Identifizierung der für die Agonisten-induzierte Phosphorylierung und Internalisierung relevanten Serine und Threonine in der C-terminalen Domäne des humanen Prostaglandin E2 Rezeptors, Subtyp EP4

Abstract

The prostanoid receptors belong to the family of G protein-coupled receptors with seven transmembrane domains. Four different subtypes have been identified for prostaglandin E2 (PGE2). The EP1-R couples to Gq and its activation leads to an increase of intracellular InsP3/Ca2+, while the EP3-R is Gi-coupled and its activation leads to an inhibition of the hormon-stimulated cAMP elevation. The EP2- and the EP4-R are both coupled to Gs and binding of PGE2 to these receptors increases intracellular cAMP levels. The sequence identity within the four receptor subtypes is less than 50%.The EP4-R shows an agonist-induced receptor desensitization. This desensitization seems to be initiated by a G protein coupled receptor kinase (GRK)-mediated phosphorylation of serine and threonine residues within the intracellular domains of the receptor. Phosphorylation is followed by the binding of beta-arrestin to the phosphorylated receptor, which leads to a sterical inhibition of receptor/G protein interaction. Desensitization is followed by internalization of the receptor. The agonist-induced phosphorylation of the human EP4-R occurs in the C-terminal domain, which contains 38 serine and threonine residues, which can serve as potential phosphorylation sites. The aim of the study was to identify the serine and threonine residues in the C-terminal domain, which are relevant for the agonist-induced phosphorylation, desensitization and internalization of the human EP4-R (hEP4-R).The hEP4-R shows an agonist-induced, possibly GRK-mediated phosphorylation. The agonist-induced phosphorylation was largly abrogated by substitution of all serine and threonine residues within the C-terminal domain of the receptor. The serine and threonine residues between Ser359 and Ser382 were identified as the major phosphorylation sites of the receptor, but a residual phosphorylation of serine and threonine residues distal to these sites could not be excluded. Probably due to the high receptor expression level the desensitization of the receptor could not be shown. Therefore the relevance of the identified phosphorylation sites for the desensitization could not be tested. The hEP4-R was internalized after agonist-exposure and was colocalized with beta-arrestin after internalization. The serine and threonine residues within the main phosphorylation sites (Ser359-Ser382) were neither sufficient nor necessary to confer the internalization of the hEP4-R. The relevant serine and threonine residues for receptor internalization were located distal from Ser382, but the proximal serine and threonine residues (Ser359-Ser382) seemed to be crucial for the receptor/beta-arrestin colocalization. The internalization of the receptor could be blocked with high sucrose concentrations and hence, most likely, was clathrin-mediated.