Abstract
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 Gq, EP2 and EP4 to Gs, and EP3 to Gi. The EP4-R, in contrast to the EP3β-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 Gi-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 EP3β-R (rEP3β-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-rEP3β-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-rEP3β-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.
Footnotes
- Received September 28, 1998.
- Accepted April 26, 1999.
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Send reprint requests to: Dr. F. Neuschäfer-Rube, Institut für Biochemie und Molekulare Zellbiologie, Humboldtallee 23, D-37073 Göttingen, Germany. E-mail:fneusch{at}gwdg.de
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The work was supported by the Deutsche Forschungsgemeinschaft through the Sonderforschungsbereich 402, Teilprojekt B6, and the Fonds der Chemischen Industrie.
- The American Society for Pharmacology and Experimental Therapeutics
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