Growth Hormone Receptor

Growth Hormone Receptor Stuart J. Frank1,* and Joseph L. Messina2 1

Department of Medicine, Division of Endocrinology and Metabolism, and Departments of Cell Biology and Physiology, University of Alabama at Birmingham, and Veterans Affairs Medical Center, 1530 3rd Avenue South, BDB 861, Birmingham, AL 35294-0012, USA 2

701 South 19th St., LHRB Room 531, Birmingham, AL 35294, USA

* corresponding author tel: (205) 934-9877, fax: (205) 934-4389, e-mail: [email protected] DOI: 10.1006/rwcy.2002.1418.

SUMMARY The growth hormone receptor (GHR) is believed to be required for all of the growth promoting and metabolic activities of growth hormone (GH). The GHR is widely expressed among species and is a single membrane-spanning receptor in the cytokine receptor superfamily. GH-induced homodimerization of the GHR causes activation of the receptorassociated cytoplasmic tyrosine kinase JAK2. Multiple signaling pathways, including the STAT, MAP kinase, and PI-3 kinase pathways are downstream of GH-induced JAK2 activation and have been linked to expression of GH-activated genes and GH-induced alterations in cell behavior. STAT5b, in particular, has been shown to mediate important sexually dimorphic effects of GH that correlate with the pulsatile pattern of GH release from the pituitary gland. Clinical disorders arising from deficient or excessive GH action are well described and in some instances are related to aspects of GHR function and/ or can be pharmacologically approached based on the accumulated knowledge concerning the GH±GHR interaction. GHBP, a high-affinity circulating GHbinding protein corresponding to the GHR extracellular domain, arises in some species by alternative RNA splicing and in others by proteolytic shedding

Cytokine Reference

from the full-length GHR. GHBP's significance in GH physiology and signaling is as yet unclear.

BACKGROUND

Discovery Early characterization of specific binding sites for GH utilized cell lines such as the human IM-9 B lymphocyte or rabbit liver (Van Obberghen et al., 1976; Waters and Friesen, 1979). While widely distributed, GHR is most abundantly expressed in liver. The first GHR cDNA clone was isolated in 1987 from human and rabbit liver after purification using anti-GHR monoclonal antibodies (Leung et al., 1987). cDNAs encoding the rodent (mouse and rat) (Baumbach et al., 1989; Smith et al., 1989), ruminant (cow and sheep) (Adams et al., 1990; Hauser et al., 1990), pig (Cioffi et al., 1990), and chicken (Burnside et al., 1991) GHRs were cloned thereafter in 1989±1991.

Alternative names Because of its role in growth promotion, GH is sometimes called somatotropin. Thus, the GHR is

Copyright # 2002 Published by Elsevier Science Ltd

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Stuart J. Frank and Joseph L. Messina

also known as the somatogenic receptor or the somatotropin receptor.

Structure The GHR is a single membrane-spanning type 1 glycoprotein member of the cytokine receptor superfamily (Bazan, 1990). The features shared with that large family of receptors include in the ligand-binding extracellular domain the characteristic placement of cysteine residues and the WSXWS-like motif, and in the cytoplasmic domain the proline-rich Box 1 element involved in association with Janus kinases. The GHR is in the subgroup of cytokine receptors that are thought to contain only one type of protein. That is, some members exist in their active (liganded) state as heterodimers or heterooligomers, but the GHR ± like PRLR, thrombopoietin receptor, EPOR, and leptin receptor ± instead forms a homodimer. Full-length GHRs from various species are in the range of 600 amino acids in length (620 residues in the mature human GHR) and contain relatively large cytoplasmic domains (350 residues in the human). Several alternatively spliced forms of the GHR encode receptors that differ from the full-length GHR. In rodents, the full-length GHR is encoded by a 4.2±4.7 kb message, while an alternatively spliced mRNA of 1.0±1.4 kb encodes a shortened GHR (Smith et al., 1989; Baumbach et al., 1989). This variant has the extracellular domain in common with the full-length receptor, but has the transmembrane and cytoplasmic domains replaced by a short hydrophilic amino acid stretch that confers secretion to this isoform, which then circulates as a highaffinity GH-binding protein (GHBP) (more below). mRNAs that predict truncated membraneanchored GHR isoforms have been described that encode the extracellular and transmembrane domains, but only have several intracellular residues (Dastot et al., 1996; Ross et al., 1997). These relatively low-abundance mRNA variants arise by alternative splicing and by frameshifting to yield the truncated receptor forms. Their exact physiological significance is as yet not known. Another variant GHR found only in humans lacks the 22 extracellular domain residues encoded by exon 3 (66 base pairs) of the human GHR gene. This interesting variant was first thought to arise from alternative splicing (Urbanek et al., 1992). A recent study suggested instead that it arises from an ancestral homologous recombination between two retroelements in intronic sequences surrounding exon 3 only in humans (Pantel et al., 2000). The allele for the exon 3 receptor is found at a frequency of 25% compared with 75% for

the full-length allele (Pantel et al., 2000). As yet, no clear difference in GH-binding affinity or function has been determined for the exon 3 GHR form.

Main activities and pathophysiological roles All known activities of GH are believed to be mediated by the GHR. The most apparent actions of GH relate to its promotion of longitudinal growth and muscle mass. Elevated levels of GH, such as result from pituitary tumors that hypersecrete the hormone, yield the clinical syndrome of acromegaly. Acromegalics manifest bony and connective tissue overgrowth and visceromegaly. If present prior to pubertal epiphyseal closure, acromegaly results in excessive height and is referred to as gigantism. GH deficiency or GH resistance due to GHR mutations results in shortness of stature. As described in the chapter on GH, the GHR also mediates the many metabolic effects of GH.

GENE

Accession numbers Human GHR: NM_000163 Rabbit GHR: AF015252 Mouse GHR: MM010284 Rat GHR: X16726 Bovine GHR: X70041 Ovine GHR: M89912 Porcine GHR: X54429 Chicken GHR: M74057

Sequence Figure 1 shows the nucleotide sequence of the human GHR full-length cDNA.

Chromosomal location and linkages The gene that encodes the human GHR is present in a single copy on chromosome 5p13-p12 (Godowski et al., 1989; Barton et al., 1989). It spans roughly 90 kb and contains nine coding exons (exons 2±10) (reviewed in Schwartzbauer and Menon, 1998; Edens and Talamantes, 1998). Exon 2 encodes some 50 UTR sequence, the signal sequence (18 residues), and the first five residues of the extracellular domain

Growth Hormone Receptor 3 Figure 1 Coding region cDNA sequence of the human GHR. Predicted leader sequence-encoding region is underlined in bold. Predicted transmembrane-encoding region is italicized and underlined in bold. Stop codon (TAG) is indicated. atggatctctggcagctgctgttgaccttggcactggaggatcaagtgatgctttttctggaagtgaggccacagcagctat ccttagcagagcaccctggagtctgcaaagtgttaatccaggcctaaagacaaattcttctaag gagcctaaattcaccaagtgc cgttcacctgagcgagagactttttcatgccactggacagatgaggttcatcatggtacaaaga acctaggacccatacagctgt tctataccagaaggaacactcaagaatggactcaagaatggaaagaatgccctgattatgtttctgctggggaaaacagctgtta ctttaattcatcgtttacctccatctggataccttattgtatcaagctaactagcaatggtggtacagtggatgaaaagtgtttctctgt tgatgaaatagtgcaaccagatccacccattgccctcaactggactttactgaacgtcagttta actgggattcatgcagatatcca agtgagatgggaagcaccacgcaatgcagatattcagaaaggatggatggttctggagtatgaa cttcaatacaaagaagtaaa tgaaactaaatggaaaatgatggaccctatattgacaacatcagttccagtgtactcattgaaa gtggataaggaatatgaagtgc gtgtgagatccaaacaacgaaactctggaaattatggcgagttcagtgaggtgctctatgtaac acttcctcagatgagccaattt acatgtgaagaagatttctactttccatggctcttaattattatctttggaatatttgggctaacagtgatgctatttgtattcttattttct aaacagcaaaggattaaaatgctgattctgcccccagttccagttccaaagattaaaggaatcgatccagatctcctcaaggaag gaaaattagaggaggtgaacacaatcttagccattcatgatagctataaacccgaattccacag tgatgactcttgggttgaattta ttgagctagatattgatgagccagatgaaaagactgaggaatcagacacagacagacttctaag cagtgaccatgagaaatca catagtaacctaggggtgaaggatggcgactctggacgtaccagctgttgtgaacctgacattctggagactgatttcaatgcca atgacatacatgagggtacctcagaggttgctcagccacagaggttaaaaggggaagcagatctcttatgccttgaccagaaga atcaaaataactcaccttatcatgatgcttgccctgctactcagcagcccagtgttatccaagcagagaaaaacaaaccacaacc acttcctactgaaggagctgagtcaactcaccaagctgcccatattcagctaagcaatccaagttcactgtcaaacatcgactttta tgcccaggtgagcgacattacaccagcaggtagtgtggtcctttccccgggccaaaagaataag gcagggatgtcccaatgtg acatgcacccggaaatggtctcactctgccaagaaaacttccttatggacaatgcctacttctgtgaggcagatgccaaaaagtg catccctgtggctcctcacatcaaggttgaatcacacatacagccaagcttaaaccaagaggacatttacatcaccacagaaagc cttaccactgctgctgggaggcctgggacaggagaacatgttccaggttctgagatgcctgtcccagactatacctccattcatat agtacagtccccacagggcctcatactcaatgcgactgccttgcccttgcctgacaaagagtttctctcatcatgtggctatgtgag cacagaccaactgaacaaaatcatgccttag

N-terminus. Exons 3±7 encode almost the entire remainder of the extracellular GH-binding domain (except when exon 3 is deleted, as above). Exon 8 encodes the 24-residue transmembrane domain and a few residues on each of the extracellular and intracellular domain sides flanking it. Exons 9 and 10 encode the remainder of the cytoplasmic domain and 2 kb of the 30 UTR. The arrangement of the mouse GHR gene exons is quite similar to that of humans with the exception of exon 4B, an extra 8 amino acid-encoding exon not found in other species, and exon 8A, an exon alternatively spliced in rodents to yield the GHBP, as above (Edens et al., 1994; Zhou et al., 1994). The significance of the exon 4B amino acids, if any, is as yet unknown. GHR mRNAs from various species exhibit substantial heterogeneity of 50 UTRs that arises from alternative exon 1 splicing, the significance of which is also as yet unknown (Schwartzbauer and Menon, 1998; Edens and Talamantes, 1998).

PROTEIN

Accession numbers Human GHR: P10912 Rabbit GHR: P19941 Mouse GHR: P16882 Rat GHR: P16310

Bovine GHR: P79108 Ovine GHR: Q38575 Porcine GHR: P19756 Chicken GHR: Q02092

Sequence Figure 2 shows the protein sequence of the human GHR full-length cDNA.

Description of protein As mentioned above, the GHR is an N-glycosylated surface receptor that spans the membrane only once and is in the cytokine receptor superfamily. The human GHR extracellular domain is predicted to extend from residues 1 to 246 (this numbering begins at the first residue of the mature GHR that results after removal of the 18 amino acid signal sequence). Its overall structure is shown in Figure 3A. Cocrystallization and structural examination of the bacterially expressed recombinant (nonglycosylated) nearly complete hGHR extracellular domain (ECD) (residues 1±238) complexed to hGH by de Vos and colleagues (1992) provided a great deal of detailed information as to the GHR's topography and the nature of its interaction with the hormone (Figure 3B). GH is composed of four antiparallel helical bundles connected by loops of variable length and possesses

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Stuart J. Frank and Joseph L. Messina Figure 2 Amino acid sequence of the human GHR. Predicted leader sequence ( 18 to 1) is underlined. Predicted transmembrane domain is italicized and underlined in bold. The Box 1 region of the cytoplasmic domain is underlined in bold. -18 51 121 191 261 331 401 471 541 611

mdlwqlll tlalagssda fsgseataai lsrapwslqs vnpglktnss kepkftkcrs peretfschw tdevhhgtkn lgpiqlfytr rntqewtqew kecpdyvsag enscyfnssf tsiwipycik ltsnggtvde kcfsvdeivq pdppialnwt llnvsltgih adiqvrweap rnadiqkgwm vleyelqyke vnetkwkmmd pilttsvpvy slkvdkeyev rvrskqrnsg nygefsevly vtlpqmsqft ceedfyfpwl liiifgifgl tvmlfvflfs kqqrikmlil ppvpvpkikgidpdllkegkleevntilai hdsykpefhs ddswvefiel didepdekte esdtdrllss dhekshsnlg vkdgdsgrtsccepdiletdfnandihegtsevaqpqrlk geadllcldq knqnnspyhd acpatqqpsv iqaeknkpqp lptegaesth qaahiqlsnp sslsnidfya qvsditpags vvlspgqknk agmsqcdmhp emvslcqenf lmdnayfcea dakkcipvaphikveshiqp slnqediyit teslttaagr pgtgehvpgs empvpdytsihivqspqgli lnatalplpd keflsscgyv stdqlnkimp

Figure 3 GHR protein structure and the GH±GHR complex. (A) Diagram of the human GHR protein structure. See text for details. Extracellular, transmembrane, and cytoplasmic domains are indicated. The extracellular subdomains 1 and 2, as well as the hinge and juxtamembrane regions, are also shown. The postions of the intrachain cysteine-mediated disulfide linkages present extracellular subdomain 1 in GHRs of all species are indicated by pairs of joined C residues. YGEFS is the WSXWS-like equivalent of the GHR. The positions of the Box 1 and UbE motifs (see text) and the conserved tyrosine residues (Y) in the cytoplasmic domain are shown. (B) Cartoon of the crystallographically-determined structure of GH bound to the soluble GHR extracellular domain. The structural features of the GHR extracellular domain are indicated, as described in the text. Sites 1 and 2 of GH, which are quite distinct, interact with very similar contact points on each GHBP molecule to result in the GH : GHBP2 complex shown. The extensive inter-GHBP dimerization interface is indicated. This diagram is based on information in de Vos et al. (1992), and is adapted from Frank (2002) with permission. B

A C C

Subdomain 1 C C C C

Extracellular Domain

GH

Hinge

1

2

Subdomain 2 YGEFS Subdomain 1

Juxtamembrane

Transmembrane Domain

Box 1 Motif

Hinge Region

Y UbE Motif

Subdomain 2 Cytoplasmic Domain Y

ECD 1

ECD 2

Y Y Y Y

Dimerization Interface

Growth Hormone Receptor 5 no axis of symmetry. Yet the GH±GHRECD complex was found in a 1 : 2 stoichiometry with each of the two different GH binding sites (site 1 and site 2) contacting a similar set of residues on each of the dimerized GHR ECDs. Further studies showed that the strength of interaction for each of these sites differed in important ways (more below). The crystal structure indicates that the ECD is divided into two sandwich subdomains, referred to as subdomain 1 (residues 1±123) and subdomain 2 (residues 128±238). Each subdomain is made up of seven strands arranged into two antiparallel sheets. Subdomains 1 and 2 are linked by a fourresidue hinge region. The remaining ECD C-terminal eight amino acids (residues 239±246) were not included in the crystal structure and there is therefore no clear indication as to the structure they adopt. The GHR's hormone binding region is largely accounted for by residues in subdomain 1 and the inter-subdomain hinge residues. In addition, each receptor within the GH±(GHRECD)2 complex interacts with the other via an extensive dimerization interface region. Six intermolecular bonds between residues in subdomain 2 of each ECD occupy a binding area of 500 AÊ2, as compared to the site 1 GHRECD and site 2 GHRECD binding areas of 1230 AÊ2 and 900 AÊ2, respectively. Other notable ECD features present in the GHR include six conserved cysteine residues (three pairs that engage in intramolecular disulfide linkages), and a WSXWS-like motif. The latter motif is conserved in cytokine receptors and has the sequence YXXFS in mammalian GHRs. Studies indicate that both the intrachain disulfide linkages and the WSXWS-like motif in the GHR are likely critical to the structural integrity of the receptor ECD without being involved in binding of GH (Fuh et al., 1990; Baumgartner et al., 1994). ECD glycosylation does not appear critical to GH binding either; bacterially expressed (nonglycosylated) GHR ECD binds GH with an affinity similar to that of glycosylated GHBP isolated from serum (Fuh et al., 1990). In contrast to the ECD, there exists no structural information regarding the GHR cytoplasmic domain. However, several features of this roughly 350 residue domain are known and quite relevant to GHR signaling and trafficking. Like other cytokine receptors, the GHR membrane proximal cytoplasmic domain harbors a proline-rich sequence (ILPPVPVP in mammalian GHRs) called Box 1, which is critical for association with JAK2. A short acidic stretch analogous to the Box 2 domains of other cytokine receptors (Murakami et al., 1991) roughly 30 residues C-terminal to Box 1 resides in a region likely required for optimal JAK2 interaction (Frank et al., 1994).

Also conserved between mammalian species are six tyrosine residues that are potential targets of GHinduced phosphorylation and are thus important in signaling (in the human, these are residues Y314, Y469, Y516, Y548, Y577, and Y609). GHR residues important for GH-induced receptor internalization and degradation include a phenylalanine at residue 346 in the rat (F327 in human), first implicated by Allevato et al. (1995), and the highly conserved sequence surrounding that phenylalanine, DSWVEFIELD, which is a so-called ubiquitindependent endocytosis (UbE) motif discovered by Strous and colleagues (Strous et al., 1996; Govers et al., 1999; van Kerkhof et al., 2001). The UbE is thought to mediate GH-induced GHR ubiquitination by allowing interactions between the receptor and ubiquitin conjugases and ligases. Though GHR ubiquitination itself is not required, the presence of an intact ubiquitination system and the UbE, perhaps by allowing ubiquitination of other associated proteins, appears to foster GHR's interaction with clathrin-coated pits and the cell's endocytic machinery. The GHR, like some other cytokine receptors, migrates aberrantly in SDS±PAGE. Its expected Mr, given its roughly 620 amino acid sequence, is roughly 70 kDa (Leung et al., 1987). However, depending on species, it migrates under reducing conditions in the 110±140 kDa range, in each species with a diffuse appearance (Argetsinger et al., 1993; Frank et al., 1994). This aberrant migration is likely only partly contributed to by glycosylation and ubiquitination (the latter of which, in any case, is only seen in response to GH) (Leung et al., 1987); rather, it likely reflects nonclassical binding of SDS by these receptors related to particular aspects of their amino acid sequences.

Relevant homologies and species differences Though GH can bind the prolactin receptor (PRLR), the reverse does not occur. Yet, among the cytokine receptors, GHR is most structurally similar to the PRLR. Sequence identity between the two receptors is less than 30%, but in data derived from cocrystallization of human GH and human PRLR ECD the 1 : 2 ligand:receptor stoichiometry and overall structural features of the PRLR ECD are very similar to those seen in the GH±GHR ECD crystal structure (Somers et al., 1994; reviewed in Behncken and Waters, 1999). Among known species, the GHRs with most similarity are the human and rabbit (84%

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Stuart J. Frank and Joseph L. Messina

identical). Other notable identity comparisons are between human and rodent (roughly 70%) and human and chicken (roughly 59%).

Affinity for ligand(s) While estimates of affinity of GH for the GHR vary, dissociation constants reported are generally in the 0.1±1.5 nM range. Variability is engendered to some extent by whether the receptor component is the extracellular ligand-binding domain alone or the fulllength receptor expressed in cells. As mentioned above, crystallographic and mutagenetic studies strongly suggest that GH binds to the GHR so as to cause formation of a tripartite GH : GHR2 complex (Cunningham et al., 1991; de Vos et al., 1992). In this model, GH site 1, which has a higher affinity for the GHR than does site 2, interacts with the first receptor, allowing the sequential interaction of site 2 with the second receptor. GHR±GHR interactions via the dimerization interface allow stabilization of this complex and substantial evidence suggests that the dimerized receptor is the activated GHR conformation (see Figure 4A). It is not yet known whether the GHR exists to any degree as a dimer in the unliganded state, as apparently does the related erythropoietin receptor (reviewed in Frank, 2002). If so, it is possible that the conformation of the receptor dimer is changed by GH (rather than the GH-induced generation of the receptor dimer) in order to achieve the active state. The sequential dimerization model may explain the bell-shaped GH concentration dependence often observed experimentally (see Figure 4B) in that at very high GH concentrations all available GHRs are occupied by site 1 interactions, thereby allowing less receptor dimers to form and less effective GH action to be seen. Site 2 GH mutants have been prepared and function as GH antagonists in both experimental and clinical situations (Fuh et al., 1992; Chen et al., 1994; Flyvbjerg et al., 1999; Trainer et al., 2000). The predominant form of pituitary-derived GH is the 22 kDa (191 residue form). Two other somewhat related molecules can also bind to the GHR. One is the product of an alternatively spliced GH transcript deleted of residues 32±46, the so-called 20K GH (20 kDa in size). 20K GH is a minority form in humans (perhaps 10% of GH in circulation). Its biological significance as compared to the 22 kDa form is unknown, but the way 20K binds to GHR is apparently different from that of 22 kDa GH (Wada et al., 1998; Uchida et al., 1999; Tsunekawa et al., 2000). 20K has much lower (one-tenth) site 1 affinity for GHR, but similar site 2 affinity. However, it is

believed that 20K induces stronger interaction between GHR dimer interfaces than does 22 kDa GH, compensating for 20K's diminished site 1 affinity and yielding similar overall affinities for the two GH forms. Indeed, expression of hGHRs mutated at certain dimerization interface residues yielded markedly reduced proliferation in response to 20K versus 22 kDa GH and 20K is less apt to form a stable 1 : 1 complex with GHR, instead engaging almost exclusively in a 1 : 2 complex. In cell culture experiments, 20K and 22 kDa GH promote proliferation of GH-dependent cells similarly at lower concentrations but 20K exhibits less self-inhibition at higher concentrations. The other non-GH ligand for the GHR is the placentally derived hormone placental lactogen (PL). PL is structurally similar to PRL and GH, but no specific PL receptor has been found. A recent report suggests a novel mechanism of PL action in which PL functionally binds to both the PRLR and GHR in a heterodimeric arrangement such that GHR is engaged by the PL site 1 and PRLR is engaged by PL site 2 (Herman et al., 2000). If validated, this would be the first such heterodimeric interaction reported for members of the subfamily of cytokine receptors that includes GHR and PRLR. Depending on the species of origin of the hormone and receptor, GH itself may not bind to the GHR. Primate GHRs cannot bind and be activated by nonprimate GH. Yet, primate GH (hGH, for example) can bind and activate both primate and nonprimate GHRs. The specificity for this longappreciated peculiarity of species-dependent GH± GHR interaction is now known to reside in differences in interacting residues contributed from both GH (residue 171 is Asp in primates and His in nonprimates) and GHR (residue 43 is Arg in primates and Leu in nonprimates). Mutagenesis experiments (summarized in Behncken and Waters, 1999) suggest that charge repulsion and steric hindrance explain the species specificity. Leucine at residue 43 of the nonprimate GHR, being shorter and without charge compared with arginine, can apparently accept either Asp171 or His171 from the GH of either primate or nonprimate, but no such tolerance apparently exists in the primate GHR arginine at residue 43.

Cell types and tissues expressing the receptor When assessed using sensitive RT/PCR methods, GHR mRNA in humans is widely distributed in various tissues, including liver, fat, muscle, kidney,

Growth Hormone Receptor 7 Figure 4 High GH concentrations and GH antagonists inhibit GH signaling. (A) Potential mechanisms of suppression of signaling by high concentrations of GH and the GH antagonist effect. Unliganded GHR, shown as a monomer, is dimerized in response to GH (upper panel). High-dose suppression (see bell-shaped dose response curve in B) for GH (middle panel) is envisioned as a reflection of GH binding to all available GHR molecules via site 1 in unproductive monomeric interactions. GH antagonist inhibition of GH signaling is viewed as the antagonist (mutated at site 2) competing for GHR binding via site 1, thus lessening productive GH-induced GHR dimerization. (This may not explain all aspects of GH antagonist effects.) (B) Concentration-dependence curves for GH alone and for GH plus GH antagonist treatment of GHR-expressing cells. At very high GH concentration, several biological and biochemical responses (e.g. proliferation, tyrosine phosphorylation, receptor disulfide linkage) are lessened. Addition of GH antagonist (mutant GH with markedly decreased site 2 binding affinity) in the presence of constant GH concentration causes inhibition of GH signaling. A and B are adapted from Frank (2002) with permission. GH 1 2 A

Active Dimer GH 1 2

H]

ow

[G

Signaling

L

GH 1 2

GH 1 2

GH 1 2

GH 2 1 No Active Dimer

High [GH] No Signaling GH 1 2

+

GHA 1 GH 1 2

GH Low an [GH ta go ] nis t

GHA 1 No Active Dimer

No Signaling

B % Maximal Response

100

Increasing [GH] alone

Increasing [GH]

50

0

Antagonist added

Constant [GH] plus increasing [antagonist] Hormone Concentration

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Stuart J. Frank and Joseph L. Messina

heart, prostate, fibroblasts, and lymphocytes (Martini et al., 1995; Hermansson et al., 1997; Ballesteros et al., 2000). The highest levels are found in liver, followed by fat and muscle. The truncated GHR isoforms (lacking most of the cytoplasmic domain) are also widely expressed, again most abundant in liver, fat, and muscle, though at much lower levels than the fulllength receptor. GHR mRNA is similarly distributed in rodent tissues. Among lymphoid cells, assessment in humans of GHR mRNA abundance as well as flow cytometry suggest that B cells are much more enriched in GHRs than are T cells (Badolato et al., 1994; Rapaport et al., 1995; Hattori et al., 2001). Although GHR is widely expressed in tissues, experimentally useful cell lines expressing GHR are scarce. Some important examples of lines expressing sufficient GHR to allow biochemical and functional studies include the human IM-9 B lymphocyte, 3T3F442A and 3T3-L1 mouse pre-adipocyte fibroblasts and adipocytes, C2C12 rat myoblasts and myotubes, human HuH7 hepatoma cells, CWSV-1 rat hepatocytes, and H4IIE rat hepatoma cells.

Regulation of receptor expression In principle, GHR abundance may be regulated by various mechanisms, including transcriptional, posttranscriptional, and posttranslational mechanisms. Many studies have examined the hormonal and ontogenic regulation of GHR gene expression at various target tissues in different species; some have produced conflicting results. Several excellent reviews that summarize much of this information include those by Schwartzbauer and Menon (1998) and Waters (1999).

Release of soluble receptors GHBP is a high-affinity soluble GH-binding protein present in the circulation of many species (reviewed in Baumann, 2001). Roughly 50% of circulating GH in humans is complexed to GHBP (Baumann et al., 1988), which is composed of a soluble form of the GHR extracellular domain. GHBP is derived by two quite independent mechanisms (see Figure 5). As

Figure 5 Mechanisms of GHBP generation. As described in the text, GH-binding protein (GHBP) is a circulating version of the extracellular domain of the GHR. In rodents (A), GHBP derives by alternative splicing of the GHR mRNA, such that a hydrophilic stretch of amino acids (black ball) replaces the transmembrane and cytoplasmic domains. In humans, rabbits, and other species (B), GHBP arises by proteolytic cleavage of the membrane GHR in the extracellular juxtamembrane region to yield the shed GHBP and the GHR remnant. Current evidence favors the transmembrane ADAM metalloprotease, TACE, as the enzyme catalyzing the GHR proteolysis and GHBP shedding.

GH 1 2

A. Alternative Splicing (rodents)

GHBP

GHR mRNA

Translation

Alternative Splicing

GH 1 2

GHR

GHR gene GHBP mRNA

B. Proteolysis (rabbits and humans)

GH 1 2

GHBP Metalloprotease Activator

GHR Proteolysis and

(eg., PMA, PDGF)

GHBP shedding

ADAM metalloprotease GHR GHR Remnant

GH 1 2

Growth Hormone Receptor 9 detailed above, in rodents and some other species, GHBP is generated by alternative splicing of the GHR mRNA such that the transmembrane and cytoplasmic domains are replaced by a short hydrophilic tail that confers secretion. In contrast, GHBP in human and rabbit is generated by regulated proteolysis of the full-length GHR and `shedding' of the extracellular domain as the GHBP. Recent data indicate that this proteolytic GHR processing is inducibly mediated by metalloprotease activity, likely TACE (TNF-cleaving enzyme). Interestingly, rodent GHRs can also be a target of this activity (Alele et al., 1998; Zhang et al., 2000; Guan et al., 2001). In addition to generating GHBP, inducible GHR proteolysis causes GHR loss and accumulation of a transmembrane/cytoplasmic domain remnant and can impact GH action. Cells are desensitized to GH stimulation after proteolysis is induced; conversely, GH-induced GHR dimerization lessens the receptor's susceptibility to proteolysis (Zhang et al., 2001; Frank, 2001). Aside from these effects of proteolysis on cellular GH sensitivity, GHBP's role(s) in GH physiology is not yet clearly known; in different model systems GHBP can both potentiate and inhibit GH action (reviewed in Baumann, 2001). In contrast to the cell surface 1 : 2 GH : GHR stoichiometry, most GHBPassociated GH in human circulation exists in 1 : 1 GH : GHBP stoichiometry. This and slightly higher affinity association of GH to GHR relative to GH± GHBP interaction have led to speculation that part of GHBP's role is as a reservoir for GH, effectively delivering it from circulation to cell surface GHR (Baumann et al., 1994). In circulation, GHBP-bound GH has a prolonged half-life relative to free GH, likely due to diminished access to GH degradation/ disposal sites within the body (Clark et al., 1996). Given the pulsatile delivery of GH into the circulation, one possible role for GHBP is to stabilize GH bioavailability (Veldhuis et al., 1993). GHBP levels vary during ontogeny and with various physiological and pathophysiological states and it may be that they reflect GHR levels in relevant target tissues. In humans, GHBP levels rise steadily during childhood, they vary little diurnally or after early adulthood, except for decline after age 60 (Silbergeld et al., 1989; Maheshwari et al., 1996). Children with idiopathic short stature (decreased height unaccounted for by genetic potential or GH or other hormonal deficiency) have lower GHBP levels than controls (Carlsson et al., 1994) perhaps reflecting a mild GH resistance state. In full-blown GH insensitivity (Laron) syndrome, both GHR and GHBP levels are usually very low. GHBP is low in type 1 diabetes and increased by insulin treatment

(Menon et al., 1992). GHBP is low in malnutrition, but not in obesity (Hochberg et al., 1992). The role, if any, of GHBP in these situations is as yet unclear.

SIGNAL TRANSDUCTION

Associated or intrinsic kinases Carter-Su and colleagues (Foster et al., 1988; CarterSu et al., 1989) were the first investigators to demonstrate that GH activates intracellular tyrosine phosphorylation and that a tyrosine kinase activity copurifies with the GHR. Yet, the GHR lacks an intrinsic kinase domain (Leung et al., 1987). Like other cytokine receptors, the GHR was found to physically and functionally associate with a member of the Janus tyrosine kinase family, JAK2 (Argetsinger et al., 1993). The JAKs are nonreceptor cytoplasmic tyrosine kinases that in mammals include JAK1, JAK2, JAK3, and TYK2 (reviewed in Ihle, 1995). All but JAK3 are ubiquitously expressed; JAK3 is expressed in a lymphoid-specific fashion. The JAK family members each have a characteristic structural organization typified by the presence of a C-terminal kinase domain, a kinase-like (pseudokinase) domain just N-terminal to the kinase domain, and the N-terminal one-half of the molecule. JAK2 associates with a number of cytokine receptors, including the PRLR, EpoR, and thrombopoietin receptor. Though association with JAK1 has also been shown, it is thought that GHR's physical association with JAK2 is critical to GH action. The GHR's proline-rich Box 1 region, which is similar to that found in some other cytokine receptors, is critical in allowing physical and functional association with JAK2, though full association may also depend on other membraneproximal receptor cytoplasmic domain regions (Frank et al., 1994; Sotiropoulos et al., 1994; VanderKuur et al., 1994). The JAK2 region required for interaction with the GHR maps to the N-terminal one-fifth of the molecule, though detailed mapping within this region is still lacking (Frank et al., 1995; Tanner et al., 1995). Removal of the JAK2 tyrosine kinase domain, while it does not support GH-induced signaling, does not impair association with the GHR, consistent with the notion that association of the GHR with JAK2 is not dependent on GH and does not require tyrosine phosphorylation of either the receptor or the kinase (Frank et al., 1995). However, the GHR±JAK2 association is likely enhanced by GH treatment (Argetsinger et al., 1993) and this enhanced association appears to depend on GH's

10 Stuart J. Frank and Joseph L. Messina ability to cause GHR dimerization (Zhang et al., 1999). Whether dimerization alone or an additional conformational change in the receptor is required for optimal JAK2 activation is still unknown (reviewed in Frank, 2002). Other tyrosine kinases are activated in response to GH. These include focal adhesion kinase (FAK) (Zhu et al., 1998a) and the Src family kinases c-src and c-fyn (Zhu et al., 1998b). For FAK, there is evidence that its interaction with the GHR±JAK2 complex may be via JAK2, though the regions of each responsible for the interaction have not been defined. A direct association of the Src family kinases with GHR or JAK2 has not been observed, but their involvement in a GH-induced signaling complex via association with FAK has been speculated. Additionally, a CORT (cloning of receptor targets) strategy using the tyrosine phosphorylated distal tail of the GHR cytoplasmic domain as bait found that csrc kinase (Csk), a tyrosine kinase that negatively regulates src by tyrosine phosphorylation, can associate with the tyrosine phosphorylated GHR (Moutoussamy et al., 1998). This may suggest a further link of the Src family kinases with the GHR. Potential roles of these non-JAK2 tyrosine kinases in GH action are mentioned below.

Cytoplasmic signaling cascades GH-induced GHR dimerization is presumed to be critical for GH signaling by promoting the apposition of JAK2 molecules that are associated with the dimerized GHRs. In this model, the closely situated JAK2 tyrosine kinase domains more readily undergo trans- and autophosphorylation, the net effect of which is to further enhance JAK2 activation. Though elements of this model are undoubtedly correct, our lack of structural information about the GHR cytoplasmic domain associated with JAK2 makes us unable to be certain about the actual JAK2 activation mechanism. In any case, several cytoplasmic signaling pathways become activated in response to GHinduced tyrosine kinase activation (see Figure 6). For a more comprehensive discussion of these pathways and their relevance, the reader is directed to several recent review articles (Carter-Su et al., 1996; Frank and O'Shea, 1999; Zhu et al., 2001). Like signaling through other cytokine receptors, GHR engagement results in activation of STATs (signal transducers and activators of transcription). STATs are latent cytoplasmic transcription factors that contain an SH2 domain, a principal tyrosine phosphorylation site, and DNA-binding and transactivation domains. The general model for their

activation by different cytokine receptor±JAK complexes is that the STAT molecule is recruited to the tyrosine phosphorylated receptor or JAK via the STAT's SH2 domain, becomes tyrosine phosphorylated by the JAK, and then homo- or heterodimerizes with another STAT and migrates to the nucleus. There the STAT dimer exerts transcriptional activation to a target gene that has in its 50 regulatory region particular DNA sequences specifically recognized by the STATs. Extensive discussion of mechanisms and consequences of STAT activation can be found in several excellent recent reviews that specifically deal with GH-induced STAT signaling (Davey et al., 1999a; Choi and Waxman, 2000; Herington et al., 2000). GH promotes the tyrosine phosphorylation and DNA-binding capacity of four different STAT molecules ± STAT1, STAT3, STAT5a, and STAT5b (Campbell et al., 1995; Gronowski and Rotwein, 1994; Gronowski et al., 1995; Ram et al., 1996; Silva et al., 1996; Smit et al., 1997; Waxman et al., 1995). Though binding sites in the GHR±JAK2 complex have not been identified for all of these, it is generally accepted that GH-induced STAT1 and STAT3 activation require little more of the GHR cytoplasmic domain than the proximal region necessary for JAK2 activation. This is in contrast to activation of STAT5a and STAT5b, which require GH-induced tyrosine phosphorylation of the receptor tail in order to become fully activated with regard to tyrosine phosphorylation, DNA binding, and transactivation of target genes (Hansen et al., 1996; Smit et al., 1996, 1997; Sotiropoulos et al., 1995, 1996; Wang et al., 1996; Yi et al., 1996). In fact, the phosphorylation of each of certain tyrosine residues in the receptor cytoplasmic domain has been shown sufficient to bind and activate STAT5 and STAT5-dependent transactivation. These include the equivalents to human tyrosines 516, 548, and 609 (Hansen et al., 1996, 1997; Smit et al., 1996, 1997). It is possible, though not yet shown, that motifs mediating association with and activation of STATs 1 and 3 might reside in JAK2, rather than the GHR. The consequences of GH-induced STAT activation are discussed below. Other pathways activated by GH also require JAK2 activation. These include the MAP kinase (ERK and JNK) cascades and the PI-3 kinase cascade; a number of adapter proteins, including IRS family members, SIRP, and SH2B- are also recruited in response to GH (Souza et al., 1994; Ridderstrale et al., 1995; Argetsinger et al., 1995, 1996; Yamauchi et al., 1998; Kim et al., 1998; Stofega et al., 1998; Carter-Su et al., 2000). GH-induced ERK1 and ERK2 activation were first shown in 1992 (Anderson, 1992; Campbell et al., 1992; Moller et al.,

Growth Hormone Receptor

11

Figure 6 GHR signaling pathways. As mentioned in the text and detailed in the review articles cited, the major signaling pathways engaged downstream of the activated GHR±JAK2 assembly include the STAT, ERK, and PI-3 kinase pathways. Some cascades leading to their activation and crosstalk between them are indicated. Tyrosine phosphorylation is indicated by Yp. Serine/threonine phosphorylation is indicated by S/Tp. Crosstalk between the GHR and the EGFR±ErbB2 system is shown with GH causing tyrosine phosphorylation of the EGFR and serine/threonine phosphorylation of ErbB-2. The former is believed to contribute to GH-induced ERK activation; the latter is thought to desensitize ErbB-2 to EGF-induced activation. Some downstream biological and biochemical outcomes of activation of these pathways are indicated in the shaded boxes. Other pathways activated and referred to in the text are in the open box.

1

EGF

GH 2

GHR

EGFR ErbB2

EGFR

2 JAK

Yp Yp

Plasma Membrane

Yp IRS 2/3

Yp

Yp

S/Tp Shc Grb2 Sos

–

JAK2

IRS 1

–

PI3K

SHP2/SIRP

+

Ras

STAT1/3

Raf

Other Pathways (src kinases, p38, JNK, SH2B)

MEK

ERK STAT5

Proliferation

c-fos Proliferation

1992; Winston and Bertics, 1992). GHR's ability to couple to ERK activation corresponds to its ability to effectively couple to a catalytically active JAK2 molecule ± only the membrane proximal GHR region is required for this pathway (Moller et al., 1992; Sotiropoulos et al., 1994; VanderKuur et al., 1994; Frank et al., 1995). Several potential upstream activators of the ERK pathway are accessed by GH. GH activates the Shc±Grb2±Sos±Ras±Raf pathway with kinetics that suggest it is a mechanism of ERK activation (VanderKuur et al., 1995, 1997). It has also been observed that GH-induced ERK activation can be inhibited by PI-3 kinase inhibitors and that expression of IRS-1 in IRS-deficient cells enhances GH-induced ERK activation in a PI-3 kinase inhibitor-sensitive fashion, findings that suggest that the PI-3 kinase pathway (or a related enzyme pathway) may function upstream of ERK activation

anti-apoptosis ? IGF-1

Spi 2.1 ALS Insulin CYP genes SOCS genes IGF-1

(Kilgour et al., 1996; Hodge et al., 1998; Liang et al., 2000). GH-induced activation of the SHP-2 protein tyrosine phosphatase may also positively modulate ERK activation (Kim et al., 1998). Another interesting route to GH-induced ERK activation is JAK2-mediated tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Yamauchi et al. (1997) showed that GH-induced phosphorylation of EGFR, likely at residue Y1068, allowed Grb-2 association at that site and augmented GH-induced ERK activation. This was unaccompanied by a change in EGFR kinase activity, implying that GH-induced ERK signaling was utilizing EGFR as an adapter molecule. As yet, the relevance of MAP kinase and PI-3 kinase activation by GH is not certain and may differ widely, depending on the cell type in question. A role in GH-induced proliferation in some cells is possible,

12 Stuart J. Frank and Joseph L. Messina as expression of IRS-1 in IRS-deficient 32D cells enhances GH-stimulated proliferation (Liang et al., 1999). Some reports suggest an anti-apoptotic effect of GH, which may relate to the PI-3 kinase/Akt or NFB pathways (Costoya et al., 1999; Jeay et al., 2000, 2001). Yet, in some cells, such as 3T3-F442A fibroblasts, GH inhibits proliferation induced by epidermal growth factor. In those cells, GH also causes a desensitization of EGF-induced activation of the EGFR family member ErbB-2, and this desensitization is mediated by ERK activation (Kim et al., 1999). Independent of its role in proliferation, ERK activation has clearly been related to c-fos activation that is acutely stimulated by GH (Hodge et al., 1998) and full activation of GH-induced STAT5 signaling may also rely on ERK activation (Pircher et al., 1999). Further, other MAP kinases, including JNK and p38, have been shown in some systems to be activated in response to GH and p38 may allow mitogenesis, as well as cytoskeletal reorganization (Zhu et al., 1998b; Goh et al., 2000; Zhu and Lobie, 2000). GH-activated signaling is modulated by several molecules. SH2-B has been shown to be a JAK2 activator; its association with JAK2 may augment signaling (Carter-Su and Rui, 1999). The SOCS proteins are activated in response to GH and negatively regulate in several ways via interactions with both GHR and JAK2 (Ram and Waxman, 1999). Other signaling molecules, such as SIRP and Grb-10, may also attenuate GH signaling

(Moutoussamy et al., 1998; Stofega et al., 2000). These are in addition to the homologous downregulation of GH signaling mediated by receptor ubiquitination, endocytosis, and degradation (reviewed in Frank, 2001).

DOWNSTREAM GENE ACTIVATION

Transcription factors activated GH activates a number of transcription factors (see Table 1). The STATs were mentioned above. STATs 1, 3, 5a, and 5b are activated in a number of systems by GH. In both humans (Winer et al., 1990; Pincus et al., 1996; Veldhuis, 1996) and, more dramatically in rodents (Jansson et al., 1985), GH secretion and therefore GH levels are pulsatile in a sex-specific manner. That is, in males GH pulses occur at roughly 3.5 hour intervals with interpulse levels being nearly undetectable (Tannenbaum and Martin, 1976). In females, the pulses are dampened and there are nearly continuous GH levels. Of the STATs, only STAT5b transcriptional activation responds to this pulsatile GH secretion. In liver and liver cell culture models, STAT5b is activated by a GH pulse, after which it is deactivated and restored for activation by the time a second pulse is given 4 hours later. Constant GH treatment

Table 1 GHR target genes, transcription factors, and relevant effects Target gene

Transcription factor/pathway

Effect relevant to GH action

IGF-I

?HNF-1, STAT5b, ?PI-3 kinase pathway

Growth mediation

c-fos

ERK pathway, TCF, SRF, STATs 1, 3

Acid labile subunit (ALS) of IGFBP-3 complex

STAT5b

Serine protease inhibitor 2.1

STAT5b

SOCS 1,2,3,CIS

STAT5b

Negative regulation of GHR/JAK2 activation

CYP2C11

STAT5b

Male-specific hepatic expression

CYP2C12

STAT5b

Female-specific hepatic expression

Egr-1

ERK pathway, TCF

Jun-B

ERK pathway, TCF ATF-2, CHOP, p38 MAPK pathway

IGF-binding protein

Mitogenesis

Some target genes affected by the activated GHR, the pathways and/or transcription factors believed to influence them, and relevant GH action effects (if known) are listed. See text for details.

Growth Hormone Receptor (mimicking more the female pattern) leads to a lower level, less pulsatile STAT5b activation profile (reviewed in Choi and Waxman, 2000; Schwartz, 2001). This pattern leads to sexually dimorphic expression of certain GH-dependent genes in liver (more below). Other transcription factors whose functions are affected by GH include the list in Table 1. It is a list that has grown significantly in recent years and includes such proteins as the ternary complex factor (p62TCF/Elk1), serum response factor (SRF), CCAAT/enhancer-binding protein (C/EBP ), C/ EBP, C/EBP homologous protein (CHOP), activating transcription factor 2 (ATF-2), yin yang 1 (YY1), hepatocyte nuclear factor 1 (HNF-1), HNF-4, and the glucocorticoid receptor (Liao et al., 1997, 1999; Hodge et al., 1998; Meton et al., 1999; Bergad et al., 2000; Lahuna et al., 2000; Zhu and Lobie, 2000; Piwien-Pilipuk et al., 2001).

Genes induced A number of genes have been shown to be activated by GH (Table 1), though the signaling pathways and transcription factors involved are not completely understood for each. GH's induction of the insulin-like growth factor I (IGF-I) gene was among the earliest appreciated and is biologically relevant (see below) (Doglio et al., 1987; Bichell et al., 1992). Transcription factors implicated only recently in GH-induced IGF-I expression include HNF-1 and STAT5 (Meton et al., 1999; Davey et al., 2001). However, pathways involved may differ between cell types, since PI-3 kinase inhibitors have been shown to either inhibit or potentiate GHinduced IGF-I transcription in hepatocytes versus myotubes, respectively (Shoba et al., 2001; Sadowski et al., 2001). Other genes thought important in GH action that are regulated at least in part by STAT5 include: the suppressors of cytokine signaling (SOCS) proteins SOCS-1, -2, -3, and CIS in liver (Adams et al., 1998; Davey et al., 1999b; Ram and Waxman, 1999; TolletEgnell et al., 1999); serine protease inhibitor (Spi) 2.1 (LeCam et al., 1987; Bergad et al., 1995); acid labile subunit (ALS) of the IGFBP-3 complex (Ooi et al., 1997); insulin (in rat insulinoma cells) (Galsgaard et al., 1996); and a number of hepatic cytochrome P450 (CYP) genes involved in the metabolism of endogenous steroids (Waxman, 1992; Waxman et al., 1995). For some in this latter group, the sexually dimorphic pulsatile GH secretion dictates a

13

STAT5b-mediated sexually dimorphic expression (reviewed in Choi and Waxman, 2000) (more below). GH-activated c-fos gene expression is mediated in part by the STAT1/3 and ERK1/2 pathways (Hodge et al., 1998; reviewed in Herington et al., 2000).

Promoter regions involved In genes such as Spi2.1 and the acid labile subunit, STAT5, when activated in response to GH, binds to a DNA enhancer sequence called a gamma-activated sequence (GAS)-like element (GLE) (Bergad et al., 1995; Ooi et al., 1998). The GLE consensus sequence is 50 -TTC-NNN-GAA-30 (Seidel et al., 1995). In these genes, STAT5 can synergistically bind to two adjacent GLEs such that two STAT5 dimers interact with the enhancer, effecting transcriptional activation. In other GH-regulated genes that may be in part influenced by STAT5 activation, such as the IGF-I gene, the promoter element(s) involved are much less clear. For the IGF-I gene, the issue of deciphering its regulatory elements is difficult both because of the gene's complexity and the relative lack of GH-responsive cell lines for study of IGF-I gene expression. To date, the major findings include the presence of a DNase1 hypersensitivity site within a 350 bp region of exon 2 of the rat IGF-I gene (Thomas et al., 1995) and the demonstration in GHR-transfected rat C6 glioma cells of GH-responsiveness of a luciferase gene driven by a 5.5 kb fragment of the rat IGF-I gene that included 412 bp of 50 flanking sequence of exon 1, exon 1, intron 1, exon 2, intron 2, and a fragment of exon 3 (Benbassat et al., 1999). There are, as yet, no clear consensus sites for transcription factor binding identified in this gene as indicative of its GH regulation. STAT1 and STAT3 are involved in GH regulation of the c-fos gene. GH treatment of tissue culture cells causes binding of homo- and heterodimers of STATs 1 and 3 to the c-sis-inducible element (SIE) of the c-fos promoter (Campbell et al., 1995; Gronowski and Rotwein, 1994; Meyer et al., 1994; Ram et al., 1996). Other regions of the c-fos enhancer are involved in GH-mediated transactivation in an apparently STAT-independent fashion. Both SRF and TCF/Elk-1 inducibly bind to the c-fos serum response element (SRE) and contribute to induction of c-fos transcription in response to GH in an ERK pathway-dependent fashion (Hodge et al., 1998; Meyer et al., 1993).

14 Stuart J. Frank and Joseph L. Messina

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Phenotypes of receptor knockouts and receptor overexpression mice Table 2 summarizes the phenotypes of mice with knockout of some molecules relevant in GHR function. The GHR global knockout (GHR / ) mouse was described in 1997 (Zhou et al., 1997). No tissue-specific knockouts have as yet been reported. As expected, the GHR / animal also had no circulating GHBP. Because its phenotype strongly resembled that of the human syndrome of GH resistance described by Laron (more below), this mouse is also referred to as the Laron mouse. The most striking features initially observed included postnatal growth retardation in both males and females, markedly reduced serum IGF-I levels, and elevation of serum GH concentration. More studies with these mice have recently uncovered interesting features, only some of which may mimic the human disorder. Reduced bone growth in GHR / mice was shown to be due to reduced chondrocyte proliferation and cortical bone growth postnatally and bone turnover was also reduced; these effects were rescued with IGF-I treatment (Sims et al., 2000). In addition, defects in both male and female reproductive function have been observed in these mice (Danilovich et al., 1999; Chandrashekar et al., 2001). Interestingly, a potentially clinically relevant observation with these mice is that they are protected against the development of diabetic nephropathy, suggesting a role for GH in mediating this pathology

(more below) (Bellush et al., 2000). In contrast to humans, a striking characteristic of Laron mice is that they live significantly longer than their GHR+/+ or GHR+/ littermates, though the reasons for this are not yet known (Coschigano et al., 2000). Targeted disruption of other important molecules implicated as mediators of GH action have recently been reported to yield phenotypes in some ways similar to the Laron mouse. Unrestricted STAT5b knockout was reported in 1997 (Udy et al., 1997). STAT5b / mice exhibit poor postnatal growth, abnormal adipose tissue development, and a loss of the sexually dimorphic hepatic expression of several GH-responsive genes. Further, they have low IGF-I concentrations in the serum without low GH levels; thus, it is thought that STAT5b deficiency renders the animals GH pulse-resistant and that STAT5b may mediate GH-induced IGF-I gene expression (Udy et al., 1997; Davey et al., 2001). Interestingly, deletion of the HNF-1 gene also leads to postnatal growth retardation, low IGF-I levels, and elevated GH (Lee et al., 1998) and HNF-1 has been implicated in GHinduced IGF-I gene expression (Meton et al., 1999). Though some of these data appear to favor GHinduced liver-derived IGF-I as a prime mediator of GH's somatogenic effects, it is informative to consider the recently described unrestricted and liver-specific IGF-I knockout mice (reviewed in LeRoith et al., 2001). Liver-specific IGF-I knockout resulted in a substantial lowering of circulating IGF-I and a raised GH level, but, surprisingly, no effect on body growth. Unrestricted IGF-I knockout, in those animals that survived, did yield significant growth retardation. Thus, while GH promotes both hepatic and peripheral IGF-I release, much of its growth effects may derive from autocrine/paracrine action of IGF-I produced extrahepatically. Tissue-specific GHR knockout studies may yield further insights into these issues.

Table 2 Phenotypes of mice with knockout of molecules important in GH action Gene knockout

Phenotypic features

GHR

Postnatal growth retardation, low IGF-I level, elevated GH level, bone growth and reproductive abnormalities

JAK2

Embryonic lethality, defective hematopoiesis

STAT5b

Postnatal growth retardation, low IGF-I level, loss of sexually dimorphic hepatic expression of several GH-responsive genes

HNF-1

Postnatal growth retardation, low IGF-I level, elevated GH level

IGF-I

Unrestricted ± variable perinatal lethality, pre- and postnatal growth retardation, other abnormalities Liver-specific ± normal growth, low IGF-I level, elevated GH level

Growth Hormone Receptor

Human abnormalities The effects of both excessive and diminished GH action are clinically seen in humans. Pituitary tumors that secrete excessive GH cause the syndrome of acromegaly, with connective tissue and bony overgrowth, visceromegaly, insulin resistance, and, if occurring prior to epiphyseal closure, excessive height (Ben-Shlomo and Melmed, 2001). Diminished GH action is most commonly caused by decreased pituitary GH secretion, but can also be seen in the GH resistance (Laron) syndrome. Laron described the index cases in the 1960s (reviewed in Laron, 1995) ± children with severe growth retardation, low IGF-I levels, and elevated circulating GH concentrations. Other features exhibited by Laron syndrome patients include obesity, small gonads and genitalia, and acromicria. A range of different GHR mutations that underlie this syndrome in kindreds worldwide have been described (reviewed in Parks et al., 1997). These may impair GHR gene expression, proper GHR cell surface expression, GH binding, or possibly GH signaling. In most cases, Laron syndrome patients lack circulating GHBP (presumably because of a lack of surface GHR to undergo shedding), but in some GHBP is present (or even increased), depending on the mutation. It is not yet clear whether more subtle GHR mutations may lead to the syndrome of idiopathic short stature (Goddard et al., 1995; Sanchez et al., 1998).

THERAPEUTIC UTILITY

Effect of treatment with soluble receptor domain To date, we are not aware of any clinical utilization of the GHR soluble extracellular domain (GHBP) in humans. In animal and in vitro experimentation, recombinant GHBP has had varying effects, as alluded to above. Coadministration of GH and recombinant GHBP to GH-deficient rodents, for instance, enhances GH's growth-promotion (Clark et al., 1996). Yet, when applied to GH-responsive cell lines, GHBP inhibits GH binding and GH-induced biological responses, likely by sequestering GH from cell surface GHR or forming inactive GH±GHBP± GHR complexes rather than GH±GHR dimer complexes (Lim et al., 1990; Mannor et al., 1991; Hansen et al., 1993). Thus, the net effect of GHBP treatment is as yet unclear.

15

Effect of inhibitors (antibodies) to receptors There have not been trials of anti-GHR antibodies for clinical purposes. Mutant recombinant GH molecules that are altered so as to markedly diminish site 2 binding affinity have been developed, however (reviewed in Kopchick and Okada, 2001) (see dashed line, Figure 4B). These molecules are unable to cause proper GHR dimerization and are therefore ineffective for promoting GH signaling. In addition, in particular when a double mutant is engineered that combines enhanced site 1 affinity with decreased site 2 affinity, this molecule acts as an antagonist of endogenous (normal) GH action, presumably by lessening the availability of GHRs for productive GH-induced dimerization. Recently a PEG (polyethylene glycol)-ylated version of such an antagonist (which, by virtue of PEGylation has a long circulating half-life) has been shown to be effective in treating the syndrome of acromegaly (reviewed in Drake et al., 2001).

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