A Ser/Thr cluster within the C-terminal domain of the rat prostaglandin receptor EP3 alpha is essential for agonist-induced phosphorylation, desensitization and internalization

Neuschaefer-Rube, F.; Hermosilla, R.; Kuna, M.; Pathe-Neuschaefer-Rube, A.; Schuelein, R.; Pueschel, G.-Paul

British Journal of Pharmacology 145(8): 1132-1142


ISSN/ISBN: 0007-1188
Accession: 011675656

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1 Two isoforms of the rat prostaglandin E-2 receptor, rEP3 alpha-R and rEP3 beta-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 rEP3 alpha-R was mutated to Ala and both isoforms and the receptor mutant (rEP3 alpha-ST341-349A-R) were stably expressed in HEK293 cells.2 All rEP3-R receptors showed a similar ligand-binding profile. They were functionally coupled to Gi and reduced forskolin-induced cAMP-formation.3 Repeated exposure of cells expressing the rEP3 alpha-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 rEP3 beta-R and the rEP3 alpha-ST341-349A-R were not affected by prior agonist-stimulation.4 Agonist-stimulation of HEK293-rEP3 alpha-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 rEP3 beta-R was not phosphorylated and the rEP3 alpha-ST341-349A-R was phosphorylated only weakly.5 These results led to the hypothesis that agonist-induced desensitization of the rEP3 alpha-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.