MOLECULAR DETERMINANTS OF CALCIUM RECEPTOR FUNCTION

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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.