Growth Hormone Receptor Dimerization/Disulfide Linkage

描述：(申请人提供)生长激素(GH)是一种强有力的促进生长和代谢的激素。生长激素受体(GHR)是GH受体的一员。 细胞因子受体超家族。GHR将GH结合在其胞外区域，并 其胞质结构域与分子相互作用的调控信号 包括酪氨酸激酶JAK2。生长激素促进生长激素受体的同源二聚化 1：2的GH：GHR复合体被认为构成了激活的GHR 集合。对GHR二聚化知之甚少，因为它发生在 细胞及其GHR二聚化对其与信号转导的影响 分子。我们之前描述了GH诱导的GHR二硫键 结果形成共价连接的GHR二聚体。二硫代生长激素受体的作用(S) 这种联系一直是个谜，但很大一部分被激活的GHR经历了这种情况 GH导致洗涤剂不能溶解的连锁和GHR被逐级计算 因为是以二硫键连接的形式。我们最近发现半胱氨酸-241(一种 未配对的胞外半胱氨酸)是GHR二硫键和GUR的关键 洗涤剂不溶。此外，这种连锁性也是一种生化指标 GHR二聚化。使用此代理，以及我们新的二聚化敏感 抗GHR胞外区抗体，抗GHR Ext我们的数据表明 GH诱导的二聚化增强了GHR与JAK2的结合。 我们假设：1)GH诱导的GHR二聚化和/或构象变化 二聚通过增强GHR-JAK2而导致信号启动 相互作用；2)尽管非共价GHR二聚化对信号是必不可少的 GH诱导的GHR功能的其他重要方面，如其 持续信令的能力，它的细胞内路线，以及它的 降解受GHR二硫键和洗涤剂不溶性的影响。 为了检验这些假设，我们的具体目标是： 1.系统研究GH诱导的GHR与GH的关系 二聚化、GHR二硫键和抗GHRext反应：使用细胞 表达正常的GHR或在GHR二聚化中突变的GHR 接口；将正常GH(22K)与20K GH变体进行比较；以及映射 抗生长激素受体EXT表位(S)，说明抗体的二聚敏感性。 2.确定GHR胞质结构域对GHR二聚化的影响 这也是生长激素诱导GHR与JAK2结合增强的基础。 3.确定GH诱导的GHR二硫键和GHR去污剂的作用 OH诱导的信号转导、细胞增殖和亚细胞OHR的不解性 路线，使用稳定表达野生型与半胱氨酸-241突变体的细胞 GHR。 结果将显著影响对GH诱导的GHR激活和 细胞因子受体信号转导。由这些研究实现的较长期目标 可能包括；激活或抑制OH的药物的机械性评估 GHR-JAK2复合体的晶体结构分析； GHR二硫键在完整动物体内的功能评价。

DESCRIPTION: (provided by applicant) Growth hormone (GH) is a potent growth-promoting and metabolic hormone. GH receptor (GHR) is a member of the cytokine receptor superfamily. GHR binds GH in its extracellular domain and signals via its cytoplasmic domain's regulated interaction with molecules including the tyrosine kinase, JAK2. GH promotes homodimerization of GHRs such that a 1:2 GH:GHR complex is believed to constitute the activated GHR assemblage. Relatively little is known about GHR dimerization as it occurs in cells and about influence of GHR dimerization on its association with signaling molecules. We previously described GH-induced GHR disulfide linkage that results in a covalently linked GHR dimer form. The role(s) of GHR disulfide linkage has been enigmatic, but a large fraction of activated GHRs undergo this linkage and GHRs rendered detergent-insoluble by GH are progressively accounted for by the disulfide-linked form. We recently found that cysteine-241 (an unpaired extracellular cysteine) is critical for GHR disulfide linkage and GUR detergent insolubility. In addition, this linkage is a biochemical proxy for GHR dimerization. Using this proxy, along with our new dimerization-sensitive anti-GHR extracellular domain antibody, anti-GHRext our data suggest that GH-induced dimerization augments association of GHR with JAK2. We hypothesize: 1) GH-induced GHR dimerization and/or conformational change pursuant to dimerization results in signal initiation by enhancing GHR-JAK2 interaction; 2) though noncovalent GHR dimerization is essential for signal initiation, other important aspects of GH-induced GHR function, such as its capacity for sustained signaling, its intracellular itinerary, and its degradation are influenced by GHR disulfide-linkage and detergent insolubility. To test these hypotheses, our specific aims are: 1. Systematically examine the relationships between GH-induced GHR dimerization, GHR disulfide linkage, and anti-GHRext reactivity by: using cells that express either normal GHR or GHRs mutated in the GHR dimerization interface; comparing normal GH (22K) to the 20K GH variant; and mapping the anti-GHRext epitope(s) accounting for the antibody's dimerization sensitivity. 2. Determine the influence of the GHR cytoplasmic domain on GHR dimerization and the basis for GH-induced enhancement of association between GHR and JAK2. 3. Determine roles of GH-induced GHR disulfide linkage and GHR detergent insolubility in OH-induced signaling, cell proliferation, and subcellular OHR routing, using cells that stably express wild-type vs. cysteine-241 mutant GHRs. Results will significantly impact knowledge of GH-induced GHR activation and cytokine receptor signaling. Longer-term goals made possible by these studies may include; mechanistic evaluation of drugs that activate or inhibit OH signaling; crystallographic structural analysis of the GHR-JAK2 complex; and evaluation of functions of GHR disulfide linkage in intact animals.