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Negative Regulation of Growth Hormone Receptor Signaling

Department of Molecular Medicine (A.F.-M., G.N., E.R.-B.), Karolinska Institute, 17176 Stockholm, Sweden; and The Walter and Eliza Hall Institute of Medical Research (C.J.G.), Victoria 3050, Australia

Address all correspondence and requests for reprints to: Amilcar Flores-Morales, Center of Molecular Medicine (CMM), L8:01, Karolinska Hospital, 17176 Stockholm, Sweden. E-mail: Amilcar.Flores{at}cmm.ki.se.

	ABSTRACT

TOP ABSTRACT INTRODUCTION NEGATIVE REGULATION OF GHR... THE SOCS FAMILY OF... GH SIGNALING AND SOCS... PHOSPHOTYROSINE PHOSPHATASES AND... SIGNAL REGULATORY PROTEIN... FUTURE PERSPECTIVES REFERENCES

 
GH has been of significant scientific interest for decades because of its capacity to dramatically change physiological growth parameters. Furthermore, GH interacts with a range of other hormonal pathways and is an established pharmacological agent for which novel therapeutical applications can be foreseen. It is easy to see the requirement for a number of postreceptor mechanisms to regulate and control target tissue sensitivity to this versatile hormone. In recent years, some of the components that take part in the down-regulatory mechanism targeting the activated GH receptor (GHR) have been defined, and the physiological significance of some of these key components has begun to be characterized. Down-regulation of the GHR is achieved through a complex mechanism that involves rapid ubiquitin-dependent endocytosis of the receptor, the action of tyrosine phosphatases, and the degradation by the proteasome. The suppressors of cytokine signaling (SOCS) protein family, particularly SOCS2, plays an important role in regulating GH actions. The aim of this review is to summarize collected knowledge, including very recent findings, regarding the intracellular mechanisms responsible for the GHR signaling down-regulation. Insights into these mechanisms can be of relevance to several aspects of GH research. It can help to understand growth-related disease conditions, to explain GH resistance, and may be used to develop pharmaceuticals that enhance some the beneficial actions of endogenously secreted GH in a tissue-specific manner.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION NEGATIVE REGULATION OF GHR... THE SOCS FAMILY OF... GH SIGNALING AND SOCS... PHOSPHOTYROSINE PHOSPHATASES AND... SIGNAL REGULATORY PROTEIN... FUTURE PERSPECTIVES REFERENCES

 
GH IS THE MAIN regulator of longitudinal growth in mammals (1). GH has diverse and pleiotropic effects on carbohydrate, lipid, nitrogen, and mineral metabolism (2, 3) and stimulates differentiation and mitogenesis in a variety of cell types in different tissues (4). GH is also important in the maintenance of the immune system (5) and heart development (6) and can act on the brain to modulate emotion, stress response, and behavior (7). GH actions are regulated mainly at two levels: the site of synthesis and secretion at the somatotrophs in the anterior pituitary gland [reviewed in Kato et al. (8)], and on the target organs.

Responsiveness to GH in target cells is primarily dependant upon the expression of the GH receptor (GHR) (9). GHR is a single membrane-spanning cell surface protein member of the class I cytokine receptor superfamily (10). Like other members of the family, GHR lacks intrinsic kinase activity and signal transduction is mediated by Janus kinase (JAK) 2, a cytoplasmic tyrosine kinase that associates to the so-called box 1 in the membrane proximal region of the GHR cytoplasmic domain (11, 12). JAK2 activation is triggered by GH-induced receptor dimerization which induces conformational changes resulting in JAK2 transphosphorylation and catalytic activation. Subsequently, the receptor and several signaling proteins are phosphorylated on key tyrosine residues, resulting in the activation of multiple signaling pathways. GHR mutations results in the Laron Syndrome that is characterized by severe postnatal growth retardation (13). This phenotype is mimicked in GHR^–/– mice, which show a 50% reduction in normal adult body weight (14). Introduction of two different GHR truncations in mice generates growth defects that are intermediate between mice expressing the wild-type receptor and GHR^–/– mice, with the length of the GHR intracellular domain positively correlating with the final length achieved by the animals (15).

JAK2 is essential for GHR signaling. Mutation of box 1 in the receptor or deletion of JAK2 renders the GHR inactive (16, 17). A multitude of signaling molecules are activated by GH, including MAPK, insulin receptor substrate 1, focal adhesion kinase, protein kinase C, Ras-like GTPases, and the signal transducer and activator of transcription (STATs) family of transcription factors [recently reviewed in Zhu et al. (18)]. The contribution of each of these pathways to the physiological actions of GH remains unclear because many of them are also activated by several additional growth factors and cytokines and in many cases, the data have been obtained only from in vitro studies. A notable exception is the transcription factor STAT5b. Analysis of STAT5b-deficient mice shows that it is directly involved in sexually dimorphic longitudinal growth and hepatic gene expression. STAT5b-deficient mice have a 27% reduction in body growth in males but not in females, elevated GH plasma levels, and reduced circulating IGF-I, as well as an inhibition of GH-induced lipolysis in adipose tissue (19). Analysis of GH-induced gene expression found that STAT5b is required for the hepatic expression of IGF-I, IGF binding protein 3, ALS, suppressors of cytokine signaling (SOCS) 1, SOCS2, SOCS3, and CIS [cytokine-inducible SH2 (Src-homology 2) protein] (20, 21, 22). STAT5b mutations have also been found in humans, resulting in severe growth deficiency comparable with that of patients with GHR-inactivating mutations (23).

The duration of GH-activated signals is a key factor in relation to the biological actions of the hormone. This is clearly illustrated in the case of hepatic GH actions where signal duration regulates gender differences in liver gene expression. The male pattern of GH secretion in rats is episodic with peaks every 3–4 h and periods when GH is undetectable (24). Consequently, intracellular activation of STAT5 is also episodic. The coordination of extracellular and intracellular signals is achieved through inactivation of GHR signals. This mechanism is GH induced and also persistent in time. Consequently, periods with low GH circulating levels are required to achieve maximal activation of STAT5. Female rats, which exhibit a more continuous GH secretion pattern with higher basal levels and smaller and intermittent peaks have reduced STAT5b activation compared with males (25). These differences in STAT5b activation are responsible for some of the gender differences in hepatic gene expression (26). Studies on primary hepatocytes and several cell lines have shown that GH-induced JAK2/STAT5b activation is transient, with maximal activation achieved within the first 30 min of stimulation, followed by a period of down-regulation (Fig. 1) (27). This period is characterized by an inability to achieve maximal JAK2/STAT5 activation by GH in the proceeding 3 h, unless GH is withdrawn from the media. The conserved control of GHR/JAK2 activation kinetic in multiple cell types emphasizes the importance of mechanisms of negative regulation for GH actions. Several studies already show that GH action can be modulated through interference with the GHR down-regulation. Phospholipase C inhibition (27), induction of the unfolded protein response (28), actin cytoskeleton depolymerization (29), and treatment with 1,25-dihydroxyvitamin D3 (30), enhance the duration of JAK2 and STAT5 phosphorylation after GH treatment, with little or no effect on its rapid and maximal activation.

View larger version (25K): [in this window] [in a new window]   Fig. 1. GH-Dependent Activation of STAT5 DNA-Binding Activity Is Transient in Different Cell Models Several cell lines were treated with GH in time course studies. Nuclear extracts were prepared and the STAT5 DNA-binding activity was analyzed by EMSA. BRL-4, Buffalo rat liver 4; INS-1E, insulinoma.

	NEGATIVE REGULATION OF GHR SIGNALING

TOP ABSTRACT INTRODUCTION NEGATIVE REGULATION OF GHR... THE SOCS FAMILY OF... GH SIGNALING AND SOCS... PHOSPHOTYROSINE PHOSPHATASES AND... SIGNAL REGULATORY PROTEIN... FUTURE PERSPECTIVES REFERENCES

The 26S proteasome complex is the location where proteolytic degradation of polyubiquitinated proteins occurs. A very strong indication that the ubiquitin-proteasome system is involved in GHR-negative regulation comes from the finding that proteasome inhibition prolongs GHR phosphorylation and JAK2/STAT5 activation in several systems (28, 46, 47). Proteasome inhibition also results in blockage of GH-induced GHR internalization and degradation, but only of naturally occurring GHR full-length proteins. Short forms, lacking residues 370–620 of the intracellular domain are internalized, and in contrast to the full-length receptor, are recycled to the membrane despite proteasomal inhibition (48).

It is apparent that GHR ubiquitination is a key control mechanism in the down-regulation of GH signaling, modulating both GHR internalization and proteasomal degradation. The mechanism responsible for GHR ubiquitination is yet to be completely defined. Protein ubiquitination is the result of the sequential action of three classes of enzymes; E1 or ubiquitin-activating enzyme, E2 or ubiquitin-conjugating enzyme, and E3 or ubiquitin ligases, which in most cases are protein complexes (49). Substrate specificity in the ubiquitination process is largely determined by E3s. Therefore, the identification of the E3 acting on the GHR/JAK2 complex is the key to understand GHR ubiquitination. A breakthrough in this field was the identification in 1997 of the SOCS family (50, 51, 52).

	THE SOCS FAMILY OF PROTEINS

TOP ABSTRACT INTRODUCTION NEGATIVE REGULATION OF GHR... THE SOCS FAMILY OF... GH SIGNALING AND SOCS... PHOSPHOTYROSINE PHOSPHATASES AND... SIGNAL REGULATORY PROTEIN... FUTURE PERSPECTIVES REFERENCES

 
SOCS proteins act as negative regulators of the main cytokine-activated signaling pathway, the JAK/STAT signal cascade (53). The SOCS family comprises at least eight proteins: CIS and SOCS-1 to SOCS-7, which display similar domain architecture. They have a variable amino-terminal region; a central SH2 domain, and a conserved carboxy-terminal motif, denominated SOCS box. In addition to the SH2 domain and the SOCS box, SOCS1 and SOCS3 contain a kinase inhibitory region (KIR) at the N-terminal domain, which has not been described for other family members (54). The SOCS box has also been identified in other proteins that lack SH2 domain but instead have other domains that can mediate protein-protein interactions (ankyrin repeats, WD40 repeats, or SPRY domains) (55).

A number of the SOCS proteins have been shown to modify cytokine action through a classic negative feedback loop. In general, SOCS proteins levels are constitutively low, but their expression is rapidly induced by stimulation with different cytokines or growth factors, including GH (reviewed in Refs.56 and 57). SOCS proteins bind the receptor/JAK complex and down-regulate the JAK/STAT pathway, as demonstrated by the reduction of JAK and STAT phosphorylation, STAT dimerization, nuclear translocation, and STAT transcriptional activity (50, 51, 58). Promiscuity and redundancy are notable features of the SOCS proteins system when tested in vitro. Any single SOCS can be induced by many cytokines in vitro and in turn act on several cytokine receptors, not necessarily the ones inducing their expression. GH induces the expression of CIS, SOCS1, 2, and 3 (59, 60, 61); all of which have shown negative actions on the GHR when forcibly overexpressed in cell lines (59, 62, 63). Evidence also indicates that growth factors that do not belong to the 4-helical bundle cytokine family (e.g. insulin, chemokines), and even steroid hormones, can induce SOCS expression (64, 65). Consequently, regulation of SOCS protein expression provides the mean for cross talk where multiple factors can regulate the activity of specific cytokines.

The analysis of genetically manipulated mice is starting to reveal the essential pathophysiological roles for SOCS proteins (66). The phenotype of SOCS2^–/– mice identifies SOCS2 as an important physiological player in the negative regulation of GH signaling (67). SOCS2^–/– mice are 30–40% larger that their littermates, with the weight gain due to an increase in bone size and a proportionate enlargement of most organs (68). Similar phenotypes have been also found in animals overexpressing GH (69), patients with gigantism (70) and in high-growth mice, which have a spontaneous deletion within the chromosome 10 resulting in a disruption and inactivation of the socs2 locus (71). IGF-I mRNA expression in SOCS2^–/– is significantly increased in some organs, without major changes in hepatic or serum IGF-I content. The role of SOCS2 in GH signaling has been further demonstrated in several key experiments. Double knockout mice, where both STAT5b and SOCS2 gene are inactivated, are not giants, and are not significantly different from STAT5b^–/– mice, a demonstration of the necessity of STAT5b for the gigantism observed in SOCS2^–/– mice (72). Accordingly, primary hepatocytes derived from SOCS2^–/– mice have mildly prolonged STAT5b activation in response to GH when compared with hepatocytes from wild-type mice, whereas the response of SOCS2^–/– fibroblasts to IGF-I is apparently unchanged. We have recently shown that the gigantism of SOCS2^–/– mice can be reversed if the animals are made GH-deficient by crossing them with dwarf mice containing the little inactivating mutation in the GHRH receptor (GHRHR^lit/lit). Moreover, SOCS2^–/–GHRHR^lit/lit mice are significantly more sensitive than (GHRHR^lit/lit) mice to exogenous GH treatment and achieve a larger size upon GH replacement therapy (73).

Shortly after the identification of the SOCS proteins, it was recognized that a SOCS box was also present in the domain of the von Hippel-Lindau tumor-suppressor gene product. Through this domain, von Hippel-Lindau associates with Elongin BC, the Cullin family member Cul2, and the ring finger Rbx1 (Roc1) to assemble an E3 ubiquitin-ligase complex, which targets hypoxia-inducible factor-1 for proteasomal degradation (74). The SOCS box of SOCS1 and SOCS3 (75, 76) also associates with the Elongin BC complex, an observation later extended to SOCS2 (73), suggesting that SOCS proteins could act to assemble ubiquitin ligases. Kamura et al. (77) have recently shown that SOCS proteins constitute a new class of ubiquitin ligases, characterized by association to cullin5-Rbx2 through a Pro-Leu-X-Pro motif within amino terminus of the SOCS box. This motif is highly conserved among members of the family with exception of SOCS1. Consequently, SOCS1 display less affinity for cullin 5 and can also associate to cullin 2 (77, 78). In line with its proposed role as ubiquitin ligase, SOCS1 induces the proteasomal degradation of guanine nucleotide exchange factor Vav (79), oncogene Tel-JAK2 (80, 81), JAK2 (Fig. 2) (82) and insulin receptor substrate 1/2 (83). Furthermore, mice lacking the SOCS box motif of SOCS1 have a phenotype similar to SOCS1^–/– mice although ameliorated, indicating that the SOCS box is essential for the complete activity of SOCS1 (84). The ubiquitin-ligase activity of other SOCS proteins has been difficult to demonstrate using overexpression systems, where the absence of the SOCS box does not appear to affect SOCS1 and SOCS3 inhibitory action on JAKs (85, 86). The most likely explanation for this phenomenon is that SOCS can achieve its inhibitory actions through alternative mechanisms. Evidence indicates that SOCS1 and SOCS3 can act as kinase inhibitors of JAK proteins, whereas other SOCS can act as binding competitors against positive regulators such as STAT proteins (87). Another layer of complexity is added by the findings that SOCS proteins themselves can be targeted for ubiquitination and proteasomal degradation, although contradictory results exist regarding the mechanisms controlling the stability of CIS, SOCS1, and SOCS3. Thus, it seems that Elongin BC complex can have dual actions: it can promote SOCS proteins degradation as has been shown for CIS (88, 89), SOCS1 (75, 90) and SOCS3 (91), whereas in other cellular systems, it can also stabilize SOCS3 (76, 92) and SOCS1 proteins (75, 76, 85).

View larger version (35K): [in this window] [in a new window]   Fig. 2. Mechanism of Negative Actions of SOCS1 on JAK2 SOCS1 interact with tyrosine phosphorylated partners through their SH2 domain and with Elongin BC through their SOCS box domain. SOCS1 binds phosphorylated JAK2 and induces its degradation by the formation of an ubiquitin ligase complex containing Elongin BC, Roc1/2, a Cullin family member and a yet unidentified ubiquitin-conjugating enzyme (E2) enzyme. A KIR is found in the N-terminal domain that can inhibit JAK2 activity independently of the SOCS domain.

	GH SIGNALING AND SOCS PROTEINS

TOP ABSTRACT INTRODUCTION NEGATIVE REGULATION OF GHR... THE SOCS FAMILY OF... GH SIGNALING AND SOCS... PHOSPHOTYROSINE PHOSPHATASES AND... SIGNAL REGULATORY PROTEIN... FUTURE PERSPECTIVES REFERENCES

Each of the SOCS members appears to have a different mechanism of action on GH signaling. SOCS1, through its SH2 domain, directly binds the Y1007 within the JAK2 activation loop and blocks the kinase activity through the KIR motif (52, 86, 95), resulting in reduced JAK2 and STAT5 phosphorylation. Interestingly, the N-terminal region of SOCS1 is interchangeable with that of SOCS3, which also contains a KIR motif (86). SOCS-1 also regulates the proteasomal degradation of activated JAK2, probably through its action as an ubiquitin ligase (82). The relevance of SOCS1 to the physiological actions of GH is unclear. SOCS1^–/– mice are growth retarded with severe fatty liver disease and monocyte infiltration in multiple tissues, which leads to premature death (96). This phenotype can be rescued through inactivation of the interferon gene. Significantly, SOCS1^–/–/interferon ^–/– mice do not show increased body weight as one would expect from a negative action of SOCS1 on GHR signaling (97). Lack of tissue and temporal coordination between regulation of SOCS1 expression and activation of GHR is a probable explanation for its apparent lack of action on the receptor in vivo. Although GH has been shown to induce SOCS1 expression in fibroblast (59) and hepatoma cell cultures (28); in liver, one of the key tissues for the growth promoting actions of GH, weak or no effects in the transcriptional activation of SOCS1 have been observed (20, 59, 98).

SOCS2, SOCS3, and CIS have also been shown to bind the tyrosine-phosphorylated GHR (62, 63). The binding site of SOCS3 to the GHR has been mapped at the membrane proximal region of the receptor, specifically phosphorylated tyrosines Y338, Y333 (63), and Y487 (62). Because the membrane proximal region of GHR is not a major STAT5 binding site, SOCS3 inhibition of STAT5 activation does not appear to operate by competitive binding, but a direct action on JAK2 is proposed (62). SOCS3 can bind JAK proteins and, at high concentrations, it can inhibit GH signaling even in the absence of GHR phosphorylation (63, 99). Similarly to SOCS1, SOCS3 can inhibit the kinase activity of JAK2 through its KIR (54). However, peptide analysis of SOCS binding to gp130 signaling systems has found that SOCS3 binds preferentially to the cytokine receptor rather than JAK2 (99). Ubiquitin ligase activity on JAK2 or GHR has not yet been demonstrated, although its significance is questionable because SOCS3 and SOCS1 (but not SOCS2 and CIS)-negative actions in vitro are insensitive to proteasomal inhibition (89). Although it is clear that GH is a potent inducer of SOCS3 expression in liver, this expression is highly transient and its relevance for GH physiological actions is yet unclear. Liver-specific SOCS3^–/– mice do not differ in size from wild-type littermates (100), indicating that SOCS3 is either of little importance for GH hepatic actions that regulate body growth or compensatory mechanisms act in SOCS3^–/– liver that negatively influence GH signaling. Indeed, it has been shown that IL-6 is able to induce the expression of SOCS2 in SOCS3 deficient but not in SOCS3 expressing livers (100).

In vitro and in vivo studies also implicate CIS in the down-regulation of GH signaling. The mechanism of CIS inhibition appears to involve direct binding to GHR. The specific sites on the GHR required for the interaction with CIS have been mapped to the membrane-distal part of the receptor, which is also the major binding site for STAT5b. It has been proposed that CIS inhibits signaling by competitively masking the STAT-binding sites in the activated receptor (89). CIS down-regulatory action can also be reversed by proteasome inhibition despite the fact that this treatment stabilizes CIS expression through inhibition of GH-induced degradation. Consequently, it is likely that CIS promotes GHR/JAK2 proteasomal degradation in addition to inhibit STAT5 activity (89, 101). Whether this effect is due to a direct ubiquitin ligase activity of CIS on the receptor, remains to be demonstrated. It is important to mention that overexpressing CIS in mice results in a phenotype similar to the one found in STAT5b^–/– mice, thus supporting the hypothesis that CIS may be an important player in the down-regulation of GH-dependent signaling in vivo (102). However, CIS actions seem to be redundant because CIS^–/– mice show no obvious growth phenotype.

Mouse genetic experiments have provided strong evidence for the role of SOCS2 as a negative regulator of GHR signaling. However, further studies have shown that SOCS2 have dual effects in GH-induced STAT5 activation; inhibition at low concentration and stimulation when expressed at high concentration. This is not a behavior that is solely observed in transfected cells. Instead of the expected growth inhibition, overexpressing SOCS2 at high levels in transgenic mice resulted in a 10% increase in body growth (103), supporting a concentration-dependent stimulatory effect on GH action. Overall, the results obtained from systems where SOCS2 is highly overexpressed have to be interpreted with caution because there is no evidence that such levels of endogenous SOCS2 protein are achieved in physiological situations. The mechanism of SOCS2’s negative actions on GH signaling is beginning to become clear. Initially, SOCS2 was found to inhibit JAK2 tyrosine phosphorylation when both are coexpressed in COS-1 cells, even in the absence of GHR expression. This effect was observed at high concentrations of JAK2, and it is still unclear whether direct binding to JAK2 is a physiological relevant mechanism whereby SOCS2 inhibits GH signaling (63). Stronger evidence indicates that binding to the GHR, through tyrosines Y595 and Y487, is required for SOCS2 inhibitory actions as receptor variants that have both residues mutated are insensitive to its negative actions (103). Because Y595 is a known binding site for STAT5 (104), it is also possible that SOCS2 can competitively inhibit STAT5b binding to the receptor. However, rapid and maximal STAT5b activation by GH does not differ in hepatocytes from SOCS2^–/– and SOCS2^+/+ mice (72). It is also possible that SOCS2 acts on IGF-I signaling and in this way regulates somatic growth, as has been suggested after the finding that SOCS2 associates with the IGF-I receptor (105). SOCS2-negative regulation of IGF-I signaling remains to be demonstrated, but if confirmed, it would place SOCS2 as a master regulator of somatic growth through dual actions on both GH- and IGF-I-dependent signaling pathways. Recent findings would support the existence of such mechanisms. It has been demonstrated that SOCS2 have inhibitory effects in both GH and IGF-I stimulation of collagen expression in intestinal mesenchymal cells (106). In contrast to SOCS1 and SOCS3 where the SOCS box is dispensable, all three domains of SOCS2 are required to inhibit GH signaling. Deletion of the N-terminal domain and mutations in the phosphotyrosine binding domain, abrogate SOCS2 down-regulatory activity. SOCS box deletion results in a SOCS2 variant that behaves as a dominant negative, enhancing GH signaling when coexpressed with the receptor. The finding that SOCS2 binds elongin B and C suggests that it may act as an ubiquitin ligase, promoting the ubiquitination and subsequent degradation of the GHR (73). Nevertheless, such activity remains to be demonstrated.

SOCS2 may also be the mean by which other signaling pathways influence GH activity. It has been known for some time that estrogen has a negative impact on GH action (107). Recent evidences indicate that estrogen inhibitory actions on GH signaling can be mediated by SOCS2 (108). Estrogen suppresses GH-dependent JAK2 phosphorylation by increasing the expression of SOCS2. Interestingly, glucocorticoids have negative effects on somatic growth and are also able to enhance GH-induced SOCS2 expression in primary hepatocytes (61). Other studies have demonstrated that SOCS2 is essential for the regulation of GH actions not directly related to somatic growth. For example, SOCS2 blocks GH-dependent inhibition of neural stem cell differentiation. Consequently SOCS2^–/– mice have fewer neurons in the developing cortex, whereas SOCS2 overexpression results in increased neural differentiation (109). Recently, Miller and co-workers (110) also demonstrated that SOCS2 inhibits intestinal epithelial cell proliferation, which is induced by GH and IGF-I.

	PHOSPHOTYROSINE PHOSPHATASES AND REGULATION OF GH SIGNALING

TOP ABSTRACT INTRODUCTION NEGATIVE REGULATION OF GHR... THE SOCS FAMILY OF... GH SIGNALING AND SOCS... PHOSPHOTYROSINE PHOSPHATASES AND... SIGNAL REGULATORY PROTEIN... FUTURE PERSPECTIVES REFERENCES

 
Activation of GH-dependent signaling pathways is based on protein phosphorylation on tyrosine, serine, or threonine residues. The obvious mechanism for deactivation of this process is the action of protein phosphatases. It has been shown that preincubation with phosphotyrosine phosphatase inhibitors induces a prolongation of the GH-dependent JAK2 and STAT5 phosphorylation (111). Considering that the GHR/JAK2/STAT5 cascade is regarded as the main GH-activated signaling pathway, the action of phosphatases on these three proteins is critical for the control of the cellular response. Several studies have resulted in the identification of three different phosphatases: 1) SHP1 (SH2 domain-containing protein-tyrosine phosphatase 1, also known as PTP-1); 2) PTP1b; and 3) PTP-H1, which are involved in the specific down-regulation of GHR signaling.

GH can activate SHP1 and induce its translocation to the nucleus where it binds to phosphorylated STAT5b, resulting in an attenuation of STAT5 activity (112). SHP1 also binds GH-activated JAK2 and controls the duration of GH-dependent JAK2 phosphorylation in the liver. Consequently, hepatic GH signaling is prolonged in mice lacking SHP1 (113). PTP1b can also regulate GH signaling. PTP1b interacts with JAK2 in a GH-dependent way and dephosphorylates the tyrosines present in the active JAK2 molecule. Fibroblasts derived from mice lacking PTP1b show JAK2 hyperphosphorylation and an enhancement of STAT3 and STAT5 phosphorylation upon GH treatment. In contrast, overexpression of PTP1b reduces GH-dependent ALS promoter activity in H4IIE cells. In vivo experiments also indicate that the absence of PTP1b reverses the hepatic GH resistance that is observed in some stress conditions such as fasting (114). PTP-1b has been shown to associate to the GH-dependent phosphorylated GHR and to induce its dephosphorylation (115). Interestingly, PTP1b is mainly located at the cytosolic surface of the endoplasmic reticulum (ER) where it exerts its phosphatase activity (116). Consequently, PTP1b action on JAK2 and/or the GHR would require previous endocytosis, a mechanism that has already been demonstrated for the epidermal growth factor and platelet-derived growth factor receptors (117). We have shown that induction of ER stress causes a prolongation in the duration of GH-dependent signaling in hepatocytes (28). Given the intracellular location of PTP1b, the interference with the down-regulation of GH signaling under ER stress could be related to the inactivation of this phosphatase.

Pasquali and co-workers (115) screened 31 tyrosine phosphatases and identified four PTPs with binding activity toward the GH-induced phosphorylated GHR. These are PTP-H1, stomach cancer-associated PTP1, T-cell-PTP, and PTP1b. By overexpression of wild-type and mutant variants of each phosphatase, it was shown that PTP-H1 in addition to PTP1b can dephosphorylate the GHR, whereas stomach cancer-associated PTP1 and TC-PTP were inactive. It is worth noting that GHR dephosphorylation in these cellular models is not complete, suggesting that there are other active phosphatases or that additional mechanisms are required for the complete silencing of GH-dependent signaling.

	SIGNAL REGULATORY PROTEIN- (SIRP-)

TOP ABSTRACT INTRODUCTION NEGATIVE REGULATION OF GHR... THE SOCS FAMILY OF... GH SIGNALING AND SOCS... PHOSPHOTYROSINE PHOSPHATASES AND... SIGNAL REGULATORY PROTEIN... FUTURE PERSPECTIVES REFERENCES

 
SIRP- belongs to a family of ubiquitously expressed transmembrane glycoproteins, which also include the SIRP-ß members. SIRP- was identified by the ability to associate with the SH2 domain of SHP-2, SHP-1, and Grb2 in response to insulin, epidermal growth factor, and platelet-derived growth factor (118). GH induces JAK2-dependent phosphorylation of SIRP-, which then enable it to bind SHP2 (119). Overexpression of SIRP- negatively regulates GH-activated signaling by inhibition of the phosphorylation of JAK2, STAT5b, STAT3, ERK1, and ERK2. This effect is not observed when SIRP 4YF (a mutant lacking four tyrosines) is overexpressed, further confirming the negative action of SIRP- on GH signaling (120). The exact mechanism of SIRP--negative control on GH signaling is not clear. It could involve binding to SHP-1, a protein tyrosine phosphatase known to act on JAK2 and STAT5 (112, 113), but it could also compete with the GHR for binding of positive regulators such as SHP-2 or JAK2 (121).

	FUTURE PERSPECTIVES

TOP ABSTRACT INTRODUCTION NEGATIVE REGULATION OF GHR... THE SOCS FAMILY OF... GH SIGNALING AND SOCS... PHOSPHOTYROSINE PHOSPHATASES AND... SIGNAL REGULATORY PROTEIN... FUTURE PERSPECTIVES REFERENCES

 
Down-regulation of GHR-activated signaling pathways occurs through a complex mechanism that engages the coordinated actions of several negative regulators. Many of the details of this process are starting to emerge. It is known to involve ligand-induced endocytosis, degradation of the GHR and signaling intermediaries, and the action of negative regulators, mainly phosphatases and SOCS proteins (Fig. 3). Nevertheless, despite the rapid accumulation of knowledge in this area, a unified model of GHR down-regulation is not yet at hand. Key issues remain to be solved. How the basal (hormone independent) mechanism acting to down-regulate the GHR relates to GH-induced mechanisms? What is the identity of the GHR ubiquitin ligases? How is the spatio-temporal relationship between the different components: ubiquitin ligases, phosphotyrosine phosphatases and kinase inhibitors? At a physiological level, issues such as tissue specificity in the activity of the down-regulatory mechanisms, their regulation by many physiological players known to affect somatic growth or interact with GH, are yet to be addressed. Today, GH is successfully used to treat growth disorders in children, but its use in adults remains controversial despite that many of its actions could be of potential benefits in several disease conditions. Taking into consideration that many of GH actions are direct, a deep understanding of GHR signaling down-regulation within specific tissues would allow scientists to rationally devise strategies to enhance specific GH effects. Pharmacological targeting of specific negative regulators has the potential to enhance the beneficial actions of GH in growth and metabolism, without the pitfalls associated to direct GH therapy.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Model of GHR-Negative Regulation GHR ubiquitination and internalization occurs constitutively, in the absence of GH and it is enhanced upon GH binding. The nature of the ubiquitin ligase (s) acting on the GHR upon hormone binding is not known yet, although the current knowledge points toward SOCS proteins in this role. SOCS1 through its interactions with Elongin BC can assemble an ubiquitin ligase with proven activity toward JAK2. It is also unknown whether different ubiquitin ligases mediate constitutive as opposed to GH-induced GHR ubiquitination. Ubiquitination of the GHR and possible other associated proteins precedes endocytosis which is mainly mediated by clathrin-coated pits. Active receptors are also found in early endosomes. GHR inactivation is achieved by the action of tyrosine phosphatases, kinases inhibitors such as SOCS-1 and proteolytic degradation. In the first two cases, it is unclear whether they act at the membrane or in the endocytic pathway. GHR is degraded both in the lysosomes and the proteasome, although the details of this process are not known. The internalized GHR could also be addressed to other intracellular compartments such as the ER where it could be targeted by PTP1b or yet unknown negative regulators. Although not depicted in the cartoon, GHR-associated proteins other than JAK2 might also play an important role in GHR endocytosis and thereby influence its inactivation.

	ACKNOWLEDGMENTS

 
We thank Dr. Leandro Fernandez for his critical comments on the manuscript. We thank all the authors that have made a contribution to the understanding of the GH signaling down-regulation. We apologize to those whose work deserves to be cited but unfortunately are not quoted because of space limitations.

	FOOTNOTES

 
This work is supported by the Swedish Research Council (529-2002-6766), the Wallenberg Foundation, the Australian Postdoctoral Research Fellowship (to C.J.G.), and Amrad Corp. Ltd.

First Published Online July 21, 2005

Abbreviations: CIS, Cytokine-inducible SH2 protein; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligases; ER, endoplasmic reticulum; GHR, GH receptor; JAK, Janus kinase; KIR, kinase inhibitory region; PTP-1, protein-tyrosine phosphatase 1; SH2, Src-homology 2; SHP1, SH2 domain-containing protein-tyrosine phosphatase 1, also known as PTP-1; SIRP-, signal regulatory protein-; SOCS, suppressors of cytokine signaling; STAT, signal transducer and activator of transcription; UbE, ubiquitin-dependent endocytosis.

Received for publication April 25, 2005. Accepted for publication July 13, 2005.

	REFERENCES

TOP ABSTRACT INTRODUCTION NEGATIVE REGULATION OF GHR... THE SOCS FAMILY OF... GH SIGNALING AND SOCS... PHOSPHOTYROSINE PHOSPHATASES AND... SIGNAL REGULATORY PROTEIN... FUTURE PERSPECTIVES REFERENCES

 

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