A Ser/Thr cluster within the C-terminal domain of the rat prostaglandin receptor EP3alpha is essential for agonist-induced phosphorylation, desensitization and internalization.
Two isoforms of the rat prostaglandin E(2) receptor, rEP3alpha-R and rEP3beta-R, differ only in their C-terminal domain. To analyze the function of the rEP3-R C-terminal domain in agonist induced desensitization, a cluster of Ser/Thr residues in the C-terminal domain of the rEP3alpha-R was mutated to Ala and both isoforms and the receptor mutant (rEP3alpha-ST341-349A-R) were stably expressed in HEK293 cells. All rEP3-R receptors showed a similar ligand-binding profile. They were functionally coupled to Gi and reduced forskolin-induced cAMP-formation. Repeated exposure of cells expressing the rEP3alpha-R isoform to PGE(2) reduced the agonist induced inhibition of forskolin-stimulated cAMP-formation by 50% and led to internalization of the receptor to intracellular endocytotic vesicles. By contrast, Gi-response as well as plasma membrane localization of the rEP3beta-R and the rEP3alpha-ST341-349A-R were not affected by prior agonist-stimulation. Agonist-stimulation of HEK293-rEP3alpha-R cells induced a time- and dose-dependent phosphorylation of the receptor most likely by G protein-coupled receptor kinases and not by protein kinase A or protein kinase C. By contrast, upon agonist-stimulation the rEP3beta-R was not phosphorylated and the rEP3alpha-ST341-349A-R was phosphorylated only weakly. These results led to the hypothesis that agonist-induced desensitization of the rEP3alpha-R isoform is mediated most likely by a GRK-dependent phosphorylation of Ser/Thr residues 341-349. Phosphorylation then initiates uncoupling of the receptor from Gi protein and receptor internalization.
Neuschäfer-Rube
Frank
F
Universität Potsdam, Institut für Ernährungswissenschaft, Biochemie der Ernährung, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany. fneusch@rz.uni-potsdam.de
Hermosilla
Ricardo
R
Kuna
Manuela
M
Pathe-Neuschäfer-Rube
Andrea
A
Schülein
Ralf
R
Püschel
Gerhard Paul
GP
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism*; D018076:DNA, Complementary; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018078:Receptors, Prostaglandin E / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D058308:Receptors, Prostaglandin E, EP3 Subtype; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D008024:Ligands; C545647:PTGER3 protein, human; D020033:Protein Isoforms; C545649:Ptger3 protein, rat; D018078:Receptors, Prostaglandin E; D058308:Receptors, Prostaglandin E, EP3 Subtype; D013912:Threonine; D012694:Serine; D000242:Cyclic AMP