Constitutively active mu-opioid receptors inhibit adenylyl cyclase activity in intact cells and activate G-proteins differently than the agonist [D-Ala2,N-MePhe4,Gly-ol5]enkephalin.
The most convincing evidence demonstrating constitutive activation of mu-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 mu-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 mu-opioid receptors by the irreversible antagonist beta-funaltrexamine would decrease spontaneous receptor activity in intact cells, revealing constitutive activity. GH3MOR cells were pretreated with increasing concentrations of beta-funaltrexamine followed by functional testing after removal of unbound drug. beta-Funaltrexamine pretreatment produced a concentration-dependent decrease in mu-opioid receptor binding with an IC50 of 0.98 nm and an Emax of 77%. Similar concentrations of beta-funaltrexamine pretreatment produced a half-maximal reduction in basal [35S]GTPgammaS binding, a decrease in basal photolabeling of G-proteins with azidoanilido-[alpha-32P]GTP, and an increase in basal adenylyl cyclase activity in intact cells. Therefore, mu-opioid receptors are constitutively active in intact cells, producing stimulation of G-proteins and inhibition of adenylyl cyclase. Importantly, photolabeling of Galpha-subunits with azidoanilido-[alpha-32P]GTP demonstrated that constitutively active mu-opioid receptors activate individual G-proteins differently than the agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin.
Liu
J G
JG
Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Ruckle
M B
MB
Prather
P L
PL
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D002460:Cell Line; D005405:D-Ala(2),MePhe(4),Met(0)-ol-enkephalin / Q000494:pharmacology*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D009271:Naltrexone / Q000031:analogs & derivatives* / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000378:metabolism / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D017450:Receptors, Opioid, mu; D010566:Virulence Factors, Bordetella; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009271:Naltrexone; D005405:D-Ala(2),MePhe(4),Met(0)-ol-enkephalin; D011189:Potassium Chloride; C024524:beta-funaltrexamine; D019204:GTP-Binding Proteins