Disulfide linkage of growth hormone (GH) receptors (GHR) reflects GH-induced GHR dimerization. Association of JAK2 with the GHR is enhanced by receptor dimerization.

The growth hormone (GH) receptor (GHR) binds GH in its extracellular domain and transduces activating signals via its cytoplasmic domain. Both GH-induced GHR dimerization and JAK2 tyrosine kinase activation are critical in initiation of GH signaling. We previously described a rapid GH-induced disulfide linkage of GHRs in human IM-9 cells. In this study, three GH-induced phenomena (GHR dimerization, GHR disulfide linkage, and enhanced GHR-JAK2 association) were examined biochemically and immunologically. By using the GH antagonist, G120K, and an antibody recognizing a dimerization-sensitive GHR epitope, we demonstrated that GH-induced GHR disulfide linkage reflects GH-induced GHR dimerization. GH, not G120K, promoted both GHR disulfide linkage and enhanced association with JAK2. Measures that diminished GH-dependent JAK2 and GHR tyrosine phosphorylation diminished neither GH-induced GHR disulfide linkage nor GH-enhanced GHR-JAK2 association. By using both transient and stable expression systems, we determined that cysteine 241 (an unpaired extracellular cysteine) was critical for GH-induced GHR disulfide linkage; however, GH-induced GHR dimerization, GHR-JAK2 interaction, and GHR, JAK2, and STAT5 tyrosine phosphorylation still proceeded when this cysteine residue was mutated. We conclude GH-induced GHR disulfide linkage is not required for GHR dimerization, and activation and GH-enhanced GHR-JAK2 association depends more on GHR dimerization than on GHR and/or JAK2 tyrosine phosphorylation.     

Physical and functional interaction of growth hormone and insulin-like growth factor-I signaling elements

GH and IGF-I are critical regulators of growth and metabolism. GH interacts with the GH receptor (GHR), a cytokine superfamily receptor, to activate the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), and initiate intracellular signaling cascades. IGF-I, produced in part in response to GH, binds to the heterotetrameric IGF-I receptor (IGF-IR), which is an intrinsic tyrosine kinase growth factor receptor that triggers proliferation, antiapoptosis, and other biological actions. Previous in vitro and overexpression studies have suggested that JAKs may interact with IGF-IR and that IGF-I stimulation may activate JAKs. In this study, we explore interactions between GHR-JAK2 and IGF-IR signaling pathway elements utilizing the GH and IGF-I-responsive 3T3-F442A and 3T3-L1 preadipocyte cell lines, which endogenously express both the GHR and IGF-IR. We find that GH induces formation of a complex that includes GHR, JAK2, and IGF-IR in these preadipocytes. The assembly of this complex in intact cells is rapid, GH concentration dependent, and can be prevented by a GH antagonist, G120K. However, it is not inhibited by the kinase inhibitor, staurosporine, which markedly inhibits GHR tyrosine phosphorylation. Moreover, complex formation does not appear dependent on GH-induced activation of the ERK or phosphatidylinositol 3-kinase signaling pathways or on the tyrosine phosphorylation of GHR, JAK2, or IGF-IR. These results suggest that GH-induced formation of the GHR-JAK2-IGF-IR complex is governed instead by GH-dependent conformational change(s) in the GHR and/or JAK2. We further demonstrate that GH and IGF-I can synergize in acute aspects of signaling and that IGF-I enhances GH-induced assembly of conformationally active GHRs. These findings suggest the existence of previously unappreciated relationships between these two hormones.     

Platelet-derived growth factor and lysophosphatidic acid inhibit growth hormone binding and signaling via a protein kinase C-dependent pathway

Growth hormone (GH) regulates body growth and metabolism. GH exerts its biological action by stimulating JAK2, a GH receptor (GHR)-associated tyrosine kinase. Activated JAK2 phosphorylates itself and GHR, thus initiating multiple signaling pathways. In this work, we demonstrate that platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA) down-regulate GH signaling via a protein kinase C (PKC)-dependent pathway. PDGF substantially reduces tyrosyl phosphorylation of JAK2 induced by GH but not interferon-gamma or leukemia inhibitory factor. PDGF, but not epidermal growth factor, decreases tyrosyl phosphorylation of GHR (by approximately 90%) and the amount of both total cellular GHR (by approximately 80%) and GH binding (by approximately 70%). The inhibitory effect of PDGF on GH-induced tyrosyl phosphorylation of JAK2 and GHR is abolished by depletion of 4beta-phorbol 12-myristate 13-acetate (PMA)-sensitive PKCs with chronic PMA treatment and is severely inhibited by GF109203X, an inhibitor of PKCs. In contrast, extracellular signal-regulated kinases 1 and 2 and phosphatidylinositol 3-kinase appear not to be involved in this inhibitory effect of PDGF. LPA, a known activator of PKC, also inhibits GH-induced tyrosyl phosphorylation of JAK2 and GHR and reduces the number of GHR. We propose that ligands that activate PKC, including PDGF, LPA, and PMA, down-regulate GH signaling by decreasing the number of cell surface GHR through promoting GHR internalization and degradation and/or cleavage of membrane GHR and release of the extracellular domain of GHR.     

Janus kinase 2 determinants for growth hormone receptor association,surface assembly,and signaling

GH signaling depends on functional interaction of the GH receptor (GHR) and the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), which possesses a C-terminal kinase domain, a catalytically inactive pseudokinase domain just N-terminal to the kinase domain, and an N-terminal half shown by us and others to harbor elements for GHR association. Computational analyses indicate that JAKs contain in their N termini ( approximately 450 residues) divergent FERM domains. FERM domains (or subdomains within them) in JAKS may be important for associations with cytokine receptors. For some cytokine receptors, JAK interaction may be required for receptor surface expression. We previously demonstrated that a JAK2 mutant devoid of its N-terminal 239 residues (JAK2-Delta1-239) did not associate with GHR and could not mediate GH- induced signaling. In this report we employ a JAK2-deficient cell line to further define N-terminal JAK2 regions required for physical and functional association with the GHR. We also examine whether JAK2 expression affects cell surface expression of the GHR. Our results suggest that FERM motifs play an important role in the interaction of GHR and JAK2. While JAK2 expression is not required for detectable surface GHR expression, an increased JAK2 level increases the fraction of GHRs that achieves resistance to deglycosylation by endoglycosidase H, suggesting that the GHR-JAK2 association may enhance either the receptor's efficiency of maturation or its stability. Further, we report evidence for the existence of a novel GH-inducible functional interaction between JAK2 molecules that may be important in the mechanism of GH-triggered JAK2 signaling.     

Growth hormone-induced alteration in ErbB-2 phosphorylation status in 3T3-F442A fibroblasts

The growth hormone receptor (GHR), a cytokine receptor superfamily member, requires the JAK2 tyrosine kinase for signaling. We now examine functional interactions between growth hormone (GH) and epidermal growth factor (EGF) in 3T3-F442A fibroblasts. Although EGF enhanced ErbB-2 tyrosine phosphorylation, GH, while causing retardation of its migration on SDS-polyacrylamide gel electrophoresis, decreased ErbB-2's tyrosine phosphorylation. GH-induced retardation was reversed by treatment of anti-ErbB-2 precipitates with both alkaline phosphatase and protein phosphatase 2A, suggesting that GH induced serine/threonine phosphorylation of ErbB-2. Both GH-induced shift in ErbB-2 migration and GH-induced MAP kinase activation were unaffected by a protein kinase C inhibitor but were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1) inhibitor, PD98059. Notably, leukemia inhibitory factor, but not interferon-gamma, also promoted ErbB-2 shift and mitogen-activated protein kinase activation. Cotreatment with EGF and GH versus EGF alone resulted in a 35% decline in acute ErbB-2 tyrosine 1248 autophosphorylation, a marked decline (approximately 50%) in DNA synthesis, and substantially decreased cyclin D1 expression. We conclude that in 3T3-F442A cells, 1) the GH-induced decrease in ErbB-2 tyrosine phosphorylation correlates with MEK1/mitogen-activated protein kinase activity and 2) GH antagonizes EGF-induced DNA synthesis and cyclin D1 expression in a pattern consistent with its alteration in ErbB-2 phosphorylation status.     

Growth hormone-induced differential desensitization of STAT5, ERK,and Akt phosphorylation

Secretion of growth hormone (GH) in adult male rats is characterized by high peak and undetectable trough levels, both of which are required for male-specific pattern of liver gene expression and GH-induced phosphorylation of STAT5. The present study suggests that regulation of GH receptor (GHR) levels in rat hepatoma cells by repeated GH stimulation determines GH responsiveness via the JAK2/STAT5 pathway. A short exposure to GH rapidly reduced GHR levels which resulted in an equal desensitization of the JAK2/STAT5 pathway. Recovery of GH-induced STAT5 phosphorylation correlated with the time-dependent recovery of GHR levels during incubation in the absence of GH. Acute GH also induced phosphorylation of ERK1/2 and Akt, and this induction was also inhibited by prior exposure to GH. However, unlike the JAK2/STAT5 pathway, the effect of GH to activate the MEK/ERK and phosphatidylinositol 3-kinase/Akt pathways did not recover following prolonged incubation in the absence of GH. Thus, GH administration desensitizes the JAK2/STAT5 pathway, possibly because of down-regulation of GHR, whereas an additional post-receptor mechanism is required for the prolonged refractoriness of the MEK/ERK and phosphatidylinositol 3-kinase/Akt pathways toward a second GH stimulation. Our study suggests that both receptor and post-receptor mechanisms are important in GH-induced homologous desensitization.     

The signal transduction of the growth hormone receptor is regulated by the ubiquitin/proteasome system and continues after endocytosis

The growth hormone receptor (GHR) intracellular domain contains all of the information required for signal transduction as well as for endocytosis. Previously, we showed that the proteasome mediates the clathrin-mediated endocytosis of the GHR. Here, we present evidence that the proteasomal inhibitor MG132 prolongs the GH-induced activity of both GHR and JAK2, presumably through stabilization of GHR and JAK2 tyrosine phosphorylation. If proteasomal inhibitor was combined with ligand in an endocytosis-deficient GHR mutant, the same phenomenon occurred indicating that proteasomal action on tyrosine dephosphorylation is independent of endocytosis. Experiments with a GHR-truncated tail mutant (GHR-(1-369)) led to a prolonged JAK2 phosphorylation caused by the loss of a phosphatase-binding site. This raised the question of what happens to the signal transduction of the GHR after its internalization. Co-immunoprecipitation of GH.GHR complexes before and after endocytosis showed that JAK2 as well as other activated proteins are bound to the GHR not only at the cell surface but also intracellularly, suggesting that the GHR signal transduction continues in endosomes. Additionally, these results provide evidence that GHR is present in endosomes both in its full-length and truncated form, indicating that the receptor is down-regulated by the proteasome.     

Growth hormone-induced phosphorylation of epidermal growth factor (EGF) receptor in 3T3-F442A cells. Modulation of EGF-induced trafficking and signaling

Growth hormone (GH) promotes signaling by causing activation of the non-receptor tyrosine kinase, JAK2, which associates with the GH receptor. GH causes phosphorylation of epidermal growth factor receptor (EGFR; ErbB-1) and its family member, ErbB-2. For EGFR, JAK2-mediated GH-induced tyrosine phosphorylation may allow EGFR to serve as a scaffold for GH signaling. For ErbB-2, GH induces serine/threonine phosphorylation that dampens basal and EGF-induced ErbB-2 kinase activation. We now further explore GH-induced EGFR phosphorylation in 3T3-F442A, a preadipocytic fibroblast cell line that expresses endogenous GH receptor, EGFR, and ErbB-2. Using a monoclonal antibody that recognizes ERK consensus site phosphorylation (PTP101), we found that GH caused PTP101-reactive phosphorylation of EGFR. This GH-induced EGFR phosphorylation was prevented by MEK1 inhibitors but not by a protein kinase C inhibitor. Although GH did not discernibly affect EGF-induced EGFR tyrosine phosphorylation, we observed by immunoblotting a substantial decrease of EGF-induced EGFR degradation in the presence of GH. Fluorescence microscopy studies indicated that EGF-induced intracellular redistribution of an EGFR-cyan fluorescent protein chimera was markedly reduced by GH cotreatment, in support of the immunoblotting results. Notably, protection from EGF-induced degradation and inhibition of EGF-induced intracellular redistribution afforded by GH were both prevented by a MEK1 inhibitor, suggesting a role for GH-induced ERK activation in regulating the trafficking itinerary of the EGF-stimulated EGFR. Finally, we observed augmentation of early aspects of EGF signaling (EGF-induced ERK2 activation and EGF-induced Cbl tyrosine phosphorylation) by GH cotreatment; the GH effect on EGF-induced Cbl tyrosine phosphorylation was also prevented by MEK1 inhibition. These data indicate that GH, by activating ERKs, can modulate EGF-induced EGFR trafficking and signaling and expand our understanding of mechanisms of cross-talk between the GH and EGF signaling systems.     

Insulin inhibits growth hormone signaling via the growth hormone receptor/JAK2/STAT5B pathway

Insulin is important for maintaining the responsiveness of the liver to growth hormone (GH). Insulin deficiency results in a decrease in liver GH receptor (GHR) expression, which can be reversed by insulin administration. In osteoblasts, continuous insulin treatment decreases the fraction of cellular GHR localized to the plasma membrane. Thus, it is not clear whether hyperinsulinemia results in an enhancement or inhibition of GH action. We asked whether continuous insulin stimulation, similar to what occurs in hyperinsulinemic states, results in GH resistance. Our present studies suggest that insulin treatment of hepatoma cells results in a time-dependent inhibition of acute GH-induced phosphorylation of STAT5B. Whereas total protein levels of JAK2 were not reduced after insulin pretreatment for 16 h, GH-induced JAK2 phosphorylation was inhibited. There was a concomitant decrease in GH binding and a reduction in immunoreactive GHR levels following pretreatment with insulin for 8-24 h. In summary, continuous insulin treatment in rat H4 hepatoma cells reduces GH binding, immunoreactive GHR, GH-induced phosphorylation of JAK2, and GH-induced tyrosine phosphorylation of STAT5B. These findings suggest that hepatic GH resistance may develop when a patient exhibits chronic hyperinsulinemia, a condition often observed in patients with obesity and in the early stage of Type 2 diabetes.     

Growth hormone receptor ubiquitination, endocytosis, and degradation are independent of signal transduction via Janus kinase 2.

The ubiquitin-proteasome system is required in growth hormone receptor (GHR) endocytosis. For cytokine receptors, which lack intrinsic tyrosine kinase activity, signal transduction is initiated by the activation of a member of the Janus kinase (JAK) family. Previously, we have shown that GHR and JAK2 tyrosine (de) phosphorylation are regulated via the ubiquitin system. In this study, we examined the role of JAK2-mediated signal transduction in GHR internalization and down-regulation. Mutation of the attachment site for JAK2, box-1, in the GHR cytoplasmic tail resulted in the complete absence of GHR and JAK2 phosphorylation. This modification did not alter the rate and extent of receptor-bound growth hormone internalization as compared with a functional GHR, nor did it change its turnover and transport to the plasma membrane. In addition, the receptor was still normally ubiquitinated and remained dependent on both an intact ubiquitin system and proteasomal action for its internalization. Thus, GHR ubiquitination, endocytosis, and degradation occur independently of GHR signal transduction via JAK2. We conclude that whereas endocytosis and degradation require the ubiquitin system, they are independent of GHR signal transduction.     

Prediction of the structure of human Janus kinase 2 (JAK2) comprising JAK homology domains 1 through 7

A theoretical model of human Janus kinase 2 (JAK2) comprising all seven Janus homology domains is presented. The model was generated by application of homology modelling approaches. The three-dimensional structure contains, starting from the N-terminus, FERM (4.1, ezrin, radixin, moesin), SH2 (Src homology region 2), tyrosine kinase-like, and tyrosine kinase domains. The predicted inter-domain orientation in JAK2 is discussed and the currently existing mutational data for Janus kinases are evaluated. Structural details of the SH2 and the FERM domains are presented. The predictions indicate that the SH2 domain is not fully functional. A number of hydrophobic amino acids of the FERM domain that are predicted to be involved in the constitutive association with the cytokine receptors are highlighted. The model gives new insights into the structure-function relationship of this important protein, and areas that could be investigated by mutation studies are highlighted.     

Interleukin-1 inhibits the induction of insulin-like growth factor-I by growth hormone in CWSV-1 hepatocytes

Sepsis results in hepatic "growth hormone (GH) resistance" with reductions in plasma IGF-I despite a two- to fourfold increase in circulating GH. In this study, we examine the effects of IL-1 on GH receptor (GHR) expression, GH signaling (via the JAK/STAT and MAPK pathways), and the induction of gene expression [IGF-I mRNA and serine protease inhibitor (Spi) 2.1] by GH in CWSV-1 hepatocytes. Incubation of cells with IL-1beta (10 ng/ml, 24 h) had no effect on the relative abundance of GHR or signaling proteins JAK2, STAT5b, and ERK1/2 in cell lysates. Baseline phosphorylation of GHR, JAK2, STAT5b, and ERK1/2 was minimal. After GH stimulation, tyrosine phosphorylation of GHR, JAK2, STAT5b, and ERK1/2 increased 2- to 10-fold. However, neither the time course nor the magnitude of GHR, JAK2, and ERK1/2 phosphorylation by GH were significantly altered by IL-1. The GH-induced translocation of STAT5b to the nucleus was not prevented by IL-1. Although phosphorylated STAT5 in nuclear extracts from GH + IL-1 cells was decreased by 24% (vs. controls) 15 min after GH stimulation, this did not result in reduced STAT5-DNA binding activity. Pretreatment with IL-1 did not significantly decrease IGF-I mRNA stability. We conclude that IL-1 only minimally affects the time course of JAK2/STAT5 and MAPK signaling by GH. Therefore, an inhibitory effect of IL-1 on IGF-I and Spi 2.1 mRNA synthesis by GH represents the most likely mechanism for IL-1-mediated GH resistance.     

Growth hormone signalling and apoptosis in neonatal rat cardiomyocytes

Growth hormone (GH) has been reported to be useful to treat heart failure. To elucidate whether GH has direct beneficial effects on the heart, we examined effects of GH on oxidative stress-induced apoptosis in cardiac myocytes. TUNEL staining and DNA ladder analysis revealed that hydrogen peroxide (H₂O₂)-induced apoptosis of cardiomyocytes was significantly suppressed by the pretreatment with GH. GH strongly activated extracellular signal-regulated kinases (ERKs) in cardiac myocytes and the cardioprotective effect of GH was abolished by inhibition of ERKs. Overexpression of dominant negative mutant Ras suppressed GH-stimulated ERK activation. Overexpression of Csk that inactivates Src family tyrosine kinases also inhibited ERK activation evoked by GH. A broad-spectrum inhibitor of protein tyrosine kinases (PTKs), genistein, strongly suppressed GH-induced ERK activation and the cardioprotective effect of GH against apoptotic cell death. GH induced tyrosine phosphorylation of EGF receptor and JAK2 in cardiac myocytes, and an EGF receptor inhibitor tyrphostin AG1478 and a JAK2 inhibitor tyrphostin B42 completely inhibited GH-induced ERK activation. Tyrphostin B42 also suppressed the phosphorylation of EGF receptor stimulated by GH. These findings suggest that GH has a direct protective effect on cardiac myocytes against apoptosis and that the effect of GH is attributed at least in part to the activation of ERKs through Ras and PTKs including JAK2, Src, and EGF receptor tyrosine kinase.     

Growth hormone receptor targeting to lipid rafts requires extracellular subdomain 2

GH receptor (GHR) is a single membrane-spanning glycoprotein dimer that binds GH in its extracellular domain (ECD). GH activates the GHR intracellular domain (ICD)-associated tyrosine kinase, JAK2, which causes intracellular signaling. We previously found that plasma membrane (PM)-associated GHR was dramatically enriched in the lipid raft (LR) component of the membrane and that localization of GHR within PM regions may regulate GH signaling by influencing the profile of pathway activation. In this study, we examined determinants of LR localization of the GHR using a reconstitution system which lacks endogenous JAK2 and GHR. By non-detergent extraction and multistep fractionation, we found that GHR was highly enriched in the LR fraction independent of JAK2 expression. Various GHR mutants were examined in transfectants harboring JAK2. LR concentration was observed for a GHR in which the native transmembrane domain (TMD) is replaced by that of the unrelated LDL receptor and for a GHR that lacks its ICD. Thus, LR association requires neither the TMD nor the ICD. Similarly, a GHR that lacks the ECD, except for the membrane-proximal ECD stem region, was only minimally LR-concentrated. Mutants with internal stem deletions in the context of the full-length receptor were LR-concentrated similar to the wild-type. A GHR lacking ECD subdomain 1 reached the PM and was LR-concentrated, while one lacking ECD subdomain 2, also reached the PM, but was not LR-concentrated. These data suggest LR targeting resides in ECD subdomain 2, a region relatively uninvolved in GH binding.     

Role of the cytokine-inducible SH2 protein CIS in desensitization of STAT5b signaling by continuous growth hormone

Growth hormone (GH)-inducible suppressors of cytokine signaling (SOCS/CIS proteins) inhibit GH receptor (GHR) signaling to STAT5b via phosphotyrosine-dependent binding interactions with the tyrosine kinase JAK2 (SOCS-1) and/or the cytoplasmic tail of GHR (CIS and SOCS-3). Presently, we investigate the mechanism of CIS inhibition and CIS's role in down-regulating GHR-JAK2 signaling to STAT5b in cells exposed to GH continuously. CIS is shown to inhibit GHR-JAK2 signaling by two distinct mechanisms: by a partial inhibition that is decreased at elevated STAT5b levels and may involve competition between CIS and STAT5b for common GHR cytoplasmic tail phosphotyrosine-binding sites; and by a time-dependent inhibition, not seen with SOCS-1 or SOCS-3, that involves proteasome action. Investigation of the latter mechanism revealed that GH stimulates degradation of CIS, but not SOCS-3. The proteasome inhibitor MG132 blocked this protein degradation and also blocked the inhibitory action of CIS, but not that of SOCS-1 or SOCS-3, on STAT5b signaling. Proteasome-dependent degradation of CIS, most likely in the form of a (GHR-JAK2)-CIS complex, is therefore proposed to be an important step in the time-dependent CIS inhibition mechanism. Finally, the down-regulation of GHR-JAK2 signaling to STAT5b seen in continuous GH-treated cells could be prevented by treatment of cells with the proteasome inhibitor MG132 or by expression of CIS-R107K, a selective, dominant-negative inhibitor of CIS activity. These findings lead us to propose that the cytokine signaling inhibitor CIS is a key mediator of the STAT5b desensitization response seen in cells and tissues exposed to GH chronically, such as adult female rat liver.     

An agonist-induced conformational change in the growth hormone receptor determines the choice of signalling pathway

The growth and metabolic actions of growth hormone (GH) are believed to be mediated through the GH receptor (GHR) by JAK2 activation. The GHR exists as a constitutive homodimer, with signal transduction by ligand-induced realignment of receptor subunits. Based on the crystal structures, we identify a conformational change in the F'G' loop of the lower cytokine module, which results from binding of hGH but not G120R hGH antagonist. Mutations disabling this conformational change cause impairment of ERK but not JAK2 and STAT5 activation by the GHR in FDC-P1 cells. This results from the use of two associated tyrosine kinases by the GHR, with JAK2 activating STAT5, and Lyn activating ERK1/2. We provide evidence that Lyn signals through phospholipase C gamma, leading to activation of Ras. Accordingly, mice with mutations in the JAK2 association motif respond to GH with activation of hepatic Src and ERK1/2, but not JAK2/STAT5. We suggest that F'G' loop movement alters the signalling choice between JAK2 and a Src family kinase by regulating TMD realignment. Our findings could explain debilitated ERK but not STAT5 signalling in some GH-resistant dwarfs and suggest pathway-specific cytokine agonists.