"Growth Hormone Receptor Dimerization/Disulfide Linkage"

“生长激素受体二聚化/二硫键”

• 批准号: 7227073
• 负责人: Stuart J Frank
• 金额: $ 25.26万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7227073
• 关键词: 1-Phosphatidylinositol 3-Kinase; Address; Affect; Antibodies; Behavior; Caveolae; Cell membrane; Cell surface; Cells; Couples; Coupling; Cytokine Receptors; Dimerization; Disulfide Linkage; Epitopes; Extracellular Domain; Extracellular Signal Regulated Kinases; Family member; Gene Expression; Glycoproteins; Growth; Growth Hormone Receptor; Hormone Antagonists; Human; JAK2 gene; Knowledge; Light; MAPK7 gene; Maps; Mediating; Membrane Microdomains; Metabolism; Mitogen-Activated Protein Kinases; Molecular; Molecular Conformation; Monoclonal Antibodies; Mutagenesis; Pathway interactions; Polymerase Chain Reaction; Protein Tyrosine Kinase; Regulation; Relative (related person); Role; STAT protein; Signal Pathway; Signal Transduction; Signaling Molecule; Somatotropin; Structure; Surface; Systems Analysis; Testing; Vertebrates; Yeasts; base; cell growth regulation; enzyme activity; hormone sensitivity; human GHR protein; human MAP kinase 7; in vivo; insight; postnatal; receptor function; tool

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. .

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产后生长和代谢。 GH信号传导与跨膜糖蛋白细胞因子受体家族成员的细胞表面GH受体（GHR）的相互作用开始。 GHR无编码固有的酶活性；相反，它与JAK2（一种细胞质酪氨酸激酶Jak2耦合在一起，其激活是在下游信号传导所必需的，从而导致GH调节细胞行为和基因表达。 GH激活转录（STAT）-5，细胞外信号调节激酶（ERK）和磷脂酰肌醇3激酶（PI3K）途径的信号转换器和激活因子。尽管对GH信号传导进行了近二十年的强烈研究，但分子机制调节了GH敏感性和JAK2活性与信号通路的差异偶联。我们最近对GHR和JAK2激活和调节的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相对丰度相关； 2）质膜GHR和JAK2富含小窝/脂质筏室； 3）GHR功能的重要方面受到单克隆抗体的抑制，该抗体以构象依赖性的方式识别细胞外结构域。我们假设：1）JAK2在确定可用于激活和介导GH诱导的信号转导的GHR水平方面起关键作用。 2）GHR及其信号分子在质膜区域内的空间定位可调节GH信号的关键方面。我们的具体目的是：1。定义JAK2在调节细胞表面GHR的可用性中的作用。我们将测试JAK2会影响JAK2缺乏和 - 重新填充细胞中GHR表面丰度的机制。我们将操纵内源表达GHR和JAK2的细胞中的JAK2水平，以定义对表面GHR的可用性和信号传导的影响。 2。定义GHR的洞穴/筏定位的机制及其对GH信号的影响。我们将定义口腔/筏定位所需的GHR区域，以及上游ERK途径激活剂对GHR及其信号分子的小窝/筏定位的影响。 3。将抗Ghrext.MAB表征为选择性GH拮抗剂。这种构象 - 敏感抗体的表位将使用随机的PCR诱变 - 酵母表达系统进行映射，并根据GHR细胞外域的已知结构进行分析。我们将测试该抗体作为GH拮抗剂的体内作用的能力。这些研究的结果将大大扩展我们对GH动作的了解和可用于操纵它的工具。 。