Activation of guanosine 5 '-[gamma-S-35]thio-triphosphate binding through M1 muscarinic receptors in transfected Chinese hamster ovary cell membranes: 2. Testing the "two-states" model of receptor activation

I suggested in the accompanying article [Mol Pharmacol 2001; 59:875-885] th at muscarinic receptors catalyzed G protein activation. Acetylcholine or ca rbamylcholine recognition facilitated not only the GDP release from recepto r-coupled inactive G proteins but also the release of G(GTP gammaS)* from t he (unstable) HRG(GTP gammaS)* complex. The two effects facilitated [S-35]G TP gammaS binding in the presence of GDP, but could be studied separately b y comparing [S-35]GTP gammaS binding in the absence and presence of GTP. Gu anyl nucleotides affected the efficiency of receptor-G protein coupling. Th e relative efficacies of partial agonists in the absence and presence of GT P should remain nonlinearly correlated if all agonists stabilize (to differ ent extents) the same active receptor conformation. The correlation between M-1 muscarinic agonists' efficacy in accelerating [S-35]GTP gammaS binding in the absence of other nucleotides and their in vivo efficacy (inositol p hosphate accumulation) was in fact very poor. This probably reflected the p resence of GTP in intact cells: pertussis toxin pretreatment (which inactiv ates the G(i/o) proteins) did not affect the agonists' efficacy profile (ev aluated in the absence of spare receptors), but the addition of GTP to the [S-35]GTP gammaS binding medium did. These results did not support the allo steric "two states" model of receptor activation, but suggested that differ ent agonists induced different receptor conformations ("induced fit").