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 motifis (where B is a basic residue and X is any nonbasic residue) in m1 muscarinic acetylcholine receptors was performed to determine the relative roles of basic amino acids in receptor coupling. This conserved motif is found in many G protein-coupled receptors and has been implicated in G protein activation. The KKAAR365 motif, located at the carboxyl-terminal third intracellular loop of m1 receptors, was mutated to AAAAA365, thereby generating a triple-substitution mutant devoid of ability to stimulate either phosphoinositide (PI) hydrolysis or cAMP accumulation. In contrast, a triple-alanine substitution of the KRTPR140 motif in the carboxyl-terminal 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 demonstrate that all three basic residues of the KKAAR365 motif participate in efficient m1 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 nucleotide-dependent conversion of agonist affinity states and correlated with an inability of full agonists to fully activate PI hydrolysis. The different combinatorial double-substitution mutants also revealed that Lys365 was necessary but not sufficient, in the context of the KKAAR365 motif, for efficient receptor coupling. This residue cannot facilitate full agonist-stimulated Pl hydrolysis in the absence of both Lys361 and Lys362. In comparison, the critical residue Lys362 was both necessary and sufficient. Substitution of nearby basic residues Lys361 and Lys365 with alanine yielded mutant AKAAA365, which exhibited partial ability to couple PI hydrolysis after full agonist stimulation. Therefore, Lys365 seems to function in a hierarchal (interdependent) manner with nearby basic residues, whereas Lys361 and Lys362 can act independent of surrounding basic residues to facilitate partial m1 receptor coupling after full agonist stimulation. In contrast, all three residues must be present for stimulation of PI hydrolysis by a partial agonist.
Lee
N H
NH
Institute for Genomic Research, Department of Molecular and Cellular Biology, Rockville, Maryland 20850, USA nhlee@tigr.org
Geoghagen
N S
NS
Cheng
E
E
Cline
R T
RT
Fraser
C M
CM
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D006224:Cricetinae; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019832:N-Methylscopolamine; D010095:Oxotremorine / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012602:Scopolamine Derivatives / Q000378:metabolism; D015290:Second Messenger Systems; D014162:Transfection
D017931:DNA Primers; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D012602:Scopolamine Derivatives; D010095:Oxotremorine; D011813:Quinuclidinyl Benzilate; D006160:Guanosine Triphosphate; D002217:Carbachol; D019204:GTP-Binding Proteins; D000409:Alanine; D019832:N-Methylscopolamine