MOLECULAR DETERMINANTS OF CALCIUM RECEPTOR FUNCTION

钙敏感受体（CaR）是一种G蛋白偶联受体，它通过改变细胞外Ca~（2+）和多价阳离子的浓度来激活细胞信号通路，参与激素分泌、生长和分化以及离子转运。 虽然已经建立了CaR表达的细胞和组织分布，并且已经描述了几种细胞类型中CaR信号传导的基本特征，但是关于CaR作为G蛋白偶联受体功能的分子细节的信息很少。 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。 目的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换为受体活化中的作用。目的3将描述急性CaR脱敏的分子机制。 脱敏/恢复的动力学将被量化，蛋白激酶/磷酸酶的调节将在功能上和CaR的蛋白激酶位点突变体中进行评估。 这些研究将建立通过激动剂调节CaR的分子机制，这是建立药理学干预的重要第一步，该药理学干预可以调节CaR在其内源性表达的广泛细胞类型中的不同功能。 作为代谢型谷氨酸、GABAB和信息素受体超家族的一员，CaR结构/功能的这些研究将可能推广到对人类生理学和病理生理学重要的广泛一类受体。

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.