Analysis of naturally occurring variants (Negishi, 1993a,b; Hasegawa, 1996), of receptor hybrids between different pro-stanoid receptors (Dorn, 1997; Kobayashi, 1997; Neuschä fer-Rube, 1997a,b), and of (Funk, 1993; Irie

Abstract

ABSTRACT Prostaglandin E 2 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 E 2 , or by the EP3-R agonist M&B28767 was investigated in COS-7 cells expressing FLAGepitope-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 E 2 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 FLAGrEP3␤-R. Agonist-induced phosphorylation of the FLAGhEP4-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. Prostaglandin E 2 receptors (EP-Rs), like most prostanoid receptors, belong to the class of G protein-coupled ectoreceptors (GPCR) with seven transmembrane domain