Defining the functional equivalence of wild-type and chemically engineered G protein-coupled receptors

The functional connection between activation of a single G protein-coupled receptor (GPCR) and a specific physiological outcome may be obscured by the presence of other closely related members of the GPCR superfamily that share spatiotemporal patterns of expression and equipotent activation by endogenous ligands. To address this issue, molecular and chemical genetic techniques have been developed to generate mutationally modified GPCRs only susceptible to activation by one or more synthetic ligands that are inactive at the equivalent wild-type receptor. This chapter provides both an overview of strategies used to generate such “receptors activated solely by synthetic ligands” (RASSLs) and, in more detail, approaches that have been used to assess the equivalence or otherwise of function of a wild-type and corresponding RASSL receptor. The human muscarinic M3 acetylcholine receptor is used as the exemplar but similar preliminary studies should be employed before further use, particularly in vivo, of such RASSLs