G-protein-coupled receptors (GPCR) participate in many disease pathways and represent the largest family of therapeutic targets. Thus, great investments are made to discover drugs modulating GPCR-mediated events. Among functional assays for screening GPCRs, the Transfluor ® imaging assay is based on redistribution of cytosolic β-arrestin to an activated GPCR and has become widely used in high-content screening. However, assessing Transfluor ® alone has limitations: relying on a single mechanistic step of β-arrestin redistribution during GPCR activation, providing no information on the stimulated GPCR's intracellular fate, and using only a single fluorescent color (green fluorescent protein). Taking full advantage of high-content imaging to screen approximately 2000 compounds, the authors multifplexed the Transfluor ® assay with an immunofluorescence-based quantification of GPCR internalization. This approach identified and classified 377 compounds interfering with agonistinduced activation of the Transfluor ® assay, receptor internalization, or both. In addition, a subset of compounds was analyzed for their performance across imaging, cell-based calcium release (fluorometric imaging plate reader [FLIPR]), and biochemical receptor binding assays (scintillation proximity assay). This indicated that the imaging assays have even better predictive power for direct inhibition of receptor binding than the FLIPR assay. In conclusion, compounds inducing unique responses can suggest novel mechanisms of action and be used as tools to study GPCR activation and internalization. (Journal of Biomolecular Screening 2008:449-455)

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