title: High-throughput confocal microscopy for beta-arrestin-green fluorescent protein translocation G protein-coupled receptor assays using the Evotec Opera.
abstract: Ligand-activated G protein-coupled receptors (GPCRs) are known to regulate a myriad of homeostatic functions. Inappropriate signaling is associated with several pathophysiological states. GPCRs belong to a approximately 800 member superfamily of seven transmembrane-spanning receptor proteins that respond to a diversity of ligands. As such, they present themselves as potential points of therapeutic intervention. Furthermore, orphan GPCRs, which are GPCRs without a known cognate ligand, offer new opportunities as drug development targets. This chapter describes a systems-based biological approach, one that combines in silico bioinformatics, genomics, high-throughput screening, and high-content cell-based confocal microscopy strategies to (1) identify a relevant subset of protein family targets, (2) within the therapeutic area of energy metabolism/obesity, (3) and to identify small molecule leads as tractable combinatorial and medicinal chemistry starting points. Our choice of screening platform was the Transfluor beta-arrestin-green fluorescent protein translocation assay in which full-length human orphan GPCRs were stably expressed in a U-2 OS cell background. These cells lend themselves to high-speed confocal imaging techniques using the Evotec Technologies Opera automated microscope system. The basic assay system can be implemented in any laboratory using a fluorescent probe, a stably expressed GPCR of interest, automation-assisted plate and liquid-handling techniques, an optimized image analysis algorithm, and a high-speed confocal microscope with sophisticated data analysis tools.
authors.affiliation: Roche Discovery Technologies, Roche Inc., Nutley, NJ, USA.
authors.forename: Ralph J
authors.initials: RJ
authors.lastname: Garippa
authors.forename: Ann F
authors.initials: AF
authors.lastname: Hoffman
authors.forename: Gabriele
authors.initials: G
authors.lastname: Gradl
authors.forename: Achim
authors.initials: A
authors.lastname: Kirsch
mesh_terms: D000465:Algorithms; D000818:Animals; D019390:Arrestins / Q000737:chemistry*; D001331:Automation; D019295:Computational Biology / Q000379:methods; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008024:Ligands; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods*; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D012984:Software; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins