title: Techniques: how to boost GPCR mutagenesis studies using yeast.
abstract: G-protein-coupled receptors (GPCRs) are the major targets of today's medicines. To elucidate the mechanism of activation of GPCRs and the interaction of these receptors with their G proteins, mutagenesis studies have proven to be a powerful tool and have provided insight into the structure and function of GPCRs. Random mutagenesis is useful in this respect particularly when combined with a robust screening assay that is based on the functional properties of the mutants. In this article, the use of random mutagenesis combined with a functional screening assay in yeast is described and compared with alternative approaches such as site-directed mutagenesis per se, alanine/cysteine scanning and another screening assay, receptor selection and amplification technology (R-SAT). Screening in yeast of randomly mutated GPCRs has proven successful in the identification of ligands for orphan receptors and in high-throughput approaches. Moreover, it has provided substantial insight into G-protein coupling and receptor activation.
authors.affiliation: Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Einsteinweg 55, 2300 CC Leiden, The Netherlands. beukers@chem.leidenuniv.nl
authors.forename: Margot W
authors.initials: MW
authors.lastname: Beukers
authors.forename: Adriaan P
authors.initials: AP
authors.lastname: Ijzerman
mesh_terms: D000818:Animals; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D016296:Mutagenesis; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism
chemical_list: D043562:Receptors, G-Protein-Coupled