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

• Published in: The Journal of biological chemistry 2001-10, Vol.276 (41), p.37779
• Main Authors: Jing-Gen Liu, Michael B. Ruckle, Paul L. Prather
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M106104200

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. GH 3 cells expressing μ-opioid receptors (GH 3 MOR) exhibit higher basal G-protein activity and lower basal cAMP levels than wild-type GH 3 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. GH 3 MOR 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 IC 50 of 0.98 n m and an E max of 77%. Similar concentrations of β-funaltrexamine pretreatment produced a half-maximal reduction in basal [ 35 S]GTPγS binding, a decrease in basal photolabeling of G-proteins with azidoanilido-[α- 32 P]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- 32 P]GTP demonstrated that constitutively active μ-opioid receptors activate individual G-proteins differently than the agonist [ d -Ala 2 , N -MePhe 4 ,Gly-ol 5 ]enkephalin.