Abstract

The µ-opioid receptor (OPRM1) signals as a classic G protein–coupled receptor by activating heterotrimeric G_i/G_o proteins resulting in adenylyl cyclase (AC) inhibition. Such AC inhibition is desensitized after prolonged agonist treatment. However, after receptor desensitization, the intracellular cAMP level remains regulated by OPRM1, as demonstrated by the intracellular cAMP level increase or AC superactivation upon removal of an agonist or addition of an antagonist. We now demonstrate that such intracellular cAMP regulation is mediated by a novel noncanonical signaling pathway resulting from OPRM1 being converted to a receptor tyrosine kinase (RTK)-like entity. This noncanonical OPRM1 signaling is initiated by the receptor recruiting and activating Src kinase within the receptor complex, leading to phosphorylation of the OPRM1 Tyr³³⁶ residue. Phospho-Tyr³³⁶ serves as the docking site for growth factor receptor-bound protein/son of sevenless, leading to the recruitment and activation of the Ras/Raf-1 and subsequent phosphorylation and activation of AC5/6 by Raf-1. Such sequence of events was established by the absence of Ras/Raf1 recruitment and activation by the OPRM1-Y336F mutant, by the presence of Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) or the absence of Src activity, by the presence of specific Raf-1 inhibitor GW5074 (5-iodo-3-[(3,5-dibromo-4-hydroxyphenyl) methylene]-2-indolinone) or the absence of Raf-1, or by the dominant negative RasN17 mutant. Src together with Ras activates Raf1 which was established by the inability of the Raf1-Tyr^340/341 mutant to activate AC. Hence, the phosphorylation of OPRM1 at Tyr³³⁶ by Src serves as the trigger for the conversion of a classic G_i/G_o-coupled receptor into an RTK-like entity, resulting in a noncanonical pathway even after the original G_i/G_o signals are blunted.