Cell-based, high-content screen for receptor internalization, recycling and intracellular trafficking

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 recepto rs (GPCRs). The internalization of GPCRs is a result of agonist stimulation . A cell-based fluorescent imaging assay is described that detects and quan tifies the presence of fluorescently labeled receptors and macromolecules i n the recycling compartment. This High Content Screening application is con ducted on the ArrayScan((R)) II System that includes fluorescent reagents, imaging instrumentation and the informatics tools necessary to screen for c ompounds that affect receptor internalization, recycling and GPCR activatio n. We demonstrate the Receptor Internalization and Trafficking application by quantifying (i) the internalization and recycling of the transferrin rec eptor 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 rang es between stimulated and unstimulated conditions and low variability acros s different screening runs. Thus, the Receptor Internalization and Traffick ing application, in conjunction with the ArrayScan II System, forms the bas is of a robust, information-rich and automated screen for GPCR activation.