Chronic morphine acts via a protein kinase Cγ-G_β- adenylyl cyclase complex to augment phosphorylation of G_β and G_βγ stimulatory adenylyl cyclase signaling

Chronic morphine augments protein kinase C (PKC) phosphorylation of G _β, which enhances the potency of G_βγ to stimulate adenylyl cyclase II (ACII) activity. The present study demonstrates an in vivo association between phosphorylated G_β and a specific PKC isoform, PKCγ. We investigated the association of G_β and PKCγ by assessing the ability of anti-PKCγ antibodies to co-immunoprecipitate G_β from ³²P-radiolabeled Chinese Hamster Ovary cells stably transfected with a μ-opioid receptor (MOR-CHO). PKCγ immunoprecipitate (IP) obtained from MOR-CHO membranes contained radiolabeled signals of ≈ 33 and 36-38 kDa that were subsequently identified as G_β(s). Chronic morphine significantly increased (≈75%) the magnitude of ³²P incorporated into G_β present in PKCγ IP. This suggests that G_β is an in vivo substrate for PKCγ, which mediates the chronic morphine-induced increment in G _β phosphorylation. In order to evaluate AC as a putative effector for phosphorylated G_βγ, its presence in IP obtained using anti-AC antibodies was evaluated. Autoradiographic analyses of AC IP also revealed the presence of phosphorylated G_β(s), the magnitude of which was significantly enhanced (≈60%) following chronic morphine treatment. This indicates that phosphorylated G_βγ associates and presumably interacts in vivo with AC, indicating that it is a target for the enhanced phosphorylated G_βγ that is generated following chronic morphine treatment. This would contribute to the previously observed shift from predominantly G_iα inhibitory to G_βγ stimulatory AC signaling following chronic morphine. The PKCγ-G_β-AC complex identified in this study provides an organizational framework for understanding the well-documented participation of PKCγ in opioid tolerance-producing mechanisms.