RECEPTOR COUPLING TO G12 AND G13

与 G12 和 G13 的受体偶联

• 批准号: 6910709
• 负责人: David Randell Manning
• 金额: $ 33.88万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6910709
• 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（由申请人提供）：异源三聚体G蛋白G12和G13因其在基因转录、Na+/H+交换以及细胞收缩性和形状变化的调节中的作用而受到相当大的关注。尽管G12和G13的重要性，但关于这些G蛋白通过激动剂激活的受体的参与仍不清楚。明确耦合到G12和/或G13的受体的身份，激动剂激活这些G蛋白的能力的变化程度，这些G蛋白在多大程度上有助于受体识别激动剂，以及信号如何在这些和其他G蛋白之间分配是重要的问题。该提案的总体目标是定义受体与G12和GI 3偶联的性质，并评估叠加形式的调节的相关性。第一个具体目标涉及激动作用的概念，因为它适用于G12和G13。我们将使用Sf 9和哺乳动物细胞表达系统来定义耦合受体GI 2和G13采用配体刺激的[35 S]GTP？S-结合和放射性配体置换试验，询问激动剂激活受体的信号是否被G12和GI 3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与协调偶联至G12和G13的受体的功能性的相关性。我们将测试的假设，不同的G蛋白赋予一个单一的受体不同的增加亲和力的激动剂，并在这样做的形状的敏感性细胞独特的耦合配置文件。我们还将研究受体刺激物的激动剂定向贩运的概念，因为它适用于Gs，Gi，Gq，G12和G13使用三态模型的隔离和完整模式。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亚基在多种细胞中响应于蛋白激酶C的活化而被磷酸化。我们建议检查人类血小板中磷酸化的位点和程度，测试Galpha 3被蛋白激酶C下游的蛋白激酶磷酸化的概念，并确定磷酸化对受体-G蛋白偶联的影响。