PT  - JOURNAL ARTICLE
AU  - Cecilea C. Clayton
AU  - Michael R. Bruchas
AU  - Michael L. Lee
AU  - Charles Chavkin
TI  - Phosphorylation of the μ-opioid receptor at tyrosine 166 (Y3.51) in the DRY motif reduces agonist efficacy
AID  - 10.1124/mol.109.060558
DP  - 2009 Jan 01
TA  - Molecular Pharmacology
PG  - mol.109.060558
4099  - http://molpharm.aspetjournals.org/content/early/2009/12/03/mol.109.060558.short
4100  - http://molpharm.aspetjournals.org/content/early/2009/12/03/mol.109.060558.full
AB  - The effects of phosphorylation of the tyrosine residue in the highly conserved DRY motif expressed in the putative 2nd cytoplasmic loop of the mu opioid receptor were assessed following expression in HEK293 cells. Tyrosine kinase activation by Epidermal Growth Factor (EGF) or hydrogen peroxide treatment effectively increased phosphorylation of the tyrosine-166 in the mu opioid receptor (MOR-Y166p) as measured by a novel phospho-selective antibody. Surprisingly, the increase in MOR-Y166p-immunoreactivity (ir) required co-activation by the opioid agonist, D-Ala2,methyl-Phe4,Gly5-ol]enkephalin (DAMGO) as demonstrated by both Western blot imaging of membrane proteins and confocal microscopy of transfected cells; MOR-Y166p-ir did not significantly increase after either DAMGO, EGF or H2O2 treatment alone. The increase in MOR-Y166p-ir was blocked by pretreatment with the opioid antagonist naloxone or the Src kinase inhibitor PP2. Consistent with these data, mutation of the tyrosine-166 to phenylalanine blocked the increased immunoreactivity, and untransfected HEK293 cells did not increase MOR-Y166p-ir following treatment. DAMGO increased [35S]GTPγS binding to membranes from cells expressing wild type MOR or MOR-Y166F receptors in a dose-dependent manner. Pretreatment of the wild type MOR expressing cells with the combination of DAMGO and EGF completely blocked subsequent DAMGO stimulation of [35S]GTPγS binding membranes; whereas [35S]GTPγS binding to membranes from cells expressing mutated MOR(Y166F) was only partially inhibited. These results suggest that G protein activation as measured by [35S]GTPγS binding can be regulated by DAMGO and EGF by convergent mechanisms and support the hypothesis that tyrosine phosphorylation within the DRY motif may reduce mu opioid receptor - G-protein coupling efficiency.The American Society for Pharmacology and Experimental Therapeutics