ALANINE SCANNING MUTAGENESIS OF CONSERVED ARGININE LYSINE-ARGININE/LYSINE-X-X-ARGININE/LYSINE G-PROTEIN-ACTIVATING MOTIFS ON M1 MUSCARINIC ACETYLCHOLINE-RECEPTORS/

Alanine scanning mutagenesis of B-B-X-X-B motifs (where B is a basic r esidue and X is any nonbasic residue) in mi muscarinic acetylcholine r eceptors was performed to determine the relative roles of basic amino acids in receptor coupling. This conserved motif is found in many G pr otein-coupled receptors and has been implicated in G protein activatio n. The KKAAR365 motif, located at the carboxyl-terminal third intracel lular loop of mi receptors, was mutated to AAAAA365, thereby generatin g a triple-substitution mutant devoid of ability to stimulate either p hosphoinositide (PI) hydrolysis or cAMP accumulation. In contrast, a t riple-alanine substitution of the KRTPR140 motif in the carboxyl-termi nal second intracellular loop, yielding mutant AATPA140, had no effect on receptor coupling to the two independent second messenger pathways . Analysis of a series of single- and double-substitution mutants demo nstrate that all three basic residues of the KKAAR365 motif participat e in efficient mi receptor coupling. The presence of second and third basic residues in this motif was absolutely critical for full agonist recognition of a high and low affinity state of the receptor. Mutation of either Lys362 or Lys365, but not Lys361, abolished guanine nucleot ide-dependent conversion of agonist affinity states and correlated wit h an inability of full agonists to fully activate Pi hydrolysis. The d ifferent combinatorial double-substitution mutants also revealed that Lys365 was necessary but not sufficient, in the context of the KKAAR36 5 motif, for efficient receptor coupling. This residue cannot facilita te full agonist-stimulated Pi hydrolysis in the absence of both Lys361 and Lys362. In comparison, the critical residue Lys362 was both neces sary and sufficient. Substitution of nearby basic residues Lys361 and Lys365 with alanine yielded mutant AKAAA365, which exhibited partial a bility to couple Pi hydrolysis after full agonist stimulation. Therefo re, Lys365 seems to function in a hierarchal (interdependent) manner w ith nearby basic residues, whereas Lys361 and Lys362 can act independe nt of surrounding basic residues to facilitate partial mi receptor cou pling after full agonist stimulation. In contrast, all three residues must be present for stimulation of PI hydrolysis by a partial agonist.