Vol. 56, Issue 2, 419-428, August 1999

Agonist-Induced Phosphorylation by G Protein-Coupled Receptor Kinases of the EP4 Receptor Carboxyl-Terminal Domain in an EP3/EP4 Prostaglandin E₂ Receptor Hybrid

Frank Neuschäfer-Rube, Martin Oppermann, Ulrike Möller, Ulrike Böer, and Gerhard P. Püschel

Institut für Biochemie und Molekulare Zellbiologie (F.N.-R., U.M., U.B., G.P.P.); and Abteilung Immunologie (M.O.), Göttingen, Germany

Prostaglandin E₂ receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G_q, EP2 and EP4 to G_s, and EP3 to G_i. The EP4-R, in contrast to the EP3-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G_i-coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E₂, or by the EP3-R agonist M&B28767 was investigated in COS-7 cells expressing FLAG-epitope-tagged rat EP3-R (rEP3-R), hEP4-R, or rEP3hEP4-Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG-rEP3-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E₂ stimulated phosphorylation of the FLAG-hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M&B28767 induced a time- and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAG-rEP3-R. Agonist-induced phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R-expressing COS-7 cells augmented the M&B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R.