G protein-coupled receptor internalization assays in the high-content screening format.
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.
Haasen
Dorothea
D
Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.
Schnapp
Andreas
A
Valler
Martin J
MJ
Heilker
Ralf
R
D000818:Animals; D001331:Automation; D019411:Clinical Laboratory Techniques; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008024:Ligands; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008954:Models, Biological; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*
D008024:Ligands; D043562:Receptors, G-Protein-Coupled