Phosphoproteomic analysis of the mouse brain mu-opioid (MOP) receptor

Many in vitro data have shown that the efficacy of several opioid drugs is correlated with differential mu-opioid (MOP) receptor phosphorylation. Label-free semiquantitative on-line nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analyses were performed to compare the endogenous MOP receptor phosphorylation patterns of mice administered with morphine, etonitazene and fentanyl. The analysis identified S363, T370 and S375 as phosphorylated residues in the carboxy-terminus. Only T370 and S375 were regulated by agonists, with a higher propensity to promote double phosphorylation for high efficacy agonists. Our study provides confirmation that differential agonist-driven multi-site phosphorylation of MOP receptor occurs in vivo and validate the use of MS to study endogenous GPCR phosphorylation.