Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast. Identification of point mutations that "silence" a constitutively active mutant M3 receptor and greatly impair receptor/G protein coupling.
The M3 muscarinic receptor is a prototypical member of the class I family of G protein-coupled receptors (GPCRs). To facilitate studies on the structural mechanisms governing M3 receptor activation, we generated an M3 receptor-expressing yeast strain (Saccharomyces cerevisiae) that requires agonist-dependent M3 receptor activation for cell growth. By using receptor random mutagenesis followed by a genetic screen in yeast, we initially identified a point mutation at the cytoplasmic end of transmembrane domain (TM) VI (Q490L) that led to robust agonist-independent M3 receptor signaling in both yeast and mammalian cells. To explore further the molecular mechanisms by which point mutations can render GPCRs constitutively active, we subjected a region of the Q490L mutant M3 receptor that included TM V-VII to random mutagenesis. We then applied a yeast genetic screen to identify second-site mutations that could suppress the activating effects of the Q490L mutation and restore wild-type receptor-like function to the Q490L mutant receptor. This analysis led to the identification of 12 point mutations that allowed the Q490L mutant receptor to function in a fashion similar to the wild-type receptor. These amino acid substitutions mapped to two distinct regions of the M3 receptor, the exofacial segments of TM V and VI and the cytoplasmic ends of TM V-VII. Strikingly, in the absence of the activating Q490L mutation, all recovered point mutations severely reduced the efficiency of receptor/G protein coupling, indicating that the targeted residues play important roles in receptor activation and/or receptor/G protein coupling. This strategy should be generally applicable to identify sites in GPCRs that are critically involved in receptor function.
Molecular Signaling Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Clarice
C
Schmidt
Bo
B
Li
Lanh
L
Bloodworth
Isolde
I
Erlenbach
Fu-Yue
FY
Zeng
Jürgen
J
Wess
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D051381:Rats; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000737:chemistry* / Q000235:genetics*; D012243:Rhodopsin / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D015398:Signal Transduction
D008024:Ligands; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D002217:Carbachol; D012243:Rhodopsin; D019204:GTP-Binding Proteins