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
2004 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 379, no Pt 3, 573-585 p.Article in journal (Refereed) Published
hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-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 beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-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 beta-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analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and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 beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.
Place, publisher, year, edition, pages
2004. Vol. 379, no Pt 3, 573-585 p.
Amino Acid Sequence, Arrestins/*metabolism, Cell Line, Endocytosis/drug effects, Humans, Ligands, Molecular Sequence Data, Mutation/genetics, Phosphopeptides/chemistry/metabolism, Phosphorylation/drug effects, Prostaglandins/*pharmacology, Protein Structure; Tertiary, Receptors; Prostaglandin E/agonists/*chemistry/genetics/*metabolism, Research Support; Non-U.S. Gov't, Serine/genetics/*metabolism, Signal Transduction/drug effects, Threonine/genetics/*metabolism
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:uu:diva-72997DOI: 10.1042/BJ20031820PubMedID: 14709160OAI: oai:DiVA.org:uu-72997DiVA: diva2:100908