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

J Biol Chem. 2001 Oct 12;276(41):37779-86. doi: 10.1074/jbc.M106104200. Epub 2001 Aug 10.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenylate Cyclase Toxin
  • Adenylyl Cyclase Inhibitors*
  • Cell Line
  • D-Ala(2),MePhe(4),Met(0)-ol-enkephalin / pharmacology*
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / pharmacology
  • GTP-Binding Proteins / metabolism*
  • Naltrexone / analogs & derivatives*
  • Naltrexone / pharmacology
  • Potassium Chloride / pharmacology
  • Protein Binding
  • Receptors, Opioid, mu / agonists
  • Receptors, Opioid, mu / metabolism
  • Receptors, Opioid, mu / physiology*
  • Virulence Factors, Bordetella / pharmacology


  • Adenylate Cyclase Toxin
  • Adenylyl Cyclase Inhibitors
  • Receptors, Opioid, mu
  • Virulence Factors, Bordetella
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
  • Naltrexone
  • D-Ala(2),MePhe(4),Met(0)-ol-enkephalin
  • Potassium Chloride
  • beta-funaltrexamine
  • GTP-Binding Proteins