Mosser_2002_J.Biol.Chem_277_922

A steady-state kinetic mechanism describing the interaction of M(2) muscarinic acetylcholine receptors and the guanine nucleotide-binding protein G(i)alpha(2)beta(1)gamma(3) are presented. Data are consistent with two parallel pathways of agonist-promoted GTPase activity arising from receptor coupled to a single or multiple guanine nucleotide-binding proteins. An aspartate 103 to asparagine receptor mutation resulted in a receptor lacking the ability to catalyze the binding of guanosine-5'-O-(3-thiotriphosphate) or guanosine triphosphate hydrolysis by the G protein. An aspartate 69 to asparagine receptor mutant was able to catalyze agonist-specific guanine nucleotide exchange and GTPase activity. A threonine 187 to alanine receptor mutation resulted in a receptor that catalyzed guanine nucleotide exchange comparable with wild-type receptors but had reduced ability to stimulate GTP hydrolysis. A tyrosine 403 to phenylalanine receptor mutation resulted in an increase in agonist-promoted GTPAse activity compared with wild type. The observation that the threonine 187 and tyrosine 403 mutants promote guanine nucleotide exchange similarly to wild type but alter GTPase activity compared with wild type suggests that the effects of the mutations arise downstream from guanine nucleotide exchange and may result from changes in receptor-G protein dissociation.