G D S G I Q E C F N R S R E Y Q L N D Y Y L D S L D R I G A G1 D 67 --NQFRVDYIl,SVMNV----PD-FDFPPEFYEHAKALWEDEGVRACYERSNEYQLID 189 --N3FRSDYIKSIAPI----TD-FEYSQEFFDHVKKI.WDDEG\CFERSNEYQLIDCAQYFLERIDSVICLVD 176 KHGMFLMAFENMtiLP----VE-PATFQLYVPALSALWRDSGIRWIFSRRSEFQLGESVKYFLDNLDRItiQLN 1 9 1 LHtiCKL.~FDTirAPMAnQGMVE-TRVFLQYLPAIRALWEDSGIQNAYDRRREFQLGEbVKYFLDNLDKLGVPD 188 - - H - - IIASLVPISQDPY- - - - KV -TTFEKRYAANIQWLWRDAGI RACY ERRREFHLLDSAWY LSHLER ITEEG 1 7 4 --E--NAQIIRNEVD----YV-TALSRDQVARIKQLWLDP~IQECYDRRREYQ~SDS~YYLTDIERI~PS 168
m-gil
--A--HAQLVREVDVE----KV-SAFENPYVDAIKSLWNDPGIQECYDRRREYQLSDSTYYLNDLDHVADPS 171 --A- -NALLIREVDVE--- -KV-TTFEHQ~~P.~AIKTLWSDPGVQECYDRRREFQLSDS~YYLTDVDRIA~G 1 71
r-gz b-atrod
YIPTVEDILRSRD~IVENKFTFKELTFKMVDVGGQRSERKKWIHCFEGVTAIIFCVELSAYDLKLYEDNQ 2 4 0 YVPTEODVLRSR~TTCIIETOFSFKDLNFRMFDVGGORSERKXWIHCFEGVTCIIFIAALSAYDMVLVEDDE 2 3 5
h-gi2 h-911 h-gl3 m-go1 ni-qo2 h-qs -1 r-golf m-gl2 m-ql3 h-g16 m-914 m- gq n-911
Y:PTQQDVLHTRVKTTGIVETllFTFKDLllFKMFDVGGQIIFCVALSAYDL\'LAEDEE
**
*
t
*
*
* *
IETQFSFKDLNFRMFDVGGPRSERKKWIHCFEGVTC
YVPNEQDVLHSRVKTEI I IFCAALSAYDMVLVEDEE 2 3 8 b-gtcone YLPNEGDVLRSRVKTTGIIETKFSV~DLNFRMFDV~QRSER~WIHCFEGVTCIIFCAALSAYDMVLVEDDE 2 3 9 r -;us t
~~
~~
240
YIPTQQDVLHTRVKTPGIVETHFTFKDLHFKMFDVGCQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAEDEE 239 YIPTQODVLRTRVKTTGIVETHFTFKDLYFKMFDVGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAEDEE 239 YQPTEQDILRTRVKTTGIVETHFTFKNLHFRLFDV~RSERKKWIHCFEDVTAIIFCVALSGYDQVLHEDET 2 4 0 YOPTEODILRTRVK'rrCIVETHFTFKNLHFRLFDVGG~RSERKKWIHCFEDVTAIIFCVALSGYDOVLHEDET 240 YVPSDQDLLRCRVLTSGIFETKFQVDKVNFHMFDVGGQRDERRKWIQCFNDVTAI IFVVASSSYNb IREDNQ 2 62 YTPTDQDLLHCRVLTSGIFETRFQVDKVNFHMFDVGGQRDERRKWIQCFNDVTAIIYVAhCSSYNMVIREDNN 249 YFPSKQDILLARKATKGIVEHDFVIKKIPFKMVDVGGQRSQRQ~QCFDGITSIL~SSSEYDQVLMEDRR 264 YIPSQQDILlARRPTKGIiIEYDFEIKNVPFKMVDVGGQRSERKRWFECFDSVTS1LFLVSSSEFM)VLMEDRQ 261 YVPTAQDVLRSRMPTPGINEYCFSVQKTNLRIVDVGGQKSERKKWIHCFEPNIALIYLASLSEYDQCLEENNQ 207
.
FVPTQQDVLRVRVPTTGIIEYPFDLENIIFRMVDVGMRSERRKWIHCFESVTSIIFLVALSEYDOVLAECDN
Y LPTQQDVLRVRVPTE I I EY PFDLQSV IFRMVDVGGQRSERRKW I HCFENVTS IMFLVALSEYDQVLVESDN YLPTQUDVLRVRVPTTCIIEYPFDLENIIFRMVDVGGQRSERRKWIHCFENVTSIMFLVALSEYDQVLVESDN
.
. t
t...
.
t
*.I)..
t.
240 244 244
1 ' S R M A E S L R L F D S I C N " W F I ~ S L I L F L N K K D L ~ E K I - - R R I P L T I C F P E ~ K G Q N - T Y E E ~ V - - - - -230 --VNRMHESLHLFNSICNIIRYFATTSIVLFLNKKDVFSEKI--KKAHLSICFPDI"GPN-TEDAGN-------- 2 9 7 V N R M H E S L H L F N S I C N H K Y F A T T S I V L F L N ~ D L F Q E ~ - - ~ V H L S I C F P E Y T G P N - T F E D A G N - - - - -300 --b-gtcone VNRMHESLHLFNSICNHKFFMTSIVLFLNKKDLFEEKI--KKVIILSICFPEYDGNN-SYEDAGN-------- 3 0 1 M N R M H E S M K L F D S I C " K W F T D T S I I L F L ~ K D L F E E K I - - T H S P L T I C F P E Y T G ~ - K Y D ~ S - - - - - - -302 11-912 r-gz
b-gtrod r-gust
h-911 h-gl3 m-go1 m-go2 11-95-1 r-golf m-gl2 m-g13 h-glG m-g14 m-gq m-911
r-gz
b-gtrod K-gUSt
b-gtcone h-gi2 h-gil h-gi3 m-go1 m-go2 h-gs-1 r-golf m-g12 m-g13 h-g16 m-914 m-qq
,,,-Gii h-gi2 m-sgi2
MNRMHESMKLFDSICNM(WFTDTSIILFLNKKDLFEEKI--KKSPLTICYPEYAGSN-TYE~-------- 3 0 1 MNRMIIESMKLFDSIC"XWFTETSIILFLNKKDLFEEKl--KRSPLTICYPEYTGSN-TEE~-------- 3 0 1 TNRMIIESIlILFDSIC"KFFIDTSIILFLNKKDLFGEKI--~SPLTICFPEYPGSN-TED~-------- 3 0 2 TNRMHESLKLFDSIC"XWFTDTSIILFLNKKDIFEEKI--KKSPLTICFPEYTGPS-AFTWIVA-------302 TNRL.QEALNLFKSIW"RWLRTISVILFLNKQDLLAEKVKSKIEDYFPEFARYT-TPEDATPEPG~DPRV 3 3 4 TNRLRESLDLFESIW"RWLRTISIILFLNKQDMLAEKVLAGKSK1EDYFPEYANYT-VPEDATPDAGEDPKV 321 TNRLVESMNIFETIVNM(LFFNVSIILFLNKMDLLVEKV--KSVSIKKHFPDFKGDPHRLEDVQR-------327 TNRLTESLNIFETIV"RVFSNVSIIl~FLNKTDLLEEKV--QWSIKDYFLEFEGDPHCLRDVQK-------- 3 2 4 ENRM(ESLALFGTILELPWFKSTSVILFLNKTDII.EEKI--PTSllLATYFPSFQGPKQDAEAAKR--------- 310 E N R M E E S K I \ L F R T I I T Y P W F L N S S V I L F L N K K D L L E E K I - - ~ S H L I S Y F P E Y ~ P K Q D ~ R D - - - - - -3-0-3 ENRMEESKI\LFRTIITYPWFQNSSVILFLNKKDLLEEKI--MYSHLVDYFP~Y~PURDAQAARE-------- 307 E ~ : R M E E S K A L F R T I I T P W F Q N S S V I L F L N K K D L L E D K I - - L H S H L V D Y F P E F D R E - - - - - - - - 307 * . t.... *
..
.
_.Y:QRQFEDLNIIM(ET-----------KE--IYSHFTCATDTSNIOFVFDAVTUVIIO"LKYIGLC --
355
._.._ Y IKVQFLELNMHRDV-- - - - - -----KE-- IYSHMTCATDTQNV~FVFDAVTDI I I~~ENLKDCGLF 350 _ _ - - YIKNQFLDLNLKKED-----------KE--IYSHMTCATDTQNVKFVFDAVTDIIIKENLKDCGLF 353
__.._ YIKSQFLDLNMRKDV-----------KE--IYSHMTCATDTQNVKFVFDAVTDIIIKENLKDCGLF 3 5 4 _._._ YIQSKFEDLNKRKDT-----------KE--IYTIIFTCATDTKQFFDAVTDVIIKNNLKDCGLF 3 5 5 __ Y I-QC. Q F E D L N K R K D T - - - - - - - - - - - K E - - I Y T H F T C A T I G L F 337 ...-_ YIQCQFEDLNRRKUT-----------KE--IYTHFTCATDTQFFDAVTDVIIK"LKECGLY 1 5 4 .__._ YIQTQFESKNRS-PN----------KE--IYCHMTCATDT"IQWFDAVTDIIIANNLRGCGLY 354 __ H I-QG Q. Y E S K N K S - A H - - - - - - - - - - - K E - - ~ S i l V T C A T ~ I Q F V F D A V T D V I I ~ N L R G C G L3 Y5 4
TRAKYFIRDEFLRISTASGU-----------GRHYCYPHFTCAVDTENIR~VFNDCRDIIQRMHLRQYELL 3 9 4 TRAKFFIRDLFLRISTATGD-----------GKHYCYPHFTCAVDTENIRRVFNDCRDIIQKMHLKQYELL 3 8 1 _ _ - - Y- L V Q C F D R K R R N R S - - - - - - - - - - - - K P - - L F H H F T T A I D T E N I R F V F I ~ ~ ~ I L Q ~ L K D I M3L7Q9 __.._ FLVECFRGKRRDQQQ-----------RP--LYHHFITAINTENIRLVFRD~DTILHDNLKQ~Q 377 __.-_ FILDmTRMY~CVDGPEGSKKGARSRR--LFSHYTCATDTQNIRKVFKDVH~S~RYLDEINLL 3 7 4 __ FILKLYQDQNPDK-E-----------KV--IYSHFTCATDTENIRFVFAAVK~ILQLNLREFNLV .-_ 355 ._.._ FILKMFVDLNPDS-D-----------KI--IYSHFTCATDTENIRFVF~~DTILOLNLKEYNLV 359 _ _ _ _ _ FILKMFVDLNPDS-D-----------KI--IYSHFTCATDTENIR~F~~~IL~LNLKEYNLV 359 t
*
t
t
t
t
_ _ _KE--IYTHFTCATDTKNVQFFDAVl!DVIIK"LKlXGLF 355 - - - -KE--IYTHFTCATDTKsrklfrstylklsgpdqhphpsFaFapFlssds~ 3 6 6
*
*
splice variant
233
G proteins in Signal Transduction
c. Phylogenetic tree:
2 a-i ' F a m i l y
12
rod
gust
L. Birnbaumer and M . Birnbaumer
234
Appendix I1 Sequence alignment and phylogenetic relationships for four mammalian and three non-mammalian G protein 0-subunits.
*, amino acids conserved in all sequences.
8'
a. Percent identity in alignment 8-1 100
p-2
p-3
p-4
90
83 80
89 88
100
100
I8
100
p-Ce
p-Dm
85 84 81 83 100
83 81
71
80 85 100
P-Lf
Subunit
86 84 78 82 88 84 100
-C. -D.
-L.
b. Alignment of sequences:
t
-3
-4
M13236 M16538 M31328 M87286 X17497 504083 X56757
-1
Number o f c o m p l e t e l y c o n s e r v e d s i t e s : 2 2 6
-2 -1
Accession #
elgegans melanogaster forbesi
M-SELEQLRQEAEQLRNQIRDARKACGDSTLTQITAGLDPVGRIQMRTRRTLRGHLAKIY~HWGTDSRL 69 M-SELDQLRQEAEQLKNQIRD~C~ATLSQI~IDPVGRIQMRTRRTLRGHLAKIYAMHWGTDSRL 69 M-GEMEQLRQEAEQLKKQIADARKACADVTLAELVSGLEWGRVQMRTRRTRRTLRGHLAKIY~HWATDSKL 69
. M-SELEQLRQEAEQLRNQIQDARKACNDATLVQITSNMDSVGRIQMRTRRTLRGHLAKIYAMHWGYDSRL
69
MTSELEALRQETEQLKNQIREARKAAADTTLAMATANVEPVGRIQMRTRRTLRGHLAKIYAMHWASDSRN 70 -hn M-NELDSLRQEAESLKNAIRDARKAACDTSLLQAATSLLQ~TSLEPIGRIQMRTRRTLRGHLAKIYAMH~~SRN 69 -Ce M-SELDQLRQEAEQLKSQIREARKSANDTTLATVASNLEPIGRIQMRTRRTLRGHLAKIYAMHWASDSRN 69 t *ff* *t* t .* . . . . . . . . . . . . . . . . . . . . **
-Lf
-2
-1
-3 -4
-Lf Rn
1I.e
E
-2 -1 -3 -4
-Lf
-hn -Ce
LVSASQDGKLIIWDSYTIT.IKVHAIPLRSSWVMTCAYAPSGNFVACGLDNICS1YSLKTREGNVRVSREL
139 139 LVSASQDGKLIVWDSY'ITNKVHAIPLRSSWVMTCAYAPSGNFVACGLD~CSIYNLKSREG~SR~L 139 LVSASQDGKLIIWDSYTPNKMHAIPLRSSWVMTCAYAPSG~ACGGLDNICSIMLKTREGD~VSREL1 3 9 LVSASQDGKLIVWDGYrPNKVHAIPLRSSWVMTCAYAPSGNWACGLDNICSIYSLKTREGNVRVSREL 1 4 0 LVSASQDGKLIVWDSHTTNKVHAIPLRSSWVMTCAYAPSGSWACGLDNMCSIMLKTREGNVRVSREL 1 3 9 LVSASQDGKLIVWDSYTHAIPLRSSWVMTCAYAPSGSFVACGGLDNICSIYSLKTREGNVRVSREL 1 3 9
LVSASQDGKLIIWDSY?TNKVHAIPLRSSWCAYAPSGNYVACGGLDNICSIYNLKTREGNVRVSREL
.**********
****
tt
********
**tt.tt***tt*.*tt.t
ttt*
t .
*+*
t
tttlt
P G H T G Y L S C C R F L D D N Q I I T S S G ~ A L W D I E T G Q Q ~ G F A G H S G D ~ S L S L A P N G R T ~ S G A C D A S I K2 0 9 AGHTGYLSCCRFLDDNQIVTSSGDTTCALWDIETGQQ~FTGHTGD~LSLAPDTRLFVSGACDASAK2 0 9 SRHMYLSCCRFLDDNNIVTSSGDTPCALWDIETGQQKTVFVGHTGDCMSLAVSPDFNLFISGACDASAK 2 0 9 AGHTGYLSCCRFLDDGQIITSSG~ALWDIETGQQTlTFTGHSGDVMSLSLSPDLK~SGACDASSK 2 0 9 PGHTGYLSCCRFIDDNQIVTSSGDMTCALWNIETGNQITSFGGH~D~SLSLAPDMRT~SGACDASAK2 1 0 P G H G Y L S C C R F L D D N Q I V T S S G D M S C G L W D I E T G ~ V T S F ~ H T G D ~ S L A P Q C K T ~ S G A C D A S209 AK PGHTGYLSCCRFLDDNQIVTSSGD~CALWDIETGQQCTAFTGHTGDVMSLSLSLSPDFRTFISGACDASAK 2 0 9
.
******** **
t
lltttt
**
*ttt
7
* +* ** *
**+****
t
LWDVRDSMCRQTFIGHESDINAVAFFPNGYAFTTGSDDATCRLFDLRADQELLmSHDNIICGITSVAFS 2 7 9 -3
-Lf
-I)m -Ce
1:
-2 -1
E
-Lf -Dm -Ce
L W D V R E G M C R Q T F T G H E S D I N A I C F F P N G N A F A T G S D D A T C R L F D L ~ ~ E ~ ~ S H D N I I C G I T S V S F S2 7 9 LWDVREGTCRQTFTGHESDINAICFFPNG~ICTGSDDASCRLFDL~DQELICFSHESIICGITSVAFS 2 7 9 LWDIRDGMCRQSFTGHISDINAVSFFPSGYAFATGSDDATCRLFDL~~ELLLYSHDNIICGITSVAFS2 7 9 LFDIRDGICKQTFTGHESDINAITYFPNGFAFATGSDDATCRLFDI~DQEIG~SHDNIICGITSVAFS2 8 0 L W D I R E G V C K Q T F P G H E S D I N A V T F F P N G Q A F A T G S D D A ~ R L F D I ~ ~ E ~ S H D N I I C G I T S V A F 2S 7 9 LWD1RM;MCKQTFPGHESDINAVAFFPSGNRFATGSDDATCRLFDI~~E~SHDNIICGITSVAFS 279
**
t
*+
*tt*l
*tt***
t*t*t
*****
**
***+**.*
RSGRLLLAGYDDFNCNIWDAMKGDRAGVLAGHDNRVSCLGVTDOGMAVATGSWDSF KSGRLLLAGYDDFNCNVWDALKADRAGVLAGHDNRVSCLG
340 340 LSGRLLFAGYDDFNCNVWDSMKSERVGILSGHDNRVGILSGHDNRVSCLG~ADG~VA~SWDSFLKIWN 340 KSGRLLLAGYDDFNCSVWDALKGGRSGVLAGHDNRVSC~VTDDG~VATGSWDSFLRIWN3 4 0 KSGRLLLGGYDDFNCNVWDVLKQERAGVLAGHDNRVSCUJSWDSFLKIWN 3 4 1 K S G R L L L A G Y D D F N C N V W D E R S G I L A G H D N R V S C L C 340 KSGRLLFAGYDDFNCNVWDSMRQERAGVLAGHDNRVSCLGSWDSFLKIWN 340
.****
****tt*
**
*
*
**ttttt*****
*ttt
********
t*
** 1
0 2 3 6 5 4
235
G proteins in Signal Transduction
c. Phylogenetic tree
I
P forbesi P-3
C.
I
P D. melanogaster
e le ga ns
L. Birnbaumer and M . Birnbaumer
236
Appendix 111 Sequence alignment and phylogenetic relationships mammalian G protein @-subunits
*, amino acids conserved in all sequences. a. Percent identity in alignment: 0
1
2
3
4
100
51
78
37
60 50
y-3
100
7-7
100
51 100
28 37
y-5 y-2 (also y-G) y-1 ( a l s o 7-T)
70
40
100
Accession I
Subunit
M37182
-
M99393
Number of c o m p l e t e l y c o n s e r v e d s i t e s : 1 5
b. Alignment of sequences: 7-7 7-3
M---SATN--------NIAQARKLVEQLRIEAGIERIKVS~SSELMSYCEQ~RNDPLLVGVPASENPF 59 MKGETPVNS~I-----SIGQARKMVEQLKIEASLCRIKVSKAAADLMTYCD~HACEDPLITPVPTSENPF 65 M - - - - A S ~ A - - - - - S I A Q A R K L V E Q L K M E A N I D R I K V S ~ D ~ Y C ~ E D P L L T P V P A S E N P F61 H - - - P V I N - - - I E D L T E K D K L K M E V D Q L K K E V T L E R M L V S P F 64 M - - - - S G S S - - - - - - - S V ~ K W Q Q L R L E A G L N R ~ V S Q ~ D L K Q F C L Q N A Q H D P L L ~ V S S S ~ P F 59
7-7 y-3
KD-KKPCIIL REKKFFCALL REKKFFCAIL K-LKGGCVIS RPQK-VCSFL
y-2 Y-1 7-5
+
y-2
y-1 7-5
+
*
+
t
t.
t
tt
***
*.*
68
75 71 73
68
c. Phylogenetic tree:
I 7-7
I
7-5
I Y-3
I
......
tsh lh
tsh lh
tsh lh
tsh lh
tsh lh
tsh lh
NLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNFTSVQGYAFN 198 N L L N L S E I L I Q ~ N L L Y I E P G A F ~ L P R L K Y L S I C N T G I R T L P D V S K I S S S E F N F I L E I C D N L Y I ~ I P G N A F Q G ~ E S I T L K L Y G N G F E E V Q S H A1F9N9 f * * * t * t*t ** * * " * * * * t * t + f * * t t * * t * * * * + **. ** **f ** * * .* *. .+
tsh lh
..
.......
..
1 1 . l l l l . . l . ~ . . . . l . . .
"
,**
f
..
..-.....,11-.....11
hum-tsh:
I..l...l.l,V...l
.....
**
.....
***
I . t l
***.******
***
. 1 . , 1 . l r . . I V I 1 = 1 = . . .
t+**t*
= . 1 . 1 , . . v 1 . . = . 1 , , .
t*
**t*
.t*
1 1 1 1 1 . 1 . 1 1
+ * * **+*. + * * * * + . .*****
D - - - Y D Y D F C - - - S P K T L Q C T P E P D A F N P C E D I M G Y A F L R V L I W L I N I L A I F L Y L L L I A
. I
.
** ****
****+***
*.
.f
t
*
tt
**
**+**.*
* * *+**.*
***tt**
**
I.*+**
. . t
* * * * + * * ,*
*****.+.*
t+*
tt
1111
CANPFLYAVFTXAFQRDFFLLLSRFGCCKHRAELYRRKEFSA----------CTFNS~GFPRSSKPSQ~LKLSIVHCQQPTPPRVLIQB
.CANPFLYAIFTKAFQRDVF;LLSKFGICKRQAQAYRGQR;PPKNSTDIQ;QKVTHDMRQGLHNMEDWELIENSHLTPKKQGQISEEYMQTVLB
.VII=,.-.~.
t
7 64 700
671 MDVESTLSQ~ILSILLLNAV~ICACWRIYFAVQNPELTAPNKDTKIAKXMAILIFTDFTCMAPISFFAISAAFKVPLITVTNSKVZLVLFYPVNS6 2 0
472 421
M31774
m u r - ~ ~ ~: 8 1 3 1 0
D---SHYDYTICGDSEDMVCTPKSDEFNPCEDIMGYKFLRIVVWNSLLALLGNVFVLLILLTSHYKLNVPRFLMCNLRFADFCMGMYLLLIA
*
.MDTETPLALAYIVFVLTLN;VAFVIVCCCHVKIYITVRNPQYNPGDKDTKIAKRVLIFTDFICMAPISFYALSAILNI;PLITVSNSK;LLVLFYPLNS
**
I=
381 334
* *
LMCNESSMQSLRQRKSVNALNSPLHQEYEENLGDSIVGYKEKSKFQDTH~AHY~FFEEQEDEIIGFGQELKNPQEETLQAF 380 SIFENFSKQC-------------------------------ESTL;REA"ETLYSAIFEENE-----------------LS~W 333
.
M G R R V P ~ L R ; L L V L k Y L V ' K ~ S Q L - H S P E L S G S R C ? E ? ~ D C A P ~ A L R C ? G ? ~ G ~ F L S L ~ L ? ~ ~ ~ P S C A F R G L N E ~ I E : S Q S 3 S L E R I E X ~9A3F C * * . . *
tsh lh
..
M--RPADLLQLV-LLCLPRDLGGMr,CSSPPCECHQE~DFR~~CKDIQR~PSLP?STQ~LKLIETHLRT:PS~FSNLPNISPI~SID~LQQtEjriSrY 98
a. TSH receptor vs. LH receptor. Number of identities in transmembrane core: 224
Phylogenetic tree is based on central core sequences and excluded central regions of the third intracellular loop, the N-termini and the C-termini. Completely conserved amino acids are highlighted by asterisks.
Appendix IV Sequence alignments and phylogenetic tree of G protein coupled receptors with DRY motif and comparison of P-adrenergic receptor (with DRY motif) to GRF receptor (lacking DRY motif)
Y
s
ri
2n
z?
0' i2 -. 3 M -.
2) %
......
..
*It.*.
tt*
I)*
PFIYAFRSPELRDAFKKMIFCSRYWQ PLIYAFRSQELRMTLKEVLLCS--WQ 297 315
acth msh X65633 X65635
human ACTW mouse MSH
. ....
334
.
.
. I
tl
..
t
** MLMASTTSAVPGHPSLPS-LPSNSSQERPLDTRDPLIJ\RRELALLS-IVRTAVALS-NGLVLAALARRGRRGHWAPIHV---FIGHLC~DLAV tt *.* *. t * * * tt.
**
88
11-11.. ..I...--. 11.11111l..IIllt . . M G Q P G N G S A F L L A P - N R S ~ P D H D V T Q Q R D E V W W G M G I V 82 D - - - Y D Y D F C - - - - S P K T - L Q C T P E P D A F N P C E D I M G Y A F L R V L I L W I N I L F C M 412
a- thr a-thr
acth
rho
alpha2
t
.. .
, . t
+
t
***
t
t
**
tt
.*
* * * *
** **
' R D P R R L L L V M C F S L C C P L ; S ~ T - P ~ S ~ T ~ * L D ~ ~ L SFLLR~PNLXYEPFW~DEE6 0 61 -KNESGLTEYRLVSINKSSPLQKQLPAFISEDASGYLTSSWLTLFVP-S-VYTGVFWS-LPLNIMAIVVFILKM--KVKXPAVVYMLHWTMVLF
t
t
86
82
151
M K H I I N S Y E N I N P S r A R N - - - N S D C P R V V L P E E I F ~ I S I V G ~ E - ~ I V L ~ V F ~ L Q - - A P ~ F - - - F I C S ~ I S7D3 ~
t
*** .* .** MGSLQPDAGNASWNGT-EAPGGGARATPYSLQVTLT--LVCLAG-LLMLLTVFG-NVLVIIAVFTSRALK--APQNL---FLVSWSMILV t * * * *t * * * **.** MNGTEGPNFYVPFS-NKTGWRSPFEAPQWLAEPWQFSM-FLLIMLGFPINFLTLWTVQHKKLR--TPLNY---ILL~V~LFM t. * * * * * II t * *
94 alpha1 MNPDLD~HlNTSAPAQWGELKD~~PNQTSSNSTLPQLD~~ISVGLVLGAFILFAIV-G-NILVILSVACNRHLR--TPTNY---FIVNWIMLLL
vP2
beta2 lh
c. Beta-2 Adrenoceptor vs. LH,type-2 Vasopressin, Alpha-I Adrenergic, Alpha-2 Adrenergic, Light (rho), ACTH and a-Thrombin Receptors. Number of idenitical rfesidues are listed at the end of the sequences.
acth msh
is
9
f
B
5
$
'LFPLMLVFILCLWHMFLLSHTRKISTLPRANM----~---K
I.....III..V.... . . ~ ~ . . l l . . l I v . l . l . . l . . . . . ...IIV......I.I. acth 272 FFLAMLALMAILYAHMFT~CQ~~~AQLHKRRRSIRQGFCLKGAATLTILLGIFFLCWGPFFLHLLLIVLCPQHPTCSCIFKNFNLFLLLIVLSSTVD 292 msh ** .* .** ..t *t.tttt t * * *. t t * * *. t t t 1
mnh
a * * *
180 192
-.llI..lllll~II..l-lII.
a ct h
.
MKHIINSYENINhPTARN--NSDCPRVVLPEEIFFTISIVGVLENLIVLLAVFK"LQAP~FFICSWISDMLGSLYKILE 80 ~L~TQEPQXSLLGSLNSNATSHLGL-ATNQSEPWCLWSIPDGLFLSLGLVSLVENVLWIAITKNRNLHSPMYYFICC~SDLMVSVSIVLE92 .* tt ** tf. tt tt ..t * * * * *
acth msh
...."""'Illllt."'111
b. ACTH Receptor vs. MSH Receptor. Number of identities in transmembrane core: 115
S F T V L P F S - A T L O J L G Y W - - - - - - - V L G R I F C D I W ~ ~ ~ C C T A S I L S - - L C A I S I D R Y I G ~ Y S L Q Y P T L ~ R R ~ I ~ L S ~ L S T V I S I G P L183 -LG
506
alpha1
.
llll.lllllIvllllllll~
A L F Q V L P Q - L A W K A T D R F - - - - - - - R G P D A L C R A V K Y L Q M S L L L S L P Q
..
111111
vp2
11111111=III1111
GLYLLLIA-SVDSQTKGQYYNHAIDWQTGSGCSAAGFFTVFASELSWT--LTVITLERWHTITYAVQLDQKLRLRI(AIPIMLGGWIFSTLMATLPL--* * ***. ** t tt ** *
1111111.1 1.1111
...
..
. .
t
*.
t1.
.
f
t
t
t
.
* *
t
~-
206
209
210
262
259
238
3. 0 239 569
a-thr
292
3
F
$
Y
Q
240
240
**
**
209
.*.
. t
acth
t
.*
rho
ttt
.tt
3 62
t
.
t
alpha2
tt
f t
272
. ..
tt
alphal
.
t
252
t.
.t*
vp2
t
.*
252 569
.It
tt
beta2 lh
alpha2
S L Y X I L E N - I L I I L R N M G Y L K - P R G S F E T T A D D I I D S L F V L S L L G S I F S - - L S V I ~ R Y I T I F H A L R Y H S I ~ R T V V V L ~ I W T F C ~ ~ I ~ 169 IFS
t
174
VFGGfiTr-LYTSLHGYF-------VFGPTGCNLEGFFATLGGEIALWS--LVVLAIER~CXPMSNFRFGENHAIMGVAF?WVMALACAAPPLVGWS
tt
* *
rho
.tt*
t
acth
**..
It
169
.
**
176
.. ... .
t
ATLVIPFS-WJEVMGYW-------YFGKTWCEIYLALDVLFCTSSIVH--LCAISLDR~SI~AIE~KRTPRRI~III~~ISAVISFPPL---
*
G L A W P F G - A A H I ~ - - - - - - - T F G N F W C E F W T S I D V L C V T A S I E T - - L C V I A ~ R ~ A I T S P F K Y Q S L L ~ V I I ~ I V S G L T S F L P I Q ~1 7 2
beta2 lh
+**
.
t
ttt
t
f f *
t
f
*
I
**
tt
**
tt*
*t
**
t*
***
tt
t
640
a-thr
acth
rho
alpha2
alphal
vP2
beta2 lh
a- thr
*.
*
*
tt
+* r
+**
*
.
*
***
*t
*+
****
t,
t
* *
* *
*
'It
*
+t
t*
**
** **
**
**
"
***
*t
**
*
*
****
IIRCLSSSAVAN-RSKKS~LFLSAAVFC-IFIICFGPTNVLLIAHYSFLSHTSTTEAAYFAYLLCVCVSSISSCIDPLIY-YYASSECQR~SILCC-
*
*
388
313
443
- - _ _L- P R A N M - - - - - - - K G A I T L T I L L G - V F I F ~ A P ~ H ~ L ~ F C P S N P Y - C A C ~ S L F Q ~ G M L I M C N A V I D P F I Y A F R S P E L R D A F K - - - - - - - 288
* *
ATTQ-----------KAEKGVTRMVIIMVIAFLICWLPYAGV~YIFTHQG----SDFGPI~TIP~F~TSA~NPVIYI~QF------------
t
acth
+
rho
+
AGGQNLEKRFTF-VLAEKRFT~LAWIG-V~~PFFF~TLTAVGCSV----PRT--LFKFFFWFGYCNSSLNPVIYTIFNHDFRRAFKKILCR--
alpha2
alphal ' H N P R S S I A V K L F K F*S*R E+ *K ~t C * ;AKT* L G*.*****. IWGI~IL~PFFIALPLGSLFSTL----KPPDA'JF~L~YFNSCLNPI;YPCSSKE~K~~RILGCQC t * + ** ** ** * 358
f
vP2
III--l--yrI-.-IIIIIII
- - _ _ - - - - - _ _ _ _ _K_I_A_K_ K M A I L I F - T D F T C M A P I S F F A I S A A F K V P L - - - - I T F Q R D F F L - -
-m--lJI-..-.--------
R T G - S P G E G A H V - S A A V A K T V R M T L V I W - ~ L ~ A P F F L V Q L W ~ W D P ~ - - - - P L E G A P ~ L M L L A S L N S C ~ P W I Y - - - A S F S S S V S S E L R S - - - - 338
1 . 1 1 1 -
R T G H G L R R S S K F - C L K E H K A L K T L G I I M G - T F ~ ~ L P F F I ~ I ~ I Q D N L - - - - I R - K ~ I L L ~ I G ~ S G F N P L I Y - C R S P D F R I ~ Q E L L 342 CL--
beta2 lh
*
371
413 700
alpha2
alphal
vp2
beta2 lh
Abbr.
M18415
504084
211687
Accession # 502960 M81310
348 297 425
---RNCMVT-TLCCGXNPLGDDEAST'I'VS-----KTETSQVAPA@
_ _ _ _ _ _ _ _ _ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _KMIFCSRYWO ____
XESSDPSSYN---SSGQLMASKMDTCSSNLN----NSIYXXLLT@
rho
acth
a-thr
a-thr
acth
rho
M62424
X65633
M12689
'*.*..+~~***."..'+.
human a-thrombin
human ACTH
bovine rhodopsin
67
61
50
.+.
...........
.rt+r+++rIII""'.'.'+
..
...........
+***++.+++~~'.'.**'."
beta2
beta2
t
t
*ttt*
..'....*+~~**..."..
.........
284
340
LPGTEDFVGHQGTVPSDNIDSQGR"DSLL@
.. 413
Accession # ' s : h-grfR: L09237; beta2-AR: J02960
L F L* I* P* L F G I H Y I I F N F* L P D N A G* *L G I *R* L P L E L G L G S F Q - G F I V A I L Y C F L N Q E C ~ 423 . . I LCWLPFF-IVNIV-HVIQDNLIRXEWILLNWIGYVNSGFNPLI-YCRSPDFRIRFQELLCLRRSSLKAYGNGYSSNG~GEQSGY~~~QEXE~LLCED 380 .vI.... ...ll...l..IIIV.........
.*.r~..*...*...**'
...l....l...V...l......
t
CYANETCCDFFTNQAYAIASSIVSFWPLVIMVFVYSRVFQWRQLQXIDXSEGRFHVQNLSQVEQDGRTGHGLRRSSXFCLXEHKALKTLGIIMGTFT
...........
beta2
**..~...**~".*""..r
f
S T V L C X V S V A A S H F A T M T N F S ~ ~ ~ L N C L ~ S T S P - - - - - - - - - - - - - - - - - - - - - - - - S S R R A F W W L V L A G W G L P V L F T G T W V S C K F E D -272 IAC * ** . * t+ t * _._ AHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINC ____ 184 ....l...VI
_ _ _ _ _ _ _ _ _ WDLDDTSPYWWIIKGPIVLSVGV"IIR~LVRK--------------------------------LEPAQGSLHTQSQYWRLSKST
h-grf
197
M G Q P G N G S A F L L A P N R S H A P D H D ~ Q Q R D ~ G M G I ~ S W I V L A I ~ G ~ L V I T A I ~ F E R L Q ~ F I T S ~ C ~ L ~ G ~ W P F 9G1A - - ~ - - - - -
.........................
f 'WSEPFPPYP;ACPVPLELLAEEESYFSTVKIIYTVGHS~SIVALFVAIT;LV~RRLHCI--P~NYVHTQLFTTFILKAGAVFLXDAAL;.H~DDTDHCSF ** ** * I
h-grf
beta2
h-grf
beta2
h-grf
109
127
70
Homology to beta2 100 70
.MDRRMWGAH;FCVLSPLPT;LGHMHPECD;.ITQLREDESACLQAAEEMP-LG~PA~LL-TAG~G~LP~~~=F~HF~SESGAVKRDCTITG 100
.......................
d. GRF Receptor vs. Beta-2 Adrenoceptor. Number of identities in transmembrane core: 43
* *
450
h-grf
human a2.C10AR
hamster a ,,-
human type-2 AVP
Receptor human p2AR murine LH/CG
_..__________________________________
LFTFKLLGEPESPGTEGDASNGGCDATTDLANGQPGFKSNMPLAPGHFB 515 ***+ t t ***
t
_ _ _ _ _ LLCC-ARGRTPPSLGPQDESCTTASSSLAK-----DTSS@ *."
---NXLLCE-DLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLLGl ---CTFNSK-NGFPRSSKPSQAALXLSIVHCQQPT--PPRVLIQ@
GDRKRIV@
alpha2
alphal
vp2
beta2 lh
3
-.
Q
242
e. Phylogenetic tree based on transmembrane core
L. Birnbaumer and M . Birnbaumer
G proteins in Signal Transduction
243
Appendix V Sequence alignment and phylogenetic tree of conserved core regions of receptors belonging to the glucagon receptor family Completely conserved amino acids are highlighted by asterisks. a. Percent homology in alignment (from CW motif to end): 0
1
0
1
2
3
4
5
6
7
8
9
10
100
30 100
31 25 100
34 30 35 100
30 25 46 38 100
28 27 34 37 38 100
30 25 37 37 42 47
30 24 34 34
30 27 44 37 52 38 39 39 100
34 27 43 42 50 42 42 40 49 100
30 23 42 39 49 36 36 38 52 49 100
2 3 4 5 6 7 8 9 10
100
41
50 49 100
Receptor human-CRF human Calcitonin human GRF human PTH/PTH-rp human-VIP rat-GIP rat -GLP-1 rat-Glucagon rat PACAP rat Secretin rat-VIP
Accession I L23332 LO0587 LO9237 LO4308 X75299 L19660 544970 LO4796 223722 X59132 225885
Number of completely conserved sites: 40 Abbreviations: CRF. corticotropin releasing factor; GRF, growth hormone releasing factor; PTH, parathyroid hormone; PTH-rp, PTH-related peptide: VIP, vasopactive peptide; GIP, gastric inhibitory peptide/glucose-dependent insulinotropic peptide; GLP-1, glucagonrelated peptide-1; PACAP, pituitary adenylate cyclase activating peptide.
b. Alignment of sequences: h-ct h-pth r-secr
MQFSGEKISGQRDLQKSKMRFTFTSRCL
28
MGTARIAPGLALLLCCPVLSSAYALVDADDVMTKEEQIFLLHRAQ 45 MLST 4
h-crf h-ct h-pth r-gip r-gluc r-glpl r-secr r-vip h-vip r-pacap h-grf h-crf h-ct h-pth r-gw r-gluc
+
58 97 115 4 MVPS-
MGGHPQLRLVKALLLLGLNPVSASLQDQHCESTS-GESLSLASNISGLQCNASVDLIG
ALFLLLNHPTPILPAFSNQTYPTIEPKPFLYWGRKKMMDAQYKCYD-RMQQLPAYQGEGPYCNR~WAQCEKRLKEVLQRPASIMESDKGWTSASTSGKPRKDKASGKLYPESEEDKEAPTGSHI-
MAVTPSLLRLAELLLGAVGRAGPRPQGATVSLSE~VQKWREYRHQCQRFLTEAPLLATLGFCNRTFDDY-
66 MLLTOLHCPYLLLLLWLSCLPK-APSAOVMDFLFEKWKLYSDO-CHHNLSLLPPPTELVCNRTFDKY69 E I I I P R L S L L L L R L L L L T H T V G V P P R L C D V R R V L L E E ~ C L Q Q L S K E K K G A L G P ~ ~ G C E G L ~73 ~58 MRASWLTCYCWLLVRVSSIHPECRFHLEIQEEETKCAELLNI~~
MRPPSPLPARWLCVW\GALAWALGPAGGQRARLQEECDWQMIEVQHKQCLEEAQLENETIGCS~NLMARVLQLSLTALLLPVAIAMHSDCIFKKEQAMCLERIQRANIMDRRMWGAHVFCVLSPLPTVLX;HMHPECDFITQLREDESACLQRAEMPKLGCPA?WDGL-
70 60
62
102 TCWPRSP---AGQLWRPCPAFFYGVRYN-'IT------ --------------NNGYRECLANCSWRRR 139 L~DTP---AGVL.SYQFCPDYFPD--FD-PS----------------------EKVTKYCDEKDVWFKH 157 LCWPLGA---PGEWAVPCPDYIYD--FN-HK----------------------GILAYRRCDRNCSWELV ICMPAG~RLPTPLPGVSCPWYLPWYRQV-AA----------------------GFVFRQCGSDGQWG--
49
109 SCWPDTP---P~ANISCP~LPWHKV-QH-----------------------RLVFKRCGPDGQWV-R 113 ACWPDGP---PGS~INVSCPWYLPWASSV-LO----------------------G~RFCTAECIWLHK 111 r-secr SCWPSSA---PARTVEVQCPKFL-LMLSN-KN----------------------GSLFRNCTQDC----96 r-vlp TCWRPAD---IGETVTVPCPKVF-SNFYS-RP----------------------GNISKNCTSDC----h-vlp ' ~ P A T P - - - R G Q V W W \ C P L F - K L F S S I - Q G - - - - - - - - - - - - - - - - - - - - - - R N V S R S C T D E C - - - - 109 r-pacap T C W K P A Q - - - V G E M V L V S C P ~ F - R I F N P ~ ~ ~ I G D S G F A D S N ~ L E I T D M G W G R N C T E ~ -121 ---100 h-grf LCWPTAG---SGEWVTLPCPDFF-SHFSS-FS---------------------GAVI(RDCTITD-----r-alol - = .
. .
f .
. . l . l l . l l l . I . l l l l l l l . l l l l
153 _ _ _VNYSECG-EI-LNEEKKS-K--V-IIYHVAVIINYLGHCISLVALLVAFVLFI.RLR------_...PENNRT-WSNYTMCN-AF-TPEKLKN-A--Y-VLYYW\I---VGHSLSIFTLVISLGIFVFFRKLTTIFP 199 213 PGHNRT-WANYSECV-KF-LTNETRE-R--E-VFDRLGMIY~GYSVS~SLTVAVLrW\YFR------95 - - - - -S - W R D H T Q C E - N P E K N G A F Q D Q K - - L - I L E R L Q W Y T V G Y S L L I L S L F R - - - - - - 168 r-gluc G P R G Q S - W R D A S Q C V M D D D E I E V ( ) K G V A - - K - M Y S S Y P V L L G L R - - - - - - r-glpl DNSSLP-WRCLSECE-ES-KQGERNSPE--E-CLLSLYIIYTVGYAL3FSAL'JIASA:LVSFR------- 170 r-secr WSETFP-HPDW\CGV-NI-NNSFNERRH--A-YLLKLI(VmTVGYSSSLLLVALSILCSFR------- 168 151 r-vip W S E T F P - D F I D A C G Y - N D - P E D E S K - - I - - T - F Y I L V K A I Y T L G Y S V S L S L ~ S I I I C L F R - - - - - - 168 h-vlp W?'HLEPGPYPIACGL-UD-~SLD--EQQ?MFYGSVLSLFR------r-DacaD W S E P F P - H Y F D A C G F - D U - Y E P E S G - - ~ D Y - Y Y I , S ~ L Y T V G Y S T S L A T L ~ ~ I L C R F R - - - - - - - 178 155 h-grf WSEPFP-PYPVACPV-PL-ELLAEE--EI-S-YFSTVKIIYTVGHSISIVAL~AITILVALR-------
,,.crf
h-ct h-pth r-glp
.
*
*
t
L. Birnbaumer and M . Birnbaumer
244 h-cr€ h-ct h-pth r-gip r-gluc r-glpl r-secr r-vip h-viu
-T - "r, a-r-d-" r
h-grf
.II..IIIIIIIIIIIII..
- - - - - - - - - SIRCLRNIIHWNLISAFILRNATWFWQLTMSPEVH-------------------------
187 LNWKYRKALSLGCQRVTLHKNMFLTYILNSMIIIIHLVEWPNGEL-----------------------V 246 - - - - - - - - - RLHCTRNYIHMHLFLSFMLRAVSIFVKDAVLYSGATLDEAERLTEEELRAIAQAPPPPATA274 148 - - - - - - - - - RLHCTRNYIHMNLFTSFMLRAILTRDQLLPPLGPYTG"PT---L------------W - - - - - - - - KLHCTRNYIHGNLFASFVLKAGSVLVIDWLLKTRYSQKIGDDLSVS~L-----------S 218 - - - - - - - - HLHCTRNYIHLNLFASFILRLSVFIKDAALKWMYS-TAAQQHQWDGLL-----------S 219 208 - - - - -- -- -R L H C T R N Y I H M H L F V S F I L R L S N F I K D A V L F S - - - - - - - - - - S D D ~ - - - - - - - - - - - C - - - - - - KI . H C.T R N Y I H L N L F L S F M L R A I S V L Y S - - - - - 194 -. ..- - - - K L t l C T K N Y I H M t I L ~ I S F l L ~ V F I K D L A L F D - - - - - - - - - - S G E S U Q - - - - - - - - - - - C 208 -_ .-.- - - KLHCTRI~FItlMNLFVSFLRAISVFIKDWILYA----------EQDSStl-----------C 218 __._ R~HCPRNYVHTQLFTTFILKAGAVFLKDAALFH_____ - ----- - - -SDDTDH--- - - -- - -- -C 195 ~~~
h-crf h-ct h-pth r-gip r-gluc r-glpl r-secr r-vip h-vip r-pacap h-grf
1.11IIIIII1.IIIIIII111.. IIl1I1II..IIVIIlIIlI. QSNVGWCRLVTAAYNYFHVF~FGEGCYLHTAIVLTYFICIGWGVPFPIIVAWAIGKL 257 RRDPVSCKILHFFHQYMMACNYFWMLCEGIYLHTLIWAVFTEKQRLR~YLLGWGFPLVP~IHAIT~ 316 A A G Y A G C R V A V T F F L Y F L A T Y W I L V E G L Y L H S L I F M R F 349 NQALAACRTAQILTQYCVGRNYTWLLVEGWLHHLLWVRRSEKGHFRCYLLLGIPWVIVRY 218 DGAVAGCRVATVIMQYGIIRNYCWLLVEGWLYSLLSITTFSEKSFFSLYLCIGWGSPLLFllIPWVWKC 288 YODSLGCRLVFLLMOYCVAANYYWLLVEGWLYTLLAFSVFSEQRIFKLYLSIGWGVPLLFVIPWGIVKY 289 278 263 278 288 SFSTVLCKVSVAASHFATMTFSWLLAEAVYLNCLLASTSPSSR~~LVLAGWGLPVLF~~SCKL 265 * * * ** ***
h-crf h-ct h-pth r-gip r-gluc r-glpl r-secr r-vip h-vip r-pacap h-grf
YYDNEK---CWFGKRP-GWTDYIYQGPMILVLLINFIFLFNIVRIL~KL~STT--SETIQY------ 315 WFNDN---CW-LSV--ETHLLYIIHGPVMAALVVNFFFFLLNIVRVLVT~RETHE--AESH~------372 TLANTG---CW-DLS--SGNKWIIQVPILASIVLNFILFA-GRCDTRQQY---403 LYENTQ---CWERNE--VKAIWWIIRTPILITILITILINFLIFIRILGILVSKLRTRQM--RCPDYR------ 275 LFENVQ---CWTSND--NMGFWWILRIPVLLAILINFFIFVRIIHLLVAKL~HQM--HYADYK-----345 LYEDEG---CWTKNS--NIIRLPILFAIGVNFLVF--CKTDIK-----346 FLENTG---CWDINA--NASVWWVIRGPVILSILINFIFFINILRILNRKLRTQETRGSETNHY------ 337 SLEM%---CWDTND--HSIPWWVIRMPILISI~FALFISIVRILLQKLTSPDVGGNDQSQY-----322 HFEDYGLLRCWDTINSSLWWIIKGPILTSILFICIIRILLQKLRPPDIRKSDS---SPY-----339 YFDDAG---CWDMND--STALWWVIKGPWGSIMVNFVLFIGIIIILVQKLQSPDMGGNESSIYLTNLRL 353 AFEDIA---CWDLDD--TSPYWWIIKGPIVLSVGVNFGLFLNIIRILVRKLEPAQGSLHTQSQY------ 328
11...IIIIIVIII.II.I1111
**
**
IIIII.l.I.vIII-..
________________________________________---------h-crf RKAVKATLVLLPLLQITYML 335 ________________________________________---------h-ct LKAVKATMILVPLLGIQFVJ 392 __-----__---_---_--_____________________---------h-pth RKLLKSTLVLNPLFGVHYIV 423 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ____---------r-gip LRLARSTLTLMPLLGVHEVV 295 ____---------r-gluc _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _FRLARSTLTLIPLLGVHEW 365 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ____---------r-glpl 366 CRLAKSTLTLIPLLGTHEVI ____---------~ r-secr _ _ - - - - - _ _ _ _ _ _ _ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ KRLAKSTLLLIPLFGIHYIV 357 __________________________________ _ _ _ _ _ _ - ~ - ~ - ~ - - - ~342 r-vip KRLAKSTLLLIPLFGVHYMV
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _SRLARSTLLLIPLFGVHYIM _ _ _ _ _ _ - ~ - ~ - - - - - ~ 359 h-vip r-pacap R V P K K T R E D P L P V P S D Q H S P P F L S C V Q K C Y C K P Q R A Q a H S R S T L L L I P L F G I H Y T V 423 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _WRLSKSTLFLIPLFGIHYII _____---------~ h-grf 344 * **
1I1II1111VIIIIIIIIIIII. FFVNPGEDE-VS--RWFIYFNSP---LESFQGFFVSVFYCFLNSEVRSAIRKRWHRWQDKHSERARVA1 1 . 1
h-crf h-ct h-pth r-gip r-gluc r-glpl r-secr r-vip h-vip r-pacap h-grf
398 455 FMATPY-TE-VS--GTLWQVQMHYEMLFNSFQGFFVAIIYCFCNGEVQAEIKKSWSRWTLALDFKRKARS 487 FAPVTEEQA-EGSLRFAKLAFEIF---LSSFQGFLVSVLYCFINKEVQSEIRRLRLSLQEQCPRPHLGQA 361 FAFVTDEHA-QGTLRSTKLFFDLF---FSSFQGLLVAVLYCFLNKEQ~LLRRWRRWQEGKALQEG~LQEE~ 431 FAFVMDEHA-RGTLRFVKLFTELS---FTSFQGFMVAVLYSWERWRLERLNIQRDS431 FAFSPEDAM-E-----VQLFFELA----LGSFQGLWAVLYCFLNGEVQLEVQ~WRQ~LQEFPLRPVA417 FMFPIGISST-----YQILFELC---VGSFQGLWAVLYCRGLCLTQPGSRDYR403 F A F F P D N F K P E - - - - - V K M V F E L V - - - V G S F Q G F V V A I L Y P K 420 FAFSPENVSKR-----ERLVFELG---LGSFQGFLYCFLNGEVQAEIKRKWRS~~RYFTMDFK484 FNFLPDNAGLG-----IRLPLELG---LGSFQGFIVAILYCFLNQ~RTEISRKW---HGHDPELLPA-- 401
h-crf h-ct h-pth r-gip r-gluc r-glpl r-secr r-vip h-vip r-pacap h-grf
RAM-S------1PTSPTRVSFNSIKQSTAVP SNRSARAAAAAAEAGDIPIYICHQEPNEPANNQGEESAEIIPLNIIEQESSAO
421 508
end end
P-RAV-P----LSSAPQEAAIRNALPSGMLHVPGDEVLESYCP SSHGSHMAPAGTCHGDPCEKLQLMSAGSSSG~CEPS~TSLASSLPRLADSPTW -SMK-P-----LKCPTSSVSSGATVGSSWMTCQNSCSO -FNN-S-----FSNATNGPTHSTKASTEQSRSIPRASIIO -LHSWS-----MSRNGSESALQIHRGSRTQSFLQSETSVIO -YRHPS-----GGSNGATCSTOVSMLTRVSPGARRSSSFOAEVSLV@ - HRHPS - - - - - LASSGVNGGTQLSILSK-SSSQLRMSSL~ADNLATW . _ . _ .WRTRAKkPITPSRSMKVLTSMCO ______
397 485 463 449 437 460 523 423
end end end end end end end end
DCGFLNGSCSGLDEEASGPERPPALLQEEWETVM'J
593
end
h-pth
FPWRPS-NK-ML--GKIYDWS---LIHFQGFFVATIYCFC~EVQ~RQWAQFKIQWNQRWGRRP
ttt
*
*** * **
GSSSYSYGPMVSHTSVTNVGPRVGLGLPLSPRLLPTATITJGHPQLPGHAKPGTPALETLETTPPAMAAPK
559
G proteins in Signal Transduction
c. Phylogenetic tree derived from pairwise comparison of common sequences (hypervariable N-termini, u p to CW motif, and C-termini, and inserts removed):
245
L. Birnbaumer and M. Birnbaumer
246
Appendix VI
Sequences and phylogenetic tree of peptides belonging to the glucagonsecretin family Phylogenetic tree is based on common sequences and excluded obvious inserts and C-terminal extensions. Completely conserved amino acids are highlighted by asterisks.
a. Percent homology in alignment 0
0 1 2
100
3
2
3
41 51 100 51 loo
1
13 24 31 100
4 5 6
4 20
37 34 30
100
7
5
6
31 48 48 51 44 100
34 37 55 27 41 41
100
7
23 24 24 36
70 48
37 100
Hormone
G I P (gastric inhibitory peptide) GLP-1 (glucagon-like peptide 1)
Glucagon GRF (growth hormone release factor) PACAP (pituitatry AC activaing peptide) PHM (peptide histidine methionine amide) Secretin V I P (vasoactive intestinal peptide)
b. Alignment of scqucnces: PHM Secretin GLP-1 Glucagon GIP GRF
PACAP VIP
C-terminus 27 amide 27 amide HAEGTFTSDVSSYLEGQRAKEFIAWLV---------------KGRG-- 31 H S q C T F T S D Y S K Y L O S R R A Q D ~ Q W ~ - - - - - - - - - - - - - - - ~ -29 --YAEGTFISDYSTAMDKIHQQDFV”LLAEKGKKNDWKHN------ 42 44 amide YADAIFTNSYRLVLCQLSARKLLQDIMSRQWESNQERGARA----RL HSDCIFTDSYSRYRKQMAMY~VffiKRYKQ----RVK-----38 amide HSDAWTONYTRLRKQMAVYLNSIL--------------N-----28 amide
HADGVFTSDFSKLLGPLSAYLESLM--------------------HSOGTFTSELSRLRDSARLQRLLWLV---------------------
I
PXM
CRF
G proteins in Signal Transduction
24 7
Appendix VII
Sequence alignment and phylogenetic tree of mammalian adenylyl cyclases Phe-503 of AC-I important for interaction with Ca2'/calmodulin is highlighted [209]. a. Percent homology in alignment
0
1
0
1
100
40 100
2 3
2
3
4
(37) (411 100
34 37 (39) 100
33 35 (37) 33 100
4
5
6
5
6
35
41
(39 57 33 100
(42) 37 35 37 100
36
66
Adenylyl Cyclase
Accession t
bovine AC-I canine AC-V human AC-VII rat AC-I1 rat AC-I11 rat AC-IV rat AC-VI
M25579 M88649 M83533 M80550 M550'76 Ma0633 M96160
Number of completely conserved sites: 191
b. Amino acid sequence alimment -
can-V rat-VI
MCSSSSAWPSAG MPLPVARSGSGR
MTEDQGFSDPEYSAEYSAEYSVSLPSDPDRGVGRTHEISVRNSGS rat- 11I rat-I1 MRRRRYLRDRAEAAA A A ~ P R W M T P P W P G A S A S A S A P G R P G R S A A A ? T A G A can-V rat-VI S S M S W F S G L L V P K V D E R K T A W G E R N G Q K R P R Q A T R A R G F C MAGAPRGRGGGGGGGGAGESGGAERAAGPGGRRGLRAC bov-I rat-111 rat-I1 rat-IV can-V rat-VI bov-I
12 12 45 15 82 82 38
CLCLPRFMRLTFVPESLENLYQTYFKRQRHETLLVLWFAAP-------107 ~ G G G E G L Q R S R D W L Y E S Y Y C M S Q Q H P L I V F L L L L I ~ G A C ~ L ~ V F F A L G L ~ E D H V A F - - - - - - 77 -MARLFSPRPPPSEDLFYETYYSLSQQYPLLILLLVI~CAIVALPAVAWASGRELTSDPSF-------61 GAPPLGGAGPGRAAGPGPRRRGRRPRGRPRGAGRRRPAGPAACCRALLQIFRS~FPSDKLERLY 152 FIRRAGPGRGVELGLRSVALFDDTEVTTPMGTAEVAPDTSPRSGPSCWHRLAQVFQSKQFPSAKLERLY 152 DEEFACPELEALFRGYTLRLEQAATLKALRVLSL~GA~~LLGA~~A~~GSHPVH---100 ----
LMVAGVGLVLDIILFVLCKKGLLPDRVSRKVVPYLLW 144 rat-I11 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _LITVPTALAIFFAIFILVCIESVFKKLLR---VFSLV ____ 111 rat-I1 __________ -L?TVLCALGGFSLLLGLASREQQLQRWTR---PLSGL _____--__----.-95- - - - - rat-IV Q R Y F F R L N Q S S L T M L M A V L V L V C L V M L A F H A A R P P L R L P H L A ~ ~ V G V I L ~ V L C N R A A F H Q D ~222 GL can-V FRQDS~ rat-VI Q R Y F F Q ~ Q S S L T L L M A V L V L L M A V L L T F H A A P A L P Q P A ~ A L L ~ A S ~ ~ ~ C ~ H S222 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - _ _ _ -CVLFLALLVVTNVRSLQVPQLQQVGQLALLFSLTFALL ----------138 bov-I _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ^ _ _ _ _ _ _ _ _ _ _ _ _
210 rat- I11 LLITAQIFSYtGLN--FSRAHAASDTVGWQAFFVFSFF--ITLPLSLSPIVIISWSCWHTLVLGVTVR rat-I1 IWICLVAMGYLFMC--FGGTVSAWDQVSFFLFII~--TMLPFNMRDAIIASILTSSSHTIVLS--VY 175 IWAALLAtGYGFLF--TGGWSAWDQVSFFLFIIFTVY--AMLPtGMRDAAAAGVISSLSHLLVLG--LY 159 rat-IV A C Y R t I A W L A V Q W G L L L P Q P R S A S E G I W W T V F F I Y T I Y T L L P V ~ ~ ~ S G V L L S ~ H ~ I R t -290 -RA can-V 290 -. -. -- rat-VI VSWVI~I~VOVGGALAANPRSPSAGLWCPVFL~ITYTLLPIRMRAAVLSGLGLSTLHLILAW--HL CCPFALGGPACA~AGAA~VPATADQGVWQLLLVTFVSYA- LLPVRSLLAIGFGLWAASHLLVTA- -TL 204 bov-I *t
rat- III rat-I1 rat-IV can-V rat-VI bov-I
QQQQDELEGMQLLREILRNVFLYLCAIIVGIMSYYMADRKHRXAFLEARQSLEVKMNLEEQSQQQEN~ 280 L S A T P G A K E H - L F W Q I W . N I I F I C G N L A G A Y H K H ~ E ~ Q Q ~ R ~ N C I K S R 1 K L E F ~ R Q Q E R L L244 L L G W R P E S Q R D - L L P Q L L F L C G N W G A Y H K A L M E R A L H Q E H L L L 228 NAQDRF-----LLKQLVSNVLIFSCTNIVGVCTHYPAEVSQRQAFQETRECIQARLHSQRENQQQERLLL 355 NNGDPF-----LWKQIX;AFLCTNAIGVCTHYPAEVHENRQQERLLL 355 V P A K R P R - - - - L W R T L G A L F L G V M r Y G I F V R I L A E R A Q R ~ F L Q A ~ C I E D R L R L E D ~ E K Q E R L270 ~
rat-I II rat-I1 rat-IV can-V rat-VI bov-I
SILPKHVADEMLKDMK------KDESQKDQQQ-FNTMYMYRHENVSILFADIVG~LSSACSAQEL~L 343 SLLPAHIAMEMKAEIIQRLQGPKAGQME~-FHNLYVKRH~SILYADIVGFTRWLSDCSPGEtM 313 SILPAYLAREMKAEIMARLQAGQSSRPENTNN-FHSLYVKRHQGVSVLYADIVGFTRLASECSPKEtVLM 297 SVLPRHVAMEMIV\DIN------AKa----EDMMFHKIYIQKHD~SILF~IEG~SWLSQCTAQEL~ 415 SVLPQHVAMEMKEDIN------TKK----ED~FHKIYIQXHDNVSILFADIEGFTS~QCTAQEL~ 415 SLLPRNVAMEMKEDFL------KPP----ERI-FHKIYIQRHDNVSILF~IVGF~LASQCTAQEL~L329
rat-I11 rat-I1 rat-IV can-V rat-VI bov-I
I~NEI~FARFDKT~MKYHOLRIKILGDCYYCICGLPDYREDHAVCSILMGU\MVEAISrSREKTK~VDMRV413 3 83 LNELFGKFDQYKEN~MRI K ILGDCYYCVSGLP I SLPNHAKNCVKMGLDMCEAIKKVHDATGVDINMRV LNELFCKFDQIAKEHECMRIKILGDCYYCVSGLPLSLPDHAINCVRMGLDMCRAIRKLRVATGVDINMRV 367 L N E L F A R F D K L A A E N H C L R I K I ~ D C Y Y C V S G L P E A R A D R V 485 LNELFARFDKLAAENHCLRIKI~DCYYCVSGLPEARADHCCVEMGVDMIEAISLVREVTGVNVNMRV 485 LNELFGKFDELATENHCRRIKILGDCYYCVSGLTQPKTDHAHCCV~GLDMIDTITSVA~T~DLNMRV399
t
* *,
(I...
t
t
t t
tt
t
.*
*
.
..*..*
f....
..
t
t
tl
* .**
t*t
.. .. . .
t
*
.
.**
.**
L. Birnbaumer and M. Birnbaumer
248
rat-I11 rat-I1 rat-IV can-V rat-VI bov-I
GVHTGTVLGGVWlQKRWQYDVWSTDVTVANKMEAGGIPGRVHISQS~CLKGEFDVEPGDGGSRCDYLD GVHSGNVLCGVIGLQKWQYDVWSHDVTLANHMEAGGVPGRVHISSVTLEHLNGAYKVEEGDGEIRDPYLK
rat -III rat-I1
rat-IV can-V rat-VI bov-I
552 EKGIETYLIIASKPEVKKTAQNGLNGSALP-NGAPASKPSSPALIETKEPNGSAHASGSTSEEAEEQEAQ 517 QHLVKTYFVINPKGERR-----SPQHLFRP-RHTLDGAKMRASV~RYLESWGAAKPFAHLHHRDSMTT 502 ELGEPTYLVIDPWAEEE-----DEKGTERGLLSSLEGH'IPFAHLSHVDSPAS 607 E H S I E T F L I L R C T - - Q K - - - - - R K E E K A M I - A K M N R Q R T N 603 EQCIETFLILGAS--QK-----RKEEKAML-VKLQRTRANF----S 1 T H N I E T F F I V P S H - - R R - - - - - K I F P G L I L - S D I K P ~ ~ F K ~ C Y L L V Q ~ H C R ~ ~ E I P F S5~3 T
r a t -I I1
ADNPSF--PNPRRRLRLQDWLDRWDASEDEHEL-NQLLNEALLER-ESAQW-KKRNTF-LLTMRFMDP
rat-I1 rat-IV can-V rat-VI bov-I
483 453 GVHSGSVLCGVIGLQKWQYDWJSHDVTLANHMEAGGVPGRVHI~ATW\LWLGAYAVE~DMEHRDPYLR 4 3 7 555 C . T U S C . R V H C C . V ~ L R K W O F D V W S " H M E A G A Y L K 555 469 t
" f
Ir
tt
****
*it*
* * *** "
*
t*
f
*
.
*.
*
*
ENGKISTTDVPMGQHNFQNRTLRTKSQKKRFEEELNERMI-NAQKQW-LKSEDIQRISLLFYNK TSTPLP--EKAFSPQWSLDRSRTPRGLHDELD~-D~FFQVIEQL-NSQKQW-KQSKDFNLLTLYFREK
GNQVSK--E--MKRMGFEDP--KDKNAQESANPE-DE-VDEFWlRAIDARSIDRLRSEHVRKFLLTFREP RTKDSK--A--FRQMGIDDSSKENRGAQDALNPE-DE-VDEFWlRAIDARSIDQLRKDHVRRFLLTFQRE CEDDDK--RRALRTASEKLRNRSSFSTNWQTTP-GTRVNRYIGRLLEARQME-LEMADLNFFTLKYKQA
616 585 567 669 667 597
66 h u m - V I I I . . .FKDSSLEHKYSQMRDEVFKSNLVCAFIVLLFITAIQSL-LPSSRVMPMTIQFSILIMLHSALVLITT 678 r a t - 1 1 1 EMETRYSVEKEKQSGAAFSCSCWLFCTAMVEIL-IDPWLMTEPnrrFWGEVLL--LILTI-----CSMA rat-I1 NIEKEYRATALPAFKYYVTCACLIFLCIFIVOILVLPKTSILGFSFGAAFLSLI--FILFVCFAGOLLOC 653 rat-IV E M E K Q Y R L S A L P A F K Y Y R A C T F L V F L S N F T I Q M L ~ R P P A L A ~ S I T F L L F L - - L L L F V C F S E H L T K C6 3 5 can-V DLEKKYSKQVDDRFGAWACASLVFLFICFVQIT-IVPHSVFMLSFYLTCFLLL--TLWF-----VSVI 7 3 1 rat-VI DLEKKYSRKVDPRFGAYVACALLVFCFICFIQFL-VFPHSALI~IYAGIFLLL--LVTVL-----ICAV 729 bov-I EREN(YHQLQDEYrTSAWLALILAALFGLWLL-IIPaSVAVLLLLVFClCFLVACVLYL-----HITR 661 ~~
hum-VIII rat-I11 rat-I1 rat-IV can-V rat-VI bov- I hum-VIII rat-I11 rat-I1 rat-IV can-V rat-VI bov-I hum-VIII rat-I11
rat-I1
rat-IV can-V rat-VI bov-I
hum-VIII rat-I11 rat-I1 rat-IV can-V rat-VI
bov-1
AEDYKCLPLILRKTCCWINETYLAR"FLGAILN--------ILWCDFDKSIPLKNLTFNS 128 A I F P R A F P K K L V A F S S W I D R T R W A R N ~ ~ W L I F I L ~ D - - - - - - - - M L S C L Q ~ G P Y ~ - - G 738 SKKASTSLMWLLKSSGIIANRPWPRISLTIVTTAIIL~VFN--------MFFLSNSEETTLPTANTSN 715 VOKGPKMLHWLPALSVLVATRPGLRVAffiTATILLV~WS--------LLFLPVSSDCPFW\PNVSS 697 799 CSCGSFFPPlALORLSRSIVRSRVHSTAVGVFSVLLVFISAI~TCSHTPLRTCMRMLNLTPSDV--'r 7 9 7 V Q C F P G C L T I Q I R T V L C I F I WLIYSVAQGCWGCLPWSWSSS - - - - - - - -PNGSLWLSSGGRDPV- -L 7 21
YSCVKLFP(;PLQSLSRKIVRSKTNSTLVGVFTITLVFLSAMNMFMCNSEDLLCWLDEHNISTSRV--N S-------------------AVFTDICSYPEYNFTGVLAMLAVLLIMIAIYALLT I-------------------ELDGGCMENPKYYNYVAVLSLIATIMLVQVSHMVKLTLMIN
A - - - - - - - - - N V S V P D N Q A S I L H A R N L F F L P Y F I Y S C I L G I L
V---------AFNTSWELPASLP---LISIPYSMHCCVffiFLSCSLFLHMSFELKLLLLLL~VASCSLF ACHVMSAANLSLGDEQGFCGTPWPSC~P~F~SVLLSLLACSVFLQISCIGKLV~------LAIEL ACH--LRQLNYSffiLEAPLCEGTAPTCSFPEYFVGSVLLS------~LGF
P - - - - - - - - - - - - - - - - - - - V P P C E S A P H A L L C G L V G T L P
179 789 776 755 863 859 763
ET-WAGLFLRYD-----------N-LNH-----SGEDFLG~E----------VSLL~FL~V~H
226
L H - T H A H V L D A Y S - - - - - - - - - - - Q V L F Q - - - - - R P G I W K L
819 803 927 924 804
L Y - A W C P V F D E Y D H K R F Q E K D S P ~ A L ~ - M Q V L S T P G L N G T D S - R L P L V P S K Y S ~ ~ S8F5 6~ F LH-SHAWLSDCLI-----------ARLYQGSLGSRPGVLKEPKL----------MGAIYFFIFFFTLLVL IY-VLWEVPRVTLFDNADLLVT~AIDF-----NN"GTSQCPEHATKVALK~PIIISVNLALYLH
IYLLLLLLGPPATIFDNYDLLLSVHGLAS-----SNETFDLYLH SY-ILVLELSGYT-----------XAMGA-----GAISGR--H
291 926 889 ARONEYYCRLDFLWKKKLROEREETETME---------NVLPAHVAPOLIGONRRNEDLYIIOSYECVCVLF 8 6~. 5 AQQVESTARLDFLWKLQATEEKEEMEELQAYNRRLLHNI LPKDVAA~FLA~~ERRNDELYY(~SCECVAVMF9 9 7 AQQVESTARLDFLWKLQhTGEKEF31EELOAYNRRLLHNILPKDVAAHFLNlERRNDELYYOSCECVAVMF 994 ARQVDVKLRLDYLWMQAEEERDDMEKVKLDNKRILFNLLPA~AQHF~SNPR~LYY~SYSQVG~ 8 7 4 GQQLEYTARLDFLWRVQAKEEINEMKELREH~NMLRNILPSHVARHFLEKDRDNEELYSQSYDAVGVMF S R H V E K W \ R T L F L W K I E V H D Q K E R W P I R R W N E A L \ P T N M F GRQSEYYCRLDFLWKM(FKKEHEE1E~ENLNRVLLENVLPAHVAEHFLARSLKNEELYHQSYDCVCVMF
~
*
**
t
tt
*t
t .
**
*
t
249
G proteins in Signal Transduction
hum-VIII ASIPGFADFYSQTEM"QGVECLRLLNEIIADFDELLGEDRFODIEKIKTIGS~VSGLSP------rat - I I I rat-I1 ASIPDFKEFYTESDVNKEGLECLRLLNEIIADFDDLLSKPKFSGVEKIKTIGST-TGLSA-------
ASLPNFADFYTEESINNGGIECLRFLNEIISDFDSLLDNPKFRVITKIKTIGSTYMAASGVTPDVNTNGF ~
~~~~
___
754
996
9 5.. 1
928 ASII\NFSEFYVELEA"EGVECLRVLNEIIADFDEIISEDRFXQLEKIKTI~STYMAASGL~------- 1060 ASIANFSEFYVELEiWNEGVECLRLLNEIIADFDEIISEERFRQL~KIKTIGS~SGLNA------- 1057 ASIPNFNDFYIELDG"MGVECLRLLNEIIADFDELMDKDFYKDLEKIKTIGSTYMAAVGWLP------937 t. t .* .t .*" ****..*....
Kilr-IV
ASIPDFKEFYSESNINtlEGLECLRLLNEIIADFDELLSKPKFSGVEKIK'~IGST-TGLNA-------
hum-VIII rat-I11 rat-I1 rat-IV can-V rat-VI bov-I
-----EKQQCEDKWGHLCALADFSLALTESIQEINKllSFNNFELRIGIStlGSWAGVI~~KPQYDIWGK 419 TSSSKEEKSDKERWQt(LADLADFALAMX~LTNINNVSFNNFMLRIGMM(GGVLAGV1GARKPHYDIWGN 1066 IPSQEHAQEPERQYMHIG~EFAYALVGKLDAIM(HSFNDFKLRVGINHGPVIAGVIGAQKPQYD1WGN 1022 TPGQDTQQDAERSCSHLGnEFAVALGSKLGVINKHSFNNFRLRVGLNHGPWAGVIGAQKPQYDIWCN 998 STYDKV---GK---THIKALnDFAMXLMDQMKYINEHSFNNFQMKIGLNIGPWAGVIGARKPQYDIWGN 1124 STIDQV---GR---SHITALADYAMRLMEQMKHINEHSFNNFQMKIGLNMGPWAGVIGARKPQYDIWGN 1121 TAGTKAMCIS---SHLSTLADFAIEMFDVLDEINYQSY"VGI~GPWAGVIGARRPQYDIWGN 1004 t * * * ..ttt.
hum-VIII rat-I11 rat-I1 rat-IV can-V rat-VI bov-I
T V N L A S R M D S T G V S G R I Q V P E E ~ L I L K ~ F A F D Y R G E I ~ G I S E Q E G K I K ~ F L L G R V Q P N P F I L P P4 8 9 TVNVASRMESTGVMGNIQWEETQVILREYGFRNRRGPIFVKG----KGELLTFFLKGRDRPAhFPNGS1132 TVNVASRMDSTGVLDKIQVTEETSLILQTLGYTCTCRGIINVXG----KGDLKTY~PISRSLSQSNL1088 TVNVASRMESTGVLGKIQVTEETARALQSLGYTCYSRGVIKVKG----KGQLCTYFLNTDI,TRTGSPSAS 1064 TVNVASRMDSTGVPDRIQVTTDMYQVLAANTlQLECHGVVKVKG----KGEMMTYFLNGGPLS 1184 TVNVSSRMDSTGVPDRIQVTTDLYQVUW(GYQLECRGVVKVKG----KGPI?TYFLNGGPSS 1180 T V N V A S R M D S T G V Q G R I Q V T E ~ H R L L R R G S Y R R I C R G K S Q T R S 1070 *." .* * *
hum-VIII rat-I11 rat-I1 bov-I
AS
Can-V rat-VI bov-I
..
.
.
..(I.
..
f...
.(I.
. .
.
( I ., .
... .
..*
RRLPGQYSWV\WU:LVQSLNRQRQKQLLNEN"lY;IKGtIYNRRTLLSPSGTEPGAQAEGTDKSDLP
SVTLPHQWDNP
LNSERKMYPFGRAGLQTRLAAGHPPVPPAAGLPVGAGPGALQGSGLI\PGPPGQllLPPGASGKEA
c. Phylogenetic tree
AC -VI11
AC-I11
I
AC-V
AC-VI AC-I
AC-IV
7 AC-I1
557 1144 1090 1134
L. Birnbaumer and M. Birnbaumer
250
Appendix VIII Sequence alignment and phylogenetic tree of CP-isoforms phosphoinositide specific phospholipases Completely conserved amino acids are highlighted by asterisks. a. Percent identity in alignment 1
2
3
4
5
6
37 100
37 35 100
41 38 48 100
42 36 45 55 100
43 37 47 60 66 100
37 53 35 39 37 38 100
0 100
0
1 2 3 4
5
6
Accession
PI-PLC drosophila PLC-21 drosophila norpA human PLC-p2 rat PLC 1 rat PLC 13 Xenopus laevis PLC rat PLC p4
p
(I
M60452 503138 M95678 L14322 M99567 L20816 L18962
Number of completely conserved sites: 197
e
b. Amino acid alignment hum- 2 rat- 1 rat- 13
MSLLNPVLLPPKVKAYLSQGERFIKh'-----DDETTVASPVIL-RVDPK MAGAQPGVHALQLKPVCVSDSLKKGTKFVKW-----DDDSTIVTPIIL-RTDPQ WETLRRGSKFIKW-----DEEASSRNLVTL-8LDPN MAGARF'CVHSLQLEPVKVPELIKGSKFIKW-----DVESSSKSLVTL-RVDTM MMSAGGTYISTASVEVPQALQDGEKFIRW-----DDDSGTCTPVTM-RVDAK
...
MTKKYEFDWIIPVPPELTPCCVFDRWFENEKETKENDFERDALFKVDEY
""l-"A ____r..
MAKPYEFNWQKEVPSFLQEGAVFDRY-----EEESFVFEPNCLFKVDEF
h-21
g
hum- 2 rat- 11 rat- 3 Xl-p rat-p4 norpA h-21
F
hum- 2 rat- 1 rat- 3
Xl-p
rat-p4 norpA h-21 hum-112 rat- 1 rat - b 3
Xl-0
ratlp4 norpA h-21
E
43 48 (31) 48 46
49 44
CYYLYWTYQSKEMEFLDITSIRDTRFGKFAKMPKSQKLRDVFNMDFPDNSFLLKTLllrVSGPDMVDLTFH 113 GFFF~DQNKETELLDLSLVKDARCGKI~PKDPKLRELLDVGNIGH-LEQRMITVWCPDLVNISHL 117 GFFLYWD-GNMEVDDLDISSIRDTRTCRYARLPKDPKIREVLGFCCPDTRLEEKLAGPDP~FL (100) G F Y L Y W T C P N M E V D I L D I S V I R D T R T C K Y A K I P K D I K M R E I ~ ~ P E Q R P E D K L ~ G N D I V N I S 118 ~L CFFLYWVDQNNELDILDIATIRDVRTGoYRKRPKDNKLRQIVTL-GPQDTLEEKTVTVCHGSDFVNMTFV 115 CFFLYWKSEGRffiDVIELCQVSDIRAGCTPKDPK-ILDKVTKKNGTNIPELDKRSLTICS~DYINITYH 118 GFFL?WKSEGKEGQVLECSLINSIRLAAIPKDPK-ILA-ALESVCKSENDLEGRILCVCSGTDLVNICFT 112
..
. .
NFVSYKENVCXAWAEDVLALVKHPLTANASRSTFLDKILVKLKMQLNSEGKIPVKNFFQMFP---ADRKR NLVAFQEEVAKEWTNEVFSWNLLAPNMSRDAFLEXAYTKLKLQ~PEGRIPLKNIYRLFS---ADRKR NFMAVQDDTVKVWSEELFKW\MNIQNAPEHV-LRKAYTKLKLQVNQffiRIPVKNILKMFS---ADKKR NFMAVQEDTAKIWTEELFKLAHNILAQNSSRNTFLQXAYTKLKLQVNQffiRIPVKNILKMFA---ADKKR
180 184 (1661 185 NFCCTRRDIAQLWTDGLIKLAYSLAQLNGSAIMFLQXAHTKLCLQVDKSCRIPVMIIKLFAQNKEDRKR 185 HVICPDAATAKSWQKNLRLITHNNRATMlCPRVNLMXliWTFA-SGKTEKL 187 YMVAENPEITKQWVEGLRSIItlNFRANNVSP~CLKKHWMKLAFLTNTSCKIPVRSITRTFA-SCKTEKV 181
f.
. ..
t
VEAALSACHLPKGKNDAINPEDFPEPWKSFLMSLCPRPEIDElFTSYHAKAKP-YElTXEHLTKFINQKQ 249 VETALEACSLPSSRNDSIPQEDFTPDWRVFLNNLCPRPEIDNIFSEFGAKSKP-YLTVDQMMDFINLKQ 253 VETLI -CCLNFNRSESIRPDEFSLEIFERFLNKLLLRPDIDKILLEIGAKGKP-YLTLEQLMDFINQKQ ( 233) VETALESCGLNFNRGDSIKPEEFTLDIFERFLNKLCLRPDIDKILLE~RKGKP-YLTLEOLMDFLNOKO254 V E K A L D ~ L P S G K V D S I S V S K F Q F E D ~ L ~ Y L T Q R S ~ E R L F D S I V G N S K ~ C M S I A Q L V255 EFL~~ VYTCIKDAGLPDDKNA~EQFTFDKFYALYHKVCPRNDIEELFTSITKGKQD-FISLEQFIQFMNDKQ 256 IFQALKELCLPSGXNDEIEPAAFTYEKFYELTQKICPRTDIEDLFKKINGDKTD-YLTVDQLVSFLNEHQ 250
-
t
t
.
hum- 2 rat- 1 rat- 3 Xl-p rat-p4 norpA h-21
RDSRLNSLLPPPARPDQVQGLIDKYEPSGINAQRGQLSPEGMFLCGPENSVLAQDKLLLLt[HD~QPLN 319 RDPRLNEILYPPLKQEQVQVLIEKYEPNSSLAKKGQMSVDGFRYLSGEENGWSPEKLDLNEDMSQPLS 323 RDPRLNEVLYPPLRSSQARLLIEKYEPNKQFLERDQMS~GFSRY~EENGILPLEAtDLSMDMTQPLS (303) RDPRLNEILFPPLKRDQVRQLIEKYEPNRQFLDRDQMSMEHENSIVPPEILDLSDD~QPLS 324 RDPRLNEILYPYANPARAKELIQQYEPNFNAQKGQLSLffiFLRYLMGDDNPIMAPSKLDLCDDMDQPMS 325 RDPRMNEILYPLYEEKRCTEIINDYELDEEKKXNVQMSLDGFKRYLMSDE~PVFLDRLDF~~DQP~326 RDPRLNEILFPFYDAKRAMQIIPnEPDEELKKKGL1SSM;FCRYLMSDENAPVFLDRLELYQ~DHP~ 320
hum- 12 rat-131 rat -133 Xl-p rat-[\4 norpA h - -21
tlYFINSSHNTILTAGQFSGLSSAE~YRQVI,LSGCRCVELDCWKGKPPL)EEPI ITHGbTM'ITDIFFKEAIE HYFINSSHNTYLTAGOLAGNSSVEMYROVLLSGCRCVELDCWKGRTAEEEPVITt1GFTMTTEISFKEVIE
~~
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.
*.
t
t i
.
t
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t
389 393
AYFINSSHNTYLTAGQLAGTSSVEMYRPALLWGCRCVELD~GRPPEEEPFITHGF~~~PLRDVLE (373) 394 HYFINSSHNTYL'PGHQL~KSSVEIYRQCLLAGCRCVELDRJNGRT--EEPVI~IGYTFVPEIFRKDVLE 393 HYYINSSHNTYLSGRQIGGKSSVEMYRQTLLAG~RCVELDC~GKGEDEEPIVTHGIIAYCTEILFK~IQ396 HYFISSSHNTYLTCROFCKSSVEMYROVLLAGCRCVELDCW~KGEDQEPIITHGKAMCTDILFKDVIQ 390
SYFITSSHNTYLTAGQLTCNSSV~RQVLLTCCRCIEL~WKGRPQDEEPFITHGF?MTTEIPFKNIE
,
.
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G proteins in Signal Transduction
P
251
hum- 2 AIAESAFKTSPYPIILSFENHVDSPRQQAKMAEYCRTIFGDMLLTEPLMFPLKPGVPLPSPEDLRGKIL 459 r a t - % l AIAECAFKTSPFPILLSFENHVDSPKQQAKMAEYCRLIFGDALLMEPLEKYPLESGVPLPSPMDLmKIL 4 6 3 r a t - 3 AIAETAFKTSPYPVILSFENHVDSAKQQAKMAEYCRSIFGEALLIDPLDKYPLSAGTPLPSPQD~GRIL (443) x1-p AIAESAFKTSPFPVILSFENHVDSSKQQAKMAEYCRNIFGDASLIDPLEKYPLQPGVALPSPQE~GKIL 464 r a t - p l AIAESAFKTSEYPVILSFENHC-NPRQQAKI~CREIFGDMLLDKPLDSHPLEPNMDLPPPAMLRRKII 462 norpA AIADCAFVSSEYPVILSFENHC-NRAQQYKW(YCDDFFGDL 465 hn-21 AIKETA~SEYPVILSFENHC-SKYQQYQMSKYCEDLFGDLLLKQALESHPLEPGRLLPSPNDLKRKIL 459
............ ..
.*
I.
t
tt
t t .
t
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rat-bd norpA
h-21 * . t
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rat+4
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SPPSAPAVWAGEEGTEL-E---------------------------.-.-----EEEVEEEEEE-ESGNL 517 TYSDSSSVFEP-SSPCA-G-------------------------.---------EADTESDDDD-DDDDC 520 ALSESSAATEP-SSPQL-GSPSSDSCPGLSMCE~~GLEKTSLEPQKSLGEEGLNRGPNVLMPDRDREDEE (539) TYSDSSSVCES-SA~LPPSESADVSLTLSNGDEKIE~P---PKYTKPRKSIDllDAYSEEEEEEEPSDP558 ~SAAGTAGHAPPLQQIRQSSKDS~SS--------------------------DSDSSSEDESLPN?TP 576 K G E L K T D D D P E E D A S A G K P P W \ A A - - - - - - - - - - - - - - - - - - - - - - - - - - - - A P A P A P ~ E523 GA ESAAPASILEDDNEEEIESAADPEEEI\HPEYKFGNEL------------------SADDFSHKEAVANSV 536
____
DEEEIKKMQSDEGTA---GL---EVTAYEEM----SSLVNYIQPTKNSFEFSAQKNRSWISS~EL~577
----- K K S S M D E G T A - - - G S - - - E A M A r r E M - - - - S N L V N Y I Q P V K G
Xl-p rat+
norpA
hn-21
575
EDEEEEETIDPKKPTTDU;TASSEVNATEEM-----STLVNYVEP~FKSFESSRK~CFEMSSFV~ (605) K K - - S D E G T A - - - S S - - - E V N A T E E M - - - - S T L V N Y V E P V K F K S F D A A K K R M ( W E M S 611 NLPSGNEPPP---EKAQKETEAGAEI----SAL~QPIHFSSFENAEKKNRCY~SSFDEKQA 634 AECCGCAEAE---AAAANYSGSTPNVtiPWLSSKVNYAQPIKFQGFDKAIEKNIAHNMSSFAESAG 585
-_-_-____ _____
.*..
......
- _ - _KKGLVTVEDEQAWMASYKYVGAlTNIHPYLS?MINYAQPVKFQCFtiVAEERNIHYNMSSFNESVG -
E
601
hum- 2 YDLLSKASVQFVDYM(RQMSR1YPKGTRMDSSNmPQMFWNAGCQMVnLNFQTMDLPMQQNMAVFEFNGQ 647 r a t - 1 L E Q L T K S P V E F V E U M ( M Q L S R I Y P K G T R V D S S N Y M P Q L F W N A G C Q ~ ~ N F Q ~ D ~ Q 1 N M G m E Y N645 GK r a t - 3 MEQLTKSPMEFVEYNKQQLSRIYPKGDRVDSSNmPQLFWNYGCQLVALNFQDLDLPMQLNAGVFEYNGR (675) L E Q L T K S P M E N E Y M U ( Q L S R 1 Y P K G T R V D S S N Y M P Q L r W G R R 681 Xl-p ~LLKERPIEFVNYNKHQLSRWPAGTRFDSSNFMPQLFWNAGCQLVALNFQrrDU\MQLNLCIFEYNAR 704 rat-p4 norpA WNYLKOSSIDNNYNKROMSRIYPKGTRADSSNYMPOVFWNAGCOMVSLNFOSSDLPMOLNOCKFEYNGC 655 LGYLK'IHAIEFVNYNKRQMSRIY PKGGRVDSSNYMPQI FWNAGCQMVSLNYQTPDLAMQLNQGKFEYNGS !A-21 67 1
...................................
E
hum- 2 SGYLLKHEFMRRPDKQFNPFSVDRIDVWA?TLSITVISGQFLSERSVRTNEVELFGLPGDP-KRR-YR 715 714 r a t - 1 SGYRLKPEFMRRPDKHFDPFTEGIVffilVANTLSVKIISGQFLSDKKVGTNEVDMFGLPVM-RRKAFK rat- 3 SCYLLKPEFMRRPDKSFDP~EVIVDGIVANALRVKVISGOFLSDRKVGIWNDMFGLPVDT-RRK-YR 17431 SGYLLKPEFMCRD?KHFDPFTEN IVDGIVANTVK IK II SGQFLSEKRVG I NEVDMFGLPVDT-KRK - FR '749' Xl-p SGYLLKPEFMRRSDRRLDPFAESTVDCIIAGTVSInnSGOFLTDKRANTFVEVDmGLPA~K-FR 774 rat+ C G Y L L K P D F M R R A D K D F D P F A D A P V ~ ~ ~ V I A A Q C S ~ I A G Q F L S D K ~ G T N E V D M F G L P S ~724 E-FR norpA h-2 1 CGYLLKPDFMRRPDRTFDPFS~P~VIMTCSVQVISGQFLSDKKIGTNEVDHYGLPTDTIRKE-FR740
E
hum- 2 rat- 1 rat- 3
Xl-p
rat+
norpA
hn-21
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rat-P4 norpA
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tt
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t
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..
.....
L
... . . . . .
LlMPALFIFLEMKDYIPGAWADLTVALANPIKFFS-----------------AH~SVKLKE~ffiLPE838 L M L P A V F W I ~ D W P D T Y A D V I ~ L S N P I R W N L M E Q R A K Q P S 853 LCLPALLTYTEASDYIPDDHQDYAEALINPIKHVSLMDQRAKQLAALIGESEAQASTEMCQETPSQQQGS (882) VCLPALLWT~DYIPDDHQY~ALTNPIKHISLMDQRAD-----------876 IALASLFLCVVVKDWPDDLSNFAEALANPIKYQSELEKRDIQLSVL~~ffiSADEDLSKSCGQKKE 913 HSLPMLFVNIELKIWPffiFEDFMAMLSDPRGFAGAAKQQNEQMKALGIEEQS-GGAARDAGKA--KEEE 8 6 0 L S L P T I F C N I V L K T N P D G L C D I V D A L S D P K K F L S I T E K ~ D Q L ~ G I E T S D I ~ V P S D T S ~ - - D 877 ~G t
t
t
*
KPFPW\SPVASQVNGALAP-------TSNGSPAA~GAREE---------------~EA--AEPR--- 8 8 1 ETRTPPAENGVNHTATLPKPPSQAPHSQPAPGSVKAPAKTE--------------DLIQSVLTEVE--907 QLSSMPVPNPLDDSPRWPPGPT----TSPTSTSLSSPGQRD---------------DLIASILS~--- ( 9 3 0 ) KIDWDPISKPPIRSRDDTPEKI----KIPIIPVPSPPAQRD---------------DHI~VLTDIQ--- 925 LRPVESLATSPKHRPSIS-----MSVD~RTDGGRGEDSISIVAPSIQHQHSLDQSVSTSIRQVE 979 FE--869 KANPAKANVTPQSSSELRPm----AA~SGQW(KG------------------------IELIP--- 916 . , . , I I . . .
L. Birnbaumer and M . Birnbaumer
252
hW-12
rat- 1 rat- 3
x1 -p
rat-p4
norpA Dm-21
_ _ _ _ _ _ _ _ _ _T A S L E E L R E L K G W K L Q R R H E K E L R E L E R R G A R R W E E L L Q R G A A P P G V G G V G A C
941 966 (9821 APSMEELKSQKSFEKLICRQYRELEQLRRKI{L~VSSLCKEQS~PLQSL-------- 978 SSQFDVDLVW\EPLEKILDHKSVKEKRLMEKKLESLRI~DKEKIKIAGQKSSPLEGKKPKFAIRJKLV 1049 PVTLESLRQEKGFPKVGKKQIKEL~RKK~ERTSVQK-----------------TQ 911 QVRIEDLKQMKAYLKHLKKQQKELNSLKKKHAKEHSTMQKLHCTQVDKIVAQYDKEKSTH 976
_ _ _ - - - - _AQTIEELKQQKSFVKLQKKHYKEMKDLVKRHHKK'ITELIKEHTPKYNEIQNDYLRRRAAL __ ----------PTPLEELRSHKLRSRQDRDLRELI{~HQR~VAL~RLLDGLAQA~E--------
_____--___
__________
_------___
4
K ~ P G K G S R K K R S L P - - - - - - - - - - - - - R E E S A G A A P G E G E E Q 994 EKSAKKDSKKKSEPS-------------SPDHGSSAIEQD~LDA~KLIDLKDKQQQQLLNLRQEQ 1023 - - - - - -G K C R P S - - - - - - - - - - - - - S S A L S R A ~ E D V K E E E K E A A Q(1030)
Xl-p
rat-P4
norpA Dm-21
F
hum- 2 rat- 1 rat- 3 Xl-p rat-P4
norpA
Dm-21
rat+
Xl-p
norpA h-21
-1
hum- 2
rat 1 rat+
Xl-p rat-p4 norpA h-21
-- --
- -- - - --R R R K P F - - - - - - - - - - - - - G R S H R G V S - - - - - - G A D O D A E R ~ O K R L L E L R E I O KRLSNKSLNCLSPHSEPGVEIPACPLDU;DSSEESAAADAGED~GGSS~---SL~R~ESRLRSACRE
1020 1115 NAAIDKLIKGKSKDD-------------IRM)A"SI-AR~ 951 EKILEKAMKKKffiSN----------CLEIKKETEIKIQTLTSDHKSKVK----EIVAQH~~SMI~H 1032
YECVLKRKEQHVAEQISKMME-LAREKQIV\ELKI\LKETSENDTKEMKKKLm(RLERIQG~--~DK 1 0 6 1 YYSEKYQKREHIKLLIQKLTD-VAEECQNNQLKKLKEICEKEKKELKKKHDKKRQEKITW(S--KDKSQ 1090 ADAETERRLEHLKQAQQRLRE-WLDAH~FKRLKELNEREKKELQKILDRKR"SISERKT--REKHK ( 1 0 9 7 ) HEQERKLKHSHLLEAVQKLQE-VAISYHSTQLKKLKEINEKEKKELQKILDRKRHNSITW(S--RERQK 1087 Y T S Q Y R E L Q E K Y H E A I Y S I E K V L K T S Q ~ Q ~ Q ~ S L D K ~ ~ Q L Q E A R ~ ~ T - -1183 VHRDR RKE~~RQHVQOSQDI--LMLTVQAAQIKQLEDRHARDIKDLNAXQAKMSADTAKEVQNDKTLXTX1020 SAEEQEIRDLHLSQQCELLRK-LLINAHEQQWQLKLSHDRESKEMRAHQAXISMENSKAISQDKSIKNK 1101
.
KEVE- -LTEINRRHITESVNSIRRLEEAQKQRHERLLAGQQQVLQQLVEEEPKLVAQLTQ (1165) KDVE--LTEINRRHINESVSSIRRLEW\QKRRQEALQTAHQFLQRIKDEEPKLOAOLDW\COAEFCOLP 1153 - -.- - - _-- ._ ._- .-NEKDRRLREKRQNNVKRFMEEKKQIGVKQXJ.AMEKLKLAH-SKQIEEFSTDVQKLMD-- - - --- MY
~- - -
--
~
AERERRVRELNSSNTKKFLEERKRW\MKQSKEMDQLKXVQE---------MQ
AEVKESVRACLRTCFPSEAKDKPEPACECPPELCEQDPLIAKADAQESRL LEILERIQEAMKGKVSEDSNHGSAPPSLASDPRKVNLKSPSS QEIRRCLLGETSEGLGDGPLVACASNGHMGSGG ~
1181 17nn A&.""
~
QEVRRYLQEDGWVGSGSGPPSSSHSSPPSITRSWGSESGEKDSLHSPGDSSDEATRL NCFLVLFHGPHHHGC~SGSSALSGNN~TLNLDAGAAGSHSIAAAAPMT
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c. Phylogcnetic trec
PLC-Pl
PLC-Pl
Dro-PLC21
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(1199) 1210 1312 1095 1176
Biomembranes Edited by Meir Shinitzky Copyright 0 VCH VerlagsgesellschaflrnbH, 1995
CHAPTER
Membrane-Associated Protein Kinases and Phosphatases DAVlD S . LESTER FDAICDERDRT, 8301 Muirkirk Road, Laurel, MD 20708, USA
Contents 254
258
264
Introduction 254 Protein phosphorylation and dephosphorylation 255 Protein kinases 258 Protein phosphatases Techniques for Studying These Activities 258 Assay conditions 258 In vivo protein kinase analyses 259 In vitro protein kinase analyses 261 In vivo protein phosphatase assays 261 In vitro protein dephosphorylation assays 261 Solubilization procedures 264 Identification of phosphoamino acid residues Membrane-Associated Protein Kinases 264 Second messenger-dependent protein kinases 264 PKA 264 CAM-PK 265 PKC
D.S. Lester
254
269
273 274 276 278 278
266 Tyrosine kinase receptors 267 G-protein receptor-activated protein kinases 267 Myristoylated oncogene tyrosine protein kinases 268 Ectokinases 269 Second messenger-independent serinelthreonine protein kinases 269 Casein kinases 269 Other serinehhreonine kinases Membrane-Associated Protein Phosphatases 271 Serinehhreonine phosphatases 271 Protein phosphatase 1 271 Protein phosphatase 2A 272 Protein phosphatase 2B 272 Protein phosphatase 2C 272 Tyrosine protein phosphatases 272 Particulate protein tyrosine phosphatases 272 Receptor protein tyrosine phosphatases Inhibitors Substrates Lipid Regulation of Membrane-Associated Protein Kinases and Phosphatases Conclusions References
Introduction Protein phosphorylation and dephosphorylation The process of reversible covalent modification of proteins is a major pathway for regulating their structural and functional state. This present review deals with one of these mechanisms, the phosphorylation-dephosphorylation of proteins, and within this broad topic, specifically, the membrane-associated protein kinases and phosphatases. Broader reviews on protein kinases and phosphatases can be read elsewhere [ 14,19,60]. The reactions involved in these enzymatic processes can be summarized as follows: Protein
+ nATP
Protein - P,
+ nH,O
Protein - P, Protein
+ nPi
+ nADP
(1) (2)
Eqn. (1) represents protein phosphorylation while Eqn. (2) is the protein dephosphorylation step. The phosphorylation occurs on serine, threonine
Membrane-associated protein kinases and phosphatases
255
or tyrosine amino acid residues. In addition, each protein molecule may have more than one phosphorylation site, and there may be different amino acid residues that can serve as phosphorylation sites for different kinases, e.g., the EGF receptor is a tyrosine kinase that autophosphorylates on tyrosine residues and can be additionally phosphorylated on a number of threonine residues [ 1161. For phosphorylation-dephosphorylation to play a role in regulation, the phosphorylated-dephosphorylated substrate must be in a relatively dynamic state. Consequently, for the kinases and phosphatases to show this kinetically determined balance, these enzymes themselves are often regulated.
Protein kinases Approximately 40-50% of total cellular protein is associated with membranes, in particular, the plasma membrane [32]. Due to the difficulty in dealing with membrane proteins, information on the regulation of phosphorylation of these proteins is limited. Indeed, the development and ultimate classification of cellular protein phosphorylation and dephosphorylation pathways is based on the ability of identified soluble protein kinases and phosphatases to modify soluble substrates such as glycogen synthetase [ 14,19,60]. The soluble enzymes are free to diffuse and migrate to their respective soluble or membrane-associated substrates. The membrane-associated kinases and phosphatases, however, must content with the physical restriction of their association with the membrane. Thus, in addition to the localization of activating agents, the membrane-associated enzymes must also deal with the physical constraints of the surrounding membrane environment. This may also provide further specificity for the interaction as it can lead to a physical means of determining whether the enzyme can interact with the protein substrate to be modified. Thus, these factors alone may be important in regulating the enzyme activity without the additional complexity of such processes as changes in synthesis or metabolism of the enzyme (section on Substrates). The role of the membrane in the regulation of the enzyme will be dealt with at a later stage of this chapter (section on Lipid Regulation). Table I summarizes many of the identified membrane-associated protein kinases. Here they are subdivided into three main categories: 1. Transmembrane protein kinases - these are the transmembrane proteins containing protein kinase activity, e.g., growth factor receptors.
Subcategory
Intracellular protein kinases
Activators
Myristoylated oncogenes (tyr)
pp60v-src
Transformation
Light
Spermidine, spermine
Receptor-associated &ARK (ser/thr) Rhodopsin kinase
Cyclic AMP Ca2+/calmodulin Ca2+/ phospholipid Diacylglycerol
?
Sangivamycin
Polyanions
Staurosporine
H-89 Calmidazolium H-7
Calpactin, EGF receptor, vinculin
0-AR, M2 muscarinic receptor, metarhodopsin Metarhodopsin, -AR
PKA-regulatory subunit PSDs MARCKS, B-50
POI
1851
[27]
1361 [931 [11,651
[79,1161
T yrphostins
CSF-1 receptor CSF-1
Autophosphorylation
[52,116]
Serine kinases
Tyrphostins
IGF-I
IGF-I receptor
[ 104,1161
PDGF
PDGF receptor
P L C r , serine kinases
EGF
EGF receptor Tyrphostins
[ 116,1171
Refs.
[26,116]
pp185, PI-3-kinase
Substrate
PLCr, ~ ~ 4 2 , MAP-kinase
Tyrphostins
Inhibitors
Genistein, lavendustin
H202
Insulin receptor Insulin, VO,,
Example
Second messenger- PKA dependent (ser/thr) CAM-PK PKC
Transmembrane Receptors kinases (tYd
Category
Categories of membrane-associated protein kinases
TABLE I
9
2
b
lo
P
Ectokinases (serlthr)
Neuronal Parasitic ?
Mn2+
Rat liver kinase Phosphorylation
Unclassified ? ?
Poly (glu,tyr)
? Heparin
Inhibitors
Notes: A schematic representation of each subcategory is listed in Fig. 1. The phosphoaminoacid specificity is in parentheses after each subcategory listing.
Extracellular
Spermine ?
CKII CKI
Casein kinase (ser/thr)
Intracellular protein kinases
Activators
Example
Subcategory
continued
Category
TABLE I,
Factor V Complement
?
Glycogen synthase
?
Substrate
r281 r691
r 1021
r71 r1011
Refs.
h
s
3
2d
4
b
z 8 8. f?
0. Q -7
258
D.S.Lester
2. Intracellular protein kinases - these are the protein kinases that upon disruption of the tissue or cellular preparations and subsequent differential centrifugation are found in the particulate fraction. These proteins require detergent to solubilize them even though they do not traverse the membrane. 3. Extracellular protein kinases - these are protein kinase activities that have been detected when radiolabelled ATP is applied extracellularly to cell preparations. Class 2 has been divided into four additional categories according to their activation properties andlor phosphoamino acid specificity.
Protein phosphatases The removal or hydrolysis of the phosphate from the substrate restores its original conformation and activity. The enzymes responsible for this reaction are the protein phosphatases, and they provide a regulatory role for the phosphorylation process. The phosphatases can generally be divided into the serinelthreonine phosphatases (PrP) and the tyrosine phosphatases (PTP). However, some of the PrP species are capable of dephosphorylating phosphotyrosine residues in vitro [99]. The membrane phosphatases, both PrP and PTP, can be subdivided into the following classes: (1) Receptor transmembrane phosphatases, which are tyrosine specific; (2) Intracellular membrane-associated protein phosphatases, which include both PrP and PTP species. Considering the observations that there are ectokinases (see above), there has been only one report of ectophosphatase activity [63] which is complicated by the high concentration of alkaline phosphatase found on the extracellular membrane surface of some cell types such as liver cells ~71.
Techniques for Studying These Activities
Assay conditions In vivo protein kinuse analyses There are three general approaches to examining protein kinase activity in tissue extracts [96]: 1. Inorganic phosphate - 32Piis incubated with intact tissue or cells. The radiolabelled compound is actively incorporated into the y-PO, of
Membrane-ussociated protein kinases and phosphatases
259
ATP. It is important to ensure that the synthesis of radiolabelled ATP reaches an equilibrium. Upon application of the desired experimental conditions, the radiolabelled sample is processed and analyzed on one[64] or two-dimensional [4] polyacrylamide gel electrophoresis. The gels are exposed to X-ray film and processed. 2. Reverse or back phosphorylation - The sample is processed into the desired cell-free fraction@) and [ Y - ~ ~ATP P ] is added in a suitable buffer in the presence of phosphatase inhibitors such as orthovanadate and NaF [99]. Samples are analyzed as described in (1). The limitation with this procedure is that many of the potential substrates may have already been phosphorylated before cellular fractionation. Thus, some of the substrates may not be identified, and it could be difficult to obtain quantitative information. 3. Dephosphorylation - The cell-free extracts are prepared as in (2), except the sample is pretreated with exogenous phosphatases such as alkaline andlor acid phosphatases. This treatment would be expected to remove much of the phosphate from prelabelled phosphoamino acids. After this treatment the samples are treated as in (2). Generally, in order to identify protein kinase activity and potential substrates, all three of the above techniques are used. To identify regulation of a protein kinase activity, the above activities can be challenged with specific pharmacological agents, some of which are listed in Table I. The ectokinases can be identified on intact cells using procedures (2) and (3) described above for cell-free extracts (i.e., reverse phosphorylation andlor dephosphorylation). [ Y - ~ ~ ATP P ] is added to intact cells. Samples are analyzed as above. With these types of analyses, it is important to verify that it is truly the external phosphate that is being incorporated into protein substrates and not the result of hydrolysis of ATP, uptake of the free Pi into the cell and subsequent incorporation into intracellular ATP pools. A more detailed analysis of what is required to verify that the researcher is dealing with an ectokinase is discussed by Ehrlich and colleagues [28]. Tyrosirie kinase activity can be monitored using an anti-phosphotyrosine antibody and immunoblotting of the relevant biological sample [20]. In vitro protein kinuse analyses Artificial or model assay systems are established in order to monitor purification of protein kinases and, ultimately, characterize the purified
260
D.S.Lester
enzyme in an environment similar to that expected in vivo. Exogenous substrates, such as histones or specific peptide substrates, or identified endogenous substrates, are used in these assays (see Table I). A specific peptide substrate can often be prepared based on the amino acid sequence of the phosphorylation site of the endogenous substrate. An example is the specific 7 amino acid sequence of the EGF receptor which is phosphorylated by protein kinase C [41]. In the case of membrane-associated kinases, often the nature of the endogenous activity is not always known. Thus, the aim of the in vitro assay, i.e., to reconstitute the endogenous kinase activity, is difficult. Protein kinase C (PKC) is the most intensely studied membrane-associated protein kinase [10,80]. Many of the discrepancies obtained in biochemical analyses can be attributed to specific properties of each of the assay conditions. The three artificial systems used for PKC are: 1. Nondefined phospholipid preparations - phospholipids are suspended in the assay buffer [56]. Ca2+ and acidic phospholipids, in particular phosphatidylserine (PS), are important for supporting PKC activity. Addition of both of these cofactors, before the assay is initiated, results in undefined lipid structures, not representative of membrane bilayers [121. 2. Detergent micelles - It is considered that the phospholipid concentrations are highly defined and in equilibrium (i.e., there is little or no exchange of lipids between vesicles) [38], However, the phospholipid exchange rate between the micelles is high, and the equilibrium can be effected by the protein binding and the assay conditions. 3. Lipid vesicles (liposomes) - These are prepared in divalent cationfree buffer often containing a neutral phospholipid support such as phosphatidylcholine (PC). This assures that the bilayer is maintained
WI.
A typical example of the difference between these systems is seen in system (1) where high Ca2+ concentrations plus PS are capable of activating PKC. Diacylglycerol (DAG) reduces the amount of Ca2+ required for activation. This is considered by many researchers to be the fundamental role for DAG in PKC activation [81]. In system (3), in the absence of DAG, PKC cannot be activated, even at higher levels of Ca2+ [65]. This more closely resembles what is considered to occur in the proposed model for PKC activation [ 101.
Membrane-associated protein kinases and phosphatases
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In vivo protein phosphutase assays Assays for identification of the phosphatases in intact cells or cellular fractions are similar to those described previously for the phosphorylation procedures. The methods used are: 1. Incorporated 32Pi- Cells are loaded with labelled phosphate as for the kinase assays. Upon equilibration of the radiolabelled ATP pool, the labelled phosphate is removed and the intact cells are maintained to monitor dephosphorylation of 32P-labelledsubstrates with time. Samples are analyzed by gel electrophoresis [68]. 2. Back phosphorylation/dephosphorylation - Cell-free extracts are incubated with [ Y - ~ ~ ATP. P] The ATP is removed and the sample monitored for reduction in the labelling of proteins. The phosphorylation patterns are monitored by gel electrophoresis as for the phosphorylation assays (procedure 1 to 3). The unbound label can be removed from intact cells by washing them or by passing them through a gel filtration separation column. For extracts the labelled ATP can be significantly diluted with unlabelled ATP or passed through a sizing column [71].
In vitro protein dephosphorylation assays The assay used for identifying the activity during purification of PrPs and PTPs is the dephosphorylation of 32P prelabelled substrates such as histone. Another less sensitive technique is the colorometric assay, using para-nitrophenyl phosphate, initially used for alkaline phosphate assays. This assay has the advantage in that kinetic analyses can be made [63].
Solubilization procedures Membrane protein kinases and phosphatases are extrinsic, peripheral or transmembrane proteins. In order to biochemically characterize their activities, it is often necessary to solubilize them. This procedure isolates the protein from its native environment, and the original activity of the enzyme is often not recovered. The approaches used for solubilization of protein kiiiases and phosphatases are listed and described in Table 11. Generally, detergents have been used for integral membrane proteins. The most commonly used is the neutral detergent, Triton X-100. Different detergents have been used for the same enzyme, but they generaliy have similar characteristics; e.g., Triton X-100 and Nonidet P-40
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TABLE II
Examples of solubilization procedures used for membrane-associated protein kinases Technique
Kinase
Classification
Ref.
Detergent
EGF receptor
[26]
Membraneassociated PKC
Transmembrane integral Integral (peripheral)
Differential detergent
Parasite ectokinase Membraneassociated PKC
Integral (peripheral) Integral (peripheral)
[421 Lester (unpublished)
Divalent cation chelators
Soluble PKC Membraneassociated CAM-PK
Extrinsic Extrinsic
1571 ~481
High pressure
Membraneassociated PKC
Integral (peripheral)
~ 4 1
~ 4 1
(NP-40), both neutral detergents, have been used to solubilize the membrane-associated PKC [46,57]. Nonionic detergents are usually considered to result in less damage to enzyme activity. However, concentrations tenfold higher than the detergents’ critical micelle concentration (CMC) are required for total solubilization [ 1051. Upon solubilization, it is difficult to remove the contaminating detergent in order to biochemically analyze the activity of the enzyme. An additional factor to consider is that the highest grade and purity of detergent should be used. An example of this can be seen in that Triton X-100 can act as an inhibitor of PKC [76]. In contrast, Triton X-100 has been used successfully in the detergent micelle assay system for characterizing many of the kinetic properties of this enzyme [38]. In the case of membrane-associated PKC (pPKC), we have found that the concentration of detergent can be reduced when the membranes are treated with a combination of two different types of detergent. Generally, ten times the CMC is required for Triton X-100 solubilization of pPKC. A combination of three times the CMC of Triton X-100 and the charged detergent, CHAPS, solubilizes pPKC in an equivalent manner (Lester and Shmeeda, unpublished data).
Membrane-associated protein kinases and phosphatases
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A detergent not often used due to the manipulations required in its use is a Triton X-100 analog, Triton X-114 [13]. It is unique in that it differentially solubilizes peripheral and integral (transmembrane) membrane proteins. The transmembrane proteins partition into the denser detergent phase, while the peripheral proteins associate with the aqueous phase. So it can also be used as a partial purification step. In addition, separation of the peripheral proteins into the aqueous phase has a less perturbing effect than their being in the detergent phase. We have successfully applied this well-described technique [ 131 to purify the membrane-associated protein kinase of the parasite, Leishmania major (Lester and Jaffe, unpublished observations). Another established procedure for extraction of specific membrane proteins requires the use of chaotropic agents [32]. An example of the use of them can be seen in relation to the solubilization of PKC activity. A variable portion of the cellular PKC activity can be extracted as a soluble fraction in a buffer containing high concentrations of divalent cation chelators 1561. These types of reagents (EGTA, EDTA) have long been considered as capable of stripping certain extrinsic membrane proteins [115]. Considering that there have been two reports of soluble PKC copurifying with associated lipids, this would suggest that the so-called “cytosolic” PKC may be associated with membranous or lipid structures [25,661. The receptor-associated kinases are soluble upon phosphorylation of their receptor substrate [40,85]. Thus, no chemical treatment is necessary to extract them. Activation is dependent upon binding of the ligand. It is not certain whether the enzyme is associated with the receptor prior to ligand activation of the G-coupled receptor [85]. A promising, less invasive, technique has been applied to purify pPKC. Originally developed by Shinitzky and colleagues [27], it has been found to extract pPKC with relatively high specific activity [68]. The technique relies on applying high pressures ( > 1,000 atm) to isolated membranes in a French press pressure cell. Specific peripheral membrane proteins are “squeezed” out of the membrane with their associated lipids [27]. The released PKC activity was found to be in two different forms [83]. The characteristics of this enzyme are discussed further in the section on Ca2+/phospholipid-dependent protein kinase, below. However, not all peripheral proteins are released; the membrane-associated Leishmania1 protein kinase could not be solubilized by pressure (Lester and Jaffe, unpublished observations).
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Ident@cation of phosphoamino acid residues To determine the amino acid (serine, threonine, tyrosine) phosphorylated or dephosphorylated, the 32P-labelledsample is acid hydrolyzed and the product examined by high voltage electrophoresis [69]. The phosphotyrosine can also be identified by antiphosphotyrosineantibodies [20] that are commercially available.
Membrane-Associated Protein Kinases For purposes of this review, the membrane-associated protein kinases will be divided into three major groups, with one being subdivided into four other classes (see Table I). The manner with which each of these protein kinase species associates with the membrane bilayer is presented as a schematic in Fig. 1.
Second messenger-dependent protein kinases These can be divided into the cyclic AMP-(PKA), the Ca2+/calmodulin-(CAM-PK) and the Ca2+/phospholipid-(PKC) dependent protein kinases. The first two are generally considered to be soluble or cytosolic.
PKA PKA is made up of two polypeptide subunits: the regulatory subunit which binds cyclic AMP, and the catalytic subunit which phosphorylates the substrates (for a review see [9]). There are two forms of this enzyme, type I and type 11. Type I1 PKA is often found to be richer in membrane fractions than the type I [45,75,95,110]; however, the significance of this differential distribution is unknown [9]. In brush border membranes, upon activation of the type I1 PKA, the catalytic subunit phosphorylates its own regulatory subunit and is released into the cytosol to phosphorylate other substrates. The phosphorylated regulatory subunit remains associated with the membrane for some reason not yet established [36].
CAM-PK The sequence of neuronal CAM-PK has been shown to be considerably homologous with the major postsynaptic density protein (mPSDP) , a structural protein considered to be associated with the membrane [93].
Membrane-associated protein kinases and phosphatases
265
EXTRA
2a
2b INTRA Membrane-associated protein kinases
Fig. 1. Categories of membrane-associated protein kinases. The different classes of membrane-associated protein kinases listed in Table I are presented schematically. The shaded region represents the hydrophobic interaction or a portion of the protein that is in contact with the hydrocarboncore of the bilayer. 1, receptor kinases; 2a, second messenger-dependent protein kinase such as PKC; 2b, receptor-associated protein kinase which is released into the cytosol upon its action; 2, the myristoylated (thick line penetrating the bilayer) oncogene tyrosine kinases; 2d, second messenger-independent protein kinases such as the casein kinases; 3, ectokinase. Extra, extracellular medium; intra, intracellular compartment.
The CAM-PK is considered to be a modified version of the mPSDP [53]. The significance of this association between CAM-PK and mPSDP is not understood.
PKC PKC is unique as a second messenger-dependent protein kinase as it is considered to be active only when associated with the membrane [ 111. Based on in vitro assays, PKC is proposed to be active when associated
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with PS, DAG and Ca2+ [11,56]. When in the cytosol, it may associate with cytoskeleton andlor other intracellular organelles [49,62], a function that is not presently understood. PKC activation is generally considered to occur when there is a change in the distribution of soluble and particulate PKC activities. Recent studies using the high pressure extraction procedure previously described suggest that there are two forms of membrane-associated PKC, one persistently active and a second inactive [84]. The inactive form is proposed to be activated upon release of DAG. The ratio between these two membrane-associated forms appears to depend upon the lipid microenvironment in which they are associated. In fact, the purified cytosolic species has been shown to have lipids copurify with it, suggesting that it may also be associated with some intracellular lipid structure [62]. The intracellular behavior of PKC is further complicated in that at least nine different isozymes, each with unique Ca2+ and phospholipid activity dependence, have been identified
~61. Tyrosine k i m e receptors (TKRs) There is considerable homology between the various growth factor receptors, and they can be crudely characterized as in Fig. 1. Activation of the growth factor TKRs occurs upon binding of their specific ligand. This results in autophosphorylation at a specific tyrosine residue located on the cytosolic side of the receptor. This is thought to be an important step in the mechanism of signal transduction by these receptors [ 1161. The significance of the TKR activation is not clear. It autophosphorylates at five tyrosine residues [44]. Autophosphorylation of the EGF-TKR is important for dimerization of the receptor, and it is also considered to play an important role in its self-regulation [ 1131. PKC is activated upon activation of a phosphatidylinositol (PI)-specific phospholipase C (PLC). In certain cells the PI-PLC is activated upon activation of the EGF-TKR, which phosphorylates a tyrosine residue of PI-PLC [114]. The EGF receptor is then phosphorylated at a threonine site by PKC [26], resulting in its inactivation [34]. This demonstrates an important cellular interaction between two membrane-associated protein kinases (Fig. 2). The truncated form of the EGF receptor, v-erb, is the TKR without the ligand binding site. It is associated with the membrane. It results in a constitutively active, ligand-independent receptor [55]. This EGF receptor form results in altered specificity in signal transduction [74]. It has been shown to induce acute leukemia in erythrocytes and to transform fibroblasts and hematopoietic cells in vitro [35].
Membrane-ussociated protein kinases and phosphatases
267
Fig. 2. Interactions between two membrane-associated protein kinases. Upon binding of EGF to its receptor, the receptor tyrosine kinase is activated which phosphorylates a tyrosine residue on phospholipaseCr (PLC). The phosphorylatedPLC is active, releasing the neutral iipid diacylglycerol which binds to and activates the Ca2+/phospholipiddependent protein kinase (PKC). The active PKC phosphorylates a threonine residue on the EGF receptor resulting in its inactivation (see [116]).
G-protein receptor-activatedprotein kinases The best studied examples of the G-protein receptorcoupled protein kinase are the rhodopsin kinase and the &-adrenergic kinase (@-ARK) [40,85]. These kinases are second-messenger-independent serine and threonine kinases that are associated with their specific substrate at multiple attachment sites [85]. The ability of the kinase to phosphorylate its receptor depends on the conformation of the sequence of the specific phosphorylation site [ 1001. Upon phosphorylation of its membraneassociated receptor substrate, the kinase dissociates and is released into the cytosol. In the case of the rhodopsin kinase, this may involve kinase autophosphorylation [ 161. As for P-ARK, it phosphorylates the agonistoccupied form of the receptor. The receptor is desensitized leading to its uncoupling from the G-protein [40]. This kinase is also capable of phosphorylating the M2-muscarinic receptor in vitro [61].
Myristoyluted oncogene tyrosine protein kinases The best known and first identified oncogene protein kinase is the pp60v-sm.It serves as a good model for myristoylated tyrosine kinases
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Fig. 3. Fatty acid association of a protein kinase and a protein phosphatase with the plasma membrane. The myristoylated oncogene tyrosine protein kinase, pp60v-8rc, requires association with a 32 kDa membrane protein to form a stable protein kinase-membrane complex (see [90]). The serine/threonine protein phosphatase is myristoylated on its a-subunit but also undergoes interactions with acidic phospholipids and subsequent binding of Ca2+. The fatty acid/acidic phospholipid/Ca2+ interaction results in a stable complex (see [99]).
such as the p56Ick. This tyrosine kinase is predominantly membranebound [90]. Expression of the pp60v-sm,the protein product of the v-src gene, transforms cells in vivo [33]. Under normal situations it is found associated with the perinuclear membrane. Viral transformation results in its transport to the plasma membrane. It is myristoylated during this transport process, which promotes its association with the plasma membrane. This kinase-membrane complex facilitates association with a 32 kDa plasma membrane protein receptor which further stabilizes this complex [91] (Fig. 3). The tyrosine kinase p56'ckassociateswith the CD4 receptor. This process is initiated by viral infection. The association of the myristoylated p56lCkto CD4 is regulated by autophosphorylation of the kinase [ 1111.
Ectokinast?s It is well established that ATP is found in the extracellular milieu; thus, it may not be surprising that there are protein kinases associated with the extracellular membrane (Fig. 1). These have been extensively characterized in cells of neuronal origin [29,109]. They are proposed to play a role in such processes as long-term potentiation and neuronal differentiation [28]. They have also been found at the surface of platelets where coagulation factors can be phosphorylated [30]. Another interesting ectokinase has been identified on the parasite Leishmania [69]. This
Membrane-associated protein kinases and phosphatases
269
is capable of phosphorylating complement protein which plays an important role in the interaction between the parasite and its host cell [42]. An ectokinase from HeLa cells has been found to be solubilized upon phosphorylation of its substrate [88].
Second messenger-independent serinekhreonine protein kinuses Casein kinases There are numerous reports of casein kinases being found in particulate fractions of various cell and tissue types [7,88,101]. The role of these kinases and their differential distribution is not understood. The lack of intensive research on these enzymes is probably because there is no evidence of hormonal regulation. In erythrocytes there is a phosphatidylinositol-4,5-biphosphatekinase which modulates the function of numerous erythrocyte membrane proteins, including the protein 4.1 and ankyrin, ultimately affecting the shape of the cell [7].
Other serinehhreonine kinuses This is a diverse collection of protein kinases with varying biochemical properties. One such protein kinase was shown to be manganese-dependent, in contrast to other protein kinases which are M?+-dependent [102]. Thus, this kinase would use MnATP as a substrate in contrast to most other kinases which use MgATP. This Mn2+-dependent kinase associates with and phosphorylates the insulin receptor [ 1021.
Membrane-AssociatedProtein Phosphatases There are many outstanding reviews of protein phosphatases [6,21, 991. In the past the data on this aspect of posttranslational modification were relatively limited. Two recent developments have greatly increased the interest and research in this area: (1) the identification of tyrosine phosphatases and their role in cellular differentiation and proliferation, and (2) the discovery of specific inhibitors of cellular phosphatase activity such as okadaic acid. The membrane-associated phosphatases can be divided into two classes: the serinelthreonine protein phosphatases (PrPs) and the tyrosine protein phosphatases (PTPs) (Fig. 4). The different types of each of these classes and their biochemical properties are summarized in Table 111.
mh4 M2'
PrP 2C
pPTP Non-receptor (protease-activated) rPTP (CD45, LAR)
Orthovanadate
Ca2+/calmodulin, Acidic phospholipids
PrP 2B
Tyrosine phosphatases
Phenothiazines
Polycations
PrP 2A
Divalent cations, anionic polycations Orthovanadate
Cell surface Ligands (?) Soluble low M, factors
Okadaic acid [0.2 nM] Calyculin A
Okadaic acid [20 a ] , Calyculin A
GSK-3
PrP 1
Inhibitor
Serineltbreonine phosphatases
Activator
Subcategory
Category
Membrane-associated protein phosphatases
TABLE III
[22,108]
[991 [94,107]
Band 3 (?) ~~56'~'
[991
[87,991
[24,991
[99,112]
Refs.
?
GAP43
PKC, pyruvate dehydroge w e
Phospholamban
Substrate
Membrane-associated protein kinases and phosphatases
EXTRA
n
2 71
INTRA Fig. 4. A schematic representationof the membrane-associatedproteinphosphatases. The different classes are summarized in Table III. The shaded regions indicate the portions of the proteins that are in contactwith the hydrocarbon core of the bilayer. 1 , PrP 1, PrP 2A, PrP 2C; 2, PrP 2B; 3, pPTP; 4, rPTP.
Serinekhreonine phosphatases Protein phosphatase 1 (Prp 1) PrP 1 binds to microsomes and can be released upon treatment with detergent [112]. Its conversion from a cytosolic to a particulate form appears to involve some form of protein-protein interaction, as it is sensitive to proteolysis [97]. The PrP 1 in cardiac sarcoplasmic reticulum may play a role in Ca2+ transport as it dephosphorylates the Ca2+ATPase activating phosphoprotein, phospholamban [59]. PrP 1 also increases the down regulation of L-type Ca2' channel activity 1431. A PrP 1 species purified from bovine brain membranes is phosphorylated by PKC in vitro, which has an effect on its activity (501.
Protein phosphutase 2A (PrP 2A) PrP 2A is largely cytoplasmic; however, a membrane-associated form has been found associated with bovine kidney mitochondria [24]. Particu-
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late forms have also been found associated with the membranes of plant cells [73] and the single-celled Paramecium [58].
Protein phosphatase 2B (PrP 2B) PrP 2B, commonly referred to as calcineurin, is a Ca2+/calmodulinactivated enzyme which has been shown to associate with the membrane in a Ca2+-dependent manner [87] (Fig. 4). It is capable of dephosphorylating phosphoserine and phosphotyrosine residues [99]. In some cases it cannot be fully extracted by detergents, which suggests that some of the enzyme may be associated with the cytoskeleton [3].
Protein phosphatase 2C (PrP 2C) PrP 2Cs are generally considered to be cytosolic enzymes [99]. However, one specific PrP2C has been partially purified from the membranes of the single celled Paramecium [58].
Tyrosine protein phosphatases (PTPs) The PrP 2A, B and C serine/threonine phosphatases are all capable of dephosphorylating phosphotyrosine residues and are all inhibited by Zn2+. However, this divalent cation is not capable of inhibiting dephosphorylation of such proteins as the autophosphorylated EGF receptor [99,116]. 'This led to the discovery of the PTPs [99,107,108]. They have been implicated as playing a major role in cellular responses to hormones and growth factors.
Particulate protein tyrosine phosphatases ( P P P ) The pPTPases are generally cytosolic or soluble. However, membrane-associated forms have been found in rabbit kidney [94], human placenta [86], rat spleen [ 1031, and erythrocytes [ 181. The final 20 amino acid residues of the carboxy-terminal segment of the T-cell PTP is highly hydrophobic (Fig. 4),and it is considered to have a role in its subcellular localization [107]. It does not play a role in the regulation of the enzyme. In vivo substrates have not been identified for these enzymes.
Receptor protein tyrosine phosphatases (rPTP) The PTP 1B isolated from human placenta was found to have considerable sequence similarity to the leukocyte common antigen, CD45 [22].
Membrane-ussociated protein kinases and phosphatases
2 73
This introduced the concept of the receptor PTPs. The extracellular domain of the PTP 1B is heavily glycosylated and has a single membrane spanning domain similar to the CD45 (Fig. 4). Another rPTP, termed the LAR, is considerably homologous with the neural adhesion molecule, N-CAM [98]. CD45 is considered to play a role in T-cell activation [31].
Inhibitors and Activators Inhibitors and activatorsof protein kinases and phosphatases have been used for two purposes: identification of the enzyme type, and of the function of the enzyme in cellular regulation. Some of the more common ones are listed in Tables I and 111. A general problem of these compounds is their lack of specificity. For many of the kinases the activation mechanism is generally specific at the cellular level. However, in the caseof the second messenger-dependent kinases, the ability to activate them in vivo depends on the penetrability of these compounds across the plasma membrane to interact with the target enzyme. Many of these compounds effect the membrane bilayer during this process [31]. Probably the best known exogenous activator of a membrane-associated protein kinase is a group of tumor promoting agents called the phorbol esters [ 11,1181. These are highly lipophilic agents that penetrate the membrane and then, according to the distribution of fluorescently labelled analogs, label membrane and intracellular organelles, excluding the nucleus [89]. The predominant phorbol ester protein receptor is considered to be PKC; however, there are other phorbol ester binding proteins [lo]. The phorbol esters have many cellular effects [5];whether all of them are due to PKC activation remains a controversial issue. None of the serinelthreonine protein kinase inhibitors are specific. This is partly due to the site of action of the majority of these inhibitors, which is the ATP binding domain in the catalytic region of the enzyme. This region of the primary protein sequence has been shown to be considerably homologous for this class of proteins [37]. Tyrosine kinase modifying reagents have relatively limitedspecificity. Compounds known as the tyrphostins are being modified and screened to develop more specific pharmacological agents [72]. Some of the protein kinases have not been significantly characterized; thus specific inhibitors have not yet been reported (Table I). As for the PrPs, the cellular activation mechanisms have been used for classification of these enzymes. The activity of PrP 1 and 2A in vivo depends on the phosphorylation state of two protein inhibitors. The
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PrP 2B is activated by Ca2+ and calmodulin, while regulation of PrP 2C is not understood (see [99]). As to examining their cellular function, the recent discovery of okadaic acid as a PrP inhibitor has greatly promoted the study of these enzymes [23]. Okadaic acid inhibits PrP 2A at subnanomolar concentrations. Inhibition of PrP 1 occurs at concentrations of 100-fold greater, while much higher concentrations have been shown to inhibit PrP 2B in certain cells [23]. Okadaic acid has not been shown to have any effect on PrP 2C. Thus, the concentration of okadaic acid that inhibits cellular phosphatase activity gives an indication of the types of phosphatases present and active in the cell. Specific inhibitors and activators of the PTPs are not known at present, but with the extensive research efforts devoted to these enzymes, it is expected that suitable pharmacological agents will be found. Nonspecific modulators are listed in Table 111.
Substrates Examination of protein kinase and phosphatase activities in various cellular tissue sources has suggested many phosphoprotein substrates, identified as bands or spots on one- and two-dimensional polyacrylamide gels. Some of these bands or spots have been purified and the proteins sequenced and their genes cloned (Tables 1and 111). A physiological role for some of these proteins has been provided. For example, the PKC substrate known as the MARCKS protein has been found to play an important role in cytoskeletal organization [106]. PKC has been shown to phosphorylate other membrane substrates such as the Na+ channel [82]. The effect on channel activity is not clear. The Ca2+/calmodulindependent protein kinase substrate PDS is also thought to play an important role in microtubule functioning [93]. In the case of the receptor tyrosine kinase receptors, autophosphorylation of the receptor is established. However, the significance of this action is still controversial [116]. Some of these receptors have been shown to activate serine/threonine protein kinases that appear to be closely associated with the receptor [51,116]. The PDGF and EGF receptors, upon activation, undergo an association with a number of important regulatory factors which has given rise to the term “signal transduction unit” (Fig. 2) (see section on tyrosine kinase receptors, above). This process is dependent on receptor autophosphorylation and leads to phosphorylation of other substrates [ 114,1161.
Membrane-associatedprotein kinases and phosphatases
2 75
A. EXTRA
CD45
B. EXTRA
C. EXTRA
M ,.,.,.;> ..>,.,;?.+ .,
a....,.
Fig. 5. Interactions between a membrane tyrosine kinase and tyrosine phosphatase. The myristoylated oncogene tyrosine protein kinase ~ ~ 5 is6 considered " ~ to be inactive when phosphorylated. Binding of a ligand to the CD45, a transmembrane receptor tyrosine phosphatase, results in activation of its phosphatase activity. This phosphatase dephosphorylates ~ ~ 5 6resulting ' ' ~ in activation of its tyrosine kinase activity. A number of substrates associated with the membrane are phosphorylated, and cellular proliferation results at a later stage (see [99,107,108]).
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The receptor-associated receptor kinases are capable of phosphorylating other receptors that undergo similar regulation pathways [85](e.g. , the @-ARKphosphorylates 0-AR, metarhodopsin 11, and the M2 muscarinic receptor [61]). The myristoylated oncogene protein kinases are capable of phosphorylating many important substrates, but there is evidence indicating that the phosphorylation of these proteins may not be critical as it does not play a role in the transduction process [go]. Substrates for other membraneassociated kinases have not been well established. Few membrane-associated substrates have been clearly established as in vivo phosphatase substrates. One of the most interesting and potentially important examples is the CD45. In T-lymphocytes that do not express CD45, the tyrosine kinase p561ckcould not be activated [77]. Additionally, when CD45 was added to the p56Ick in vitro, tyrosine kinase was increased. This indicated that CD45 phosphatase activity plays an important role in pp56lCkactivation and subsequent cellular proliferation. However, this is in conflict with the increase in cellular tyrosine kinase activity upon p56lCkactivation. It has been proposed that a tyrosine site on CD45 may be phosphorylated, possibly by the p56lCk,resulting in feedback regulation (inhibition) of rPTP activity (see [108]). A possible model for this interaction is presented in Fig. 5.
Lipid Regulation of Membrane-Associated Protein Kinases and Phosphatases Generally there is a poor understanding of the interaction(s) between the membrane bilayer and the membrane-associated protein kinases or protein phosphatases. This is not unexpected due to our limited understanding of protein-lipid interactions. The membrane receptor kinases and phosphatases are generally not considered to be lipid regulated as they are transmembrane spanning proteins. However, it may not be surprising to find that they have specific annulus lipid compositions [32]. Undoubtedly, the most famous and best understood of a membraneassociated protein kinase is PKC. PKC is activated by the lipid diacylglycerol in the presence of the acidic phospholipid PS and Ca2+ [ 111. There is some evidence of cooperativity of the PS-dependence [39,78]. However, the types of analyses required to establish cofactor cooperativity for lipid-dependent enzymes, i.e., Hill plots, are not trivial [l]. It is also
Membrane-associated protein kinases and phosphatases
2 77
difficult to determine which are the physiological lipids that activate the enzyme for the following reasons: 1. There are at least nine different isozymes each with different biochemical characteristics. 2. The different assay systems used have their own unique characteristics (see section on in vitro protein kinase analyses, above). 3. The exogenous substrates used in these in v i m assays have their own cofactor dependence [81. 4. There is a lack of biophysical data regarding the protein and its lipid interactions [ 1181. 5. The in vivo activation process is not clearly understood. 6. The enzyme can be activated by cis-fatty acids alone, i.e., in the absence of DAG and PS [75]. The generally accepted models for PKC activation are subject to the difficulties described in 1-3. A recent review on the biophysical studies relating to lipid regulation demonstrates the shortcomings of the techniques used for these studies [ 1181. Biophysical and biochemical studies of the membrane-associated and cytosolic species suggest that the subcellular localization of PKC depends on the lipid environment [66,83]. This is supported by in vitro biochemical studies showing that the enzyme can be activated by different membrane bilayer compositions [69,70]. There is also some evidence for hydrophobic and electrostatic interactions [ 151, which are considered to be important in order for the enzyme to be converted from a loosely associated membrane protein (extrinsic) to a tightly associated form (integral). Another example of lipid regulation of an enzyme active in phosphorylation/dephosphorylation pathways is PrP 2B, calcineurin. It physically associates with acidic phospholipids. This interaction is further enhanced by the addition of Ca2+ [87] (Fig. 4). These lipids also significantly stimulate the dephosphorylating activity. Activation of PrP 2B by phosphatidylinositol and calmodulin has been shown to be synergistic [47]. In addition, PrP 2B is myristoylated at the N-terminus of its A-subunit [2]. But it is likely that, as for pp60'-sm, fatty acylation is not sufficient to account for the membrane localization (Fig. 3). While in the case of the a membrane-association protein (32 K) appears to be involved in membrane anchoring, the PrP 2B appears to undergo strong electrostatic interactions with phospholipids which appear to be further stabilized by the binding of Ca2+ (Fig. 3).
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There are other scattered examples of lipid-regulated kinases and phosphatases, e.g., pig brain membrane protein phosphatase [ 1171, and the insulin receptor kinase [ 1041. These studies demonstrate that it is the acidic phospholipids which are most potent in exerting some regulatory effect, These phospholipids are capable of inducing both hydrophobic and electrostatic effects.
Conclusions
In most studies of membrane-associated protein kinases and phosphatases, the emphasis has been on the protein and its activity. The lipid contribution, except in the case of PKC,has been neglected. While it is often acknowledged that the lipid bilayer may not have a significant function in regulating the activities of these enzymes, it cannot be doubted that it has an important role in their subcellular localization. This contribution of the membrane would be expected to have a major impact in determining which substrates can be modified by these regulatory enzymes and subsequently exert their ultimate cellular effect. The plasma membrane and membranes of intracellular organelles are vital players in cellular compartmentilization. This reason alone substantiates the need for continuing efforts to elucidate these protein-lipid interactions in the context of protein kinase and phosphatase interactions with membrane lipids. References 1 Adams, G. and M. Delbruck, (1968), In: Structural Chemistry and Molecular Biology, A. Rich and N. Davidson (Eds.), W.H. Freeman, San Francisco, p. 198. 2 Aitken, A., C.B. Klee and P. Cohen, (1986), Eur. J. Biochem. 139:663. 3 Alexander, D.R., J.M. Hexham and M.J. Crumpton, (1988), Biochem. J. 256:885. 4 Alkon, D.L., S. Naito, M. Kubota, C. Chen, B. Bank, J. Smallwood, P. Gallant and H. Rasmussen, (1988), J. Neurochem. 51:903. 5 Ashendel, C.L., (1985), Biochim. Biophys. Acta 822:219. 6 Ballou, L.M. and E.H. Fischer, (1986), In: The Enzymes, Vol. 17, P.D. Boyer and E.G. Krebs (Eds.). Academic Press, New York, p. 312. 7 Bazenet, C.E., J.L. Brockman, D. Lewis, C. Chan and R.A. Anderson, (1990), J. Biol. Chem. 265:7369. 8 Bazzi, M.D. and G.L. Nelsesteun, (1991), Biochemistry 26:1974. 9 Beebe, S.J. and J.D. Corbin, (1986), In: The Enzymes, Vol. 17, P.D. Boyer and E.G. Krebs (Eds.). Academic Press, New York, p. 43. 10 Bell, R.M. and D.J. Burns, (1991), J. Biol. Chem. 266:4661. 11 Bell, R.M., (1986), Cell 45:631.
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Biomembranes Edited by Meir Shinitzky Copyright 0 VCH VerlagsgesellschaflrnbH, 1995
CHAPTER
6
Phospholipases in Signal Transduction DANIELA CORDA, MARC0 FALASCA, MARIA DI GIROLAMO TIZIANA CACCIAMANI
and
Laboratory of Cellular and Molecular Endocrinology, Istituto di Ricerche Farmacologiche “Mario Negri, ” Consonio Mario Negri Sud, 66030 Santa Maria Imbaro, Chieti, Italy
Contents Abbreviations Introduction Modulation of Phospholipase C Isoforms Localization and substrate specificity Stimulatory regulation of PLC Inhibitory regulation of PLC Modulation of PLA, Molecular forms of PLA, Stirnulatory regulation of PLA, Inhibitory regulation of PLA, PLA, activity and ras-induced transformation Biological activity of PLA, metabolites Phospholipase D Conclusions Acknowledgements References
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Abbreviations AMF ARF CAMP DAG EGF G protein GAP GDP GDPOS Gi Gpi GPS GroPIns4P Gs Gt GTP GTPyS Ig InsP, LPA LPC LPI MAP kinase PA PDGF PKC PLA 1 PLA, PLC PLD PtdCho PtdIns PtdInsP PtdInsP, PtdSer
-
-
-
-
-
-
-
mixture of AlCl,, MgCl, and N a F ADP-ribosylating factor adenosine 3',5'-cyclic-monophosphate diacylglycerol epidermal growth factor guanine nucleotide binding protein GTPase activating protein guanosine-5'-diphosphate guanosine 5'-0-(2-thiodiphosphate) inhibitory G-protein, coupled to adenylyl cyclase inhibitory G-protein, coupled to phospholipases stimulatory G-protein, coupled to phospholipases glycerophosphoinositol4phosphate stimulatory G-protein, coupled to adenylyl cyclase transducin guanosine-5 '-tr iphosphate guanosine 5'-0-(3-thiotriphosphate) immunoglobulin
inositol-1,4,5-trisphosphate
lysophosphatidic acid lysophosphatidylcholine lysophosphatidylinositol microtubule-associated protein 2 kinase phosphatidic acid platelet derived growth factor protein kinase C phospholipase A, phospholipase A, phospholipase C phospholipase D phosphatidylcholine phosphatidylinositol phosphatidylinositol-4-phosphate
- phosphatidylinositol-4,5-bisphosphate
- phosphatidylserine
Phospholipases in signal transduction
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Introduction Besides their important structural function of forming the lipid bilayer, membrane phospholipids are able to modulate the activity of membrane proteins and are essential components of several signalling pathways. Receptors for hormones, growth factors and neurotransmitters may be coupled to different phospholipases (phospholipase A,, C and D) acting on membrane phospholipids to form second messengers such as diacylglycerol, inositol trisphosphate, arachidonic acid and lysophospholipids (reviewed in [ 1-81). The mechanism of activation of the different phospholipases can be direct, involving the molecular interaction between the receptor and the enzyme (e.g., tyrosine kinase receptor and PLCy), it can be mediated by a family of membrane-associated G proteins which are directly coupled to the enzyme (e.g., the activation of PLCP by a, and Pr subunits), or it can be due to the receptor-mediated increase in cytosolic Ca2+, which leads to an increase in the activity of the enzyme (e.g., PLA,). Several reviews have recently appeared which summarize the various aspects of phospholipase structure, function and regulation [ 1, 7-19]. This chapter focuses mostly on recent findings that outline the role of the subunits of G proteins in the modulation of phospholipases. G proteins are a highly homologous family of heterotrimers which bind GTP and consist of a,/3 and y subunits [20-251. The activation of a G protein results in the exchange of GDP for GTP on the a-subunit followed by the dissociation of the a-GTP-bound subunit from By. Much attention was initially dedicated to the a-subunits that were supposed to be the only active components in coupling receptors to the different effectors. At least 18 a-subunits have so far been identified [21,22,25]. The G protein heterotrimer usually takes the name of the specific asubunit that composes it. Thus, G, is the G protein that upon hormonal stimulation releases the a,stimulator of adenylyl cyclase activity and is the substrate of the cholera toxin-induced ADP-ribosylation [20,24,25]. Gi was initially identified as the negative regulator of adenylyl cyclase, which was also a specific substrate of pertussis toxin [20,24,25]. Later, the Gi family was related to the regulation of phospholipases and ion channels [20,24-261. It is now clear that the fly-subunits also play a role in modulating effectors such as adenylyl cyclase, ion channels and phospholipase A, and C [20,22,26-281. Molecular cloning has further enabled the division of a-subunits into four families (Gi, G,, G, and
286
D. Corda et al.
GI2)according to their sequence homology [21]. Moreover four p- and six y-subunits have so far been identified by molecular cloning [22]. The structure and function of the different G proteins is discussed in detail elsewhere in this book (see also Chapter 4 for a comprehensive list of references).
Modulation of Phospholipase C One of the most important phospholipases in signal transduction is phospholipase C (PLC) that hydrolyze the inositol lipid phosphatidylinositol-4,5-bisphosphate(PtdInsP,) to form inositol- 1,4,5trisphosphate (InsP3) and diacylglycerol (DAG) [3, lo]. Interest in the metabolism of the inositol lipids arose from the initial observations made by Hokin and Hokin [29] who showed that in agonist-stimulated exocrine pancreas, the increase in lipid radiolabelling associated with secretion was mostly evident in phosphatidylinositol (PtdIns) and phosphatidic acid (PA). Although this finding was extended to other systems and proven to be a general pattern of cell response to hormone stimulation, its biological significance remained obscure until Michell [30] proposed that the activation of the inositol lipid hydrolysis was related to increase in intracellular Ca2+. The metabolism of inositol lipids was elucidated in the following years. It was shown that the agonist-dependent PLC was specific for PtdInsP, and that two second messengers originated from its action: InsP, and DAG [2]. InsP, was identified as the agent capable of mobilizing Ca2+ from intracellular stores [2,3 11. Recently, the purification and cloning of the InsP, receptor and its localization in specific intracellular compartments has contributed to the understanding of the mechanism of action of InsP3 in regulating Ca2+ mobilization and homeostasis [3,32]. The other product of the PtdInsP,-specific PLC, DAG, has been shown to activate a family of protein kinase C isoenzymes in a transient manner, whereas the sustained activation of these enzymes has been related to the DAG generated from phosphatidylcholine (PtdCho) by the action of phospholipase D [4,5,8,19,33,34]. Numerous receptors have been reported to be coupled to PLC [ 13,141. Based on the characteristics of the receptor involved and on the identification of different isoforms of PLC (see below), it is now possibie to identify at least two major pathways of regulation: one related to G-
Phospholipases in signal transduction
287
protein-linked receptors, the second related to proteins having intrinsic tyrosine kinase activity (Table I; [ 12,14,15,35]). In the following we will focus only on the G-protein-mediated modulation; the reader is referred to recent reviews for the tyrosine kinase-dependent activation of this enzyme [ 12,14,15,35].
Phospholipase C isoforms Several forms of PtdInsP2-specificPLC have been identified in various tissues by either purification or immunological analysis of purified proteins or molecular cloning [ 10,151. The currently accepted nomenclature proposed by Rhee, which divides the PLC isoforms into three families - p, y and 6 - is based on the comparison and homology of the deduced amino acid sequences (Table 11; [ 10,121). The range of molecular masses for the three PLC families are 150-154 kDa for PLCP, 145-148 kDa for PLCy and 85-88 kDa for PLCG [ 10,151. There are two additonal PLC families purified and partially characterized, although not yet cloned: the a and E with M, of 57-70 kDa and 85-88 kDa, respectively [12,14]. It has not yet been clarified whether these lower M, species are proteolytic fragments of the /3, y or 6 families, based on the sensitivity of PLC in general, and PLC-6, in particular, to proteolytic cleavage [ 12,14,36,37]. Within the sequence of the p, y and 6 families, there are two regions named X ( 170 amino acids) and Y (- 240 amino acids), which share homologies of 70% and 60%, respectively [15]. These regions which might represent the catalytic domains are separated by a short sequence in PLCP and PLCG (50-70 amino acids) and by a longer sequence in PLCy (- 400 amino acids) [ 151. This domain of PLCy contains highly conserved regions homologous to the src domains SH, and SH, [ 15,381. The SH, and SH, are involved in protein-protein interactions [38-401. In particular, the SH, domain interacts with specific phosphorylated tyrosine in the cytosolic portion of the tyrosine kinase receptors (EGF, PDGF, etc.) [41-43]. SH, domains are shared by several proteins that are substrates and are able to directly interact with growth factor receptors (GTPase activating protein (GAP), PLCy, PI3 kinase) [38-401. The PLCP and PLCG families differ also in the C terminal portion following the Y region; these portions are composed of 450 and 10 residues, respectively [12,15]. Each PLC family is formed by several subtypes. For example, the three isoforms of PLCp that have been identified so far
-
D. Corda et al.
288
TABLE I
Agonists stimulating PLC activity in different cell types Agonist Adrenaline
Pertussin toxin'
+
+/-
-
Angiotensin II
+ +-
ANP
+ +
ATP
+/+/-
+ + Bradykinin
Endothelin
Enkephalin
RlMCT cells Endothelial cells HL60 (differentiated) cells Fibroblasts HL60 cells FRTL-5 cells Rat renal cortex cells
-
1321N1 (astrocytoma) cells Myometrium PC12 cells (M5) L cell& (M2) CHO cellsb (MI) SBcellsb
+ EGF
Renal mesangial cells Adrenal glomerulosa cells Liver Pituitary cells (anterior) cells Vascular smooth muscle cells 73 15c cells
Pulmonary endothelial cells Neuroblastoma-glioma cells Renal (MDCK) cells 1321N1 (astrocytoma) cells NG108-15 cells Sensory neurons (DRG)
Cholecystokinin
Fat cells Adipocytes (brown) FRTL-5 thyroid cells Liver (qARs)CHO cellsb
+ +
-
+/-
Carbachol
Cell type
-
Pituitary cells
+ +-
Liver Rat hepatocytes 3T3 fibroblasts
+
+/-
Vascular smooth muscle cells Rat mesangials cells C6 glioma cells
+
NG108-15 cells
+/-
Refs.
Phospholipases in signal transduction
289
TABLE I, continued MetLeuPhe
HL60 (differentiated) cells HL60 cells
Basophils Gastrin
Gastric cells
GRH
Pituitary gonadotrophs
Histamine
Astrocytoma cells HeLa cells Pulmonary endothelial cells 1321N1 cells
5-Hydroxytryptamine
HeLa cells Vascular smooth muscle cells Ventricular myocytes
Leukotriene B4
Macrophage
LH
L cellsb
Neuropeptide Y
Sensory neurons
Neurokinin-1
U373MG cells
Oxytocin
Myometrium
PAF
Macrophage Renal mesangial cells
PGE,
Renal mesangial cells
PGF2,
NIH 3T3 cells
Thrombin
Fibroblasts (CCL39) Platelets Osteosarcoma cells
Thromboxane A,
Platelets Astrocytoma
TRH
Pituitary GH3 cells
Vasopressin
Renal tubular cells Adrenal glomerulosa Liver Glomerular mesangial cells Glomerulosa cells
Note: The table is only a partial list of studies reported so far. More examples can be found in recentreviews [11,13,14]. 'Sensitivity t o pertussis toxin pretreatment. bReceptors overexpressed in the specific cell system.
D. Corda et al.
290 TABLE II
G protein- and receptor-dependent regulation of PLC isoenzymes PLC isoform
Activators
Refs.
PI
Gqm
1671 [73-751 [45,76,78,85] [45,78,79] [851
aq, all
raq, roll1 rCY14,ra16
Pr
02
Pr req, roll], r q 4 ,
[8 1-85] ray16
[78,851
Pr
raq, T a l l , ra16
[45,851 [82,851
71
PTK rEGF PTKrPDGF PTKs TCR in T cells mIgM in B cells CD4-CD5 in B cells IgE in RBL-2H3 cells FcyRI, in U937 cells Fcy, FcyIIIA in NK
t2W ~301 [23 1-2331 [234 PSI ~361 12371 [238-2401
72
PTKs IG in B cells Fcy, FcyIIIA in NK
[241-2431 [238-2401
61
Ca2+
1851
62
?
6,
?
P3
Note: G , CY and @yrefer to the native isolated heterotrimers and subunits: r a refers to the recombinant subunit. PTKrEGF, PTKrPDGF, epidermal growth factor receptor- and platelet-derived growth factor receptor-intrinsic tyrosine kinase; PTKs, soluble tyrosine kinases coupled to the indicated receptor; TCR, T-cell antigen receptor. See text for details and for the rank-order potency of the activators.
are related. to different mechanisms of activation [ 12,14,28,44,45]. The amino acid sequence identity within each family is higher than among families and is found also in regions different from the X and Y domains. Detailed information on the different members of PLC families can be found in several recent reviews [ 10-151.
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Localization and substrate-specificity PtdIns-specific PLC activities have been characterized in the plasma membrane, in the cytosol and in the nuclear membrane of several tissues [45-551. However, most of the PLC isoforms thus far purified are from the cytosol [ 12-14]. Membrane-associated activity can be extracted by high salt, suggesting that the association between PLC and the membrane is mediated by ionic interactions. Although PLCy is predominantly cytosolic, PLCP seems to be equally distributed between the cytosol and the membrane compartments [46,47,51]. Moreover, PLCPl has been shown to be the isoenzyme preferentially associated with the nuclear membrane in Swiss 3T3 fibroblasts and rat liver cells [53-553. All PLC isoforms hydrolyze the three common inositol phospholipids: phosphatidylinositol (PtdIns),phosphatidylinositol-4-phosphate(PtdInsP) and PtdInsP2. In vitro studies showed that the selectivity for PtdInsP, over PtdIns decreases in the order PLCPl> PLCGl> PLCyl [46]. All PLC isoforms appear to utilize PtdInsP2 as the preferred substrate at pM Ca2+ concentrations [46,47,52]. However, the PLCG family can also efficiently utilize PtdIns at mM Ca2+ [46-481. The hydrolysis of PtdIns, PtdInsP and PtdInsP, by PLC isoforms also leads to the formation of cyclic inositol phosphates [56,57]. The physiological role of these compounds has not yet been elucidated, although it has been related in some studies to the control of intracellular Ca2+ levels and cell proliferation [57]. Moreover, it has been proposed that cyclic inositol phosphate is the first product of PLC activity, which is either released or converted to the non-cyclic compound [%I. PLC activity has been reported to be Ca2+-dependent [12,14]. A putative Ca2+ binding site is present on the C terminal part of the Y region where the aminoacid sequence is homologous to the Ca2+ binding domains of protein kinase C (PKC) and phospholipase A, (PLA,) [59]. The pH optimum for PLC activity is 5.2-5.5. Detergents and lipids might affect the enzyme activity (for a review, see [60]). For example, deoxycholate and octylglucoside have been shown to stimulate PLC activity, whereas an inhibitory effect was observed in the presence of Triton X-100 [60]. Membrane lipids can also affect this enzymatic activity; PtdCho inhibits the hydrolysis of inositol phospholipids, whereas phosphatidylserine (PtdSer), DAG and free fatty acids could partially reverse this inhibition [60].
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Stirnulatory regulation of PLC The involvement of a specific G protein (G,, G phospholipase) in the agonist-dependent activation of PLC has been suggested by a series of observations: the PLC activation requires GTP; non-hydrolyzable analogues of guanine nucleotides and aluminum fluoride (a G protein activator) are able to stimulate this activity; and in some cell types, pertussis toxin (which ADP-ribosylates G, and Go, inhibiting their function in this way) prevents PLC activation (reviewed in [61]). A large number of agonists has been shown to modulate PLC activity in a Gp-dependent manner (Table I). Based on the different sensitivities to pertussis toxin pretreatment, two G, proteins appeared to activate PLC in the different cell systems (Table I and [61]). The mechanism by which G proteins sensitive and insensitive to pertussis toxin could modulate the activity of PLC is beginning to be clarified (see below). Two main approaches have been taken: first, the biochemical, based on purification and in vitro reconstitution, and the second based on coexpression of the three elements by cDNA transfection (for reviews see [8,14,25,35]). G protein a-subunits of an apparent M, of 42 kDa, pertussis toxin insensitive and able to stimulate PLC activity, were first purified from bovine liver membranes [62] and subsequently from rat and bovine brain [63,64]. The sequences of these a-subunits were found to be identical to those of two newly identified G protein a-subunits, a, and al which are members of a family of G proteins named G,, characterized by a low homology (<50%) with the known a-subunits [21,62-64]. Accordingly, G, were not recognized by most antibodies available for the known G proteins and showed a cross-reactivity only with the antipeptide antibody raised against residues 160-169 of ail[62]. The G, family comprises five members: G,, G l l , G14,GI5and GI6 [21]. G, and G,,, which share 88% homology, are widely distributed, whereas G15and GI6 are expressed in cells of hematopoietic origin [2 1,65,66]. G proteins have a low affinity for GTP (half maximal activation of PL& requires 4 p M GTPTS), require millimolar concentration of M$+, are inhibited by GDPflS and by excess of fly-subunits and are not substrates for pertussis toxininduced ADP-ribosylation [67,68]. Several studies have confirmed that a, and a l l are the subunits that activate PLC (reviewed in [8,35]). G, protein activated by AIF; was able to stimulate partially purified PLCP, whereas purified By, a0,ail and
,,
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ai2 were completely ineffective [64].Gutowski et al. [69], using an antibody raised against the last 12 amino acid residues of the C-terminal region of a, (X384, which recognizes a, and a l l )could inhibit the bradykinin stimulated PLC activity in NG108-15 cell membranes, the angiotensin 11-effect in rat liver and the histamine stimulation in 1321N1 cells. In turkey erythrocytes, the G protein activating the 150 kDa PLC purified from the same system was identified as a component of the G, family, being recognized by antibodies raised against the C-terminal region of a, and a l l [68,70,71]. This protein was later cloned and identified as a1 [72]. Taylor et al. [73] showed that G, (purified from bovine liver) specifically activates PLCP1, whereas it did not affect PLCy, and PLCG, (all purified from bovine brain). By Mono Q chromatography, it was shown that two proteins, immunologically identified as a, and a l l , are the specific activators of PLCP, in bovine liver [74]. Using purified proteins, Berstein et al. [75] could show that receptor-G-protein-enzyme are the components sufficient for in vitro reconstitution of PLC activity. Thus, PtdInsPz hydrolysis was shown to be agonist- and GTPyS-dependent in vesicles in which purified recombinant M 1 muscarinic receptor, PLCP, from bovine brain and G,,], from bovine liver were reconstituted [75]. Similar results were obtained by a cotransfection approach. Wu et al. [76] showed that transfection of cDNA encoding for a, and a1 in COS-7 cells increased the AIFY-stimulated formation of InsP,. Cotransfection of PLCP, and allla, cDNAs resulted in a more pronounced increase. Instead, transfection with cDNAs for ao,at, a, and a I 2did not affect InsP, formation. Single point mutations that constitutively activate G proteins (gln-209 to leu), resulted in high basal levels of InsP,, which could not be further increased by AIF; [76]. Moreover, in a cell-free system consisting of membranes from transiently transfected COS-7 cells as a source of a Gal subunit, purified PLCP, and radiolabelled phospholipid vesicles, the enzyme activity was indeed stimulated in a GTPyS-dependent manner by increasing concentration of membranes enriched in a l l ;this stimulation was prevented by pretreatment of the membrane with an antibody specific for all-subunits [76]. Using the same approach, Wu et al. [77] also described the PLC domains involved in the interaction with the a, subunit. By coexpressing in COS-7 cells a, and mutants of PLCP1, it was shown that the region involved in the aq-dependent activation is localized in the sequence 1030-1 142. More-
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over, peptides from this region inhibited the activation of the enzyme In order to test the ability of the different a-subunits to stimulate the purified PLC isoenzymes, the cDNA clones of four members of the G, family were transfected in COS-7 cells and the membranes used in an in v i m assay [78]. All four of the Gar-subunits tested were able to stimulate PLCP, in the following order of efficacy: Gaq=Gall >Gal6>Ga14 [78]. PLCP, was initially reported to be stimulated only by Gal6 and G a l l [78]. Subsequently, it was shown that Gaq was also effective and that the extent of activation towards the PLCP isoforms was: PLCP, > PLCP, S- PLC& [45]. In order to examine the coupling of G, to receptors, Wu et al. [79] cotransfected in COS-7 cells the cDNAs of three subtypes of a,-adrenergic receptors and different a-subunits. PLCP activity was increased in a norepinephrine-dependent manner by all receptor subtypes when they were coexpressed with a, or al [79]. With the same approach it was shown that a 1 4 and a16 were coupled to the a l B adrenergic receptor and poorly coupled to the a l Areceptor; the alC receptor was coupled only to the a14 subunit [79]. The data mentioned so far characterize the pertussis toxin-independent activation of PLC at the molecular level. As mentioned above, there is also evidence of a pertussis toxin-dependent mechanism of PLC activation. Thus, although it has been proposed that G proteins sensitive to the toxin such as Gi and Go are involved in this modulation, no evidence has been reported on the possibility that aior a. might directly activate the enzyme. Recently, the pertussis toxin-dependent activation of PLC has been related to a direct activation due to the Py subunits of G proteins [28,80]. Accordingly, Camps et al. [28] first showed that a cytosolic preparation of PLC from HL-60 granulocytes was activated by G protein fly subunits either obtained from transducin (flyt) or from bovine brain. This effect was additive to the stimulation obtained with GTPyS and, most importantly, reversed by the addition of GDP-bound at [28]. Using anion-exchange chromatography, the same authors isolated two isoforms of PLC from HL-60 granulocytes of which only one was stimulated by Pr [28]. This was later identified as PLCP, [81]. The sensitivity of the PLC isoforms to stimulation follows the order: PLP, > PLCP, > PLCP, > PLCG,, with virtually no action on PLCy, [82]. Katz et al., using recombinant Py dimers, showed that Plyl and Ply2 were able to stimulate PLCP, but not PLCP,, and that this effect was suppressed by coexpression with Ga2 [83]. They also suggest-
,
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ed that a2 could act as a “scavenger” of free fly. This mechanism could explain the pertussis toxin-sensitive activation of PLC. Thus, the toxin, by ADP-ribosylating the heterotrimeric G protein, would uncouple it from the activated receptor and prevent the release of the active Py subunit. By coexpressing the M2 muscarinic receptor which activates PLC in a pertussis toxin-dependent manner, together with PLCP,, p l y l , and either aiz or ai3,it was shown that, indeed, the muscarinic agonist carbachol could stimulate the enzyme and that pretreatment with the toxin prevented this stimulation [83]. Wu et al. (84), in cotransfection experiments in COS-7 cells, showed that P l y I , P2y5and Plys were able to stimulate PLCP, whereas P2y1was not. Using chimeric forms of PLCP,, they could also show that the by subunits interact with the N-terminal region of the enzyme, therefore, at a different site from that involved in the interaction with the aqsubunits [77,84]. Hepler et al. confirmed that Py subunits purified from brain stimulate the three isoforms of PLCP (PLCp3 2 PLCP, > PLCP,) [85]. The ability of recombinant auqand a 1 expressed and purified from Sf9 cells, to stimulate PLCP, and PLCp, was much greater than that of 07,whereas PLCP, was activated to a
Fig. 1 . Molecular mechanisms involved in the stimulatory regulation of PLC. The stirnulatory regulation might involve the direct interaction (1) of an aq(al1or a1dsubunit with the PLCP, isoform or (2)the direct interaction of the fly subunit with the PLC& and PLCP, isoforms. (3) and (4) show the activationof PLCy by tyrosine kinases. and indicate the stirnulatory and inhibitory pathways, respectively. See text for details and relevant references.
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similar extent by fly, a9 and a l l subunits [85]. Moreover, the effect of (Y and fly were not additive [85].The rank order of potency of isoenzyme stimulation by activated a, and a l l was PLCfl, =PLCfl, > PLCfl2 [851. To summarize, the agonist-dependent stimulation of PLC activity involves both the a and fly subunits of different G proteins (see scheme in Fig. 1). It is now accepted that the a-subunits of the G, family are involved in the pertussis toxin-insensitive pathway of PLC activation. Instead, the fly subunits are clearly involved in the pertussis toxin-sensitive pathway, although it is not yet clear from which specific G proteins these subunits are released. Both a-and fly-subunits are coupled to the fl isoforms of PLC but with different specificity toward each isoenzyme. It also appears that the different subtypes of fl- and y-subunits play an important role in the specific coupling between the activated receptor on one side and the specific PLC on the other. Taken together, the information on the mechanism of interaction between G proteins and PLC isoenzymes can be considered quite well detailed, but future studies will have to address the physiological significance of these molecular interactions in intact cells and their role in the regulation of cell function.
Inhibitory regulation of PLC Evidence is gradually accumulating that G proteins are also involved in the inhibitory modulation of PLC. Table I11 summarizes the cellular systems in which G proteins have been proposed to act as dual modulator of PLC activity (reviewed in [7,86]). The inhibitory modulation of PLC has been suggested to account for the action of several agonists [86,87]. Two mechanisms might be involved: a direct coupling of the inhibitory receptor to PLC via an inhibitory G protein (Gpi), or an indirect secondary effect due to Ca2+ or other second messengers generated by the inhibitory receptors (see scheme in Fig. 2). Enjalbert et al. [88] first proposed that in pituitary cells the dopamineinduced inhibition of PLC was mediated by a Gpi..In this system dopamine inhibited the angiotensin 11-stimulated inositol trisphosphate accumulation in a pertussis toxin-dependent manner [88]. This study, however, did not conclusively prove that the effect of dopamine consisted of direct inhibition of the enzyme [MI, as it was later shown that the inhibition was largely mediated by a decrease in cytosolic Ca2' due to dopamine-induced closure of a Ca2+ channel [89]. Similarly, Delahunty et al. [90] proposed that in a rat pituitary cell line (GH3) the inhibitory
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/
Ca2+
Fig. 2. Molecularmechanismsinvolvedinthe inhibitory regulationofPLC. The inhibitory regulation might involve (1) the direct interaction of an cYi-subunit with the enzyme. The indirect mechanism (2) involves the reduced effect of the stirnulatory 06. or aq-subunits, upon interaction with the 0-y subunit madeavailable by the inhibitory stimulus. Alternatively, (3) a decrease in the intracellular levels of C a + + due to the closure of a Ca++ channel might inhibit the enzyme. The stimulation due to the direct interaction(4) of Q $ ~or uqwith PLC is also indicated. and - indicate the stirnulatory and inhibitory pathways, respectively. See text for details and relevant references. Taken from (71.
+
TABLE Ill
Studies reporting evidence for the G,;-mediated inhibition of PLCa Cell type
Agent
Refs.
Pituitary Cerebral cortex Pituitary GH3 Cerebral cortex Pituitary Thyroid FRTL5 HUVECs-endothelium Parotid acini Cerebellum Astrocytoma
Dopamine NaF Adenosine GTPyS GTPyS, GDPPS Carbachol NaF, GTPyS Carbachol Opioids Adenosine
[88,2441 [2451
'Modified from Table I1 in [7]. See text for details.
[XI
"WW
~461 [911 [931 I2471 ~481 1941
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effect of adenosine on PLC activity was mediated by a pertussis toxin-sensitive G protein, since no other second messenger could mimic the adenosine effect. These experiments performed in intact cells cannot exclude the agonist-dependent activation of more than one pathway involved in the regulation of the enzyme. The most compelling evidence for a direct modulation of PLC by Gpi would be the demonstration of a receptormediated, GTP-dependent inhibition of the enzyme obtained either in permeabilized cells or cell-free systems where second messengers and other factors can be controlled. Such evidence has been recently reported by Bizzarri et al. [91], who showed that in permeabilized thyroid cells the muscarinic agonist carbachol inhibited both the basal and stimulated level of inositol trisphosphate in a GTP- and pertussis toxin-dependent manner. Since CAMP,Ca2+ and diacylglycerol do not play a role in this phenomenon, it was proposed that the inhibition of PLC in thyroid cells is indeed due to its direct coupling to a Gpisensitive to pertussis toxin [91]. A dual regulation of PLC has been reported in cerebralcortical membranes by Litosh [92], who observed that nanomolar concentrations of guanine nucleotides induced inhibition of PLC, whereas micromolar concentrations elicited a stimulation of the enzyme. Similarly, Van Geet et al. [93] reported that NaF and GTPyS induced a biphasic effect in human umbilical vein endothelial cells, consisting of PLC stimulation which turned into an inhibition at higher doses of the two compounds. The same effect was observed in membranes and permeabilized cells, and was thus consistent with the activation of an inhibitory G protein with a lower affinity for GTP [93]. Another example of receptor-mediated inhibition of PLC was analyzed by Nakahata et al. [94] in astrocytoma cell membranes. In this system, adenosine inhibited the GTPyS-dependent accumulation of inositol phosphates. Also this inhibition, similar to that reported in thyroid cells [91], was prevented by pertussis toxin. In astrocytoma cell membranes, the inhibition of the GTPyS-stimulated PLC was reconstituted in the presence of a purified preparation of GJG, from brain [94]. Despite the detailed information on the activation of the different isoforms of PLC (see above), very little is known on the nature of the Gpi protein and on the molecular mechanism that leads to its inhibitory interaction with PLC. As indicated in the scheme in Fig. 2, several mechanisms can be hypothesized: either the inhibition could be due to a direct interaction with a specific a-or By-subunits, or, alternatively, the
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fly-subunit released by the receptor-activated G could decrease the P activity of the stimulatory apby associating with It. In line with the latter possibility, Moriarty et al. [95] reported that exogenous &-subunit added to Xenopus oocytes decreased the PLC activity stimulated by acetylcholine, possibly by reconstituting the troy heterotrimer. Similar results were obtained by Boyer et al. [96] in turkey erythrocytes where the PLC was stimulated by AIF;. In neither case was a direct role of the &subunit completely ruled out. The direct role of the fly-subunit was instead excluded in the adenosine-induced inhibition of PLC in astrocytoma cells where the &subunit released by the activation of an adrenergic receptor did not affect PLC activity [94]. In this case, one should consider the possibility that the Py released is not the one specifically required for the inhibition of PLC. Recently, Litosh et al. reported that in solubilized bovine brain membranes in which the PLC activity is inhibited by nanomolar concentrations of GTPyS, purified ai, both in the GTP- and GDP-bound form, inhibited the enzyme activity by 14%, whereas a, was ineffective [97]. The &-subunit also did not affect the basal PLC activity but slightly decreased that stimulated by GTPyS [97]. It is not clear from these data whether an activated subunit directly interacts with PLC and if so, which one. However, they support the studies reported above on the role of a pertussis toxin-sensitive G protein in the inhibition of PLC [91,94]. Litosh et al. [97] also showed that antibodies to PLCP,, but not to PLCy, or PLCG,, prevented the GTPySinduced inhibition of PLC, suggesting that this is one of the isoforms that can be under dual regulation by G and Gpi. PS Altogether, the data on the inhibitory regulation of PLC are scarce. The elucidation of the molecular mechanism involved will have to wait for reconstitution studies which will employ the purified components of this system (receptor, G protein subunit, various PLC isoforms).
Modulation of PLA, PLA, catalyzes the cleavage of the sn-2 fatty acyl chain of many different phospholipids, concomitantly releasing free fatty acids and lysophospholipids. The latter, as discussed later, may serve as cellular mediators or may be precursors of other second messengers. A well known example relates to free arachidonic acid that is metabolized to pro-inflammatory lipid mediators such as prostaglandins, leukotrienes
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and hydroxyeicosatetraenoic acid 198-1001. In mammalian cells, the sn-2 position of phosphatidylcholine,phosphatidylethanolamineand phosphatidylinositol is occupied, to a significant extent, by arachidonic acid [98, 991. Thus. the activation of PLA, in these systems results in a specific increase in arachidonic acid release, It should be mentioned that other pathways might lead to the formation of arachidonic acid [loll. For example, DAG, formed concomitantly with inositol trisphosphate by the hormonal activation of PLC, serves as a substrate of a DAG-lipase that can liberate arachidonic acid [26]. The receptor-mediated generation of arachidonic acid has been attributed in several cell systems to the activation of PLA, (rather than PLC) based on the observation that specific inhibitors of DAG-lipase and PLC did not interfere with arachidonic acid release 1261. These systems have been selected to analyze the molecular mechanism involved in the modulation of PLA, and the potential role played by G proteins [7,26, 27,98,102,103].
Molecular f o r m of PLA, Two groups of PLA, have so far been identified in mammalian cells: the low molecular mass or secretory forms and the high molecular mass or cytosolic forms [16,17]. The secretory PLA, is characterized by a mass of 14 kDa, is prevalent in digestive organs and homologous to the PLA, of snake venoms 11041. These enzymes have been extensively characterized and structurally defined; they require millimolar Ca2+ concentrations for activation and do not show selectivity for the fatty acid in the sn-2 position [ 16,17,104]. The cytosolic PLA,, on the other hand, has a mass of 85 kDa, is selective for arachidonic acid and requires submicromolar Ca2+ concentration for maximal activity [9,16,17,105]. This enzyme has originally been purified from a monocytic tumor cell line (U937), a macrophage-like cell line (RAW 264.7) and rat kidney as a 110 kDa protein [106-1091. Following molecular cloning, a mass of 85 kDa was predicted from the 749 amino acid sequence, which also revealed 12 possible sites of Ser/Thr phosphorylation and four of Tyr phosphorylation [ 110,1111. Indeed, it has been reported that the 85 kDa PLA, is phosphorylated at Ser and, as a consequence, activated by the microtubule-associated protein 2 (MAP) kinase (a cytosolic serine-kinase, involved in cell growth regulation) [ 112,1131.
-
-
-
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The cytosolic PLA, sequence has no homology with that of the secretory form of the enzyme [ 110,lllJ. The only homology found is in its amino-terminal portion where a consensus sequence for Ca2+/phospholipid binding has been identified, homologous to that of the Ca2+dependent forms of protein kinase C and of PLCy. This domain is believed to be involved in protein translocation to the membrane [ 110, 1111. Thus, an amino-terminal fragment of 138 amino acids, which includes the homologous region, has been shown to associate with the plasma membrane in a Ca2+-dependentmanner [ 1101. Similarly, in RAW 264.7 cells, a cytosolic PLA, was found associated with the membrane at cytosolic Ca2+ levels (230-450 nM) typically induced by hormonal stimulation [ 1051.
Stimulatory regulation of PLA, Receptor-dependent PLA, stimulation can be either due to the direct action of a G protein or to the PLC-dependent activation of PKC, which then, either directly or indirectly, activates the enzyme [7,16,17,26,27]. The role of G proteins was initially proposed following the observation that arachidonic acid release was inhibited in cells pretreated with pertussis toxin, thus under conditions of G protein inactivation [ 114, 1151. In addition, agents able to directly activate G proteins, such as guanine nucleotides or fluoride, were able to induce arachidonic acid release in several cells [7,98,102]. Table IV lists some of the cellular systems in which the G protein-mediated activation of PLA, has been demonstrated. Additional examples have been reviewed recently [26,27, 98,102,103]. A well documented demonstration that G proteins might act by directly interacting with PLA, was first obtained by Axelrod and coworkers [ 1141 in a thyroid cell line (FRTLS). In this system, the adrenergic stimulation of arachidonic acid release was shown to be independent of the hormonal stimulation of PLC and was potently inhibited by pertussis toxin [114, 1151. In addition, in permeabilized cells, GTPyS directly stimulated the release of arachidonic acid [ 1141. As shown in Table IV, several studies have confirmed this observation in other systems, leading to the conclusion that the G protein-mediated activation of PLA, is a rather general phenomenon.
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302 TABLE IV
Studies reporting evidence for the G-protein-mediated modulation of PLA2 activity Cell type
Stimulationa Thyroid FRTLS Thyroid FRTLS Thyroid FRTL5 Retina Brain Fibroblast Fibroblast Neutrophils Keratinocytes RBL-2H3 (basophils) Kidney Granulocytes Leukocytes LAN-5 neuroblastoma Inhibitionb Retina Macrophages Pancreas
Agent
Refs.
NorepinephrinelGTPyS Carbachol TSH
[ 1141
or
Carbachol Bradykinin Mastoparan met-Leu-Phe Bradykinin IgE EGF GTPyS GTPyS Interferon-y
P491 ~501 161 12511
GTPyS PT GTPyS
'The table has been modified from Table I in [7]. Additional studies have been reviewed recently [16,26,27,98,102,103]. bThis mechanism has been deduced from a GTPyS dependent-stimulation or pertussis toxin-mediated inhibition of a putative GPispecific for PLA, (see also Fig. 3).
The molecular mechanism involved in the activation of PLA, has not yet been elucidated, although both the 07-and a-subunits have been proposed to directly activate the enzyme. In dark-adapted G,-depleted rod outer segment membranes, free & - s u b u n i was t able to activate PLA, and the addition of free at-subunit inhibited this effect [116]. In the same system, addition of at alone was slightly stimulatory. Similarly, a, from brain activated the retina PLA2, and this effect was additive to that induced by P-y [117]. The by-subunit has also been involved in the
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PLA2-dependent activation of atrial K + channels [ 118-1201, whereas the Na' channel regulation in A6 cells (Xenopus Zaevis renal tubular cells) has been related to the activation of PLA, by the ai3-subunit [121]. Also the az-subunit has been proposed to mediate the activation of PLA,. Chimeras of G, and G, proteins (where the C-terminal of the as protein had been replaced with the 38 C-terminal amino acids of the az) overexpressed in Chinese hamster ovary cells, prevented the hormonal activation of PLA,, evidencing the relevance of the C-terminal of a2 in PLA, activation [122]. Following the discovery of the GI, family, Xu et al. [ 1231 have shown that overexpression of an activated form of the a12subunit in NIH 3T3 fibroblasts, correlated with increased arachidonic acid release. It was not indicated, however, whether this effect was due to a direct or indirect coupling of the a12-subunitto the enzyme [123]. The G protein-mediated activation of PLA, has also been involved in the action of the epidermal growth factor (EGF), the IgE and the interferon? receptors [124-1261. Taken together, these studies suggest that multiple mechanisms of PLA, activation might take place, possibly related to the cellular systems and type of enzyme involved. The scheme in Fig. 3 presents the possible interactions between G protein subunits and PLA, based on the studies reported above.
1,
'UUU
yl
hhhhhhhhhhhAhhhhhhAhAhhhhhhhhhhhhhfihhhh YYYYY YYY YYYY yyy vyyyyy Y Y y y Y Y Y Y ~ Y Y Y YYY Y
--
I,
7 ' '"2
Y
-w(-,\ .
Arachidonic Acid
I
Metabolites
Fig. 3. Molecular mechanisms involved in the dual regulation of P L 4 by G proteins. The stimulatory regulation may involve the direct interaction of (1) the %, mJ and or,, or (2) the byt subunits. The inhibitory regulation might either be due to the interaction of the cu, subunit (3) made available by the inhibitory stimulus with the stimulatory flyt or to the direct interaction of an inhibitory or-subunit (4) with the enzyme. and - indicate the stirnulatory and inhibitory pathways, respectively. See text for details and relevant references. Taken from [7].
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Inhibitory regulation of PLA, Although some reports have addressed the possibility that G proteins are involved also in the inhibitory regulation of PLA,, the evidence reported so far is indirect and the molecular mechanism undefined. In rod outer segments GTPyS was shown to inhibit the activity of light-stimulated PLA, [ 1271. A mechanism of dual regulation of PLA, by G proteins similar to the one described for the regulation of adenylyl cyclase has been proposed on the basis of the cholera and pertussis toxin action in the macrophage RAW264.7 cell line [128]. Consistent with a dual regulation of the enzyme, in pancreatic membranes GTPyS induced an increase in arachidonic acid release under resting conditions whereas it inhibited the Ca2+-induced release [ 1291. The possible dual regulation of PLA, by G proteins is schematically represented in Fig. 3.
PLA, activity and ras-induced transformation Several studies have recently addressed the possible correlation between the expression of p21, the small G protein encoded by the ras oncogene, and an altered metabolism of membrane phospholipids. The role of PLA, in ras-transformation was first analyzed in NIH-3T3 fibroblasts where it was shown that microinjection of the purified ras oncoprotein induced morphological changes that correlated with the production of lysophospholipids, hence with a putative activation of PLA, (the enzymatic activity leading to lysophospholipid formation) [ 1301. Moreover, ultrastructural analyses have dem.onstratedthe colocalization of ras protein and PLA, in the ruffles of ras-transformed kidney cells [131]. In fibroblasts and epithelial thyroid cells transformed by the ras oncogene, the expression of p21 correlated with a pronounced increase in glycerophosphoinositol, a phosphoinositide derivative formed by the action of PLA, and PLA, on membrane phosphoinositides [132-1341. A parallel increase in the levels of arachidonic acid was also reported, confirming that ras-transformed cells are characterized by an enhanced basal activity of PLA, [ 1341. In thyroid cells, the membrane phosphoinositides were shown to be the preferential substrates of this enzyme [ 1351. Increased levels of glycerophosphoinositol were also reported in cell lines transformed by membrane-associated or cytosolic oncogenes (such as
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src, met, trk, mos and raf), suggesting that the ras-induced PLA, activa-
tion could be a common step in the action of different oncogenes [ 132, 1361. The interrelationship between ras and PLA, has been also proposed in a recent study where the resistance to melittin, an amphipathic peptide from bee venom that activates PLA,, was shown to correlate with decreased expression of ras oncoprotein and reversion of transformed cells to a normal phenotype [ 1371. The mechanism linking ras-induced transformation to PLA, activation has not been elucidated. As mentioned above, PKC or other cellular kinases might be involved in the activation of PLA, [26,27]. Indeed, in scrape-loading experiments it was proposed that the oncogenic p21-mediated increase in arachidonic acid release was due to PKC activation [ 1381. The activity of the cytosolic 85 kDa PLA, was increased by hormones and growth factors that induced the phosphorylation of the PLA, serine residues [139-1411. As mentioned above, PLA, has been shown to be a substrate of MAP kinase [ 112,1131. Inasmuch as MAP kinases are downstream of the ras-mediated signalling [ 142,1431, one of the mechanisms involved in the action of oncogenic p21 could be the activation of PLA, by this kinase. Indeed, in rat-1 fibroblasts, the expression of the dominant negative ras mutant N-17 resulted in the attenuation of the EGF-induced arachidonic acid release and MAP kinase phosphorylation [ 1441. Another factor that could contribute to the altered activity of PLA, in ras-transformed cells is a defect at the level of the G proteins coupled to the enzyme. In ras-transformed thyroid cells, a partial decrease (about 30 %) in glycerophosphoinositol levels was observed upon pretreatment with pertussis toxin (Valitutti and Corda, unpublished observations), suggesting that the phosphoinositide-specific PLA, in this system is, at least in part, controlled by a G protein sensitive to the toxin. In the same cell system, ras-induced transformation has been associated with altered G protein ADP-ribosylation, since an inhibitor of the ADP-ribosylation reaction (identified in normal cells) appeared to be inactive upon rasinduced transformation [ 1451. Moreover, pertussis and cholera toxin, which in normal thyroid cells markedly increase CAMP levels, were much less effective in transformed cells, again suggesting an altered G protein function [146]. Similarly, in fibroblasts transformed by ras and other membrane associated or cytosolic oncogenes, the ability of G proteins to stimulate PLC was significantly impaired [136].
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Biological activity of the PLA, metabolites The activation of PLA2 leads to the formation of arachidonic acid and lysophospholipids. While much is known on the role of arachidonic acid in the regulation of cell function [17,27,100], the regulatory role of lysophospholipids and of their derivativeshas only been recently analyzed [ 135,147- 1511. Lysophosphatidic acid (LPA), by far the most studied lysophospholipid, has been related to a wide spectrum of biological activities [ 147149,152-1541. In fibroblasts, LPA is highly mitogenic and its activity is higher than that of phosphatidic acid (PA), a well known mitogen in quiescient fibroblasts [ 1481. In the same study, other lysophospholipids were found inactive [1481. The LPA-induced cell proliferation was prevented by pertussis toxin pretreatment of the cells, suggesting that the action of 1,PA is mediated by Gi. In quiescent fibroblasts LPA activates the normal p21, product of the ras protooncogene; also this effect involves a Gi protein since it was inhibited by pertussis toxin [153]. Three different pathways have been related to the action of LPA: the activation of a pertussis toxin-sensitive Gi protein mediating the inhibition of adenylyl cyclase, the stimulation of phosphatidylinositol hydrolysis by the activation of a pertussis toxin-insensitive G protein and the GTPdependent release of arachidonic acid [ 1481. These findings are consistent with the presence of a membrane receptor able to interact with G proteins. Indeed, a putative membrane receptor specific for LPA has been recently identified [149]. Apart from the stimulation of cell proliferation, LPA has been proposed to be involved in inflammatory responses (being released by activatedplatelets, [ 155]), platelet aggregation [ 156,1571, smooth-muscle contraction [ 158,1591, neuronal cell rounding and neurite retraction [ 1541. Other lysophospholipids that displayed regulatory functions are lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI). In rat islets of Langerhans, both lysophospholipids stimulated calcium mobilization and insulin release [ 160-1631. LPC greatly potentiates the activation of T-lymphocytes and the differentiation of HL-60 cells, probably by enhancing the DAG-induced PKC activation [ 164-1661. This effect required the presence of phosphatidylserine (PtdSer), DAG and calcium [ 1661.
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In PC12 and Swiss 3T3 cells, lysophospholipids bound to albumin, in particular LPA, appear to mediate the biological effects usually attributed to serum factors, such as the stimulation of stress fiber and focal adhesion formation [ 1521. In thyroid cells, LPA, LPI and LPC are potent mitogens [ 1501. Interestingly, ras-transformed thyroid cells are characterized by a specific increase (two- to three-fold) in the basal levels of LPI, suggesting the existence of a constitutively activated PLA, specific for membrane phosphoinositides [ 1351. Surprisingly, LPI and LPC were the only mitogenic lysophospholipids in ras-transformed thyroid cells [ 1351. Thus, the increased basal levels of LPI in this system might play a role in the hormone-independent regulation of cell growth [ 1351. Other metabolites derived from the action of PLA, are the glycerophosphoinositols [132-1341. Recently, glycerophosphoinositol-4-phosphate has been shown to be an endogenous inhibitor of adenylyl cyclase [151]. Its mechanism of action involves a direct interaction with G,, the G protein that activates adenylyl cyclase. Glycerophosphoinositol-4-phosphate is therefore able to inhibit the cellular functions that are modulated by an increase in CAMP. This has been directly demonstrated in thyroid cells [151]. This compound can therefore be considered a mediator of a novel mechanism of cross-talk between PLA, and adenylyl cyclase.
Phospholipase D PLD exists in a dormant form in many cell membranes. Activation of this enzyme, ensured by binding of an agonist to a receptor, leads to the liberation of PA mostly from hydrolysis of neighbouring PtdCho. PA can be further hydrolyzed to DAG or LPA which can then enter into the routes of signal transduction described above. Alternatively, a series of similar pathways were attributed to PA itself. The notion of PLD involvement in signal transduction is relatively new and not yet fully understood (for short reviews, see [257-2601). Only recently, the activation of this enzyme has been related to the small G protein ARF (ADP-ribosylating factor) [261,262]. Thus, it has been directly demonstrated that the cytosolic factors activating PLD in a GTP-dependent manner are ARFl and ARF3 [261,262]. Since ARF plays a central role in vescicular traffic, a possibility is that the PLD metabolites formed by PLD-ARF interaction are important mediators of vescicle formation and/or movement (262-2621.
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Conclusions The interactions between G protein subunits and either PLA, or PLC isoforms appear to have a high degree of complexity. In general, it seems that the scheme of dual regulation originally proposed for the G protein modulation of adenylyl cyclase can be considered valid for these enzymes as well (Figs. 1-3), and that the contribution of a-and &-subunits has to be defined in each case. Moreover, the G proteins and phospholipase isoforms present in the different tissues might determine the prevalent mechanism of regulation of the system under study.
Acknowledgements We wish to thank Ms. D. Spadano and Ms. M.G. Mencuccini for help with the bibliography, Ms. S. Falcone for typing the manuscript and Ms. R. Bertazzi for preparing the figures. Work in the author’s laboratory was supported in part by the Italian Association for Cancer Research (AIRC), the Agenzia per la Promozione dello Sviluppo nel Mezzogiorno and Convenzione CNR (PR-3) and the Italian National Research Council (No 93.02354.PF39, ACRO and Convezione CNR- Consorzio Mario Negri Sud). M.F. is the recipient of a fellowship from the Centro di Formazione e Studi per il Mezzogiorno (FORMEZ).
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Biomembranes Edited by Meir Shinitzky Copyright 0 VCH VerlagsgesellschaflrnbH, 1995
Index A6 cells (Xenopus laevis renal tubular cells) 303 Acetylcholine 6, 20, 21, 25, 63-65, 69, 72, 119, 130, 299 Acetylcholine receptor 25, 34, 52, 64, 68, 143 Activin receptor 15 Adenocarcinomas 182
Adenosine triphosphate (ATP) 8, 10, 33, 63, 70, 112-115, 121, 134, 135, 145,258, 259, 261, 268, 269, 273
al-adrenergic receptor
20,
34, 119, 130
a2-adrenergic receptor 186, 193, 217
a l - and 02-adrenergic receptors 197 P-adrenergic receptor 20, 130-132, 166, 181, 186, 189, 192, 193, 197, 198
Adrenergic receptor k i n a s e (PARK) 21, 90, 130, 267, 276
Adenosine triphosphate (ATP) gated channels 70 Adenosine triphosphate (ATP) receptors 63, 70 Adenylate cyclase 8, 10, 27, 28,
Adrenergic receptor kinase (PARK) phosphorylated receptor 90 Adrenocorticotropin ( A C T H )
33, 52, 113-116, 118, 119, 124, 130, 132, 145, 155, 207, 212, 215, 216 Adenylyl cyclase 78, 79, 155, 168, 169, 176-178, 181, 185, 188-190, 193, 201, 205-207, 209-217, 220,221, 285, 304, 306-308
Adrenocorticotropin ( A C T H ) receptor 34 Aldosterone 6 Alkaline phosphatase 258, 259 AMPA 75 Angiotensin 20, 118, 130, 188,
Adhesion receptors (integrins) 25
Adipocytes 146 Adipocytes (brown) 288 Adrenal cortical carcinomas 182 Adrenal glomerulosa cells 288, 289
Adrenaline 288 Adrenergic receptors 20, 21, 34, 42, 78, 79, 84, 86, 87, 89, 90, 115, 119, 130, 186, 189, 193, 197, 204, 217, 294, 299
114, 117
293, 296
Angiotensin receptor 5 1 Angiotensin I1 119, 121, 288 Angiotensin I1 receptor 34 Antidiuretic hormone (ADH) 20, 114, 117, 119, 121, 128, 130, 141, 142, 188, 189, 199, 289 Astrocytoma cells 289, 297-299 ATPase 271
Basal ganglia 178 Basophils 289, 302 BDNF 48 Bombesin 20, 80, 121, 128
320
Index
Bombesin receptor 34 Bradykinin 20, 119, 121, 302 Bradykinin receptor 34 Brain 302 Calciosomes 123 Calcitonin 85, 114, 118, 189, 190
Calcium/calmodulin
(Ca/CaM)
21 1
Calmodulin 214, 272, 274, 277 Calmodulin-dependent CAMkinase 10 CAM-kinase 10, 11, 40 Cardiac myocytes 178 Cardiac ventricle cells 207 Casein kinases 39, 265, 269 CD4 268 CD4 receptor 268 Ceramides 34 Cerebellum 297 Cerebral cortex 297 Chinese hamster ovary cells 303 Cholecystokinin (CCK) 139 Cholecystokinin (CCK) receptor 139, 140
Conductance 63, 65 Corticosterone 6 Corticotropin-releasing hormone 119
Cortisol 6 Critical micelle concentration (CMC) 262 Cytokine receptors 24-26, 33, 40, 44
Cytokines 6, 13, 14, 23-25, 37, 43, 49
D1A-dopamine receptor 198 Depolarization 63 Desensitization 63 Detergents 258, 260-263, 271, 272
Diabetes insipidus 199 Diacylglycerol (DAG) 8, 9, 11, 26, 34, 35, 37-40,
79-82,
119, 120, 122-125, 128, 129, 155, 213, 260,266, 267, 276, 277, 285, 286,291, 300, 306, 3 07 Diacylglycerol (DAG) lipase 34, 38, 119, 130, 300 Diacylglycerol (DAG) kinase 3 8 Dopamine 6, 20, 21, 84, 121, 296, 297 Dopamine receptor 21, 83, 84, 89, 119 Drosophila 46
Ectokinases 258, 259, 262, 265, 268, 269
Ectophosphatase 258 Endocrine.cells 178 Endothelial cells 206, 288, 298 Endothelin 20, 288 Enkephalin 288 Epidermal growth factor (EGF) 6, 17, 19, 44, 47, 96, 121, 128, 130, 131, 134, 136,266, 267, 287, 288, 302, 303, 305
Epidermal growth factor (EGF) receptor 17-19, 34, 45-47, 50, 51, 133-136, 255, 260, 262, 266, 272, 274, 290
Epidermal growth factor receptor threonine kinase (ERT kinase) 44
Epinephrine 6, 20, 21, 112 Epithelial cells 145, 171, 178 Epithelial thyroid cells 304 Erythrocytes 114, 115, 145, 266, 269, 272, 293, 299
Erythroleukemia cells 115 Estradiol 6 Extracellular signal-regulated kinases'(ERKs) 44 Fat cells 288 Fibroblast growth factor 19, 44, 50 Fibroblast growth factor receptor 50
Index Fibroblasts 266, 288, 289, 291, 302, 304-306 Fluorescence 87, 164
Follicle stimulating hormone (FSH) 20, 85, 114, 117, 190 Follicle stimulating hormone and TSH receptors 190 FQrmyl-Met-Leu-Phe (fMLP) receptor 215, 220 FRTL-5 thyroid cells 288 G proteins 6, 8-15, 19-30, 34, 36, 37, 39-42, 44, 51, 52, 63, 78, 85, 86, 89, 90, 113, 115, 116, 118, 119, 125, 128, 130, 132, 155, 160, 161, 163, 164-170, 172, 176-183, 185191, 193, 197-199, 203-205, 207, 211,212, 214-221,231, 234, 236,267, 285-287,290, 292-296, 298-308 G-CSF 23 G-CSF receptor 23 G-protein coupled receptor kinases 189 G-protein coupled receptors 63, 78, 79, 83-87, 89, 90, 267 y-aminobutyric acid (GABA) 63, 70-72, 80 y-aminobutyric acid (GABA) and glycine receptors 26, 70-74 Gastric cells 289 Gastrin 139, 140, 289 Gastrin receptor 140 GC 33 Glomerulosa cells 289 Glucagon 189- 19 1 Glucagon or PTH receptor 219 Glutamate 63, 80, 85 Glutamate receptors 26, 63, 7377, 85, 191 Glycerol-3-phosphate (G3P) 124 G 1y cero p hosp hoc hol i ne (G P C) 124 Glycine 63, 7 1 Glycine receptors 70-74
321 Glycine-gated chloride ion channels 74 Glycogen synthetase 255 Glycosyl-phosphatidylinositol (GPI) 125-127 GM-CSF 23, 25 GM-CSF receptor 23-25 Granulocytes 302 GRH 289 GRH receptor 3 4 Growth factors 4-6, 14, 19, 30, 32, 33, 37, 40, 43-45, 49, 50, 121, 128, 136, 140, 272 Growth factor receptors 33, 41, 51, 255, 266
Growth factor receptor tyrosine kinase 30, 32, 37-39, 43, 44, 50, 266 Growth hormone (GH) 23, 25, 138, 140, 141, 143, 182
Growth hormone (GH) receptor 23-25, 142, 143
Growth
hormone-N
(hGH-N)
141 GTP 1555 216 GTPase 13, 19, 25-28, 30-32, 37, 39, 46, 52, 160, 164, 180-183, 185, 197, 215, 216, 220
GTPase activating protein (GAP) 287
HCG 114 HeLa cells 215, 288, 289, 306 Hematopoitic cells 266 HGF 17 HGF receptor 50 HGH-V 141 Hill plot 110-1 12, 276 Histamine 20, 121, 289, 293 Histamine receptor 34, 119 HUVECs-endothelium 297 Hydropathy 83 Hydrophobicity 65 Hyperpolarization 63 Hypothalamic releasing hormone receptors 84
322 IgE receptor 34 IGF 6 , 136, 140, 203 IGF receptor 16, 17, 203-204 Immune responses 34 Inositol 3-phosphate (IP) 11, 119 I n o s i t o l - l , 4 , 5 trisphosphate (IP3) 9 , 11, 35, 38, 79-82, 120-123 Inos itol-l,4,5 trisphosphate (IP3) receptor 286 Insulin 6 , 19, 96, 97, 107-109, 114, 121, 125-128, 130, 132136, 138-143, 145, 146, 306 Insulin receptor 131, 132, 142, 144, 269 Insulin receptor kinase 134, 135, 278 Insulin-like growth factor I (IGF-I) 130 Interferon 23-25, 121, 302 Interferon receptors 24, 25, 34, 303 Interleukin receptors 23-25,217 Interleukins 6 , 23-25, 34, 51 Ion channel receptors 64 Ion channels 8 Ion transport 65 Kainate receptors 75 Keratinocytes 302 L cells 289 LAN-5 neuroblastoma 302 Leukocytes 302 Ligand-gated ion channel 63-66, 69, 70, 73-75, 77 Ligand-gated ion channel receptors 63, 77 Lipoxygenases 35 Low-density lipoprotein (LDL) receptor 142 Low-density lipoproteins (LDL) 13 1 Luteinizing hormone (LH) 20, 114, 117, 190, 289
Index Luteinizing hormone-CG 20, 85 Luteinizing hormone-CG receptor 188, 203 Lymphocytes 108, 276 Lymphokines 23 Lysophosphatidylcholine 37, 124 Lysophospholipids 34, 37, 285, 304, 306, 307 Lysosome5 136 Macrophages 289, 300, 302, 304 McCune-Albrights Syndrome 182 Melanocyte stimulating hormone (MSH) 201 Melanocyte stimulating hormone (MSH) receptor 199, 201, 202 Melanocytes 201, 202 Micelles 260, 262 Michaelis-Menten equation 106 Microsomes 27 1 Microtubule-associatedprotein-2 (MAP-2) kinases 19, 32, 33, 39, 44, 45, 182, 300, 305 Muscarinic receptors 20, 8 1, 84, 89, 119, 130, 188, 197, 198,204, 216, 267, 276, 293, 295 Myelin basic protein (MBP) kinases.44 Neuroblastoma-glioma cells 288 Neuroendocrine cells 207 Neuroepithelium 21 1 Neurokinin-1 289 Neuromedin K 20 Neurons 178, 179 Neuropeptide Y 289 Neuropeptides 82, 289 Neurotensin 20, 82, 121 Neutrophils 179, 220, 302 NG108-15 cells 288 Nerve growth factor (NGF) 17, 19, 31, 44, 48, 96, 121, 136,
Index 140 Nerve growth factor (NGF) receptor 17, 24, 49, 50 Nicotine 64 Nicotinic acetylcholine receptor (nAChR) 25, 64, 68 NIH 3T3 cells 289, 303, 304 NMDA glutamate receptor 75, 77 Norepinephrine 20, 21, 192, 302 Oncogene protein kinases 265, 267, 268, 275, 276 Oocytes 299 Opiate 84 Opioid receptors 193 Osteosarcoma cells 289 Ovarian granulosa 182 Oxytocin 20, 117, 121, 289 P2-Purinergic receptor 34 PAF 34, 289 PAF receptor 34 Pancreas 302 Pancreatic cell 178 Pancreatic islet cells 207 Parathyroid hormone (PTH) 144, 145 Parathyroid hormone receptor 145 Parotid acini 297 Particulate protein tyrosine phosphatases (pPTP) 272 PC12 cells 288, 307 Peptide hormones 190, 191 Peripheral nerve cells 171 Permeability 63, 69, 77 Phorbol ester protein receptor 273 Phosphatases 15, 39, 41, 221, 254, 255, 258, 259, 261,268, 269, 271-276, 278 Phosphatidate phosphatase 39 Phosphatidic acid (PA) 38, 306 Phosphatidic acid phosphohydro-
323 lase (PAP) 124, 128, 129 Phosphatidylcholine (PC) 38, 39, 124, 128, 129, 260 Phosphatidylcholine phospholipase (PC PLipase) 129 Phosphatidylinositides 38 P hosp ha t idyl inosi t ol (PI) 37, 38, 119, 120, 122, 125-128, 130, 266, 277 Phosphatidylinositol4,5-biphosphate kinase 269 Phosphatidylinositol4,s-bisphosphate (PIP2) 9, 11, 37, 38, 119-122, 129 Phosphatidylinositol4-monophosphate (PIP) 37, 38, 120, 122 Phosphatidylserine (PS) 40, 123, 260 Phosphodiesterase (PDE) 33, 34, 113, 119, 155, 168 Phosphoinositides 8, 9, 26, 34, 38, 41, 119, 121-123, 125 Phospholipases 8-12, 18, 19, 23, 27-29, 32, 34-40,52, 78, 79, 118, 119, 122-129, 155, 168, 188, 197, 198, 205,207, 212-217,220, 2 2 1 , 2 6 6 , 2 6 7 , 285-287, 290-308 Phospholipid vesicles 293 Phospholipids 8 , 34-40, 119, 120, 122-125, 128, 163, 203, 204, 260, 264, 266, 268, 276-278, 285, 291, 299, 304 Phosphorylation 221 PI-3 kinase 18, 19, 38, 52, 287 Pituitary 297 Pituitary and thyroid adenomas 181 Pituitary cells 288, 289, 297 Pituitary gonadotrophs 289 Pituitary lobe hormone (melanocyte stimulating hormone, MSH) 201 Platelet activating factor (PAF) receptor 217
324
Platelet-derived growth factor (PDGF) 6, 17, 19, 44, 50, 121, 128, 130, 274, 287, 290 Platelet-derived growth factor (PDGF) receptor 17-19, 34, 46, 290 Platelet prostaglandin receptors 189 Platelets 171, 178, 289, 306 Progesterone 6 Prolactin (PRL) 140 Prostaglandins 299 Protein kinases 8-12, 15, 21, 25, 32, 33, 36-44, 53, 54, 65, 86, 119, 123, 129, 130, 131, 135, 136, 176, 212-214, 221, 254, 255, 258-269,271, 273, 274, 276-278, 286, 291, 301, 305, 306 Protein kinase analyses 258 Protein phosphatases 254, 255, 258,261, 268,269, 271-273, 276, 278 Protein tyrosine kinase (PTK) 15, 25, 53, 130, 131, 213 Protein tyrosine kinase receptors 39 Protein-lipid interactions 276, 27 8 Pulmonary endothelial cells 288, 289 Raf kinases 19, 32, 33, 39, 43, 45, 46, 48, 53 Rat hepatocytes 288 Rat liver cells 291 Rat mesangials cells 288 Rat renal cortex cells 288 Rat-1 fibroblasts 305 Receptor kinases 6 , 9, 10, 1319, 26, 32-34, 37-40, 43, 45-48, 50, 53, 54, 133 Receptor protein tyrosine phosphatases (rPTP) 6, 272, 273 Renal mesangial cells 288, 289
Index Renal tubular cells 289 Retina 302 Retinal cone cells 171 Retinal rod cells 171 Rhodopsin 34 Rhodopsin'kinase 267 Rhodopsin receptors 191, 198 Ribosomal S6 protein kinase (RSK) 19, 32, 33, 39, 45 Ribosomal S6 protein kinasekinases (RSK kinases) 44 Scatchard plot 107-109 Sensory neurons 288, 289 Serinelthreonine and tyrosine kinases 8, 10, 11, 13-18, 23, 25, 33, 36, 39-41, 43, 49, 51-53, 54, 65, 128, 132, 134, 136, 142, 174, 255, 259, 267-269, 273-276, 287, 290, 295 Serinelthreonine phosphatases (PrP) 258, 268, 269, 271, 272 Serotonin 20, 21, 63, 121 Serotonin receptors 20, 21, 52, 69, 84, 119 Signal peptide 191 Somatostatin 82 Sperm cells 171 Sphingosine 34 Src kinase 18, 19 Steroid/thyroid hormone receptor 6 Substance K 20, 121 SubstanceP 20, 119, 121 Substance P-R 34 Swiss 3T3 cells 307 T-cell antigen receptor (TCR) 290 T-lymphocytes 306 Tachykinin receptor 84 Tachykinins 20, 82, 84 Testosterone 6
325
Index T G F a 17 TGFP 49 TGFP receptor 15, 204 Theca cell 182 Thrombin 121, 289 Thrombin receptor 34, 190, 191 Thyroid adenomas 182 Thyroid cells 297, 298, 301, 302, 304, 305, 307
Thyroid hormone 6, 144 Thyroid-stimulating hormone (TSH)-receptor 143, 144, 189, 190, 203 Thyrotropin (TSH) 20, 85, 114, 117, 119, 136, 144, 190, 302
Thyrotropin-releasing hormone 119, 121, 128
TKR 266 T N F 23, 24 T N F receptor 24, 34 Transducin 294 Translocation 36, 41, 42, 44, 50, 53, 146 TRH 20, 289
TRH receptor 84 Tyrosinase 201 Tyrosine kinase receptors (TKRs) 43, 266, 274, 285, 287
Tyrosine
phosphatases
(PTP)
14, 15, 258, 269, 272, 275
Vascular smooth muscle cells 288
Vasoactive intestinal peptide (VIP) 83, 118, 121 Vasoactive intestinal polypeptide 83, 85 Vasopressin (antidiuretic hormone, ADH) 20, 114, 117, 119, 121, 128, 130, 188, 189, 199, 289 Vasopressin receptor 189, 198, 199 Vasopressine-la receptor 34 Ventricular myocytes 289 Vesicles 90, 123, 136, 137, 163, 203, 204, 260
Biomembranes Edited by Meir Shinitzky Copyright 0 VCH VerlagsgesellschaflrnbH, 1995
Shinitzky, M. (ed.)
Biomembranes Physical Aspects
1993. VIII, 371 pages with 104 figures and 47 tables. Hardcover. DM 198.00. ISBN 3-527-30022-8
From the Contents: Chapter 1 Bilayers, Monolayers, Multilayers and Non-Lamellar Lipid Phases Derek Marsh Chapter 2 Lipid Phase Transitions Dennis Chapman
Chapter 3 Micelles and Liposomes Dov Lichtenberg Chapter 4 Fluidity, Dynamics and Order B. Wieb van der Meer Chapter 5 Membrane Lipid-Protein Interactions Abraham H. Parola Chapter 6 Lateral and Rotational Diffusion in Biological Membranes Yoav I. Henis Chapter 7 Electrical Properties of Biomembranes Leslie M. Loew
Date of information: November 1994
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Shinitzky, M. (ed.)
Biomembranes Structural and Functional Aspects 1994. VII, 383 pages with 68 figures and 39 tables. Hardcover. DM 198.00. ISBN 3-527-30022-8
From the Contents: Chapter 1 Membrane Lipids and Aging Hilary R. Shmeeda et al. Chapter 2 Membrane-bound Enzymes Giorgio Lenaz and M. Degli Espoti Chapter 3 Ion Channels in Biological Membranes General Principles Rami Rahamimoff et al. Chapter 4 Anion Exchangers of Mammalian Cell Membranes Z. Ioav Cabantchik Chapter 5 Diversity of Transport Mechanisms in Bacteria Bert Poolman et al.
Date of information: November 1994
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Ibelgaufts, H
Dictionary of Cytokines 1995.XXI, 778 pages with 110 figures, 108 in color. Hardcover. DM 184.-. ISBN 3-527-30042-2 Cytokines are hormone-like secretedproteinsregulating the survival, proliferation, and differentiation of cells by maintaining an intricate intercellular communication network. Their clinical use offers exciting ways of diagnosis and treatment oriented towards the molecular pathophysiology of individual diseases to an extent previously thought to be impossible. The Dictionary ofcytokines provides concise yet comprehensive coverage of such diverse topics as
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alternativenomenclature sources and targets of cytokines proteincharacteristics gene organization and chromosomal location relatedfactors and factorfamilies receptor structure andexpression biological activities in vitro and in vivo assays systems cytokine studies in transgenic animal models.
This 800+ page dictionary comprises ca 3200 extensively crossreferenced entries, ca 14000 primary references and reviews, and more than 100 full color illustrations. It guides the user through the zoo of factors, thejungle of interactions, the morasses of acronyms, and the desert of synonyms. The book will be an invaluable aid for the growing number of clinicians, scientists and advanced students of immunology,hematology, oncology, endocrinology,biochemistry, cell biology and molecular biology confronted with the necessity of coming to terms with this key area of interdisciplinary research. Date of information: November 1994
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