Quantification of G-protein coupled receptor internalization using G-protein coupled receptor-green fluorescent protein conjugates with the ArrayScan(TM) high-content screening system

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent homologou s desensitization and internalization. Desensitization, defined as a decrea se in the responsiveness to ligand, is accompanied by receptor aggregation on the cell surface and internalization via clathrin-coated pits to an intr acellular endosomal compartment, In this study, we have taken advantage of the trafficking properties of GPCRs to develop a useful screening method fo r the identification of receptor mimetics, A series of studies were underta ken to evaluate the expression, functionality, and ligand-dependent traffic king of GPCR-green fluorescent protein (GFP) fusion conjugates stably trans fected into HEK 293 cells. These GPCR-GFP expressing cells were then utiliz ed in the validation of the ArrayScan(TM) (Cellomics(TM), Pittsburgh, PA), a microtiter plate imaging system that permits cellular and subcellular qua ntitation of fluorescence in whole cells. These studies demonstrated our ab ility to measure the internalization of a parathyroid hormone (PTH) recepto r-GFP conjugate after ligand treatment by spatially resolving internalized receptors. Internalization was time- and dose-dependent and appeared to be selective for PTH, Similar results were obtained for a beta(2)-adrenergic r eceptor (beta(2) AR)-GFP conjugate stably expressed in HEK 293 cells. The i nternalized GFP-labeled receptors were visualized as numerous punctate "spo ts" within the cell interior, An algorithm has been developed that identifi es and collects information about these spots, allowing quantification of t he internalization process. Variables such as the receptor-GFP expression l evel, plating density, cell number per field, number of fields scanned per well, spot size, and spot intensity were evaluated during the development o f this assay, The method represents a valuable tool to screen far receptor mimetics and antagonists of receptor internalization in whole cells rapidly .