Enhanced stimulatory adenylyl cyclase signaling during opioid dependence is associated with a reduction in palmitoylated Gsα

Chronic opioid treatment of stably mu-opioid receptor transfected human mammary epidermoid A431 carcinoma cells (clone A431/mu 13) results in sensitization of adenylyl cyclase (AC), a cellular adaptation associated with drug dependence. Up-regulation of AC is characterized by significantly increased levels of both basal and post-receptor-stimulated effector activities, which develop without any apparent change in the quantity of stimulatory G proteins and the maximum catalytic activity of AC. Here, we report that detergent extracts from membranes of chronically morphine-treated (10 mu M; 2 days) A431/mu 13 cells display higher stimulatory AC activities as assessed in the S49cyc(-) reconstitution assay. This finding is most likely due to an increased functional activity of G(S alpha) because the addition of exogenous G(beta gamma) subunits, which per se stimulate AC in S49cyc(-) membranes, failed to affect the difference in reconstitutive AC activity. Moreover, both chemical depalmitoylation by hydroxylamine and inhibition of palmitoyl-CoA transferase in vivo by tunicamycin treatment increased the reconstitutive activity of detergent extracts and eliminated the differences between native and opioid-dependent cells, indicating that the increase in stimulatory activity is due to depalmitoylation of G(S alpha). Indeed, metabolic labeling studies with [H-3]palmitic acid revealed that chronic opioid treatment reduces considerably the fraction of palmitoylated G(S alpha) in the plasma membrane. Furthermore, high affinity [H-3]forskolin binding experiments demonstrated that depalmitoylated G(S alpha) is able to associate directly with AC during the state of opioid dependence even without preceding receptor activation. These results suggest that post-translational palmitoylation of G(S alpha) provides a potential regulator of transmembrane signaling. Moreover, accumulation of the depalmitoylated form of G(S alpha) in the plasma membrane as reported herein may contribute to the increase in stimulatory AC signaling, as is characteristic for the state of opioid dependence.