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

1 Two isoforms of the rat prostaglandin E 2 receptor, rEP3 α ‐R and rEP3 β ‐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 α ‐R was mutated to Ala and both isoforms and the receptor mutant (rEP3 α ‐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 α ‐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 β ‐R and the rEP3 α ‐ST341‐349A‐R were not affected by prior agonist‐stimulation. 4 Agonist‐stimulation of HEK293‐rEP3 α ‐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 β ‐R was not phosphorylated and the rEP3 α ‐ST341‐349A‐R was phosphorylated only weakly. 5 These results led to the hypothesis that agonist‐induced desensitization of the rEP3 α ‐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.British Journal of Pharmacology (2005) 145 , 1132–1142. doi: 10.1038/sj.bjp.0706282