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-coupled receptors is binding of agonist to receptors at the cell surface. The mechanism of the resulting receptor activation is not clear, but models based on the ternary complex model are capable of explaining most of the observations that have been reported in G protein-coupled receptors. This model suggests that a single agonist/receptor/G protein complex capable of activating G protein is formed as the result of agonist 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 and coupling to physiological effector systems of the m2 muscarinic acetylcholine receptor site-directed mutant Y403F (residue 403 mutated from tyrosine to phenylalanine) expressed in Chinese hamster ovary cells and compare our results with results reported for the homologous Y506F mutation in the m3 muscarinic receptor [J. Biol. Chem. 267:19313-19319 (1992)]. The mutation in the m2 muscarinic receptor reduced absolute agonist affinities more dramatically than in the m3 muscarinic receptor. Unlike the results reported for the m3 subtype mutant, in which coupling to physiological effector systems was reduced, coupling to effector systems for the mutant in the m2 subtype was robust. In the Y403F m2 muscarinic receptor, the difference between the two agonist binding affinities was greater than in the wild-type receptor, whereas in the m3 subtype, the effect of the mutation was to decrease this difference. 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 achieved by the relative affinity of agonist for two receptor states and does not require the existence of multiple states in conformational equilibrium.

Porcine m2 muscarinic acetylcholine receptor-effector coupling in Chinese hamster ovary cells.

The relationship between porcine m2 muscarinic receptor coupling to inhibition of cAMP formation and stimulation of phosphatidylinositol metabolism in Chinese hamster ovary cells was examined. Reduction of the number of receptors per cell with the slowly dissociating antagonist (-)-quinuclidinyl benzilate caused a decrease in maximal response with no effect on EC50 for coupling to phosphatidylinositol metabolism. Inhibition of cAMP formation showed the opposite dependence with no effect on maximal response but an increase in EC50 value as receptor density decreased. Pilocarpine appeared to be a partial agonist at low cell receptor density but displayed full agonism at higher receptor density. These results are compatible with a two-state model describing m2 muscarinic receptor acting via two different G proteins. This model is compatible with observations of negative antagonism where antagonists stimulated cAMP formation in adenylyl cyclase inhibition assays, and can also be used to estimate receptor affinities for G proteins in systems which display negative antagonism.

Kinetic and biophysical analysis of the m2 muscarinic receptor.

The recombinant Pm2 muscarinic receptor expressed in Chinese hamster ovary (CHO) cells was used as a model system to examine receptor-effector coupling and ligand binding. In CHO cells, equilibrium binding studies and the dependence on receptor number per cell of the maximum response and EC50 values for agonist stimulation of phosphatidylinositol metabolism and inhibition of cAMP formation were consistent with a modified ternary complex model of signal transduction that included a physiologically noncompetent receptor state. Detailed kinetic studies of oxotremorine M (Oxo-M) binding to CHO cell membranes suggested that agonist interactions at the high affinity class of binding sites are complicated and depend on receptor expression levels. At low levels of expression, kinetic data were consistent with a special case of a mechanism in which Oxo-M shifts the equilibrium between two receptor conformations while at high levels of expression, it was necessary to evoke receptor-receptor interactions to explain the kinetic data. Far ultraviolet circular dichroism studies of the purified recombinant receptor showed a high content of alpha-helical secondary structure and small changes in secondary structure upon antagonist, but not agonist, binding.