EXPRESSION OF THE MU-OPIOID RECEPTOR IN CHO CELLS - ABILITY OF MU-OPIOID LIGANDS TO PROMOTE ALPHA-AZIDOANILIDO [P-32] GTP LABELING OF MULTIPLE G-PROTEIN ALPHA-SUBUNITS

The identities of heterotrimeric G proteins that can interact with the mu-opioid receptor were investigated by alpha-azidoanilido[P-32] GTP labeling of alpha subunits in the presence of opioid agonists in Chine se hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expresse d mu-opioid receptor cDNA (MOR-1), This clone expressed 1.01 x 10(6) m u-opioid receptors per cell and had higher binding affinity and potenc y to inhibit adenylyl cyclase for the mu-opioid-selective ligands [D-A la(2), N-MePhe(4), Gly-ol]-enkephalin and [N-MePhe(3), D-Pro(4)]-morph iceptin, relative to the delta-selective opioid agonist [D-Pen(2), D-P en(5)]-enkephalin or the kappa-selective opioid agonist U-50, 488H. mu -Opioid ligands induced an increase in alpha-azidoanilido [P-32]GTP ph otoaffinity labeling of four G alpha subunits in this clone, three of which were identified as G(i3)alpha G(i2)alpha, and G(o2)alpha) The sa me pattern of simultaneous interaction of the mu-opioid receptor with multiple G alpha subunits was also observed in two other clones, one e xpressing 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 de pendent, and blocked by naloxone and by pretreatment of these cells wi th pertussis toxin. A greater agonist-induced increase of alpha-azidoa nilido [P-32]GTP incorporation into G(i2)alpha (160-280%) and G(o2)alp ha (110-220%) than for an unknown G(alpha (G(?)alpha) (60%) Or G(i3)al pha (40%) was produced by three different mu-opioid ligands tested. In addition, slight differences were also found between the ability of v arious mu-opioid agonists to produce half-maximal labeling (ED(50)) of any given G alpha subunit, with a rank order of G(i3)alpha > G(o2)alp ha > G(i2)alpha = G(?)alpha In any case, these results suggest that th e activated mu-opioid receptor couples to four distinct G protein alph a subunits simultaneously.