Growth Hormone Receptor Dimerization/Disulfide Linkage

描述（由申请人提供）：生长激素（GH）调节人类和其他脊椎动物的出生后生长和代谢。生长激素信号开始于其与细胞表面生长激素受体（GHR）的相互作用，GHR是一种跨膜糖蛋白细胞因子受体家族成员。GHR不编码内在的酶活性；相反，它与JAK2偶联，JAK2是一种细胞质酪氨酸激酶，其激活是导致GH调节细胞行为和基因表达的下游信号所必需的。GH激活信号转导和转录激活因子(STAT)-5、细胞外信号调节激酶（ERK）和磷脂酰肌醇-3激酶（PI3K）通路。尽管近二十年来对生长激素信号传导进行了深入的研究，但调节生长激素敏感性的分子机制以及JAK2活性对信号传导途径的差异偶联在很大程度上尚不清楚。我们最近对GHR和JAK2激活和调控的令人兴奋的见解包括：1)JAK2的细胞水平与成熟细胞表面GHR的相对丰度相关；2)质膜GHR和JAK2富集于小泡/脂筏隔室；3) GHR功能的重要方面被单克隆抗体抑制，单克隆抗体以构象依赖的方式识别细胞外结构域。我们假设：1)JAK2在决定可激活的GHR水平和介导gh诱导的信号转导中起关键作用。2) GHR及其信号分子在质膜区域内的空间定位调节了GH信号传导的关键方面。我们的具体目标是：1。定义JAK2在调节细胞表面GHR可用性中的作用。我们将测试JAK2在JAK2缺陷和-充满细胞中影响GHR表面丰度的机制。我们将在内源性表达GHR和JAK2的细胞中操纵JAK2水平，以确定对表面GHR可用性和信号传导的影响。2. 定义GHR的洞穴/筏定位机制及其对GH信号传导的影响。我们将定义小泡/筏定位所需的GHR区域，以及上游ERK通路激活剂对GHR及其信号分子的小泡/筏定位的影响。3. anti-GHRext的特点。单克隆抗体作为选择性生长激素拮抗剂。这种构象敏感抗体的表位将使用随机PCR诱变酵母表达系统进行定位，并根据已知的GHR胞外结构域结构进行分析。我们将测试这种抗体在体内作为生长激素拮抗剂的能力。这些研究的结果将大大扩展我们对生长激素作用的认识和可用的工具来操纵它。

DESCRIPTION (provided by applicant): Growth hormone (GH) regulates postnatal growth and metabolism in humans and other vertebrates. GH signaling begins with its interaction with cell surface GH receptor (GHR), a transmembrane glycoprotein cytokine receptor family member. GHR encodes no intrinsic enzyme activity; rather, it couples to JAK2, a cytoplasmic tyrosine kinase whose activation is required for downstream signaling resulting in GH's regulation of cellular behavior and gene expression. GH activates the signal transducer and activator of transcription (STAT)-5, extracellular signal-regulated kinase (ERK), and phosphatidylinositol-3 kinase (PI3K) pathways. Despite intense study of GH signaling for nearly two decades, molecular mechanisms modulating GH sensitivity and differential coupling of JAK2 activity to signaling pathways are largely unclear. Our recent exciting insights into GHR and JAK2 activation and regulation, include: 1) the cellular level of JAK2 correlates with the relative abundance of mature, cell surface GHR; 2) plasma membrane GHR and JAK2 are enriched in caveolae/lipid raft compartments; and 3) important aspects of GHR function are inhibited by a monoclonal antibody that recognizes the extracellular domain in a conformationally-dependent fashion. We hypothesize: 1) JAK2 serves critical roles both in determining the level of GHR available for activation and in mediating GH-induced signal transduction. 2) Spatial localization of GHR and its signaling molecules within regions of the plasma membrane regulates critical aspects of GH signaling. Our specific aims are: 1. Define JAK2's role in regulating availability of cell surface GHR. We will test mechanisms by which JAK2 affects GHR surface abundance in JAK2-deficient and -replete cells. We will manipulate JAK2 levels in cells endogenously expressing GHR and JAK2 to define effects on surface GHR availability and signaling. 2. Define mechanisms of caveolar/raft localization of GHR and its impact on GH signaling. We will define the GHR region(s) required for caveolae/rafts localization and the impact of upstream ERK pathway activators on caveolae/raft localization of GHR and its signaling molecules. 3. Characterize anti-GHRext.mAb as a selective GH antagonist. The epitope for this conformation- sensitive antibody will be mapped using a random PCR mutagenesis-yeast expression system and analyzed in light of the known structure of the GHR extracellular domain. We will test this antibody's ability to act in vivo as a GH antagonist. Results of these studies will significantly expand our knowledge of GH action and tools available to manipulate it. .