ABSTRACT
The interaction between nuclear receptors (NRs) and their coactivators is a key step in transcription regulation. AlphaScreen
(Amplified Luminescent Proximity Assay) is a nonradioactive homogeneous proximity assay that takes advantage of the receptor/coactivator interaction to identify and characterize compounds modulating NR activity. In these sections, estrogen receptor
α
(ER
α
) and
retinoic acid receptors
γ
(RAR
γ
) were chosen as models to demonstrate the different steps leading to the development of an AlphaScreen assay for NRs. The assays presented are performed with either a full-length receptor or a tagged receptor ligand binding domain (LBD) each interacting with a biotinylated peptide derived from a coactivator interacting domain sequence. The assays can differentiate between ligands that act as agonists vs. antagonists, since only agonists will allow recruitment of the coactivator sequence-derived peptide. The assay configuration offers flexibility to screen for antagonists targeting the ligand binding site in the ligand binding pocket of the LBD or the interaction interface between receptor and coactivator.
