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

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.