Intensive mutational analysis of G protein-coupled receptors in yeast

Expression of G protein-coupled receptors (GPCRs) in yeast makes possible a genetic procedure for determining the range of amino-acid substitutions that are compatible with function in particular receptor regions. The regions of interest are targeted for intensive random mutagenesis, providing multiple amino-acid substitutions per allele. Genetic screening of the mutagenized receptors in yeast allows the identification of rare functional alleles, which can then be recovered, sequenced, and further characterized. Procedures for random oligonucleotide-directed mutagenesis, creation, and screening of mutational libraries in yeast, as well as quantitative assay of receptor function, are described.