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.
Schmidt
Clarice
C
Molecular Signaling Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Li
Bo
B
Bloodworth
Lanh
L
Erlenbach
Isolde
I
Zeng
Fu-Yue
FY
Wess
Jürgen
J
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