Rapid identification of functionally critical amino acids in a G protein-coupled receptor.
G protein-coupled receptors (GPCRs) comprise one of the largest protein families found in nature. Here we describe a new experimental strategy that allows rapid identification of functionally critical amino acids in the rat M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR. This approach involves low-frequency random mutagenesis of the entire M3R coding sequence, followed by the application of a new yeast genetic screen that allows the recovery of inactivating M3R single point mutations. The vast majority of recovered mutant M3Rs also showed substantial functional impairments in transfected mammalian (COS-7) cells. A subset of mutant receptors, however, behaved differently in yeast and mammalian cells, probably because of the specific features of the yeast expression system used. The screening strategy described here should be applicable to all GPCRs that can be expressed functionally in yeast.
Li
Bo
B
Molecular Signaling, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA.
Scarselli
Marco
M
Knudsen
Christopher D
CD
Kim
Soo-Kyung
SK
Jacobson
Kenneth A
KA
McMillin
Sara M
SM
Wess
Jürgen
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D051381:Rats; D043587:Receptor, Muscarinic M3 / Q000737:chemistry* / Q000235:genetics*; D012441:Saccharomyces cerevisiae
D000596:Amino Acids; D043587:Receptor, Muscarinic M3