ANALYSIS OF A PORCINE EP3-RECEPTOR - CLONING, EXPRESSION AND SIGNAL-TRANSDUCTION

A cDNA clone, encoding a complete porcine EP3 receptor, was isolated f rom a porcine heart cDNA library. The deduced amino acid sequence reve aled a protein of 387 amino acid residues with an estimated molecular weight of 43 kD and strongest homology to the human EP3-II receptor (8 4% identity on protein level). Ligand binding studies with transfected COS-7 cells, expressing the porcine receptor. showed displacement of [H-3]PGE(1) with the EP3-specific agonist M&B 28.767, the EP1/EP3-agon ist sulprostone but not with the EP2-specific agonist butaprost. Stimu lation of transfected CHO cells with M&B 28.767 resulted in inhibition of forskolin-induced cAMP formation, suggesting coupling to an inhibi tory G protein. Agonist-induced translocation of the transcription fac tor NF kappa B into the nucleus of transfected CHO cells was demonstra ted by Western blot analysis, indicating that these EP3 receptors modu late NF kappa B-dependent cellular signal transduction. Analysis of th e genomic organization identified the major transcription initiation s ite at about 160 bp upstream of the ATG start codon. The 800-bp 5' fla nking region contains a variety of putative fis-acting regulatory elem ents, including binding sites for AP2, SP1 and MyoD (E-box). The prese nt data will now allow further studies on EP3 receptor-mediated signal transduction and its regulation.