Analysis of a porcine EP3-receptor: cloning, expression and signal transduction

Abstract

A cDNA clone, encoding a complete porcine EP₃ receptor, was isolated from a porcine heart cDNA library. The deduced amino acid sequence revealed a protein of 387 amino acid residues with an estimated molecular weight of 43 kD and strongest homology to the human EP_3-II receptor (84% identity on protein level). Ligand binding studies with transfected COS-7 cells, expressing the porcine receptor, showed displacement of [³H]PGE₁ with the EP₃-specific agonist M & B 28.767, the EP₁/EP₃-agonist sulprostone but not with the EP₂-specific agonist butaprost. Stimulation of transfected CHO cells with M & B 28.767 resulted in inhibition of forskolin-induced cAMP formation, suggesting coupling to an inhibitory G protein. Agonist-induced translocation of the transcription factor NFκB into the nucleus of transfected CHO cells was demonstrated by Western blot analysis, indicating that these EP₃ receptors modulate NFκB-dependent cellular signal transduction. Analysis of the genomic organization identified the major transcription initiation site at about 160 bp upstream of the ATG start codon. The 800-bp 5’ flanking region contains a variety of putative cis-acting regulatory elements, including binding sites for AP2, SP1 and MyoD (E-box). The present data will now allow further studies on EP₃ receptor-mediated signal transduction and its regulation.