Preservation of Gi coupling of a chimeric EP3/I-type prostaglandin (IP) receptor.
For the EP3 subtype of prostaglandin E receptors, different C-terminal splice variants are known, which are coupled to distinct heterotrimeric GTP-binding proteins (G-proteins). To test the hypothesis that the C-terminal domain is essential for the G-protein-coupling specificity of the EP3 receptor, we exchanged the carboxyl-terminal tail of a porcine Gi-coupled EP3 receptor isoform for the corresponding C-terminal part of a Gs-coupled prostaglandin receptor. The porcine EP3 receptor was truncated at a lysine (K350) residue at the end of the seventh transmembrane region, representing the splicing site of the different EP3 receptor isoforms. The wild-type C-terminus (37 amino acids) was substituted by the C-terminal tail (89 amino acids) of the human I-type prostaglandin receptor (hIP-R). The G-protein coupling of the resulting chimeric receptor protein was studied in transfected Chinese hamster ovary (CHO) cells. Stimulation of the chimeric receptor protein with the EP3 receptor-specific agonist M&B 28.767 did not increase adenosine 3',5'-cyclic monophosphate (cAMP) formation but did reduce the forskolin-stimulated cAMP formation, indicating Gi coupling. Furthermore, the chimeric receptor did not show constitutive activity as demonstrated for the C-terminally truncated EP3 receptor. Thus, coupling specificity of the EP3 receptor is not exclusively mediated by the carboxyl-terminal tail, and constitutive activity of a C-terminally truncated EP3 receptor can be suppressed by the hIP-R C-terminus.
Meyer-Kirchrath
J
J
Institut für Pharmakologie und Klinische Pharmakologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Hasse
A
A
Schrör
K
K
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000096:biosynthesis* / Q000235:genetics; D018078:Receptors, Prostaglandin E / Q000096:biosynthesis* / Q000235:genetics; D058308:Receptors, Prostaglandin E, EP3 Subtype; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
C545647:PTGER3 protein, human; C113936:PTGIR protein, human; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D018078:Receptors, Prostaglandin E; D058308:Receptors, Prostaglandin E, EP3 Subtype; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D019204:GTP-Binding Proteins