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.
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analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and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.
Neuschäfer-Rube
Frank
F
Universität Potsdam, Institut für Ernährungswissenschaft, Biochemie der Ernährung, Arthur-Scheunert-Allee 114-116, 14558 Bergholz-Rehbrücke, Germany. fneusch@rz.uni-potsdam.de
Hermosilla
Ricardo
R
Rehwald
Mathias
M
Rönnstrand
Lars
L
Schülein
Ralf
R
Wernstedt
Christer
C
Püschel
Gerhard Paul
GP
D000595:Amino Acid Sequence; D019390:Arrestins / Q000378:metabolism*; D002460:Cell Line; D004705:Endocytosis / Q000187:drug effects; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011453:Prostaglandins / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D018078:Receptors, Prostaglandin E / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058309:Receptors, Prostaglandin E, EP4 Subtype; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D000071557:beta-Arrestins
D019390:Arrestins; D008024:Ligands; C545650:PTGER4 protein, human; D010748:Phosphopeptides; D011453:Prostaglandins; D018078:Receptors, Prostaglandin E; D058309:Receptors, Prostaglandin E, EP4 Subtype; D000071557:beta-Arrestins; D013912:Threonine; D012694:Serine