MOLECULAR-CLONING AND CHARACTERIZATION OF THE 4 RAT PROSTAGLANDIN E-2PROSTANOID RECEPTOR SUBTYPES

We have characterized the rat prostanoid EP1, EP2, EP3 alpha and EP4 r eceptor subtypes cloned from spleen, hepatocyte and/or kidney cDNA lib raries. Comparison of the deduced amino acid sequences of the rat EP r eceptors with their respective homologues from mouse and human showed 91% to 98% and 82% to 89% identity, respectively. Radioreceptor bindin g assays and functional assays were performed on EP receptor expressin g human embryonic kidney (HEK) 293 cells. The K-D values obtained with prostaglandin E-2 for the prostanoid receptor subtypes EP1, EP2, EP3 alpha and EP4 were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for various prostanoids at the prostanoid rec eptor subtypes EP2, EP3 alpha and EP4 receptor subtypes was prostaglan din E-2 = prostaglandin E-1 > iloprost > prostaglandin F-2 alpha > pro staglandin D-2 > U46619. The rank order at the prostanoid EP1 receptor was essentially the same except that iloprost had the highest affinit y of the prostanoids tested. Of the selective ligands, butaprost was s elective for prostanoid EP2, M&B28767 and sulprostone were selective f or EP3 alpha and enprostil displayed dual selectivity, interacting wit h both prostanoid receptor subtypes EP1 and EP3 alpha. All four recept ors coupled to their predominant signal transduction pathways in HEK 2 93 cells. Notably, using a novel aequorin luminescence assay to monito r prostanoid EP1 mediated increases in intracellular calcium, both ilo prost and sulprostone were identified as partial agonists. Finally, by Northern blot analysis EP3 transcripts were most abundant in liver an d kidney whereas prostanoid EP2 receptor mRNA was expressed in spleen, lung and testis and prostanoid EP1 receptor mRNA transcripts were pre dominantly expressed in the kidney. The rat prostanoid EP1 probes also detected additional and abundant transcripts present in all the tissu es examined. These were found to be related to the expression of a nov el protein kinase gene and not the prostanoid EP1 gene [Batshake, B., Sundelin, J., 1996. The mouse genes for the EP1 prostanoid receptor an d the novel protein kinase overlap. Biochem. Biophys. Res. Commun. 227 , 1329-1333]. (C) 1997 Elsevier Science B.V.