Abstract

An exceptionally conserved sequence that is shared among most G protein-coupled neurotransmitter receptors is an aspartate-arginine-tyrosine triplet that is located at the amino terminus of the putative second cytoplasmic domain, where the arginine residue is invariant. Using the m1 subtype of muscarinic acetylcholine receptors as an example, we induced a point mutation of the arginine residue at position 123 into asparagine. This mutation resulted in a precipitous decrease in the coupling of m1 receptors to activation of phosphoinositide hydrolysis, in spite of the expression of the wild-type and mutant receptors at similar concentrations in Chinese hamster ovary cells. There were no significant effects on antagonist or partial agonist binding. In marked contrast, whereas binding of the full agonist carbachol to wild-type receptors exhibited high and low affinity components, this agonist bound to a single low affinity state in asparagine-123 mutant cells. Furthermore, agonist-induced enhancement of the specific binding of guanosine-5'-O-(3-[35S] thio)triphosphate was not observed in membranes of cells expressing the mutant receptor. A similar mutation in the m2 muscarinic receptor resulted in a significant but smaller decrease in its coupling to inhibition of cAMP formation. On the other hand, a point mutation of tyrosine-124 in the m1 receptor sequence produced less marked changes in agonist-induced phosphoinositide hydrolysis and no effects on agonist or antagonist binding to the receptor. Taken together, our data demonstrate for the first time that this highly conserved arginine residue plays an important role in coupling of muscarinic receptors to signal transduction mechanisms.