Analysis of a porcine EP3-receptor: cloning, expression and signal transduction.
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 NFkappaB into the nucleus of transfected CHO cells was demonstrated by Western blot analysis, indicating that these EP3 receptors modulate NFkappaB-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.
Meyer-Kirchrath
J
J
Institut für Pharmakologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Kuger
P
P
Hohlfeld
T
T
Schrör
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding; D011869:Radioligand Assay; D018078:Receptors, Prostaglandin E / Q000235:genetics* / Q000378:metabolism; D058308:Receptors, Prostaglandin E, EP3 Subtype; D012045:Regulatory Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013552:Swine / Q000235:genetics*; D014158:Transcription, Genetic
D008024:Ligands; C545647:PTGER3 protein, human; D018078:Receptors, Prostaglandin E; D058308:Receptors, Prostaglandin E, EP3 Subtype; D005576:Colforsin; D004247:DNA; D000242:Cyclic AMP