FUNCTION ANALYSIS OF THE PROSTAGLANDIN EP3 RECEPTOR

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DESCRIPTION (Adapted from the Applicant's Abstract): Molecular cloning has identified at least four E-Prostanoid (EP) receptor subtypes, designated EP1, EP2, EP3 and EP4 which mediate the effects of Prostaglandin E2 (PGE2). The EP3 receptor modulates water and ion transport in the kidney, gastric acid secretion, neurotransmitter release, and hepatic glucose metabolism. In the kidney PGE2 production modulates salt and water transport in the medullary thick ascending limb and collecting duct. Importantly, evidence suggests that some EP3 effects are mediated from the basolateral surface of the nephron, while others are mediated by apical receptors. The EP3 receptor is unique among the EP receptor family in that it exists as multiple isoforms generated by alternative splicing of a common precursor mRNA transcribed from a single gene. Although the EP3 receptor has classically been characterized as a Gi coupled receptor that signals by lowering intracellular cAMP levels, recent studies suggest that EP3 alternative splice variants couple through a variety of signal transduction pathways. The proposed studies will focus on the functional differences of the alternatively spliced PGE2 receptor subtypes thereby providing insight into the roles of the multiple PGE2 receptors in normal and pathophysiological states. The principal hypothesis of this proposal is that differential expression of EP3 receptor splice variants in individual cell types results in differential activation of signaling pathways that determine the physiologic response of a target cell to PGE2. In Specific Aim #1 the principal investigator will determine the repertoire and tissue distribution of EP3 receptor splice variant messenger RNAs. Immuno-localization will be performed, including subcellular receptor targeting of selected EP3 receptor proteins using receptor selective antibodies in relevant tissues as well as in cell culture models. In Specific Aim #2 selected cDNAs encoding rabbit EP3 receptor splice variants will be expressed in mammalian cell lines and their ligand binding and signal transduction properties characterized. The third Specific Aim will determine the effects of site-specific amino acid substitutions on G-protein activation receptor phosphorylation and receptor desensitization. Completion of these studies will not only elucidate the significance of these multiple EP3 splice variants, but will also increase our understanding of the structure and function of G-protein coupled receptors.