THE ROLE OF THE ASPARTATE-ARGININE-TYROSINE TRIAD IN THE M1 MUSCARINIC RECEPTOR - MUTATIONS OF ASPARTATE-122 AND TYROSINE-124 DECREASE RECEPTOR EXPRESSION BUT DO NOT ABOLISH SIGNALING

An Asp-Arg-Tyr triad occurs in a majority of rhodopsin-like G protein- coupled receptors. The fully conserved Arg is critical for G protein a ctivation, but the function of the flanking residues is not well under stood. We expressed in COS-7 cells mi muscarinic receptors that were m utated at Asp122 and Tyr124. Most mutations at either position strongl y attenuated or prevented the expression of binding sites for the anta gonist [H-3]N-methylscopolamine. However, sites that were expressed di splayed unaltered affinity for the antagonist. Receptor protein, visua lized with a carboxyl-terminally directed antibody, was reduced but ne ver completely abolished. The effects of these mutations were partiall y reversed by the deletion of 129 amino acids from the third intracell ular loop of the receptor. In several cases, comparison of immunocytoc hemistry with binding measurements suggested the presence of substanti al amounts of inactive, presumably misfolded, receptor protein. Some o f the variants that bound [H-3]N-methylscopolamine underwent small cha nges in their affinities for acetylcholine. All retained nearly normal abilities to mediate an acetylcholine-induced phosphoinositide respon se. We propose that Asp122 and Tyr124 make intramolecular contacts who se integrity is important for efficient receptor folding but that they do not participate directly in signaling. The role of these residues is completely distinct from that of Arg123, whose mutation abolishes s ignaling without diminishing receptor expression.