MOLECULAR DETERMINANTS OF CALCIUM RECEPTOR FUNCTION

钙受体功能的分子决定因素

• 批准号: 6386369
• 负责人: GERDA E BREITWIESER
• 金额: $ 30.72万
• 依托单位: SYRACUSE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386369
• 关键词: G protein; biological signal transduction; calcium; disulfide bond; fluorescent dye /probe; glutamate receptor; human tissue; immunochemistry; immunoprecipitation; intermolecular interaction; phosphoprotein phosphatase; phosphorylation; polymerase chain reaction; protein kinase; protein structure function; receptor binding; receptor coupling; receptor expression; single cell analysis; site directed mutagenesis; western blottings

The calcium sensing receptor (CaR) is a G protein-coupled receptor transducer alterations in extracellular Ca2+ and polyvalent cation concentrations into activation of cellular signaling pathways involved in hormone secretion, growth and differentiation, and ion transport. While the cell and tissue distributions of CaR expression have been established, and the basic features of CaR signaling in several cell types have been described, there is little information about the molecular details of CaR function as a G protein-coupled receptor. The long term objective of this study is to define the molecular mechanism(s) of CaR activation and regulation. Human CaR will be studied in either transiently or stably transfected HEK-293 cells. Aim 1 addresses the kinetics of agonist-mediated CaR activation. Fluorescence single cell imaging of intracellular Ca2+ will be used to characterize the basic features and regulation of CaR activation, including agonist dose/responses and the origins of CaR cooperativity. Aim 2 will determine the contributions of disulfide bonds to CaR function/regulation. We will determine the contributions of disulfide bond-mediated dimerization to CaR function as well as the role(s) of disulfide bonds in transducing agonist binding into receptor activation. Aim 3 will characterize the molecular mechanism(s) involved in acute CaR desensitization. The kinetics of desensitization/recovery will be quantified, and modulation by protein kinases/phosphatases will be assesesed both functionally and in protein kinase site mutants of CaR. These studies will establish the molecular mechanism(s) of CaR regulation by agonist, an important first step in establishing pharmacological interventions which can modulate the disparate functions of CaR in the wide range of cell types in which it is endogenously expressed. As a member of the superfamily encompassing metabotropic glutamate, GABAB, and pheromone receptors, these studies of CaR structure/function will likely be generalizable to a broad class of receptors important to human physiology and pathophysiology.

钙传感受体 (CaR) 是一种 G 蛋白偶联受体转导器，可改变细胞外 Ca2+ 和多价阳离子浓度，激活参与激素分泌、生长和分化以及离子转运的细胞信号传导途径。 虽然 CaR 表达的细胞和组织分布已经确定，并且几种细胞类型中 CaR 信号传导的基本特征已经被描述，但关于 CaR 作为 G 蛋白偶联受体功能的分子细节信息很少。 本研究的长期目标是确定 CaR 激活和调节的分子机制。 将在瞬时或稳定转染的 HEK-293 细胞中研究人 CaR。 目标 1 解决激动剂介导的 CaR 激活的动力学。 细胞内 Ca2+ 的荧光单细胞成像将用于表征 CaR 激活的基本特征和调节，包括激动剂剂量/反应和 CaR 协同性的起源。 目标 2 将确定二硫键对 CaR 功能/调节的贡献。 我们将确定二硫键介导的二聚化对 CaR 功能的贡献以及二硫键在将激动剂结合转导为受体激活中的作用。目标 3 将描述急性 CaR 脱敏的分子机制。 将量化脱敏/恢复的动力学，并在功能上和 CaR 的蛋白激酶位点突变体中评估蛋白激酶/磷酸酶的调节。 这些研究将建立激动剂调节 CaR 的分子机制，这是建立药理学干预措施的重要第一步，该干预措施可以调节 CaR 在内源性表达的多种细胞类型中的不同功能。 作为包含代谢型谷氨酸、GABAB 和信息素受体的超家族的一员，这些对 CaR 结构/功能的研究可能会推广到对人类生理学和病理生理学重要的一类广泛的受体。