RECEPTOR COUPLING TO G12 AND G13

与 G12 和 G13 的受体偶联

• 批准号: 7087747
• 负责人: David Randell Manning
• 金额: $ 33.08万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7087747
• 关键词: G protein; biological signal transduction; clinical research; genetic transcription; human tissue; immunoprecipitation; ligands; phosphorylation; protein kinase C; protein transport; radiotracer; receptor coupling; receptor expression; sodium hydrogen exchanger; spectrometry; tissue /cell culture

DESCRIPTION (provided by applicant): The heterotrimeric G proteins G12 and G13 have received considerable attention for their roles in the regulation of gene transcription, Na+/H+ exchange, and changes in cell contractility and shape. Despite the importance of G12 and G13, much regarding the engagement of these G proteins by agonist-activated receptors remains unclear. The identity of receptors unequivocally coupled to G12 and/or G13, the extent to which agonists vary in capacity to activate these G proteins, the extent to which these G proteins contribute to recognition of agonists by receptors, and how signals are allocated among these and other G proteins are important questions. The overall objective of this proposal is to define the properties of receptor coupling to G12 and GI3 and to evaluate the relevance of superimposed forms of regulation. The first specific aim addresses the concept of agonism as it applies to G12 and G13. We will use Sf9 and mammalian cell expression systems to define the coupling of receptors to GI2 and G13 employing ligand-stimulated [35S]GTP?S-binding and radioligand displacement assays, asking whether signals from agonist-activated receptors are interpreted differently by G12 and GI3 than by other G proteins. The second specific aim addresses the relevance of Gs, Gi, and Gq to the functionality of receptors coordinately coupled to G12 and G13. We will test the hypothesis that different G proteins impart to a single receptor different increases in affinities for agonists and in so doing shape the sensitivity of cells uniquely according to coupling profiles. We will also examine the concept of agonist-directed trafficking of receptor stimulus as it applies to Gs, Gi, Gq, G12, and G13 using isolated and intact modes of a three-state model. The third specific aim is to define the underlying basis and impact of G12 and G13 phosphorylation in the intact cell. The alpha subunits of G12 and G13 are phosphorylated in a variety of cells in response to activation of protein kinase C. We propose to examine the sites and extent of phosphorylation in human platelets, test the notion that Galpha3 is phosphorylated by a protein kinase downstream of protein kinase C, and determine the impact of phosphorylation on receptor-G protein coupling.

描述(申请人提供)：异三聚体G蛋白G12和G13因其在基因转录调控、Na+/H+交换以及细胞收缩和形状改变中的作用而备受关注。尽管G12和G13很重要，但关于这些G蛋白与激动剂激活的受体的结合仍不清楚。明确地与G12和/或G13偶联的受体的身份，激动剂激活这些G蛋白的能力的不同程度，这些G蛋白对受体识别激动剂的贡献程度，以及信号如何在这些G蛋白和其他G蛋白之间分配是重要的问题。这项建议的总体目标是确定G12和GI3受体偶联的特性，并评估重叠形式的调控的相关性。第一个具体目标涉及激励性概念，因为它适用于12国集团和13国集团。我们将使用Sf9和哺乳动物细胞表达系统，通过配体刺激的[35S]GTP、S结合和放射性配体置换实验来确定受体与GI2和G13的偶联，询问来自激动剂激活的受体的信号是否被G12和GI3与其他G蛋白不同地解释。第二个具体目的是解决Gs、GI和GQ与G12和G13协同偶联的受体功能的相关性。我们将检验这一假设，即不同的G蛋白赋予单个受体不同的激动剂亲和力的增加，并在这样做的过程中，根据偶联曲线独特地塑造细胞的敏感性。我们还将使用三态模型的孤立和完整模式来研究受体刺激激动剂定向运输的概念，因为它适用于Gs、GI、GQ、G12和G13。第三个具体目标是确定完整细胞中G12和G13磷酸化的潜在基础和影响。G12和G13的α亚基在不同的细胞中被蛋白激酶C激活而被磷酸化。我们建议研究人类血小板中磷酸化的位置和程度，测试Galpha3被蛋白激酶C下游的蛋白激酶磷酸化的观点，并确定磷酸化对受体-G蛋白偶联的影响。