title: Identification of phosphorylation sites in the COOH-terminal tail of the μ-opioid receptor.
abstract: Phosphorylation is considered a key event in the signalling and regulation of the μ opioid receptor (MOPr). Here, we used mass spectroscopy to determine the phosphorylation status of the C-terminal tail of the rat MOPr expressed in human embryonic kidney 293 (HEK-293) cells. Under basal conditions, MOPr is phosphorylated on Ser(363) and Thr(370), while in the presence of morphine or [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO), the COOH terminus is phosphorylated at three additional residues, Ser(356) , Thr(357) and Ser(375). Using N-terminal glutathione S transferase (GST) fusion proteins of the cytoplasmic, C-terminal tail of MOPr and point mutations of the same, we show that, in vitro, purified G protein-coupled receptor kinase 2 (GRK2) phosphorylates Ser(375), protein kinase C (PKC) phosphorylates Ser(363), while CaMKII phosphorylates Thr(370). Phosphorylation of the GST fusion protein of the C-terminal tail of MOPr enhanced its ability to bind arrestin-2 and -3. Hence, our study identifies both the basal and agonist-stimulated phospho-acceptor sites in the C-terminal tail of MOPr, and suggests that the receptor is subject to phosphorylation and hence regulation by multiple protein kinases.
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, UK.
authors.forename: Ying-Ju
authors.initials: YJ
authors.lastname: Chen
authors.forename: Sue
authors.initials: S
authors.lastname: Oldfield
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Butcher
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Tobin
authors.forename: Kunal
authors.initials: K
authors.lastname: Saxena
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Gurevich
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Benovic
authors.forename: Graeme
authors.initials: G
authors.lastname: Henderson
authors.forename: Eamonn
authors.initials: E
authors.lastname: Kelly
mesh_terms: D024506:5' Flanking Region / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D051381:Rats; D017450:Receptors, Opioid, mu / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D017450:Receptors, Opioid, mu; D011494:Protein Kinases