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.     

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.     

SOCS3 promotes apoptosis of mammary differentiated cells

Growth and function of the mammary gland is regulated by cytokines and modulated by suppressor of cytokine signalling (SOCS) proteins. In vitro experiments demonstrated that SOCS3 can inhibit PRL induction of milk protein gene expression and STAT5 activation. We explored the SOCS3 expression pattern during mouse mammary development and its regulation by PRL and GH in wild-type and STAT5a-null mammary tissue. Our results suggest that, in vivo, PRL stimulates SOCS3 expression in stromal adipocytes, independently of STAT5a stimulation. In mammary epithelial cells, SOCS3 expression appears to be related to STAT3 activation. Together, our results are consistent with a role of SOCS3 in the mammary gland by promoting apoptosis of differentiated cells (adipocytes during gestation and epithelial cells during involution).     

Role of the cytokine-induced SH2 domain-containing protein CIS in growth hormone receptor internalization

The cytokine-inducible SH2 domain-containing protein CIS inhibits signaling from the growth hormone (GH) receptor (GHR) to STAT5b by a proteasome-dependent mechanism. Here, we used the GH-responsive rat liver cell line CWSV-1 to investigate the role of CIS and the proteasome in GH-induced GHR internalization. Cell-surface GHR localization and internalization were monitored in GH-stimulated cells by confocal immunofluorescence microscopy using an antibody directed against the GHR extracellular domain. In GH na?ve cells, GHR was detected in small, randomly distributed granules on the cell surface and in the cytoplasm, with accumulation in the perinuclear area. GH treatment induced a rapid (within 5 min) internalization of GH.GHR complexes, which coincided with the onset of GHR tyrosine phosphorylation and the appearance in the cytosol of distinct granular structures containing internalized GH. GHR signaling to STAT5b continued for approximately 30-40 min, however, indicating that GHR signaling and deactivation of the GH.GHR complex both proceed from an intracellular compartment. The internalization of GH and GHR was inhibited by CIS-R107K, a dominant-negative SH2 domain mutant of CIS, and by the proteasome inhibitors MG132 and epoxomicin, which prolong GHR signaling to STAT5b. GH pulse-chase studies established that the internalized GH.GHR complexes did not recycle back to the cell surface in significant amounts under these conditions. Given the established specificity of CIS-R107K for blocking the GHR signaling inhibitory actions of CIS, but not those of other SOCS/CIS family members, these findings implicate CIS and the proteasome in the control of GHR internalization following receptor activation and suggest that CIS-dependent receptor internalization is a prerequisite for efficient termination of GHR signaling.     

Structural basis for c-KIT inhibition by the suppressor of cytokine signaling 6 (SOCS6) ubiquitin ligase

The c-KIT receptor tyrosine kinase mediates the cellular response to stem cell factor (SCF). Whereas c-KIT activity is important for the proliferation of hematopoietic cells, melanocytes and germ cells, uncontrolled c-KIT activity contributes to the growth of diverse human tumors. Suppressor of cytokine signaling 6 (SOCS6) is a member of the SOCS family of E3 ubiquitin ligases that can interact with c-KIT and suppress c-KIT-dependent pathways. Here, we analyzed the molecular mechanisms that determine SOCS6 substrate recognition. Our results show that the SH2 domain of SOCS6 is essential for its interaction with c-KIT pY568. The 1.45-Å crystal structure of SOCS6 SH2 domain bound to the c-KIT substrate peptide (c-KIT residues 564–574) revealed a highly complementary and specific interface giving rise to a high affinity interaction (K_d = 0.3 μm). Interestingly, the SH2 binding pocket extends to substrate residue position pY+6 and envelopes the c-KIT phosphopeptide with a large BG loop insertion that contributes significantly to substrate interaction. We demonstrate that SOCS6 has ubiquitin ligase activity toward c-KIT and regulates c-KIT protein turnover in cells. Our data support a role of SOCS6 as a feedback inhibitor of SCF-dependent signaling and provides molecular data to account for target specificity within the SOCS family of ubiquitin ligases.     

Multimeric Growth Hormone Receptor Complexes Serve as Signaling Platforms

Growth hormone (GH) signaling is required for promoting longitudinal body growth, stem cell activation, differentiation, and survival and for regulation of metabolism. Failure to adequately regulate GH signaling leads to disease: excessive GH signaling has been connected to cancer, and GH insensitivity has been reported in cachexia patients. Since its discovery in 1989, the receptor has served a pivotal role as the prototype cytokine receptor both structurally and functionally. Phosphorylation and ubiquitylation regulate the GH receptor (GHR) at the cell surface: two ubiquitin ligases (SCF^βTrCP2 and CHIP) determine the GH responsiveness of cells by controlling its endocytosis, whereas JAK2 initiates the JAK/STAT pathway. We used blue native electrophoresis to identify phosphorylated and ubiquitylated receptor intermediates. We show that GHRs occur as ∼500-kDa complexes that dimerize into active ∼900-kDa complexes upon GH binding. The dimerized complexes act as platforms for transient interaction with JAK2 and ubiquitin ligases. If GH and receptors are made in the same cell (autocrine mode), only limited numbers of ∼900-kDa complexes are formed. The experiments reveal the dynamic changes in post-translational modifications during GH-induced signaling events and show that relatively simple cytokine receptors like GHRs are able to form higher order protein complexes. Insight in the complex formation of cytokine receptors is crucially important for engineering cytokines that control ligand-induced cell responses and for generating a new class of therapeutic agents for a wide range of diseases.     

The SOCS Box Encodes a Hierarchy of Affinities for Cullin5: Implications for Ubiquitin Ligase Formation and Cytokine Signalling Suppression

The SOCS (suppressors of cytokine signalling) family of proteins inhibits the cytokine-induced signalling cascade in part by promoting the ubiquitination of signalling intermediates that are then targeted for proteasomal degradation. This activity relies upon an interaction between the SOCS box domain, the adapter complex elonginBC and a member of the Cullin family, the scaffold protein of an E3 ubiquitin ligase. In this study, we dissected this interaction in vitro using purified components. We found that all eight SOCS proteins bound Cullin5 but required prior recruitment of elonginBC. Neither SOCS nor elonginBC bound Cullin5 when in isolation. Interestingly, the affinity of each SOCS-elonginBC complex for Cullin5 varied by 2 orders of magnitude across the SOCS family. Unexpectedly, the most potent suppressors of signalling, SOCS-1 and SOCS-3, bound most weakly to the E3 ligase scaffold, with affinities 100- and 10-fold lower, respectively, than the rest of the family. The remaining six SOCS proteins all bound Cullin5 with high affinity (K_d of ∼ 10 nM) due to a slower off-rate and hence a longer half-life of the complex. This difference in affinity may reflect a difference in mode of action as only SOCS-1 and SOCS-3 have been shown to suppress signalling using both SOCS box-dependent and SOCS box-independent mechanisms. This is not the case with the other six SOCS proteins, and our data imply the existence of two distinct subclasses of SOCS proteins with a high affinity for Cullin5, the E3 ligase scaffold, possibly reflecting complete dependence upon ubiquitination for suppression of cytokine signalling.     

Negative regulation of growth hormone receptor signaling

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.     

The SOCS box-adapting proteins for ubiquitination and proteasomal degradation

The suppressor of cytokine signalling (SOCS) box was first identified in the SH2-containing SOCS box family (cytokine-inducible SH2-containing protein, SOCS1-7) and is a 40-amino acid motif, which functions to recruit an E3 ubiquitin ligase complex consisting of the adapter proteins elongins B and C, Rbx2 and the scaffold protein Cullin5. The SOCS box is found in a diverse array of intracellular signalling molecules, many of which contain different protein interaction domains such as SPRY and WD40 domains, leucine and ankyrin repeats or other functional domains such as GTPases. In general, the SOCS box-containing proteins are thought to act as substrate-recognition modules to mediate the polyubiquitination and subsequent degradation of substrate proteins by the 26S proteasome.     

The E3 ubiquitin ligase HOIL-1 induces the polyubiquitination and degradation of SOCS6 associated proteins

The suppressor of cytokine signaling (SOCS) proteins are thought to exert their function through the recruitment of interacting-proteins to the ubiquitin/proteasome degradation pathway. All SOCS proteins bind an Elongin BC E3 ubiquitin ligase complex through the common Socs-box. Here, we show that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), another E3 ubiquitin ligase, interacts with SOCS6. The Ubl domain of HOIL-1 and the SH2 and Socs-box domains of SOCS6 are required for the interaction. HOIL-1 expression stabilizes SOCS6 and induces the ubiquitination and degradation of proteins associated with SOCS6. These data suggest that SOCS proteins may interact with different E3 ubiquitin ligases in addition to a common Elongin BC E3 complex.     

βTrCP controls GH receptor degradation via two different motifs

The physiological roles of GH are broad and include metabolism regulation and promotion of somatic growth. Therefore, the responsiveness of cells to GH must be tightly regulated. This is mainly achieved by a complex and well-controlled mechanism of GH receptor (GHR) endocytosis. GHR endocytosis occurs independently of GH and requires the ubiquitin ligase, SCF (βTrCP) that is recruited to the ubiquitin-dependent endocytosis (UbE) motif in the cytoplasmic tail of the GHR. In this study we report that, in addition to the UbE motif, a downstream degron, DSGRTS, binds to βTrCP. The WD40 residues on βTrCP involved in the interaction with this sequence are identical to the ones necessary for binding the classical motif, DSGxxS, in inhibitor of NFκB signalling, and β-catenin. Previously, we showed that this motif is not involved in GH-induced endocytosis. We show here that the DSGRTS sequence significantly contributes to GHR endocytosis/degradation in basal conditions, whereas the UbE motif is involved both in basal and GH-induced conditions. These findings explain the high rate of GHR degradation under basal conditions, which is important for regulating the responsiveness of cells to GH.     

Unbiased identification of substrates for the Epac1-inducible E3 ubiquitin ligase component SOCS-3

The anti-inflammatory effects of the prototypical second messenger cAMP have been extensively documented in multiple cell types. One mechanism by which these effects are achieved is via Epac1 (exchange protein directly activated by cAMP 1)-dependent induction of SOCS-3 (suppressor of cytokine signalling 3), which binds and inhibits specific class I cytokine receptors. One important aspect of SOCS-3 functionality is its role as the specificity determinant within an E3 ubiquitin ligase complex which targets cellular substrates for polyubiquitylation and proteasomal degradation. In the present review, we describe key inhibitory processes that serve to reduce cytokine receptor signalling, focusing primarily on SOCS protein function and regulation. We also outline a strategy we have developed to identify novel ubiquitylated substrates for the Epac1-inducible SOCS-3 E3 ubiquitin ligase complex following purification of the ubiquitinome. It is anticipated that identifying substrates for the Epac1-regulated SOCS-3 E3 ubiquitin ligase, and assessment of their functional significance, may pinpoint new sites for therapeutic intervention that would achieve therapeutic efficacy of cAMP-elevating drugs while minimizing the adverse effects usually associated with these agents.     

Growth hormone-stimulated insulin-like growth factor-1 expression in rainbow trout (Oncorhynchus mykiss) hepatocytes is mediated by ERK, PI3K-AKT, and JAK-STAT

Growth hormone (GH) initiates many of its growth-promoting actions by binding to GH receptors (GHR) and stimulating the synthesis and secretion of insulin-like growth factor-1 (IGF-1) from the liver and other sites. In this study, we used hepatocytes isolated from rainbow trout as a model system in which to determine the molecular signaling events of GH in fish. GH directly stimulated the phosphorylation of ERK, protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K), JAK2, and STAT5 in hepatocytes incubated in vitro. Activation of ERK, Akt, JAK2, and STAT5 was rapid, occurring within 5-10 min, and was concentration dependent. GH-induced ERK activation was completely blocked by the ERK pathway inhibitor, U0126, and the JAK2 inhibitor, 1,2,3,4,5,6-hexabromocyclohexane (Hex), and was partially blocked by the PI3K inhibitor LY294002. GH-stimulated Akt activation was completely blocked by LY294002 and Hex, but was not affected by U0126; whereas, STAT5 activation by GH was blocked only by Hex, and was not affected by either U0126 or LY294002. GH stimulated hepatic expression of IGF-1 mRNA as well as the secretion of IGF-1, effects that were partially or completely blocked by U0126, LY294002, and Hex. These results indicate that GHR linkage to the ERK, PI3K/Akt, or STAT pathways in trout liver cells requires activation of JAK2, and that GH-stimulated IGF-1 synthesis and secretion is mediated through the ERK, PI3K/Akt, and JAK-STAT pathways.