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

Phosphorylation sites of KOPR ( 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...

Source: Biochemical Journal - February 9, 2016 Category: Biochemistry Authors: Chen, C., Chiu, Y.-T., Wu, W., Huang, P., Mann, A., Schulz, S., Liu-Chen, L.-Y. Tags: Biomolecules, ChemBio Research articles Source Type: research