"Growth Hormone Receptor Dimerization/Disulfide Linkage"

描述(申请人提供)：生长激素(GH)调节人类和其他脊椎动物出生后的生长和新陈代谢。GH信号始于与细胞表面GH受体(GHR)的相互作用，GHR是跨膜糖蛋白细胞因子受体家族成员。GHR不编码固有的酶活性；相反，它与JAK2偶联，JAK2是一种细胞质酪氨酸激酶，其激活是下游信号传递所必需的，从而导致GH对细胞行为和基因表达的调节。GH激活信号转导和转录激活子(STAT)-5、细胞外信号调节激酶(ERK)和磷脂酰肌醇-3激酶(PI3K)途径。尽管近二十年来对生长激素信号进行了深入的研究，但调节生长激素敏感性的分子机制以及JAK2活性与信号通路的差异化耦合在很大程度上还不清楚。我们最近对GHR和JAK2的激活和调控有了令人兴奋的见解，包括：1)JAK2的细胞水平与成熟的细胞表面GHR的相对丰度相关；2)质膜GHR和JAK2在小窝/脂筏中富含；以及3)GHR功能的重要方面被一种识别细胞外结构域的单抗以构象依赖的方式抑制。我们假设：1)JAK2在决定可供激活的GHR水平和介导GH诱导的信号转导中发挥关键作用。2)GHR及其信号分子在质膜区域的空间定位调控GH信号的关键环节。我们的具体目标是：1.明确JAK2的S在调节细胞表面GHR可利用性中的作用。我们将测试JAK2在JAK2缺失和缺失细胞中影响GHR表面丰度的机制。我们将操纵内源性表达GHR和JAK2的细胞中JAK2的水平，以确定对表面GHR可用性和信号转导的影响。2.明确GHR的腔隙/RAFT定位机制及其对GH信号的影响。我们将确定定位小窝/RAFT所需的GHR区域(S)，以及上游ERK通路激活物对GHR及其信号分子定位的影响。3.确定抗GHRext.mAb为GH选择性拮抗剂。这种构象敏感抗体的表位将使用随机PCR突变-酵母表达系统进行映射，并根据GHR胞外区的已知结构进行分析。我们将测试这种抗体在体内作为生长激素拮抗剂的能力。这些研究的结果将极大地扩展我们对生长激素作用和可用于操纵它的工具的了解。。

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. .