Expression of the mu-opioid receptor in CHO cells: ability of mu-opioid ligands to promote alpha-azidoanilido[32P]GTP labeling of multiple G protein alpha subunits

J Neurochem. 1995 Jun;64(6):2534-43. doi: 10.1046/j.1471-4159.1995.64062534.x.


The identities of heterotrimeric G proteins that can interact with the mu-opioid receptor were investigated by alpha-azidoanilido[32P]GTP labeling of alpha subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed mu-opioid receptor cDNA (MOR-1). This clone expressed 1.01 x 10(6) mu-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the mu-opioid-selective ligands [D-Ala2,N-MePhe4, Gly-ol]-enkephalin and [N-MePhe3,D-Pro4]-morphiceptin, relative to the delta-selective opioid agonist [D-Pen2,D-Pen5]-enkephalin or the kappa-selective opioid agonist U-50,488H. mu-Opioid ligands induced an increase in alpha-azidoanilido[32P]GTP photoaffinity labeling of four G alpha subunits in this clone, three of which were identified as Gi3 alpha, Gi2 alpha, and Go2 alpha. The same pattern of simultaneous interaction of the mu-opioid receptor with multiple G alpha subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of alpha-azidoanilido[32P]GTP incorporation into Gi2 alpha (160-280%) and Go2 alpha (110-220%) than for an unknown G alpha (G? alpha) (60%) or Gi3 alpha (40%) was produced by three different mu-opioid ligands tested. In addition, slight differences were also found between the ability of various mu-opioid agonists to produce half-maximal labeling (ED50) of any given G alpha subunit, with a rank order of Gi3 alpha > Go2 alpha > Gi2 alpha = G? alpha. In any case, these results suggest that the activated mu-opioid receptor couples to four distinct G protein alpha subunits simultaneously.

Publication types

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

MeSH terms

  • Affinity Labels
  • Animals
  • Azides*
  • CHO Cells / metabolism*
  • Cricetinae
  • GTP-Binding Proteins / classification
  • GTP-Binding Proteins / metabolism*
  • Guanosine Triphosphate / analogs & derivatives*
  • Ligands
  • Phosphorus Radioisotopes
  • Receptors, Opioid, mu / genetics
  • Receptors, Opioid, mu / metabolism*
  • Transfection


  • Affinity Labels
  • Azides
  • Ligands
  • Phosphorus Radioisotopes
  • Receptors, Opioid, mu
  • GTP gamma-4-azidoanilide
  • Guanosine Triphosphate
  • GTP-Binding Proteins