Prostaglandin E2 (PGE2) is the major renal cyclooxygenase metabolite of arachidonic acid and a potent modulator of a wide variety of cellular responses including vascular tone as well as water and ion transport. Recently it has become apparent that most of PGE2's effects are mediated via specific guanine nucleotide regulatory protein coupled receptors. In the cortical collecting duct (CCD), PGE2 modulates water reabsorption by either increasing or decreasing levels of cAMP and inhibits sodium transport via its recently described ability to elevate intracellular calcium levels in this segment of the nephron. The molecular basis for these multiple effects of PGE2 remain unclear, but may be accounted for by multiple PGE2 receptors. Based on the activity of PGE2 analogs and their ability to act as either relaxors or constrictors of smooth muscle, four different subtypes of PGE2 receptor have been proposed, designated EP1, EP2, EP3, and EP4. We propose that multiple receptor subtypes are present on renal epithelial cells as well and may specifically account for the various effects of PGE2 in the CCD. We have isolated four putative EP receptor subtypes from a rabbit renal cortex cDNA library using a homology based cloning strategy. We will characterize these renal EP receptor clones defining the ligand binding and signal transduction properties of each subtype using mammalian cell lines transfected with each receptor subtype. In vivo studies will address questions of receptor regulation by drugs which alter PGE2 synthesis, and will also examine the distribution of the receptor subtypes at the animal, organ and cellular levels. We will isolate the gene(s) encoding these cDNAs to determine their molecular organization, which will provide insight into the mechanism of receptor regulation. The completion of the project should allow the isolation of a PGE2 receptor gene, expression of the receptor in heterologous systems, and detailed characterization of its hormone binding and signaling properties, and this in turn will address the functional significance of the cloned EP receptor subtypes.
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