Phosphorylation sites of KOPR following treatment with the selective agonist U50,488H were identified after affinity purification, SDS-PAGE, in-gel digestion with Glu-C and LC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated S356, T357, T363 and S369 in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pS356/pT357, pT363, and pS369, respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pT363, pS369 and pS356/pT357 antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. S369 phosphorylation affected T363 phosphorylation and vice versa and T363 or S369 phosphorylation was important for S356/T357 phosphorylation, revealing 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 S356/T357, T363 and S369 by immunoblotting. Using SILAC (stable isotope labeling by amino acids in cell culture) and LC-MS/MS, we found that compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at T363 and S369 with U/E ratio of 2.5 and 2, respectively. Both induced double phosphorylation at T363+S369 and T357+S369 with ratios of U/E=3.3 and 3.4, respectively. Only U50,488H induced triple phosphorylation at S356+T357+S369. An unphosphorylated KOPR(354–372) fragment containing all the phosphorylation sites was detected with a ratio of C/E/U =1/0.7/0.4, indicating that ~60% and ~30% of the mKOPR 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.
