The C‐terminal domain of the G_s‐coupled EP₄ receptor confers agonist‐dependent coupling control to G_i but no coupling to G_s in a receptor hybrid with the G_i‐coupled EP₃ receptor

Abstract

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. EPiR are coupled to G q , EP 2 and EP 4 R to G s and EP 3 R to G;. 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 Gj -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 Gj-coupled rat EP 3 ßR 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 hEP4R 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 receptorspecific ligands as cells transfected with the native rat EP 3 ßR. 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 Gj 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 3ß 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.

Agonist-Induced Phosphorylation by G Protein-Coupled Receptor Kinases of the EP4 Receptor Carboxyl-Terminal Domain in an EP3/EP4 Prostaglandin E2Receptor Hybrid

“…Competition binding studies with membrane preparations of transfected cells showed that all receptors had a similar affinity for PGE 2 (FLAG-rEP3␤-R, K d ϭ 11 Ϯ 1 nM; FLAGhEP4-R, K d ϭ 5.9 Ϯ 3 nM; FLAG-rEP3hEP4-Ct-R, K d ϭ 12 Ϯ 4 nM; not shown). The K d values were in the same range as those of the untagged wild-type receptors (Neuschä fer- Rube, 1994Rube, , 1997a, which indicates that the amino-terminal FLAG-tag did not alter the receptor binding properties. FLAG-rEP3␤-R and FLAG-rEP3hEP4-Ct-R were expressed to a comparable very high level (FLAG-rEP3␤-R, B max ϭ 7.2 Ϯ 0.3 pmol/mg of protein; FLAG-rEP3hEP4-Ct-R, B max ϭ 5 Ϯ 1 pmol/mg of protein) whereas FLAG-hEP4-R expression was somewhat lower (B max 0.9 Ϯ 0.2 pmol/mg of protein).…”

“…As previously shown, the carboxyl-terminal domain of the EP4-R is not only necessary but also sufficient to confer rapid agonist-induced desensitization in a hybrid receptor with the nondesensitizable rEP3␤-R (Fig. 1) (Neuschä fer-Rube, 1997a). By contrast, the third intracellular loop of the EP4-R was neither necessary nor sufficient to mediate agonist-induced desensitization (Neuschä fer-Rube, 1997a).…”

Localization of Functional Prostaglandin E2 Receptors EP3 and EP4 in the Nuclear Envelope

“…Indirect Immunofluorescence of EP 3␣ and EP 4 Receptors in HEK 293 Cells-To assess whether this perinuclear distribution of EP 3␣ and EP 4 receptors applies generally to cells, the localization of these receptors was studied after transfection of cDNA for EP 3␣ and EP 4 into HEK 293 cells that do not normally express prostanoid receptors (39); ectopically expressed EP receptors in HEK 293 cells bind PGE 2 and are functional (39,40). Immunoreactivity for EP 3␣ and EP 4 receptors was seen on the plasma membrane (Fig.…”

A Ser/Thr cluster within the C‐terminal domain of the rat prostaglandin receptor EP3α is essential for agonist‐induced phosphorylation, desensitization and internalization

“…Of the two mouse EP3‐R isoforms, mEP3 α ‐R and mEP3 β ‐R, only the mEP3 α ‐R underwent agonist‐induced desensitization and internalization (Negishi et al ., 1993). The importance of the C‐terminal domain for EP3‐R desensitization was also shown in studies using a receptor hybrid consisting of the N‐terminal main portion of the nondesensitizable rEP3 β ‐R up to the end of the seventh transmembrane domain and the C‐terminal portion of the Gs‐coupled, desensitizable human EP4‐R (hEP4‐R) (Neuschäfer‐Rube et al ., 1997a). For this receptor hybrid, which was exclusively coupled to Gi (Neuschäfer‐Rube et al ., 1997b), the C‐terminal domain of EP4‐R was necessary and sufficient to confer agonist‐induced receptor desensitization.…”