SITE-DIRECTED MUTAGENESIS ON THE M2 MUSCARINIC ACETYLCHOLINE-RECEPTOR- THE SIGNIFICANCE OF TYR403 IN THE BINDING OF AGONISTS AND FUNCTIONAL COUPLING

The first step in the transmembrane signal mediated by G protein-coupl ed receptors is binding of agonist to receptors at the cell surface. T he mechanism of the resulting receptor activation is not clear, but mo dels based on the ternary complex model are capable of explaining most of the observations that have been reported in G protein-coupled rece ptors. This model suggests that a single agonist/receptor/G protein co mplex capable of activating G protein is formed as the result of agoni st binding. Extensions of this basic model differ primarily in whether an equilibrium between active and inactive conformations is required to explain experimental results. We report results on ligand binding a nd coupling to physiological effector systems of the m2 muscarinic ace tylcholine receptor site-directed mutant Y403F (residue 403 mutated fr om tyrosine to phenylalanine) expressed in Chinese hamster ovary cells and compare our results with results reported for the homologous Y506 F mutation in the m3 muscarinic receptor [J. Biol Chem. 267:19313-1931 9 (1992)]. The mutation in the m2 muscarinic receptor reduced absolute agonist affinities more dramatically than in the m3 muscarinic recept or. Unlike the results reported for the m3 subtype mutant, in which co upling to physiological effector systems was reduced, coupling to effe ctor systems for the mutant in the m2 subtype was robust. In the Y403F m2 muscarinic receptor, the difference between the two agonist bindin g affinities was greater than in the wild-type receptor, whereas in th e m3 subtype, the effect of the mutation was to decrease this differen ce. A prediction of the ternary complex model is that relative binding affinities will affect the steady state concentration of the agonist/ receptor/G protein complex and, as the result, the extent of G protein coupling. These results can best be rationalized by this model, which suggests that the activation of G protein-coupled receptors is achiev ed by the relative affinity of agonist for two receptor states and doe s not require the existence of multiple states in conformational equil ibrium.