"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。GH信号转导开始于其与细胞表面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不编码内在酶活性;相反，它与JAK 2偶联，JAK 2是一种细胞质酪氨酸激酶，其活化是导致GH调节细胞行为和基因表达的下游信号传导所需的。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（PI 3 K）通路。尽管近二十年来对GH信号传导的深入研究，但调节GH敏感性的分子机制以及JAK 2活性与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JAK 2激活和调节的令人兴奋的见解包括：1）JAK 2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JAK 2富集在小窝/脂筏区室中; 3）GHR功能的重要方面受到单克隆抗体的抑制，该抗体以构象依赖的方式识别胞外结构域。我们假设：1）JAK 2在确定可用于激活的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GH信号传导的关键方面。我们的具体目标是：1.定义JAK 2在调节细胞表面GHR可用性中的作用。我们将测试JAK 2在JAK 2缺乏和JAK 2充满的细胞中影响GHR表面丰度的机制。我们将操纵内源性表达GHR和JAK 2的细胞中的JAK 2水平，以确定对表面GHR可用性和信号传导的影响。2.定义GHR的小窝/筏定位机制及其对GH信号传导的影响。我们将定义GHR区域（S）所需的小窝/筏定位和上游ERK通路激活剂对小窝/筏定位的GHR及其信号分子的影响。3.将抗GHRext.mAb表征为选择性GH拮抗剂。该构象敏感性抗体的表位将使用随机PCR诱变-酵母表达系统作图，并根据GHR胞外结构域的已知结构进行分析。我们将测试这种抗体作为GH拮抗剂在体内发挥作用的能力。这些研究的结果将显着扩大我们的知识GH行动和工具可用于操纵它。

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. .