Agonist-induced phosphorylation by G protein-coupled receptor kinases of the EP4 receptor carboxyl-terminal domain in an EP3/EP4 prostaglandin E(2) receptor hybrid.
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 EP3beta-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 FLAG-epitope-tagged rat EP3beta-R (rEP3beta-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-rEP3beta-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 FLAG-rEP3beta-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.
Institut für Biochemie und Molekulare Zellbiologie, Göttingen, Germany.
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Neuschäfer-Rube
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Oppermann
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Möller
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U
Böer
G P
GP
Püschel
D000818:Animals; D019556:COS Cells; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D018078:Receptors, Prostaglandin E / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058308:Receptors, Prostaglandin E, EP3 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
D017931:DNA Primers; D004791:Enzyme Inhibitors; C545646:Ptger2 protein, rat; C545649:Ptger3 protein, rat; C545652:Ptger4 protein, rat; D018078:Receptors, Prostaglandin E; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058308:Receptors, Prostaglandin E, EP3 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D011993:Recombinant Fusion Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019311:Staurosporine