生长激素受体及其介导的信号转导

生长激素受体(growth hormone receptor,GHR)是细胞因子／造血因子受体超级家族的一员。它通过二聚体的形式和生长激素(growth hormone,GH)相结合,然后诱发Janus激酶2(Janus kinase 2,JAK2)等细胞因子酪氨酸磷酸化并通过4条不同的途径将信号传入细胞内从而产生一系列的生理效应。现在了解GHR的结构特征、组织分布的基础上,对其介导的信号转导途径作进一步的阐明。     

胰岛素对生长激素的分泌和细胞内信号传导的影响（英文）

生长激素(growth hormone,GH)在行使其功能时需要经历一系列的过程,包括从垂体分泌和进入血液循环到达靶器官或细胞(受体前过程)以及和生长激素受体(GH receptor,GHR)结合并引发细胞内信号转导(受体后过程)。胰岛素可以直接或间接地影响这些过程。GH从垂体的生长激素分泌细胞中分泌需要依赖于下丘脑释放的生长激素释放激素(GH-releasing hor-mone,GHRH)和生长激素抑制素(somatostatin,SS),在生理或病理条件下,胰岛素可以对这两种激素以及GH分泌细胞施加不同影响,从而干预GH的分泌及循环水平。血糖、血脂以及饮食习惯都可以改变胰岛素对GH的影响。胰岛素还能通过影响GHR的敏感性,以及影响胰岛素样生长因子-1(insulin-like growth factor 1,IGF-1),进而影响GH。受体后过程也是GH行使功能的重要一环,细胞内信号转导依赖于信号通路完成。GH信号转导通路和胰岛素的信号通路有部分交叉,这使得两者的信号可以相互作用,胰岛素通过这种作用对GH的信号转导产生影响。还有很多因素可以改变胰岛素对GH的影响,包括细胞因子信号抑制物、GHR敏感性以及JAK2蛋白和胰岛素受体底物间的相互作用,且随着胰岛素浓度升高和作用时间延长,胰岛素对GH的影响趋向于增强。但胰岛素的浓度和时间对GH分泌和细胞内信号转导的具体影响还未完全阐明。胰岛素和SS的关系也有待进一步研究。     

晚期糖基化终产物受体的结构和功能

晚期糖基化终产物受体（RAGE）是一种膜蛋白,属于免疫球蛋白家庭,由400多个氨基酸组成,分子量为35kD,分胞包段、跨膜段和胞内段,在单核巨噬细胞、血管内皮细胞、肾系膜细胞、神经细胞及平滑肌细胞等细胞中普扁表达。RAGE作为信号转导受体介导晚期糖基化终产物（AGE）和其配体在细胞表面结合,激活细胞内多种信号转导机制,在糖尿病慢性并发症、透析相关性淀粉样变（DRA）、阿尔采末病（AD）、动脉粥样硬化等疾病发生中起重要作用。对RAGE结构和功能的认识可能为这些疾病的防治提供新的靶位,因此具有重要意义。     

生长激素和生长激素受体的多样性

生长激素及其受体对动物生长发育起着重要的作用。转录过程选择性剪接和存在多种降解途径可能是GH或GHR产生多样性的原因。随着GH结构形态的改变,其功能也在发生变化。GH基因的多样性对鸡的抗病选择性反应与产蛋性能有相关,GH和GHR基因的多样性会影响奶牛的产奶生产性能。GHR的分子多样性可能导致动物生长发育模式的变异,例如动物的矮小病。     

生长激素受体基因敲除小鼠模型

由脑垂体合成并分泌的生长激素(GH)不仅控制机体生长发育,还在许多代谢疾病中起关键调控作用。GH的生物学功能通过与其表面受体(GHR)结合而启动,基于分子生物学技术构建各种GHR敲除小鼠模型成为揭示GH调控机制的基础。利用Cre-LoxP重组酶系统,迄今已在小鼠全身或组织特异性(如肝,骨骼肌,脂肪,巨噬细胞和胰岛β细胞等)敲除ghr基因,并从中探索GH/GHR信号转导及其与其他信号通路的相互作用。本文综述并讨论了这些ghr基因敲除小鼠模型的表型特征和应用,从不同方面介绍了GH/GHR相关信号通路研究现状。     

中华鲟生长激素受体的分子克隆和功能分析

为研究生长激素对中华鲟生长的调控机制,克隆了中华鲟生长激素受体cDNA.csGHRcDNA的可读框编码了611个氨基酸残基的跨膜蛋白质,含有GHR的所有特征结构域.序列对比发现其他种属GHR中高度保守的氨基酸残基在csGHR中发生了替换.我们利用CHO细胞分析了csGHR的生物功能和csGHR分子中高度保守性氨基酸残基替换的生物意义.csGHR稳定表达细胞中共转染的受丝氨酸蛋白酶抑制剂2.1（Spi1.2）启动子驱动的荧光素酶报告基因受海鲤生长激素（seabream GH,sbGH）诱导表达,并且sbGH诱导稳定表达细胞显著增殖.csGHR稳定表达细胞培养液中检测到中华鲟生长激素结合蛋白质,并且csGHBP的生成需要金属蛋白酶活性的参与.csGHR配体结合域的Asp突变为Glu显著提高csGHR介导的上述生物活性,而Asp突变为Ala则明显降低csGHR的生物活性.这些结果表明,克隆的csGHR具有完全生物功能,并且csGHBP可能通过csGHR蛋白酶解而生成.这些发现将有助于全面了解中华鲟生长调控机制.     

前列腺素核受体系统信号转导及基因表达调控

脂肪酸和前列腺素等脂代谢的产物不仅通过膜受体起作用,也可以通过与核受体结合来调节基因表达.前列腺素I₂(PGI₂)既可以与G蛋白偶联的细胞表面IP受体起作用,也可以通过核受体过氧化物酶体增殖因子活化受体（PPARs）发挥生物学功能.前列腺素E₂(PGE₂)的受体（EPs）不仅仅在质膜上有,最近在核膜上也发现了EPs受体.前列腺素核受体介导的信号转导途径与膜受体介导的信号途径不同,对于基因转录的调控机制也不同.     

新的生长激素异形体基因的发现及全长cDNA的克隆

在通过大规模 ESTS技术对垂体基因表达谱的研究中 ,从垂体组织产生了 72 2 2个 ESTS,有385个 ESTs是代表生长激素 (GH)基因的 ,其中 1个为中间缺失 1 38bp的 GH异形体基因 ,并经巢式 RT- PCR及测序证实 ;该基因编码 1 71个氨基酸的前肽 ,去除信号肽后 ,其成熟肽由 1 45个氨基酸组成 ;经生物信息学处理 ,其分子量大小约 1 7k D;与正常生长激素分子内有 2个 GH受体结合位点不同 ,该新的 GH异形体分子内仅有一个生长激素受体的结合位点 .研究结果揭示 :正常垂体内存在着新的 GH异形体基因 ,该基因可能编码外周血中 1 6k D的生长激素 ;其功能可能为 2 2k D GH的生理拮挤剂 .     

转接蛋白NTAL/LAB在淋巴细胞发育和活化中的作用

由膜受体介导的信号转导过程在淋巴细胞发育、分化和活化过程中起着重要的作用,转接蛋白是淋巴细胞信号转导中发挥承上启下作用的分子。它们被相应的蛋白酶磷酸化后,可以通过招募具有特定氨基酸基序的蛋白质,将信号转导通路中不同阶段的分子募集在一起,从而实现信号的整合和分流。NTAL／LAB是新发现的跨膜转接蛋白,体外B细胞受体、FcγRI和FcεRI受体的交联可以引起该分子的迅速磷酸化,提示NTAL／LAB可能参与了这些受体介导的信号转导途径,从而影响淋巴细胞的发育和功能。本文将对NTAL／LAB在主要淋巴细胞发育和分化中的作用作一介绍。     

植物G蛋白偶联受体及其在信号转导中的作用

G蛋白偶联受体(G protein-coupled receptors,GPCRs)是具有7个跨膜螺旋的蛋白质受体,是人体内最大的蛋白质超家族.GPCRs能调控细胞周期,参与多种植物信号通路以及影响一系列的代谢和分化活动.简要介绍了GPCR和G蛋白介导的信号转导机制,GPCRs的结构和植物GPCR及其在植物跨膜信号转导中的作用,并对GPCR的信号转导机制及植物抗病反应分子机制的研究提出展望.     

Role of the growth hormone (GH) receptor transmembrane domain in receptor predimerization and GH-induced activation

The GH receptor (GHR) mediates GH effects by activating the GHR-associated cytoplasmic tyrosine kinase, Janus kinase 2. Recent studies indicate that GHRs exist as dimers independently of GH binding. Some authors suggest that receptor predimerization is mediated by the transmembrane domain (TMD) and that GH binding initiates signaling by triggering changes in the orientation of the two GHRs within the dimer. In this study, we investigate the role of GHR TMD in GH-independent receptor dimerization and ligand-induced activation. We prepared a GHR mutant, GHR(LDLR), in which the TMD is replaced with the TMD of the human low-density lipoprotein receptor (LDLR). The resultant chimera has a TMD two residues shorter than the native GHR TMD; thus, in addition to possessing a different TMD, the altered GHR(LDLR) TMD helical register may change positions of the GHR extracellular domain (ECD) and intracellular domain relative to the TMD when compared with the wild-type (WT) receptor. When each was coexpressed with an intracellular domain-truncated GHR mutant, GHR(1-274-Myc), both WT GHR and GHR(LDLR) were specifically coprecipitated with GHR(1-274-Myc), indicating that the GHR TMD was not required for GHR heterodimerization with GHR(1-274-Myc). We further examined the contribution of the so-called "dimerization interface," a GHR ECD region that is critical for GH-induced signaling, to receptor predimerization. Coimmunoprecipitation experiments with either WT GHR, a dimerization interface mutant (GHR-H150D), or a control mutant (GHR-T147D) with GHR(1-274-Myc) showed dramatically reduced coprecipitation of GHR-H150D with GHR(1-274-Myc) when compared with WT GHR or GHR-T147K. This result suggests that, in contrast to some recent models, the dimerization interface contributes to GHR predimerization. We also compared WT GHR with GHR(LDLR) and GHR(LDLRDelta4) (a chimera in which the LDLR TMD has an internal deletion of four residues) with regard to response to GH stimulation. Although the chimeras had similar GH dose responses and time courses for signaling as WT GHR, they were markedly less sensitive to inhibition of signaling by a conformation-sensitive GHR ECD monoclonal antibody. Further, the chimeras were much less sensitive to inducible metalloprotease cleavage than was WT GHR, implying that the ECD conformations of the chimera receptors differ from WT GHR. Collectively, our data indicate that the composition and/or length of the TMD affect some aspects of GHR function, but do not affect receptor predimerization or GH-induced GHR activation. Further, they suggest that the GHR ECD-TMD is more flexible than previously thought in terms of the ability to achieve the active conformation in response to GH.     

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.     

Growth hormone (GH)-induced dimerization inhibits phorbol ester-stimulated GH receptor proteolysis

Growth hormone (GH) initiates its cellular action by properly dimerizing GH receptor (GHR). A substantial fraction of circulating GH is complexed with a high-affinity GH-binding protein (GHBP) that in many species can be generated by GHR proteolysis and shedding of the receptor's ligand-binding extracellular domain. We previously showed that this proteolysis 1) can be acutely promoted by the phorbol ester phorbol 12-myristate 13-acetate (PMA), 2) requires a metalloprotease activity, 3) generates both shed GHBP and a membrane-associated GHR transmembrane/cytoplasmic domain remnant, and 4) results in down-regulation of GHR abundance and GH signaling. Using cell culture model systems, we now explore the effects of GH treatment on inducible GHR proteolysis and GHBP shedding. In human IM-9 lymphocytes, which endogenously express GHRs, and in Chinese hamster ovary cells heterologously expressing wild-type or cytoplasmic domain internal deletion mutant rabbit GHRs, brief exposure to GH inhibited PMA-induced GHR proteolysis (receptor loss and remnant accumulation) by 60-93%. PMA-induced shedding of GHBP from Chinese hamster ovary transfectants was also inhibited by 70% in the presence of GH. The capacity of GH to inhibit inducible GHR cleavage did not rely on JAK2-dependent GH signaling, as evidenced by its continued protection in JAK2-deficient gamma2A rabbit GHR cells. The GH concentration dependence for inhibition of PMA-induced GHR proteolysis paralleled that for its promotion of receptor dimerization (as monitored by formation of GHR disulfide linkage). Unlike GH, the GH antagonist, G120K, which binds to but fails to properly dimerize GHRs, alone did not protect against PMA-induced GHR proteolysis; G120K did, however, antagonize the protective effect of GH. Our data suggest that GH inhibits PMA-induced GHR proteolysis and GHBP shedding by inducing GHR dimerization and that this effect does not appear to be related to GH site 1 binding, GHR internalization, or GHR signaling. The implications of these findings with regard to GH signaling and GHR down-regulation are discussed.     

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.     

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.     

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.     

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.     

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.     

Studies with a growth hormone antagonist and dual-fluorescent confocal microscopy demonstrate that the full-length human growth hormone receptor, but not the truncated isoform, is very rapidly internalized independent of Jak2-Stat5 signaling.

We have investigated trafficking of two negative regulators of growth hormone receptor (GHR) signaling: a human, truncated receptor, GHR1-279, and a GH antagonist, B2036. Fluorescent-labeled growth hormone (GH) was rapidly internalized by the full-length GHR, with >80% of the hormone internalized within 5 min of exposure to GH. In contrast, <5% of labeled GH was internalized by cells expressing truncated GHR1-279. Using another truncated receptor, GHR1-317 fused to enhanced green fluorescent protein (EGFP), we have exploited fluorescence energy transfer to monitor the trafficking of ligand-receptor complexes. The data confirmed that internalization of this truncated receptor is very inefficient. It was possible to visualize the truncated GHR1-317-EGFP packaged in the endoplasmic reticulum, its rapid movement in membrane bound vesicles to the Golgi apparatus, and subsequent transport to the cell membrane. The GH antagonist, B2036, blocked Jak2-Stat5-mediated GHR signaling but was internalized with a similar time course to native GH. The results: 1) demonstrate the rapid internalization of GH when studied under physiological conditions; 2) confirm the hypothesis that internalization of cytoplasmic domain truncated human GHRs is very inefficient, which explains their dominant negative action; and 3) show that the antagonist action of B2036 is independent of receptor internalization.