title: Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.
abstract: Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated following U50,488H and etorphine respectively. Thus KOPR internalization requires receptor phosphorylation above a certain threshold, and higher-order KOPR phosphorylation may be disproportionally important.
authors.affiliation: Center for Substance Abuse Research and Department of Pharmacology, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, U.S.A.
authors.forename: Chongguang
authors.initials: C
authors.lastname: Chen
authors.affiliation: Center for Substance Abuse Research and Department of Pharmacology, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, U.S.A.
authors.forename: Yi-Ting
authors.initials: YT
authors.lastname: Chiu
authors.affiliation: Center for Substance Abuse Research and Department of Pharmacology, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, U.S.A.
authors.forename: Wenman
authors.initials: W
authors.lastname: Wu
authors.affiliation: Center for Substance Abuse Research and Department of Pharmacology, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, U.S.A.
authors.forename: Peng
authors.initials: P
authors.lastname: Huang
authors.affiliation: Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich-Schiller-University, 07747 Jena, Germany.
authors.forename: Anika
authors.initials: A
authors.lastname: Mann
authors.affiliation: Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich-Schiller-University, 07747 Jena, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schulz
authors.affiliation: Center for Substance Abuse Research and Department of Pharmacology, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, U.S.A. lliuche@temple.edu.
authors.forename: Lee-Yuan
authors.initials: LY
authors.lastname: Liu-Chen
mesh_terms: D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer / Q000494:pharmacology*; D018712:Analgesics, Non-Narcotic / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004705:Endocytosis / Q000187:drug effects*; D005048:Etorphine / Q000494:pharmacology; D051379:Mice; D010766:Phosphorylation; D017473:Receptors, Opioid, kappa / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D018712:Analgesics, Non-Narcotic; D017473:Receptors, Opioid, kappa; D005048:Etorphine; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer
keywords: SILAC; endocytosis threshold; phospho-κ opioid receptor-specific antibody; phosphorylation sites; κ opioid receptor