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- Title
Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.
- Authors
Chongguang Chen; Wenman Wu; Peng Huang; Mann, Anika; Schulz, Stefan; Yi-Ting Chiu; Lee-Yuan Liu-Chen
- 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)cyclohexyl] 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 theKOPR 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.
- Subjects
DEPHOSPHORYLATION; PHOSPHORYLATION; CHEMICAL reactions; EXTERNALIZING behavior; ENDORPHIN receptors
- Publication
Biochemical Journal, 2016, Vol 473, Issue 4, p497
- ISSN
0264-6021
- Publication type
Article
- DOI
10.1042/BJ20141471