Abstract

The effects of phosphorylation of the tyrosine residue in the highly conserved DRY motif expressed in the putative second cytoplasmic loop of the μ-opioid receptor were assessed after expression in human embryonic kidney (HEK) 293 cells. Tyrosine kinase activation by epidermal growth factor (EGF) or hydrogen peroxide treatment effectively increased phosphorylation of the tyrosine-166 in the μ-opioid receptor (MOR-Tyr166p) as measured by a novel phosphoselective antibody. We were surprised to find that the increase in MOR-Tyr166p immunoreactivity (ir) required coactivation 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-Tyr166p-ir did not significantly increase after either DAMGO, EGF, or H₂O₂ treatment alone. The increase in MOR-Tyr166p-ir was blocked by pretreatment with the opioid antagonist naloxone or the Src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Consistent with these data, mutation of the tyrosine-166 to phenylalanine blocked the increased immunoreactivity, and untransfected HEK293 cells did not increase MOR-Tyr166p-ir after treatment. DAMGO increased guanosine 5′-O-(3-[³⁵S]thio)triphosphate ([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 μ-opioid receptor–G-protein coupling efficiency.