Protein-tyrosine phosphatase H1 controls growth hormone receptor signaling and systemic growth

Several protein-tyrosine phosphatases (PTPs) have been implicated in the control of growth hormone receptor (GHR) signaling, but none have been shown to affect growth in vivo.We have applied a battery of molecular and cellular approaches to test a family-wide panel of PTPs for interference with GHR signaling. Among the subset of PTPs that showed activity in multiple readouts, we selected PTP-H1/PTPN3 for further in vivo studies and found that mice lacking the PTP-H1 catalytic domain show significantly enhanced growth over their wild type littermates. In addition, PTP-H1 mutant animals had enhanced plasma and liver mRNA expression of insulin-like growth factor 1, as well as increased bone density and mineral content. These observations point to a controlling role for PTP-H1 in modulating GHR signaling and systemic growth through insulin-like growth factor 1 secretion.     

生长激素受体信号相关因子在斑马鱼中的比较表达分析及GHR信号通路体内研究模型的建立(英文)

GHR信号通路在动物出生后的生长中扮演着重要角色。实验利用斑马鱼模型研究了GHR信号相关基因在成体组织、胚胎发育以及幼体期的表达情况,这些基因包括gh、ghra、ghrb、jak2a、jak2b、stat5.1、stat5.2、igf1、c-fos、socs1和socs2。值得关注的是,上述的大部分基因都存在母源性表达,且它们的合子表达均起始在体节早期之前。这说明在有功能性的脑垂体形成之前和完善的循环系统建立之前,GH及GH信号相关因子就已经存在于早期胚胎中,因此GH很可能是控制胚胎发育的一系列自分泌/旁分泌生长因子中的一员。同时,我们发现成体组织的socs表达水平与GH信号靶基因igf1和c-fos的表达呈某种程度的负相关。我们利用实时定量PCR技术和荧光素酶分析技术,通过注射GH和GHR表达载体,在斑马鱼胚胎中分析了它们促进GH信号靶基因c-fos和igf1转录活性以及GH应激启动子spi2.1活性的能力。由此,研究利用斑马鱼胚胎建立一个体内研究模型来评估发育过程中的GH信号激活(GHSA)。在受精后1天(dpf)和3dpf斑马鱼胚胎中,单独过表达gh或ghr均可以显著刺激GHSA,这表明在1dpf的斑马鱼胚胎中即存在功能性的GH和GHR蛋白表达,而这一时期是在功能性垂体的形成之前的。gh以及ghr的协同过表达则可以显著放大gh或ghr单独过表达的GHSA效果。     

Disruption of growth hormone receptor gene causes diminished pancreatic islet size and increased insulin sensitivity in mice

Growth hormone, acting through its receptor (GHR), plays an important role in carbohydrate metabolism and in promoting postnatal growth. GHR gene-deficient (GHR(-/-)) mice exhibit severe growth retardation and proportionate dwarfism. To assess the physiological relevance of growth hormone actions, GHR(-/-) mice were used to investigate their phenotype in glucose metabolism and pancreatic islet function. Adult GHR(-/-) mice exhibited significant reductions in the levels of blood glucose and insulin, as well as insulin mRNA accumulation. Immunohistochemical analysis of pancreatic sections revealed normal distribution of the islets despite a significantly smaller size. The average size of the islets found in GHR(-/-) mice was only one-third of that in wild-type littermates. Total beta-cell mass was reduced 4.5-fold in GHR(-/-) mice, significantly more than their body size reduction. This reduction in pancreatic islet mass appears to be related to decreases in proliferation and cell growth. GHR(-/-) mice were different from the human Laron syndrome in serum insulin level, insulin responsiveness, and obesity. We conclude that growth hormone signaling is essential for maintaining pancreatic islet size, stimulating islet hormone production, and maintaining normal insulin sensitivity and glucose homeostasis.     

Growth hormone receptor gene deficiency causes delayed insulin responsiveness in skeletal muscles without affecting compensatory islet cell overgrowth in obese mice

Growth hormone (GH), acting through its receptor (GHR), is essential for somatic growth and development and maintaining metabolic homeostasis. GHR gene-deficient (GHR(-/-)) mice exhibit drastically diminished insulin-like growth factor-I (IGF-I) levels, proportional growth retardation, elevated insulin sensitivity, and reduced islet beta-cell mass. Unlike the liver, which is mostly unaffected by changes in IGF-I level, skeletal muscles express high levels of IGF-I receptor (IGF-IR). The net result of a concurrent deficiency in the actions of both GH and IGF-I, which exert opposite influences on insulin responsiveness, has not been evaluated. We studied insulin-stimulated early responses in the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and p85 subunit of phosphatidylinositol 3-kinase. Upon in vivo insulin stimulation, skeletal muscles of GHR(-/-) mice exhibit transient delayed responses in IR and IRS-1 phosphorylation but normal levels of p85 association with IRS-1. This is in contrast to normal/elevated insulin responses in hepatocytes and indicates tissue-specific effects of GHR gene deficiency. In addition to stimulating normal islet cell growth, GH may participate in islet cell overgrowth, which compensates for insulin resistance induced by obesity. To determine whether the islet cell overgrowth is dependent on GH signaling, we studied the response of male GHR(-/-) mice to high-fat diet (HFD)-induced obesity. After 17 wk on a HFD, GHR(-/-) mice became more significantly obese than wild-type mice and exhibited increased beta-cell mass to a slightly higher extent. These data demonstrate that GH signaling is not required for compensatory islet growth. Thus, in both muscle insulin responsiveness and islet growth compensation, normal levels of GH signals do not seem to play a dominant role.     

Growth hormone receptor ubiquitination coincides with recruitment to clathrin-coated membrane domains

Endocytosis of the growth hormone receptor (GHR) depends on a functional ubiquitin conjugation system. A 10-amino acid residue motif within the GHR cytosolic tail (the ubiquitin-dependent endocytosis motif) is involved in both GHR ubiquitination and endocytosis. As shown previously, ubiquitination of the receptor itself is not required. In this paper ubiquitination of the GHR was used as a tool to address the question of at which stage the ubiquitin conjugation system acts in the process of GHR endocytosis. If potassium depletion was used to interfere with early stages of coated pit formation, both GHR endocytosis and ubiquitination were inhibited. Treatment of cells with methyl-beta-cyclodextrin inhibited endocytosis at the stage of coated vesicle formation. Growth hormone addition to methyl-beta-cyclodextrin-treated cells resulted in an accumulation of ubiquitinated GHR at the cell surface. Using immunoelectron microscopy, the GHR was localized in flattened clathrin-coated membranes. In addition, when clathrin-mediated endocytosis was inhibited in HeLa cells expressing a temperature-sensitive dynamin mutant, ubiquitinated GHR accumulated at the cell surface. Together, these data show that the GHR is ubiquitinated at the plasma membrane, before endocytosis occurs, and indicate that the resident time of the GHR at the cell surface is regulated by the ubiquitin conjugation system together with the endocytic machinery.     

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.     

Growth hormone receptor is a target for presenilin-dependent gamma-secretase cleavage

Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.     

Different signaling roles of two conserved residues in the cytoplasmic hairpin tip of Tsr, the Escherichia coli serine chemoreceptor

Bacterial chemoreceptors form ternary signaling complexes with the histidine kinase CheA through the coupling protein CheW. Receptor complexes in turn cluster into cellular arrays that produce highly sensitive responses to chemical stimuli. In Escherichia coli, receptors of different types form mixed trimer-of-dimers signaling teams through the tips of their highly conserved cytoplasmic domains. To explore the possibility that the hairpin loop at the tip of the trimer contact region might promote interactions with CheA or CheW, we constructed and characterized mutant receptors with amino acid replacements at the two nearly invariant hairpin charged residues of Tsr: R388, the most tip-proximal trimer contact residue, and E391, the apex residue of the hairpin turn. Mutant receptors were subjected to in vivo tests for the assembly and function of trimers, ternary complexes, and clusters. All R388 replacements impaired or destroyed Tsr function, apparently through changes in trimer stability or geometry. Large-residue replacements locked R388 mutant ternary complexes in the kinase-off (F, H) or kinase-on (W, Y) signaling state, suggesting that R388 contributes to signaling-related conformational changes in the trimer. In contrast, most E391 mutants retained function and all formed ternary signaling complexes efficiently. Hydrophobic replacements of any size (G, A, P, V, I, L, F, W) caused a novel phenotype in which the mutant receptors produced rapid switching between kinase-on and -off states, indicating that hairpin tip flexibility plays an important role in signal state transitions. These findings demonstrate that the receptor determinants for CheA and CheW binding probably lie outside the hairpin tip of the receptor signaling domain.     

Growth hormone induces transforming growth factor-β1 in podocytes: Implications in podocytopathy and proteinuria

Pituitary growth hormone (GH) is essential for growth, metabolism, and renal function. Overactive GH signaling is associated with impaired kidney function. Glomerular podocytes, a key kidney cell type, play an indispensable role in the renal filtration and express GH receptors (GHR), suggesting the direct action of GH on these cells. However, the precise mechanism and the downstream signaling events by which GH leads to diabetic nephropathy remain to be elucidated. Here we performed proteome analysis of the condition media from human podocytes and confirmed that GH-induces TGF-β1. Inhibition of GH/GHR stimulated-JAK2 signaling abrogates GH-induced TGF-β1 secretion. Mice administered with GH showed glomerular manifestations concomitant with proteinuria. Pharmacological inhibition of TGF-βR1 in mice prevented GH-induced TGF-β dependent SMAD signaling and proteinuria. Conditional deletion of GHR in podocytes protected mice from streptozotocin-induced diabetic nephropathy. GH and TGF-β1 signaling components expression was elevated in the kidneys of human diabetic nephropathy patients. Our study identifies that GH induces TGF-β1 in podocytes, contributing to diabetic nephropathy.     

Functional promiscuity of squirrel monkey growth hormone receptor toward both primate and nonprimate growth hormones

Primate growth hormone (GH) has evolved rapidly, having undergone approximately 30% amino acid substitutions from the inferred ancestral eutherian sequence. Nevertheless, human growth hormone (hGH) is physiologically effective when administered to nonprimate mammals. In contrast, its functional counterpart, the human growth hormone receptor (hGHR), has evolved species specificity so that it responds only to Old World primate GHs. It has been proposed that this species specificity of the hGHR is largely caused by the Leu --> Arg change at position 43 after a prior His --> Asp change at position 171 of the GH. Sequence analyses supported this hypothesis and revealed that the transitional phase in the GH:GHR coevolution still persists in New World monkeys. For example, although the GH of the squirrel monkey has the His --> Asp substitution at position 171, residue 43 of its GHR is a Leu, the nonprimate residue. If the squirrel monkey truly represents an intermediate stage of GH:GHR coevolution, its GHR should respond to both hGH and nonprimate GH. Also, if the emergence of species specificity was a result of the selection for a more efficient GH:GHR interaction, then changing residue 43 of the squirrel monkey growth hormone receptor (smGHR) to Arg should increase its binding affinity toward higher primate GH. To test these hypotheses, we performed protein-binding assays between the smGHR and both human and rat GHs, using the surface plasmon resonance methodology. Furthermore, the effects of reciprocal mutations at position 43 of human and squirrel monkey GHRs are measured for their binding affinities toward human and squirrel monkey GHs. The results from the binding kinetic assays clearly demonstrate that the smGHR is in the intermediate state of the evolution of species specificity. Interestingly, the altered residue Arg at position 43 of the smGHR does not lead to an increased binding affinity. The implications of these results on the evolution of the GH:GHR interaction and on functional evolution are discussed.     

Medaka receptors for somatolactin and growth hormone: phylogenetic paradox among fish growth hormone receptors

Somatolactin (SL) in fish belongs to the growth hormone/prolactin family. Its ortholog in tetrapods has not been identified and its function(s) remains largely unknown. The SL-deficient mutant of medaka (color interfere, ci) and an SL receptor (SLR) recently identified in salmon provide a fascinating field for investigating SL's function(s) in vivo. Here we isolated a medaka ortholog of the salmon SLR. The mRNA is transcribed in variable organs. Triglycerides and cholesterol contents in the ci are significantly higher than those in the wild type, providing the first evidence of SL's function in suppressing lipid accumulation to organs. Interestingly, phylogenetic comparisons between the medaka SLR and growth hormone receptor (GHR), which is also isolated in this study, in relation to GHRs of other fish, suggested that all GHRs reported from nonsalmonid species are, at least phylogenetically, SLRs. An extra intron inserted in medaka and pufferfish SLRs and flounder and sea bream GHRs also supports their orthologous relationship, but not with tetrapod GHRs. These results may indicate lineage-specific diversification of SLR and GHR functions among fish or just an inappropriate naming of these receptors. Further functional and comparative reassessments are necessary to address this question.     

Selective role of a distinct tyrosine residue on Tie2 in heart development and early hematopoiesis

The development of the cardiovascular system and the development of the early hematopoietic systems are closely related, and both require signaling through the Tie2 receptor tyrosine kinase. Although endothelial cells and hematopoietic cells as well as their precursors share common gene expression patterns during development, it remains completely unknown how Tie2 signaling coordinately regulates cardiovascular development and early hematopoiesis in vivo. We show here that mice with a targeted mutation in tyrosine residue 1100 in the carboxyl-terminal tail of Tie2 display defective cardiac development and impaired hematopoietic and endothelial cell development in the paraaortic splanchnopleural mesoderm similar to that seen in Tie2-null mutant mice. Surprisingly, however, unlike Tie2-null mutant mice, mice deficient in signaling through this tyrosine residue show a normal association of perivascular cells with nascent blood vessels. These studies are the first to demonstrate the physiological importance of a single tyrosine residue in Tie2, and they suggest that multiple tyrosine residues in the receptor may coordinate cardiovascular development and early hematopoietic development.     

A single amino acid substitution in the exoplasmic domain of the human growth hormone (GH) receptor confers familial GH resistance (Laron syndrome) with positive GH-binding activity by abolishing receptor homodimerization.

Growth hormone (GH) elicits a variety of biological activities mainly mediated by the GH receptor (GHR), a transmembrane protein that, based on in vitro studies, seemed to function as a homodimer. To test this hypothesis directly, we investigated patients displaying the classic features of Laron syndrome (familial GH resistance characterized by severe dwarfism and metabolic dysfunction), except for the presence of normal binding activity of the plasma GH-binding protein, a molecule that derives from the exoplasmic-coding domain of the GHR gene. In two unrelated families, the same GHR mutation was identified, resulting in the substitution of a highly conserved aspartate residue by histidine at position 152 (D152H) of the exoplasmic domain, within the postulated interface sequence involved in homodimerization. The recombinant mutated receptor protein was correctly expressed at the plasma membrane. It displayed subnormal GH-binding activity, a finding in agreement with the X-ray crystal structure data inferring this aspartate residue outside the GH-binding domain. However, mAb-based studies suggested the critical role of aspartate 152 in the proper folding of the interface area. We show that a recombinant soluble form of the mutant receptor is unable to dimerize, the D152H substitution also preventing the formation of heterodimers of wild-type and mutant molecules. These results provide in vivo evidence that monomeric receptors are inactive and that receptor dimerization is involved in the primary signalling of the GH-associated growth-promoting and metabolic actions.     

生长激素受体的研究进展

生长激素（GH）在促进动物生长、发育等代谢过程中起着重要作用,GH发挥生理作用的第一步是与靶细胞膜表面的生长激素受体（CHR）结合。现已基本阐明了CHR的结构及由CHR介导的信号转导途径,对GHR基因表达调节的机制也有了一定的了解。GHR是由约620个氨基酸组成的单链跨膜糖蛋白,其胞外区、跨膜区及胞区内分别由约245、25及350个氨基酸组成。由GHR介导的信号转导途径主要有：①酪氨酸激酶系统;②蛋白激酶C途径;③胰岛素受体底物途径。营养状况及GH等内分泌因子对GHR的表达也有调节作用。