Effects of partial agonists and Mg2+ ions on the interaction of M2 muscarinic acetylcholine receptor and G protein Galpha i1 subunit in the M2-Galpha i1 fusion protein.

We have expressed a M(2)-Galpha(i1) fusion protein in insect cells, in which the G protein alpha(i1) subunit was fused with a mutant of the muscarinic receptor M(2) subtype without glycosylation sites and the central part of the third intracellular loop. The M(2)-Galpha(i1) fusion protein showed GTP-sensitive, high-affinity agonist binding. Displacement curves by GDP of [(35)S]GTPgammaS binding shifted to the right in the presence of muscarinic agonists. The extent of the shift was greater for full agonists (120-150 fold) than for partial agonists (25-35 fold), and virtually no shift was observed for antagonists. The affinity for GDP decreased with increasing MgCl(2) concentration in the presence of an agonist but was not affected by MgCl(2) in the presence of an antagonist. These results indicate that the apparent affinity for GDP of the M(2)-Galpha(i1) fusion protein bound to a ligand represents the efficacy of the given ligand, and that Mg(2+) is required for the agonist-bound M(2) to interact with Galpha(i1), reducing its affinity for GDP. We propose that the agonist-M(2)-Galpha(i1) complex represents the transition state for the GDP-GTP exchange reaction catalyzed by agonist-bound receptors, and that the complex has different affinities for GDP depending on the species of the ligand bound to M(2) receptors.