Determination of sites of U50,488H-promoted phosphorylation of the mouse kappa opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization

作者：Chen Chongguang; Chiu Yi Ting; Wu Wenman; Huang Peng; Mann Anika; Schulz Stefan; Liu Chen Lee Yuan^*

来源：Biochemical Journal, 2016, 473(4): 497-508.

DOI：10.1042/BJ20141471

• endocytosis threshold
• kappa opioid receptor
• phospho-kappa opioid receptor-specific antibody
• phosphorylation sites
• SILAC

Phosphorylation sites of KOPR (kappa 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 theKOPR 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 similar to 60% and similar to 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.

• 出版日期2016-2-15