Development of assays for nuclear receptor modulators using fluorescently tagged proteins

This chapter describes a method for designing cell-based assays to screen for nuclear receptor modulators. The basic strategy consists in following the movement of the receptors from the cytoplasm into the nucleus in response to ligand binding or analogous activating events. The receptors are tagged with green fluorescent protein for automated, fluorescent detection. In the case of constitutively nuclear receptors, they are engineered for cytoplasmic retention in the absence of an activating signal by fusing them to specific regions of the glucocorticoid receptor, which is found predominantly in the cytoplasm of cultured cells. The resulting chimeras respond to ligands or receptor modulators by translocating into the nucleus. This movement is monitored easily by automated fluorescent microscopy and serves as the basis for screening libraries. Finally, secondary assays built into the cell system can differentiate between modulators that stimulate, inhibit, or do not affect the transcriptional activity of the receptor under study. This approach has been validated for both the estrogen receptor and the retinoic acid receptor and should be applicable to any member of the superfamily, facilitating the identification of new ligands and selective receptor modulators.