Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site

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Abstract
HEP4-R (human prostaglandin E2 receptor, subtype EP4) is a Gs-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C-terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced β-arrestin complex formation, which is stabilized by phosphorylation. Subsequently β-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist-induced phosphorylation, interaction with β-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370–382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389–Ser-390–Thr-391–Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of β-arrestin1. However, phosphorylation greatly enhanced the stability of the β-arrestin1–receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both β-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.

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