High-content screening of known G protein-coupled receptors by arrestin translocation.

G protein-coupled receptors (GPCRs) have proven to be one of the most successful target classes for drug discovery. Accordingly, many assays are available to screen GPCRs, including radioactive-binding assays, second messenger signaling assays, and downstream reporter assays. One of the more novel approaches is the Transfluor technology, a cell-based assay that uses a detectable tag on a cytosolic protein, called arrestin, that is involved in the desensitization or inactivation of GPCRs. Monitoring the translocation of GFP-tagged arrestin from the cytosol to activated GPCRs at the plasma membrane measures the pharmacological effect of test compounds that bind the receptor target. Moreover, the Transfluor assay provides further, high-content information on the test compound itself and its effects on cell processes due to the fluorescent imaging of whole cells used in this screen. Screening known GPCRs with Transfluor against large compound libraries is best accomplished in cell lines stably expressing an optimum level of the target receptor. This chapter describes how to generate a clonal cell line stably expressing the known GPCR with suitable Transfluor properties. It then describes the steps involved in performing a Transfluor screen and discusses high content data resulting from the screen.