Constitutively Active μ-Opioid Receptors Inhibit Adenylyl Cyclase Activity in Intact Cells and Activate G-proteins Differently than the Agonist [d-Ala2,N-MePhe4,Gly-ol5]Enkephalin

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Abstract
The most convincing evidence demonstrating constitutive activation of μ-opioid receptors is the observation that putative inverse agonists decrease basal G-protein activity in membrane preparations. However, it is not clear whether constitutively active receptors in isolated membranes have any physiological relevance in intact cells. GH3 cells expressing μ-opioid receptors (GH3MOR) exhibit higher basal G-protein activity and lower basal cAMP levels than wild-type GH3 cells, indicative of constitutively active receptors. This study determined whether alkylation of μ-opioid receptors by the irreversible antagonist β-funaltrexamine would decrease spontaneous receptor activity in intact cells, revealing constitutive activity. GH3MOR cells were pretreated with increasing concentrations of β-funaltrexamine followed by functional testing after removal of unbound drug. β-Funaltrexamine pretreatment produced a concentration-dependent decrease in μ-opioid receptor binding with an IC50 of 0.98 nm and anEmax of 77%. Similar concentrations of β-funaltrexamine pretreatment produced a half-maximal reduction in basal [35S]GTPγS binding, a decrease in basal photolabeling of G-proteins with azidoanilido-[α-32P]GTP, and an increase in basal adenylyl cyclase activity in intact cells. Therefore, μ-opioid receptors are constitutively active in intact cells, producing stimulation of G-proteins and inhibition of adenylyl cyclase. Importantly, photolabeling of Gα-subunits with azidoanilido-[α-32P]GTP demonstrated that constitutively active μ-opioid receptors activate individual G-proteins differently than the agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin.