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JuIy 20,1993 Alternative Splicing Generates Multiple Isoforms of a Rabbit Prostaglandin E2 Receptor (Alternative spIicing/Dinoprostone/Kidney/Receptor) Running Title: Multiple EP3 Receptor Isoforms Richard M. Breyer*$§, Ronald B. Emeson$, Matthew D. Breyer *:~, Linda S. Davis*, Richard M. Abromson*, & Suzanne M. Ferrenbach* *Division of Nephrology, ~Depart~ent of Pharmacology gVeterans Administration Medical Center, and Var~derbilt University School of Medicine Nashville, Tennessee USA 37232-2372 Telephone: (615) 343-8496 FAX: (615) 343-7156 The nucleotide seqhences reported in this paper have been submitted to the G.enbank/EMBL Data Bank with accession numbers 1111, 2222, 3333 and 4444 § .Author to whom correspondence should be addressed 40008910

P~. Breyer ~t ~L 2 SUltrY Four cDNA clones homologous with a murine prostaglandin E2 receptor have been isolated from a rabbit kidney cortex cDNA library. These cDNAs encode related proteins which differ only in their carboxyl- terminal sequences. Southern blot analysis of rabbit genornic DNA has indicated that these receptor cDNAs represent alternatively spliced variants derD,,ed from a single gene. Transient expression of a novel full length cDNA in COS1 cells confirmed the ligand binding profile typical of an EP3 receptor subtype. Ribonuclease protection assays have indicated that the gene encoding these receptors is most highly expressed in kidney, adrenal, and stomach, with lower but significant expression in uterus, lung, heart, ileum, spleen and brain. Moreover, each of the cloned isoforms is expressed in the kidney, hz situ hybridization analyses of rabbit kidney demonstrated that the level of expression is highest in the outer medulla with lesser expression in the cortex and no detectable expression in the inner medulla. The ligand binding profile and tissue distribution of these receptors is consistent with a functional role for this family of EP3 recept6rs in mediating the renal actions of prostagland/ns as wetl as the effects of prostaglandins on gastric acid secretion and adrenal function. 40008911

In situ Hybridization and Localization of mRNA for the Rabbit Prostaglandin EP3 Receptor. Matthew D. Breyer, Harry R. Jacobson, Linda S. Davis, and Richard M. Breyer Department of Veterans Affairs Medical Center, Nashville Division of Nephrology and Department of Medicine Vanderbilt University F427-ACRE Bldg. Nashville, TN. 37212 SHORT TITLE: In situ hybridization of EP3 receptor Address correspondence to: Dr. Matthew D. Breyer Division of Nephrology and Department of Medicine Vanderbilt University F427oACRE Bldg. Department of Veterans Affairs Medical Center Nashville, TN. 37212 pH (615) 343-9867 FAX: (615) 343-7156 40008912

ABSTRACT: The physiologic effects of PGE2 appear to be mediated by at least three different "E-prostanoid" receptors designated EP1, EP2, and EP3. These receptors are differentially activated by structural PGE anal6gs (such as misoprostol) and each couples to a different signal transduction mechanism. Studies demonstrating that inhibition of water absorption in the collecting duct is mediated by a Gi coupled mechanism, suggests that an EP3 receptor is involved the renal effects of PGE2. We used in situ hybridization to determine the tissue distribution of the rabbit EP3 receptor. [o~-35S] UTP labeled antisense RNA, comprising transmembrane domains 4 through 7, was hybridized to tissue sections. Specific labeling of kidney, stomach and adrenal was observed. In the kidney, medullary thick ascending limb and cortical and medullary collecting ducts were intensely labeled, while no labeling of glomeruli, proximal tubules, or cortical thick ascending limbs was observed. The adrenal gland labeled exclusively in the medulla. In the stomach the gastric epithelial crypts were the predominant site of hybridization, without evidence of labeling of the smooth muscle. These results suggest an important role for the EP3 receptor in mediating PGE2 effects in these tissues. 40008913