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 Ser356, Thr357, Thr363 and Ser369 in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer356/pThr357, pThr363 and pSer369 respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr363-, pSer369- and pSer356/pThr357-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser369 phosphorylation affected Thr363 phosphorylation and vice versa, and Thr363 or Ser369 phosphorylation was important for Ser356/Thr357 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 Ser356/Thr357, Thr363 and Ser369 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 Thr363 and Ser369 with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr363+Ser369 and Thr357+Ser369 with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser356+Thr357+Ser369. 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.
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Wild type Neuroligin3 (red) localizes to the cell surface in PC12 Tet-on cells, in contrast to proteins within the endoplasmic reticulum (calnexin, green). See pp. 423–434 for further details. Image kindly provided by Ulbrich et al. - PDF Icon PDF LinkFront Matter
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Research Article|
February 09 2016
Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization
Chongguang Chen;
Chongguang Chen
*Center for Substance Abuse Research and Department of Pharmacology, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, U.S.A.
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Yi-Ting Chiu;
Yi-Ting Chiu
*Center for Substance Abuse Research and Department of Pharmacology, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, U.S.A.
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Wenman Wu;
Wenman Wu
*Center for Substance Abuse Research and Department of Pharmacology, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, U.S.A.
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Peng Huang;
Peng Huang
*Center for Substance Abuse Research and Department of Pharmacology, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, U.S.A.
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Anika Mann;
Anika Mann
†Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich-Schiller-University, 07747 Jena, Germany
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Stefan Schulz;
Stefan Schulz
†Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich-Schiller-University, 07747 Jena, Germany
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Lee-Yuan Liu-Chen
Lee-Yuan Liu-Chen
1
*Center for Substance Abuse Research and Department of Pharmacology, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, U.S.A.
1To whom correspondence should be addressed (email [email protected]).
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Publisher: Portland Press Ltd
Received:
December 12 2014
Revision Received:
November 25 2015
Accepted:
December 03 2015
Accepted Manuscript online:
December 03 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2016 Authors; published by Portland Press Limited
2016
Biochem J (2016) 473 (4): 497–508.
Article history
Received:
December 12 2014
Revision Received:
November 25 2015
Accepted:
December 03 2015
Accepted Manuscript online:
December 03 2015
Citation
Chongguang Chen, Yi-Ting Chiu, Wenman Wu, Peng Huang, Anika Mann, Stefan Schulz, Lee-Yuan Liu-Chen; Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization. Biochem J 15 February 2016; 473 (4): 497–508. doi: https://doi.org/10.1042/BJ20141471
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