Modulating μ-Opioid Receptor Phosphorylation Switches Agonist-dependent Signaling as Reflected in PKCε Activation and Dendritic Spine Stability

A new role of G protein-coupled receptor (GPCR) phosphorylation was demonstrated in the current studies by using the mu-opioid receptor (OPRM1) as a model. Morphine induces a low level of receptor phosphorylation and uses the PKC epsilon pathway to induce ERK phosphorylation and receptor desensitization, whereas etorphine, fentanyl, and [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) induce extensive receptor phosphorylation and use the beta-arrestin2 pathway. Blocking OPRM1 phosphorylation (by mutating Ser(363), Thr(370) and Ser(375) to Ala) enabled etorphine, fentanyl, and DAMGO to use the PKC epsilon pathway. This was not due to the decreased recruitment of beta-arrestin2 to the receptor signaling complex, because these agonists were unable to use the PKC epsilon pathway when beta-arrestin2 was absent. In addition, overexpressing G protein-coupled receptor kinase 2 (GRK2) decreased the ability of morphine to activate PKC epsilon, whereas overexpressing dominant-negative GRK2 enabled etorphine, fentanyl, and DAMGO to activate PKC epsilon. Furthermore, by overexpressing wild-type OPRM1 and a phosphorylation-deficient mutant in primary cultures of hippocampal neurons, we demonstrated that receptor phosphorylation contributes to the differential effects of agonists on dendritic spine stability. Phosphorylation blockage made etorphine, fentanyl, and DAMGO function as morphine in the primary cultures. Therefore, agonist-dependent phosphorylation of GPCR regulates the activation of the PKC pathway and the subsequent responses.