The C-terminal domain of the G(s)-coupled EP(4) receptor confers agonist-dependent coupling control to G(i) but no coupling to G(s) in a receptor hybrid with the G(i)-coupled EP(3) receptor

Prostaglandin E(2) 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, EP(1)R are coupled to G(q), EP(2), and EP(4)R to G(s) and EP(3)R to G(i). Different C-terminal splice variants of the bovine EP(3)R are coupled to different G-proteins, A mouse EP(3)R whose C-terminal domain had been partially truncated no longer showed agonist-induced G(i)-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 rEP(3)hEP(4)R, containing the N-terminal main portion of the G(i)-coupled rat EP(3 beta)R including the 7th transmembrane domain and the intracellular C-terminal domain of the G(s)-coupled human EP(4)R, was generated by PCR, HEK293 cells transiently transfected with the chimeric rEP(3)hEP(4)R cDNA expressed a plasma membrane PGE(2) binding site with a slightly lower K-d value for PGE(2) but an identical binding profile for receptor-specific ligands as cells transfected with the native rat EP(3 beta)R, In HepG(2) cells stably transfected with the chimeric rEP(3)hEP(4)R cDNA PGE(2) did not increase cAMP formation characteristic of G(s) coupling but attenuated the forskolin-stimulated cAMP synthesis characteristic of G(i) 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(3 beta)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.