The C-terminal domain of the Gs-coupled EP4 receptor confers agonist-dependent coupling control to Gi but no coupling to Gs in a receptor hybrid with the Gi-coupled EP3 receptor.
Prostaglandin E2 receptors (EPR) belong to the family of G-protein-coupled receptors with 7 transmembrane domains. They form a family of four subtypes, which are linked to different G-proteins. EP1R are coupled to Gq, EP2 and EP4R to Gs and EP3R to Gi. Different C-terminal splice variants of the bovine EP3R are coupled to different G-proteins. A mouse EP3R whose C-terminal domain had been partially truncated no longer showed agonist-induced Gi-protein activation and was constitutively active. In order to test the hypothesis that the C-terminal domain confers coupling specificity of the receptors on the respective G-proteins, a cDNA for a hybrid rEP3hEP4R, containing the N-terminal main portion of the Gi-coupled rat EP(3beta)R including the 7th transmembrane domain and the intracellular C-terminal domain of the Gs-coupled human EP4R, was generated by PCR. HEK293 cells transiently transfected with the chimeric rEP3hEP4R cDNA expressed a plasma membrane PGE2 binding site with a slightly lower Kd value for PGE2 but an identical binding profile for receptor-specific ligands as cells transfected with the native rat EP(3beta)R. In HepG2 cells stably transfected with the chimeric rEP3hEP4R cDNA PGE2 did not increase cAMP formation characteristic of Gs coupling but attenuated the forskolin-stimulated cAMP synthesis characteristic of Gi coupling. This effect was inhibited by pre-treatment of the cells with pertussis toxin. Thus, the hybrid receptor behaved both in binding and in functional coupling characteristics as the native rat EP(3beta)R. Apparently, the intracellular C-terminal domain did not confer coupling specificity but coupling control, i.e. allowed a signalling state of the receptor only with agonist binding.
Neuschäfer-Rube
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Institut für Biochemie und Molekulare Zellbiologie, Georg-August-Universität, Göttingen, Germany.
Hänecke
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Blaschke
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Jungermann
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Püschel
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D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D051379:Mice; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D018078:Receptors, Prostaglandin E / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D058308:Receptors, Prostaglandin E, EP3 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D008024:Ligands; C545647:PTGER3 protein, human; C545650:PTGER4 protein, human; C545648:Ptger3 protein, mouse; C545649:Ptger3 protein, rat; C545651:Ptger4 protein, mouse; C545652:Ptger4 protein, rat; D018078:Receptors, Prostaglandin E; D058308:Receptors, Prostaglandin E, EP3 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs