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

Abstract

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.