Growth Hormone Receptor structure, post-biogenesis behavior and degradation

Historical text by DatBTrue, written between 2008 and 2016 and reproduced verbatim. It records what was argued then; it is not dosing guidance and not medical advice. Read the disclaimer.

The following was originally created primarily for my benefit and serves as a basic summary of Growth Hormone Receptor structure, post-biogenesis behavior and degradation.

It is not intended for a wider audience and is not a Datbtrue copyrighted article. It represents the current state of knowledge in the aforementioned area (October, 2008) and was derived primarily from an unpublished paper by Stuart J. Frank & Serge Y. Fuchs on growth hormone receptor abundance and function and from Growth hormone receptor; mechanism of action, Andrew J. Brooks, Jong Wei Wooh, Kathryn A. Tunny, Michael J. Waters, The International Journal of Biochemistry & Cell Biology 40 (2008) 1984–1989

These unrefined notes are for educational use only.

GHR Structure

The growth hormone receptor (GHR) is a member of the cytokine receptor superfamily. Cytokines is a general category encompassing signaling proteins and glycoproteins (often cellular membrane proteins) that similar to hormones and neurotransmitters facilitate cellular communication. [12,13] It is a type I cytokine receptor as is the prolactin receptor, which in essence means it is connected to Janus kinase (JAK) which acts as its primary mediator of signaling events.

Physically the GHR is composed of 620 residues (a residue is an individual amino acid in a peptide chain). 350 of these residues reside inside the cell and make up what is known as the intracellular domain. 246 of these residues reside outside the cell and make up what is known as the extracellular domain. The remaining 24 residues reside in the membrane of the cell and make up what is known as the transcellular domain. [13-16]

The GHR extracellular domain consists of two fibronectin type III beta sandwich domains connected by a short flexible linker.

The intracellular domain comprises Box 1 and Box 2 motifs which bind the tyrosine kinase, Janus kinase 2 (JAK2) and several tyrosine residues that act as substrates for phoshorylation by JAK2 and thus become binding sites for SH2 domain proteins.

A Kinase is a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific target molecules (substrates) in a process called “phosphorylation”.

Since cytokine receptors such as GHR possess no catalytic kinase activity, they rely on the Janus kinase family of tyrosine kinases to phosphorylate and activate downstream proteins involved in their signal transduction pathways.

Phosphorylation of a substrate by tyrosine kinases acts as a switch to trigger binding to an SH2 domain-containing protein. STAT & PI3K proteins possess an SH2 “docking” protein and once bound begin to exert their respective effects.

The growth hormone (GH) molecule intiates this phosphorylation process by binding to one of the two GHR domains via its site of stongest attraction. For the 22kDa form of GH it would be site 1. Once bound via GH site 1 to one of the two GHR domains the GH molecule binds to the second GHR domain via the GH molecule location known as site 2.

Note that the result of the GH molecule binding with the two receptor domains does not have the effect of connecting the two receptor domains for they were already connected by a short flexible linker. Rather the binding of the GH molecule to the GHR causes a repositioning of the intracellular domains, resulting in the activation of associated tyrosine kinases and thus signal transduction. [17]

[Diagram not preserved: it lived in a forum attachment that was not archived.]

JAK2

The GHR does not possess built-in tyrosine kinase activity and relies on associated kinases for signal transduction. The Janus kinase JAK2 provides a major component of signal transduction by GH. JAK2 associates with the GHR intracellular domain proline rich Box 1 motif via its amino terminal (JH5-7) FERM domain. JAK2 phosphorylates tyrosine residues on the associated GHR intracellular domain which provide docking sites for Src homology 2 (SH2) domain proteins, in particular STAT5a and 5b. The STATs 1, 3, 5a, and 5b are subsequently phosphorylated by JAK2, homo- or heterodimerise, translocate to the nucleus, bind to STAT responsive elements and activate transcription. [18]

[Diagram not preserved: it lived in a forum attachment that was not archived.]

In the GHR unliganded state (unbound by the Gh molecule) it is thought that the JAK2 pseudokinase JH2 domain interacts with and autoinhibits the JH1 kinase domain resulting in an inactive JAK2. GH binding causes structural reorientation of the receptor GH domains , which results in a structural re-orientation of JAK2 and disruption of the ability of pseudokinase JH2 domains to inhibit the JH1 kinase domain, leading to JAK2 activation. There are several mechanisms for the termination of the JAK2 mediated signal which involve phosphatases, suppressors of cytokine signalling (SOCS) proteins that block signalling via their SH2 domains and receptor downregulation. [19]

In essence GH binding to the GHR triggers activation of JAK2 which causes the GHR and JAK2 tyrosine phosphorylation which induces signaling systems. The primary signaling systems are:

STATs (most notably STAT5b) ERKs PI3-kinase [23-25]

GH-induced STAT5b activation requires receptor tyrosine phosphorylation and promotes gene transcription (eg., IGF-1, acid-labile subunit (ALS) of the IGF binding protein complex, SOCS proteins, hepatic P450 enzymes, and serine protease inhibitor[26–36]).

Unlike STAT5b, GH-induced ERK and PI3K activation does not require the entire GHR cytoplasmic domain, but only JAK2 coupling [23,37,38 ,39-41]. ERK activity is critical for GH-induced c-fos transcription [42], enhances GH-stimulated proliferation [43], and mediates crosstalk with EGF signaling [44-46]. GH-induced PI3K activity is implicated in antiapoptosis and/or proliferation and likely contributes to GH-induced ERK, p70 S6 kinase, and phosphodiesterase activity [42,43,47-50].

[Diagram not preserved: it lived in a forum attachment that was not archived.]

GH sensitivity is substantially affected by the abundance of GHR available for ligand engagement at particular target cells and tissues. Surface GHR availability is regulated at several levels, including transcriptional, post-transcriptional, and post-translational.

For a thorough review of transcriptional and post-transcriptional events see references 51 & 52.

GHR regulation at the post-translational will be reviewed herein.

Movement of newly synthesized GHR to the cell surface

GHR is synthesized as a non-glycosylated precursor that is transported from the endoplasmic reticulum (ER) to the Gogli apparatus. GHR dimerizes in the ER early in the process of biogenesis thus accounting for the receptor dimers detected at the cell surface even in the absence of GH engagement. [53, 60,61] In the process of transport through the Golgi, the GHR acquires carbohydrate in a characteristic fashion, in which high-mannose sugars added in the ER are ultimately removed during the transition from the early to late Golgi to yield the mature glycosylated GHR that populates the cell surface.

[Diagram not preserved: it lived in a forum attachment that was not archived.]

JAK2 apparently associates with GHR early in the biosynthetic pathway and not only acts as a GHR chaperone as it makes its way to the surface but fosters GHR maturation [59,62]

Studies suggest that in cells that lack JAK2 the nascent precursor GHR is a target for endoplasmic reticulum associated degradation (ERAD) and represent the first example of ERAD-associated cleavage of a cytokine receptor family member that stems from a lack of its cognate JAK. ERAD is a process whereby proteins that fail to fold properly or otherwise fail quality control mechanisms in the ER undergo retrotranslocation and proteasomal degradation in the cytosol [67–69]. JAK2, by virtue of its association with the GHR, rather than via its kinase activity, apparently “chaperones” the dimerized precursor so as to avoid quality control and proceed with efficient processing to mature GHR in the secretory pathway.

How does JAK2 exert this chaperone effect? Multiple possibilities exist, including the notion that a receptor region that might otherwise be seen as defective or unfolded to the quality control apparatus is hidden by JAK2 binding. In a similar fashion, JAK2 binding might allosterically (i.e. shape change) alter a GHR site outside of the region that interacts with JAK2 to make that site appear less defective.

In essence JAK2 association affects endoplasmic reticulum to cell surface GHR trafficking. In cells harboring GHR and JAK2 molecules that can associate, GHR moves from the ER to the Golgi, matures, and reaches the cell surface efficiently. In cells lacking JAK2 or with GHR molecules that cannot associate with JAK2 (by virtue of mutation of the receptor Box 1 region), GHR primarily undergoes endoplasmic reticulum-associated degradation (ERAD) while a small amount inefficiently matures and traffics to the cell surface (without association with JAK2).

[Diagram not preserved: it lived in a forum attachment that was not archived.]

Proteolysis (degradation) of the GHR

Over the last decade, it has become appreciated that GHR, like some other surface receptors, is a target for regulated sequential proteolysis, the first step of which (alpha-secretase cleavage) occurs in the proximal extracellular domain stem region 8–9 residues (depending on species) outside the plasma membrane [65,80,81]. This alpha- secretase cleavage results in loss of full-length GHR, appearance of a cell-associated transmembrane domain (TMD)/intracellular domain (ICD)-containing receptor fragment (the “remnant”), and a soluble GHR extracellular domain (ECD) (called GH binding protein (GHBP)[82,83]).

GHR alpha- secretase cleavage is constitutive, but can be further induced in various cell types by a proteinkinase C activator (the phorbol ester, PMA), platelet-derived growth factor (PDGF), or serum [65,80,81,84– 87]. This cleavage is catalyzed mainly by the extracellular domain of the transmembrane metalloprotease, TACE (tumor necrosis factor-alpha converting enzyme; ADAM-17) [66,88].

Importantly, inducible alpha- secretase cleavage likely regulates GH sensitivity; that is, GH-induced signaling is dampened after cells are exposed to stimuli that promote GHR alpha-secretase cleavage, but not in the presence of metalloprotease inhibitors or if noncleavable receptor mutants are expressed, suggesting that metalloproteolysis modulates GH responsiveness in part by regulating surface GHR levels [65,83,84].

Further, recent in vivo experiments indicate that administration of bacterial endotoxin leads to downregulation of hepatic GHR abundance and hepatic insensitivity to GH at least in part by inducing receptor proteolysis, suggesting that this may constitute a physiologically-relevant mechanism of regulation of GH action [89]. Notably, GH itself does not promote GHR alpha-secretase cleavage; indeed, GH inhibits subsequent GHR proteolysis, apparently by altering GHR conformation, rather than by causing signaling [66].

Recent studies have shown that the alpha-secretase-generated GHR TMD/ICD remnant is further cleaved by an enzyme activity termed gamma-secretase within the TMD, which liberates the ICD, a protein termed the “GHR stub” [87]. gamma-secretase consists of four molecules, including presenilin, which forms the aspartyl protease core and facilitates a process known as regulated intramembrane proteolysis (RIP) [90].

In essence inducible alpha-secretase cleavage generates remnant, which is converted to stub by gamma-secretase. The stub is labile and can accumulate in either the cytosol or the nucleus; proteasome inhibition prevents stub degradation.

In summary GHR undergoes sequential TACE and gamma-secretase cleavage. Surface GHR undergoes constitutive and inducible cleavage in the extracellular domain stem region by TACE in a process called “alpha-secretase” cleavage. This yields the shed GHBP and the GHR remnant. Remnant is then cleaved by gamma-secretase within the membrane to yield the GHR stub (soluble intracellular domain), which localizes to the nucleus, where it may affect gene expression.

[Diagram not preserved: it lived in a forum attachment that was not archived.]

Surface GHR stability

Once at the cell surface, the GHR could, in principle, achieve several fates. If engaged by GH, signaling is triggered and the receptor undergoes ligand-dependent downregulation. However, in the natural milieu, GH is released from the pituitary gland in a pulsatile fashion such that GH levels are quite low in periods between pulses. Thus, it is critical to understand factors that govern GHR abundance independent of GH. It is believed that mature GHRs are cleared from the cell surface by constitutive or inducible proteolytic shedding (discussed above) and by constitutive downregulation (discussed below).

JAK2 affects the fate of the cell surface GHR and in cells lacking JAK2, the ratio of mature (cell surface):precursor GHR was substantially reduced in comparison to JAK2-replete cells [25,62]. This finding is partly explained by the chaperone effect of JAK2 during GHR biogenesis [63]. However, notable JAK2-dependent differences in the constitutive fate of mature GHRs are found as well [62]. In the context of a stable reconstitution system, the half-life (t1/2) of the receptor was estimated by anti-GHR immunoblotting after 0–4 h of treatment with cycloheximide (CHX) to inhibit new protein synthesis. The results of such a “CHX chase” assay indicated that the precursor GHR abundance dropped precipitously and to a similar degree with increasing duration of CHX treatment both in cells that did or did not express JAK2. For the mature receptor, however, there was a dramatic effect of JAK2. As measured by this assay, the GHR t1/2 increased from roughly 1 hr in cells that lack JAK2 to roughly 4 h in cells expressing JAK2 [62].

Thus, in the absence of GH, it appears that, in addition to its role in shepherding the GHR through the secretory pathway and lessening the degree to which it is targeted for ERAD, JAK2 also extends the receptor’s presence at or near the cell surface, presumably by interfering with constitutively active cellular machinery that functions to internalize and downregulate the receptor.

In summary JAK2 association affects the constitutive (GH-independent) fate of surface GHR. In cells harboring GHR and JAK2 molecules that can associate, surface GHR is downregulated at a low constitutive rate and its half-life is long. In cells that lack JAK2 or have GHR and JAK2 molecules that cannot associate, GHR undergoes enhanced constitutive downregulation and exhibits a short half-life.

[Diagram not preserved: it lived in a forum attachment that was not archived.]

GH-induced GHR downregulation

Like many surface receptors, GHR undergoes important trafficking events in response to binding of its ligand. The net effect is substantial GH-induced receptor downregulation, which serves to limit or alter the receptor’s signaling capacity and perhaps thereby further emphasize the physiologic effects of pulsatile GH release from the pituitary gland. Work as early as the 1970s–1980s and since that time suggested that GH-induced GHR downregulation proceeds via clathrin coated pit-mediated endocytosis and lysosomal degradation [91,98–100].

[Diagram not preserved: it lived in a forum attachment that was not archived.]

GH ultimately causes its receptor to be degraded in lysosomes. However GH-induced receptor ubiquitination (inactivation by an attaching ubiquitin) depends on both JAK2 activity and the ability of the receptor to be tyrosine phosphorylated.

Several binding partners have been shown to associate via their SH2 domains with the tyrosine phosphorylated intracellular domain [107–111, 114]. One of them, the protein tyrosine phosphatase, SHP-2, may contribute modestly to GH-induced GHR downregulation [108]. More recently, it has been appreciated that the SOCS family protein CIS (cytokine inducible SH2 domain-containing protein), which interacts with tyrosine phosphorylated GHR [109,110] and is likely linked to Cullin5-based E3 ubiquitin ligase complex that can recruit proteins to the proteasome for degradation [112], promotes GH-induced GHR internalization and thus can desensitize GH signaling [113].

REFERENCES:

[1] – [11] – UNUSED

[12] J.F. Bazan, Structural design and molecular evolution of a cytokine receptor superfamily, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 6934–6938.

[13] D.W. Leung, S.A. Spencer, G. Cachianes, R.G. Hammonds, C. Collins,W.J. Henzel, R. Barnard, M.J. Waters, W.I. Wood, Growth hormone receptor and serum binding protein: purification, cloning and expression, Nature 330 (1987) 537–543.

[14] S.J. Frank, J.L. Messina, Growth hormone receptor, in: J.J. Oppenheim, M. Feldman (Eds.), Cytokine Reference On-Line, Academic Press, Harcourt, London, UK, 2002, pp. 1–21, Website,www.academicpress.com/cytokinereference, 24-hour free access.

[15] C. Carter Su, J. Schwartz, L.S. Smit, Molecular mechanism of growth hormone action, Annu. Rev. Physiol. 58 (1996) 187–207.

[16] S.J. Frank, J.J. O’Shea (Eds.), Recent Advances in Cytokine Signal Transduction: Lessons from Growth Hormone and other Cytokines, 1999, Greenwich, CT.

[17] Brown, R. J., Adams, J. J., Pelekanos, R. A.,Wan,Y., McKinstry,W. J., Palethorpe, K., et al. (2005). Model for growth hormone receptor activation based on subunit rotation within a receptor dimer. Nat. Struct. Mol. Biol., 12(9), 814–821.

[18] Ihle, J. N., & Gilliland, D. G. (2007). Jak2: Normal function and role in hematopoietic disorders. Curr. Opin. Genet. Dev., 17(1), 8–14.

[19] Waters, M. J., Hoang, H. N., Fairlie, D. P., Pelekanos, R. A., & Brown, R. J. (2006). New insights into growth hormone action. J. Mol. Endocrinol., 36(1), 1–7.

[20] Zhu, T., Ling, L., & Lobie, P. E. (2002). Identification of a JAK2- independent pathway regulating growth hormone (GH)-stimulated p44/42 mitogen-activated protein kinase activity. GH activation of Ral and phospholipaseDis Src-dependent. J. Biol. Chem., 277(47), 45592–45603.

[21] Rowlinson, S. W., Yoshizato, H., Brown, R. J., Behncken, S. N., Barclay, J. L., Brooks, A. J., et al. An activation-related conformational change determines signalling pathway choice for the growth hormone (GH) receptor. Manuscript submitted for publication.

[22] Fresno Vara, J. A., Caceres, M. A., Silva, A., & Martin-Perez, J. (2001). Src family kinases are required for prolactin induction of cell proliferation. Mol. Biol. Cell, 12(7), 2171–2183.

[23] S.J. Frank, J.L. Messina, Growth hormone receptor, in: J.J. Oppenheim, M. Feldman (Eds.), Cytokine Reference On-Line, Academic Press, Harcourt, London, UK, 2002, pp. 1–21, Website,www.academicpress.com/cytokinereference, 24-hour free access.

[24] C. Carter Su, J. Schwartz, L.S. Smit, Molecular mechanism of growth hormone action, Annu. Rev. Physiol. 58 (1996) 187–207.

[25] S.J. Frank, J.J. O’Shea (Eds.), Recent Advances in Cytokine Signal Transduction: Lessons from Growth Hormone and other Cytokines, 1999, Greenwich, CT.

[26] L.H. Hansen, X. Wang, J.J. Kopchick, P. Bouchelouche, J.H. Nielsen, E.D. Galsgaard, N. Billestrup, Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation, J. Biol. Chem. 271 (1996) 12669–12673.

[27] L.S. Smit, D.J. Meyer, N. Billestrup, G. Norstedt, J. Schwartz, C. Carter-Su, The role of the growth hormone (GH) receptor and JAK1 and JAK2 kinases in the activation of Stats 1, 3, and 5 by GH, Mol. Endocrinol. 10 (1996) 519–533.

[28] A. Sotiropoulos, S. Moutoussamy, F. Renaudie, M. Clauss, C. Kayser, F. Gouilleux, P.A. Kelly, J. Finidori, Differential activation of Stat3 and Stat5 by distinct regions of the growth hormone receptor, Mol. Endocrinol. 10 (1996) 998–1009.

[29] X. Wang, C.J. Darus, B.C. Xu, J.J. Kopchick, Identification of growth hormone receptor (GHR) tyrosine residues required for GHR phosphorylation and JAK2 and STAT5 activation, Mol. Endocrinol. 10 (1996) 1249–1260.

[30] W. Yi, S.O. Kim, J. Jiang, S.H. Park, A.S. Kraft, D.J. Waxman, S.J. Frank, Growth hormone receptor cytoplasmic domain differentially promotes tyrosine phosphorylation of signal transducers and activators of transcription 5b and 3 by activated JAK2 kinase, Mol. Endocrinol. 10 (1996) 1425–1443.

[31] P.L. Bergad, H.M. Shih, H.C. Towle, S.J. Schwarzenberg, S.A. Berry, Growth hormone induction of hepatic serine protease inhibitor 2.1 transcription is mediated by a Stat5-related factor binding synergistically to two alphaactivated sites, J. Biol. Chem. 270 (1995) 24903–24910.

[32] H.W. Davey, M.J. McLachlan, R.J. Wilkins, D.J. Hilton, T.E. Adams, STAT5b mediates the GH-induced expression of SOCS-2 and SOCS-3 mRNA in the liver, Mol. Cell Endocrinol. 158 (1999) 111–116.

[33] H.W. Davey, T. Xie, M.J. McLachlan, R.J. Wilkins, D.J. Waxman, D.R. Grattan, STAT5b is required for GH-induced liver IGF-I gene expression, Endocrinology 142 (2001) 3836–3841.

[34] G.B. Udy, R.P. Towers, R.G. Snell, R.J. Wilkins, S.H. Park, P.A. Ram, D.J. Waxman, H.W. Davey, Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 7239–7244.

[35] G.T. Ooi, K.R. Hurst, M.N. Poy, M.M. Rechler, Y.R. Boisclair, Binding of STAT5a and STAT5b to a single element resembling a alpha-interferon-activated sequence mediates the growth hormone induction of the mouse acid-labile subunit promoter in liver cells, Molecular. Endocrinology. 12 (1998) 675–687.

[36] J.Woelfle, J. Billiard, P. Rotwein, Acute control of insulin-like growth factor-I gene transcription by growth hormone through Stat5b, J. Biol. Chem. 278 (2003) 22696–22702

[37] A. Sotiropoulos, M. Perrot-Applanat, H. Dinerstein, A. Pallier, M.C. Postel-Vinay, J. Finidori, P.A. Kelly, Distinct cytoplasmic regions of the growth hormone receptor are required for activation of JAK2, mitogen-activated protein kinase, and transcription, Endocrinology 135 (1994) 1292–1298.

[38] J.A. Vanderkuur, X. Wang, L. Zhang, G.S. Campbell, G. Allevato, N. Billestrup, G. Norstedt, C. Carter-Su, Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase, J. Biol. Chem. 269 (1994) 21709–21717.

[39] C. Moller, A. Hansson, B. Enberg, P.E. Lobie, G. Norstedt, Growth hormone (GH) induction of tyrosine phosphorylation and activation of mitogen-activated protein kinases in cells transfected with rat GH receptor cDNA, J. Biol. Chem. 267 (1992) 23403–23408.

[40] L.S. Argetsinger, G.W. Hsu, M.G.J. Myers, N. Billestrup, M.F. White, C. Carter-Su, Growth hormone, interferon-alpha, and leukemia inhibitory factor promoted tyrosyl phosphorylation of insulin receptor substrate-1, J. Biol. Chem. 270 (1995) 14685–14692.

[41] L.S. Argetsinger, G. Norstedt, N. Billestrup, M.F. White, C. Carter-Su, Growth hormone, interferon-alpha, and leukemia inhibitory factor utilize insulin receptor substrate-2 in intracellular signaling, J. Biol. Chem. 271 (1996) 29415–29421

[42] C. Hodge, J. Liao, M. Stofega, K. Guan, C. Carter-Su, J. Schwartz, Growth hormone stimulates phosphorylation and activation of elk-1 and expression of c-fos, egr-1, and junB through activation of extracellular signal-regulated kinases 1 and 2, J. Biol. Chem. 273 (1998) 31327–31336.

[43] L. Liang, T. Zhou, J. Jiang, J.H. Pierce, T.A. Gustafson, S.J. Frank, Insulin receptor substrate-1 enhances growth hormone-induced proliferation, Endocrinology 140 (1999) 1972–1983.

[44] S.O. Kim, J.C. Houtman, J. Jiang, J.M. Ruppert, P.J. Bertics, S.J. Frank, Growth hormone-induced alteration in ErbB-2 phosphorylation status in 3T3-F442A fibroblasts, J. Biol. Chem. 274 (1999) 36015–36024.

[45] Y. Huang, Y. Chang, X.Wang, J. Jiang, S.J. Frank, Growth hormone alters epidermal growth factor receptor binding affinity via activation of ERKs in 3T3-F442A cells, Endocrinology 145 (2004) 3297–3306

[46] J.A. Costoya, J. Finidori, S. Moutoussamy, R. Searis, J. Devesa, V.M. Arce, Activation of growth hormone receptor delivers an antiapoptotic signal: evidence for a role of Akt in this pathway, Endocrinology 140 (1999) 5937–5943.

[47] S. Jeay, G.E. Sonenshein, P.A. Kelly, M.C. Postel-Vinay, E. Baixeras, Growth hormone exerts antiapoptotic and proliferative effects through two different pathways involving nuclear factor-kappaB and phosphatidylinositol 3-kinase, Endocrinology 142 (2001) 147–156.

[48] E. Kilgour, I. Gout, N.G. Anderson, Requirement for phosphoinositide 3-OH kinase in growth hormone signalling to the mitogen-activated protein kinase and p70s6k pathways, Biochem. J. 315 (1996) 517–522.

[49] S.J. MacKenzie, S.J. Yarwood, A.H. Peden, G.B. Bolger, R.G. Vernon, M.D. Houslay, Stimulation of p70S6 kinase via a growth hormone-controlled phosphatidylinositol 3-kinase pathway leads to the activation of a PDE4A cyclic amp-specific phosphodiesterase in 3T3-F442A preadipocytes, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 3549–3554.

[50] L. Liang, J. Jiang, S.J. Frank, Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation, Endocrinology 141 (2000) 3328–3336.

[51] G. Schwartzbauer, R.K. Menon, Regulation of growth hormone receptor gene expression, Mol. Genet. Metab. 63 (1998) 243–253.

[52] F. Talamantes, R. Ortiz, Structure and regulation of expression of the mouse GH receptor, J. Endocrinol. 175 (2002) 55–59.

[53] J. Gent, P. van Kerkhof, M. Roza, G. Bu, G.J. Strous, Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 9858–9863.

[54] – [58] – UNUSED

[59] K. He, X.Wang, J. Jiang, R. Guan, K.E. Bernstein, P.P. Sayeski, S.J. Frank, Janus kinase 2 determinants for growth hormone receptor association, surface assembly, and signaling, Mol. Endocrinol. 17 (2003) 2211–2227.

[60] R.J. Brown, J.J. Adams, R.A. Pelekanos, Y.Wan,W.J. McKinstry, K. Palethorpe, R.M. Seeber, T.A. Monks, K.A. Eidne, M.W. Parker, M.J. Waters, Model for growth hormone receptor activation based on subunit rotation within a receptor dimer, Nat. Struct. Mol. Biol. 12 (2005) 814–821.

[61] M.J. van den Eijnden, L.L. Lahaye, G.J. Strous, Disulfide bonds determine growth hormone receptor folding, dimerisation and ligand binding, J. Cell. Sci. 119 (2006) 3078–3086.

[62] K. He, K. Loesch, J.W. Cowan, X. Li, L. Deng, X. Wang, J. Jiang, S.J. Frank, JAK2 enhances the stability of the mature GH receptor. Endocrinology 145 (2005) 4755–4765.

[63] K. Loesch, L. Deng, X. Wang, K. He, J. Jiang, S.J. Frank, Endoplasmic reticulumassociated degradation of growth hormone receptor in Janus kinase 2-deficient cells, Endocrinology 148 (2007) 5955–5965

[64] – UNUSED

[65] X. Wang, K. He, M. Gerhart, Y. Huang, J. Jiang, R.J. Paxton, S. Yang, C. Lu, R.K. Menon, R.A. Black, G. Baumann, S.J. Frank, Metalloprotease-mediated GH receptor proteolysis and GHBP shedding. Determination of extracellular domain stem region cleavage site, J. Biol. Chem. 277 (2002) 50510–50519.

[66] K. Loesch, L. Deng, J.W. Cowan, X.Wang, K. He, J. Jiang, R.A. Black, S.J. Frank, JAK2 influences growth hormone receptor metalloproteolysis, Endocrinology 147 (2006) 2839–2849.

[67] K. Romisch, Endoplasmic reticulum-associated degradation, Annu. Rev. Cell. Dev. Biol. 21 (2005) 435–456.

[68] A. Ahner, J.L. Brodsky, Checkpoints in ER-associated degradation: excuse me, which way to the proteasome? Trends. Cell. Biol. 14 (2004) 474–478.

[69] A. Schmitz, V. Herzog, Endoplasmic reticulum-associated degradation: exceptions to the rule, Eur. J. Cell. Biol. 83 (2004) 501–509.

[70] – [79] – UNUSED

[80] J. Alele, J. Jiang, J.F. Goldsmith, X. Yang, H.G. Maheshwari, R.A. Black, G. Baumann, S.J. Frank, Blockade of growth hormone receptor shedding by a metalloprotease inhibitor, Endocrinology 139 (1998) 1927–1935.

[81] X. Wang, K. He, M. Gerhart, J. Jiang, R.J. Paxton, R.K. Menon, R.A. Black, G. Baumann, S.J. Frank, Reduced proteolysis of rabbit growth hormone (GH) receptor substituted with mouse GH receptor cleavage site, Mol. Endocrinol. 17 (2003) 1931–1943.

[82] G. Baumann, Growth hormone binding protein 2001, J. Pediatr. Endocrinol. Metab. 14 (2001) 355–375.

[83] G. Baumann, S.J. Frank, Metalloproteinases and the modulation of GH signaling, J. Endocrinol. 174 (2002) 361–368.

[84] R. Guan, Y. Zhang, J. Jiang, C.A. Baumann, R.A. Black, G. Baumann, S.J. Frank, Phorbol ester- and growth factor-induced growth hormone (GH) receptor proteolysis and GH-bindingprotein shedding: relationship to GH receptor downregulation, Endocrinology 142 (2001) 1137–1147.

[85] Y. Zhang, R. Guan, J. Jiang, J.J. Kopchick, R.A. Black, G. Baumann, S.J. Frank, Growth hormone (GH)-induced dimerization inhibits phorbol ester-stimulated GH receptor proteolysis, J. Biol. Chem. 276 (2001) 24565–24573.

[86] J. Jiang, X. Wang, K. He, X. Li, C. Chen, P.P. Sayeski, M.J. Waters, S.J. Frank, A Conformationally-sensitive GHR (Growth Hormone (GH) Receptor) antibody: impact on GH signaling and GHR proteolysis. Mol. Endocrinol. 18 (2004) 2981–2996.

[87] J.W. Cowan, X. Wang, R. Guan, K. He, J. Jiang, G. Baumann, R.A. Black, M.S. Wolfe, S.J. Frank, Growth hormone receptor is a target for presenilin-dependent gamma-secretase cleavage, J. Biol. Chem. 280 (2005) 19331–19342.

[88] – [89] – UNUSED

[90] M.S. Wolfe, R. Kopan, Intramembrane proteolysis: theme and variations, Science 305 (2004) 1119–1123.

[91] G.J. Strous, P. van Kerkhof, The ubiquitin–proteasome pathway and the regulation of growth hormone receptor availability, Mol. Cell. Endocrinol. 197 (2002) 143–151

[92] – [97] – UNUSED

[98] P. Roupas, A.C. Herington, Intracellular processing of growth hormone receptors by adipocytes in primary culture, Mol. Cell. Endocrinol. 57 (1988) 93–99.

[99] M.A. Lesniak, J. Roth, Regulation of receptor concentration by homologous hormone. Effect of human growth hormone on its receptor in IM-9 lymphocytes, J. Biol. Chem. 251 (1976) 3720–3729.

[100] N. Hizuka, P. Gorden, M.A. Lesniak, E. Van Obberghen, J.L. Carpentier, L. Orci, Polypeptide hormone degradation and receptor regulation are coupled to ligand internalization. A direct biochemical and morphologic demonstration, J. Biol. Chem. 256 (1981) 4591–4597.

[101] – [106] – UNUSED

[107] S. Moutoussamy, F. Renaudie, F. Lago, P.A. Kelly, J. Finidori, Grb10 identified as a potential regulator of growth hormone (GH) signaling by cloning of GH receptor target proteins, J. Biol. Chem. 273 (1998) 15906–15912.

[108] M.R. Stofega, J. Herrington, N. Billestrup, C. Carter-Su, Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GH-promoted tyrosyl phosphorylation of GH receptor, JAK2, and STAT5B, Mol. Endocrinol. 14 (2000) 1338–1350.

[109] P.A. Ram, D.J. Waxman, SOCS/CIS protein inhibition of growth hormonestimulated STAT5 signaling by multiple mechanisms, J. Biol. Chem. 274 (1999) 35553–35561.

[110] L. Du, G.P. Frick, L.R. Tai, A. Yoshimura, H.M. Goodman, Interaction of the growth hormone receptor with cytokine-induced Src homology domain 2 protein in rat adipocytes, Endocrinology 144 (2003) 868–876.

[111] S.O. Kim, J. Jiang, W. Yi, G.S. Feng, S.J. Frank, Involvement of the Src homology 2- containing tyrosine phosphatase SHP-2 in growth hormone signaling, J. Biol. Chem. 273 (1998) 2344–2354.

[112] J.G. Zhang, A. Farley, S.E. Nicholson, T.A. Willson, L.M. Zugaro, R.J. Simpson, R.L. Moritz, D. Cary, R. Richardson, G. Hausmann, B.J. Kile, S.B. Kent,W.S. Alexander, D. Metcalf, D.J. Hilton, N.A. Nicola, M. Baca, The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 2071–2076.

[113] T. Landsman, D.J.Waxman, Role of the cytokine-induced SH2 domain-containing proteinCIS in growth hormone receptor internalization, J. Biol. Chem. 280 (2005) 37471–37480.

[114] W. Yi, S.O. Kim, J. Jiang, S.H. Park, A.S. Kraft, D.J. Waxman, S.J. Frank, Growth hormone receptor cytoplasmic domain differentially promotes tyrosine phosphorylation of signal transducers and activators of transcription 5b and 3 by activated JAK2 kinase, Mol. Endocrinol. 10 (1996) 1425–1443.