application) Interaction of ANP with its guanylyl cyclase-A receptor (Npra) plays a central role in the pathophysiology of hypertension and cardiovascular disorders. Gaining insight into the intricacies of ANP/Npra signaling pathway is pivotal importance for understanding both receptor biology and the diseased state arising from the abnormal hormone receptor interplay. The long-term objective of this proposal is directed at elucidating at the molecular level, the nature and mode of functioning of Npra. The recombinant Npra expressed in transfected cells lacking the endogenous receptor provides the useful system to accomplish this goal. The insights gained from the domain mapping of cDNA clone and biochemical analysis of the receptor will be used to elucidate those receptor areas that can be analyzed further by deletion and site directed mutagenesis in transfected cells to learn what structural components are involved in the functioning of Npra. This proposal has three major, intimately linked goals, all involving the primary focus on the ANP receptor as an approach to gain increased understanding of the normal and abnormal control of cellular and physiological processes. The PI hopes to learn what structural components are involved in ligand binding, activation of protein kinase like domain, cyclase active site and receptor endocytosis, down-regulation and desensitization. He will analyze the molecular determinants in receptor sequence that mediate the overall functional ability of the Npra, critical for the hormone dependent signaling processes. There are three specific aims to investigate this structural/functional relationship: 1) To determine the molecular regulatory mechanisms of the protein kinase like domain of Npra by site directed mutagenesis. The focus will be to test the hypothesis that the mutations in the Gly531-x1-Gly533-x2-x3-x4Gly537 (residues of kinase domain affect the function of the receptors). In addition, the proposal that the receptor contains intrinsic protein kinase activity will also be investigated, 2) To determine the molecular determinants in guanylyl cyclase catalytic domain of Npra by site directed mutagenesis. The objective would be to test the hypothesis that the conserved residues in the cyclase catalytic site (Glu837, Lys838, Lys840, Pro850, His851 and Val853) are critical for the generation of cyclic GMP after hormone binding. This region plays a critical role in the physiological response of ANP in the vascular smooth muscle and mesangial cells, and 3) To study the molecular determinants mediating post-binding events and metabolic turnover of Npra. The main objective of this aim is to test the hypothesis that the conserved residues in protein kinase like guanylyl cyclase and ligand binding domains of Npra determine the molecular mechanism of endocytosis, down-regulation and desensitization relevant to receptor function and intracellular signal transduction by site directed mutagenesis using transfected 293 cells.
Showing the most recent 10 out of 30 publications
