Growth Hormone Receptor Dimerization/Disulfide Linkage

描述：（由申请人提供）生长激素（GH）是一种有效的 促生长和代谢激素。生长激素受体（GHR）是一个成员， 细胞因子受体超家族GHR在其胞外结构域结合GH， 通过其胞质结构域与分子的调节相互作用发出信号 包括酪氨酸激酶JAK 2。GH促进GHR的同源二聚化， 1：2 GH：GHR复合物被认为构成活化的GHR 集合关于GHR二聚化的了解相对较少，因为它发生在 GHR二聚化对其与信号传导相关性的影响 分子。我们之前描述了GH诱导的GHR二硫键， 导致共价连接的GHR二聚体形式。GHR二硫化物的作用 连接一直是谜，但很大一部分激活的GHR进行这种连接。 键和GHR呈现洗涤剂不溶性的GH逐步占 由二硫键连接的形式。我们最近发现，半胱氨酸-241（一种 未配对的细胞外半胱氨酸）对于GHR二硫键和GUR 洗涤剂不溶性此外，这种联系是一种生化代理， GHR二聚化。使用这个代理，沿着我们的新的二聚敏感 抗GHR胞外域抗体，抗GHRext，我们的数据表明， GH诱导的二聚化增强了GHR与JAK 2的结合。 我们假设：1）GH诱导GHR二聚化和/或构象变化 根据二聚化，通过增强GHR-JAK 2 相互作用; 2）尽管非共价GHR二聚化对于信号传导是必需的， 起始，GH诱导的GHR功能的其他重要方面，如其 持续信号传导的能力，它的细胞内行程， 降解受GHR二硫键和去污剂不溶性的影响。 为了验证这些假设，我们的具体目标是： 1.系统地研究GH诱导的GHR 二聚化、GHR二硫键和抗GHRext反应性：使用细胞 表达正常GHR或在GHR二聚化中突变的GHR 接口;将正常GH（22 K）与20 K GH变体进行比较;以及映射 抗GHRext表位，其解释了抗体的二聚化敏感性。 2.确定GHR胞质结构域对GHR二聚化的影响 以及GH诱导增强GHR和JAK 2之间关联的基础。 3.确定GH诱导的GHR二硫键和GHR去污剂的作用 在OH诱导的信号传导、细胞增殖和亚细胞OHR中的不溶性 路由，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GH诱导的GHR激活的知识， 细胞因子受体信号传导。这些研究使长期目标成为可能 可能包括;激活或抑制OH的药物的机制评价 GHR-JAK 2复合物的晶体学结构分析;和 完整动物中GHR二硫键的功能评价。

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.