A remarkable potential exists for current and future development of therapeutic drugs acting at GPCRs. As one of the initial steps in GPCR drug development, in vitro assays are required to characterize the pharmacology of new ligands acting at distinct GPCRs. This is routinely accomplished by first employing cellular models to establish high affinity, selectivity, and efficacy of a test compound for a specific GPCR involved in a disease process of interest. However, several limitations are encountered when native cell lines or isolated tissues expressing low levels of endogenous GCPRs are employed for receptor characterization. To overcome many of these issues, cells are routinely transfected with cDNA of a desired GPCR to create cell lines stably expressing a sufficient receptor density to allow for adequate pharmacological studies. Although several commercial suppliers offer stably transfected cell lines expressing various GPCRs, as well as kits to examine several signal transduction pathways regulated by GPCRs, the cost and specialized equipment required to conduct these essential studies are often out of reach for many laboratories. Therefore, the purpose of this chapter is to provide a brief, simple, economical, and straightforward guide for the production of a cell line stably expressing a GPCR of interest and the methods required to characterize the basic pharmacology of ligands acting at that receptor. Specifically, methods will describe how to stably transfect cells and to conduct receptor binding studies for determination of receptor density and ligand affinity. Finally, methods will be presented to subsequently characterize the functional signaling of the expressed GPCR, from G-protein activation to regulation of two distinct intracellular effectors.
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