Phosphorylation Validation of a Cell-Based Screen for Insulin Receptor Modulators by Quantification of Insulin Receptor

Stimulation of a cell with insulin initiates a signal transduction cascade that results in cellular activities that includephosphorylation of the receptor itself. Measurement of the degree of phosphorylation can serve as a marker for receptor acti -vation.ReceptorphosphorylationhasbeenmeasuredusingWesternblotanalysis,whichisverylowthroughputandnoteasilyquantifiable. The goal of this project was to develop a cell-based assay to measure receptor phosphorylation in high through -put. This report describes a cell-based assay for insulin receptor phosphorylation that is robust and amenable to high-volumescreening in a microwell format. ( Journal of Biomolecular Screening 2003:439-446)Key words: insulin, receptor phosphorylation, cell-based assays, fluorescence resonance energy transferINTRODUCTION