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