G protein-coupled receptor internalization assays in the high-content screening format.

Abstract High‐content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease‐modeling systems. This chapter describes the measurement of G protein‐coupled receptor (GPCR) internalization in the HCS format using a high‐throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand‐induced internalization of GPCRs labeled previously with either a fluorophore‐conjugated ligand or an antibody directed against an N‐terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high‐throughput screening format to determine the cellular activity of GPCR ligands.