title: Cell-based, high-content screen for receptor internalization, recycling and intracellular trafficking.
abstract: A variety of physiologically important receptors are internalized and then recycled back to the plasma membrane by the endocytic recycling compartment. These include the transferrin receptor and many G-protein coupled receptors (GPCRs). The internalization of GPCRs is a result of agonist stimulation. A cell-based fluorescent imaging assay is described that detects and quantifies the presence of fluorescently labeled receptors and macromolecules in the recycling compartment. This High Content Screening application is conducted on the ArrayScan II System that includes fluorescent reagents, imaging instrumentation and the informatics tools necessary to screen for compounds that affect receptor internalization, recycling and GPCR activation. We demonstrate the Receptor Internalization and Trafficking application by quantifying (i) the internalization and recycling of the transferrin receptor using a fluorescently labeled ligand and (ii) the internalization of a physiologically functional model GPCR, a GFP-parathyroid hormone receptor chimera. These assays give high signal-to-noise ratios, broad dynamic ranges between stimulated and unstimulated conditions and low variability across different screening runs. Thus, the Receptor Internalization and Trafficking application, in conjunction with the ArrayScan II System, forms the basis of a robust, information-rich and automated screen for GPCR activation.
authors.affiliation: Cellomics Inc., Pittsburgh, PA, USA.
authors.forename: R N
authors.initials: RN
authors.lastname: Ghosh
authors.forename: Y T
authors.initials: YT
authors.lastname: Chen
authors.forename: R
authors.initials: R
authors.lastname: DeBiasio
authors.forename: R L
authors.initials: RL
authors.lastname: DeBiasio
authors.forename: B R
authors.initials: BR
authors.lastname: Conway
authors.forename: L K
authors.initials: LK
authors.lastname: Minor
authors.forename: K T
authors.initials: KT
authors.lastname: Demarest
mesh_terms: D000818:Animals; D019556:COS Cells; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002460:Cell Line; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D018016:Receptors, Parathyroid Hormone / Q000235:genetics / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011503:Transglutaminases / Q000037:antagonists & inhibitors
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D018016:Receptors, Parathyroid Hormone; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011503:Transglutaminases; C008542:monodansylcadaverine; D002103:Cadaverine