Techniques: How to boost GPCR mutagenesis studies using yeast

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.