Abstract
Recent studies have revealed that in G protein-coupled receptor signalings switching between G protein- and beta-arrestin (betaArr)-dependent pathways occurs. In the case of opioid receptors, the signal is switched from the initial inhibition of adenylyl cyclase (AC) to an increase in AC activity (AC activation) during prolonged agonist treatment. The mechanism of such AC activation has been suggested to involve the switching of G proteins activated by the receptor, phosphorylation of signaling molecules, or receptor-dependent recruitment of cellular proteins. Using protein kinase inhibitors, dominant negative mutant studies and mouse embryonic fibroblast cells isolated from Src kinase knock-out mice, we demonstrated that mu-opioid receptor (OPRM1)-mediated AC activation requires direct association and activation of Src kinase by lipid raft-located OPRM1. Such Src activation was independent of betaArr as indicated by the ability of OPRM1 to activate Src and AC after prolonged agonist treatment in mouse embryonic fibroblast cells lacking both betaArr-1 and -2. Instead the switching of OPRM1 signals was dependent on the heterotrimeric G protein, specifically Gi2 alpha-subunit. Among the Src kinase substrates, OPRM1 was phosphorylated at Tyr336 within NPXXY motif by Src during AC activation. Mutation of this Tyr residue, together with mutation of Tyr166 within the DRY motif to Phe, resulted in the complete blunting of AC activation. Thus, the recruitment and activation of Src kinase by OPRM1 during chronic agonist treatment, which eventually results in the receptor tyrosine phosphorylation, is the key for switching the opioid receptor signals from its initial AC inhibition to subsequent AC activation.