The Cellular Distribution of Fluorescently Labeled Arrestins Provides a Robust,Sensitive,and Universal Assay for Screening G Protein-Coupled Receptors

Abstract

G protein-coupled receptors (GPCRs) have proven to be a rich source of therapeutic targets; therefore, finding compounds that regulate these receptors is a critical goal in drug discovery. The Transfluor™ technology utilizes the redistribution of fluorescently labeled arrestins from the cytoplasm to agonist-occupied receptors at the plasma membrane to monitor quantitatively the activation or inactivation of GPCRs. Here, we show that the Transfluor technology can be quantitated on the INCell Analyzer system (INCAS) using the vasopressin V₂ receptor (V₂R), which binds arrestin with high affinity, and the β₂-adrenergic receptor (β₂AR), which binds arrestin with low affinity. U2OS cells stably expressing an arrestin-green fluorescent protein conjugate and either the V₂R or the β₂AR were plated in 96-well plastic plates and analyzed by the INCAS at a screening rate of 5 min per plate. Agonist dose-response and antagonist dose-inhibition curves revealed signal-to-background ratios of approximately 25:1 and 8:1 for the V₂R and β₂AR, respectively. EC₅₀ values agreed closely with K _d values reported in the literature for the different receptor agonists. In addition, small amounts of arrestin translocation induced by sub-EC₅₀ doses of agonist were distinguished from the background noise of untreated cells. Furthermore, differences in the magnitude of arrestin translocation distinguished partial agonists from full agonists, and Z′ values for these ligands were >0.5. These data show that the Transfluor technology, combined with an automated image analysis system, provides a direct, robust, and universal assay for high throughput screening of known and orphan GPCRs.