Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype.

Abstract

A functional cDNA clone for mouse EP3 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of the human thromboxane A2 receptor and cross-hybridization screening.The mouse EP3 receptor consists of 365 amino acid residues with putative seven-transmembrane domains.The sequence revealed significant homology to the human thromboxane A2 receptor.Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGE2 binding.The binding was displaced with unlabeled PGs in the order of PGE2 = PGEl > iloprost > PGF2, > PGD2.The EP3-selective agonists, M&B 28,767 or GR 6379912, potently competed for the ['H]PGE2 binding, but no competition was found with EP1-or EP2-selective ligands.PGEz and MQB 28,767 decreased forskolin-induced CAMP formation in a concentration-dependent manner in Chinese hamster ovary cells permanently expressing the cDNA.Northern blot analysis demonstrated that the EP3 mRNA is expressed abundantly in kidney, uterus, and mastocytoma P-815 cells and in a lesser amount in brain, thymus, lung, heart, stomach, and spleen.Eicosanoids comprising various oxygenated metabolites of arachidonic acid such as prostaglandins (PGs)' and leukotrienes exert a variety of biological activities for maintenance of local homeostasis in the body (1, 2).These metabolites act on a cell surface receptor specific for each member to exert their actions.Among them, PGEz in particular, produces a broad range of biological actions in diverse tissues.PGE,