Development of a coactivator displacement assay for the orphan receptor estrogen-related receptor-γ using time-resolved fluorescence resonance energy transfer

The estrogen-related receptor-γ (ERRγ) is a constitutively active orphan receptor that belongs to the nuclear receptor superfamily and is most closely related to the estrogen receptors. Although its physiological ligand is unknown, ERRγ has been shown to interact with synthetic estrogenic compounds such as 4-hydroxytamoxifen (4-OHT), tamoxifen, and diethylstilbestrol (DES). To assess how coregulator proteins interact with ERRγ in response to ligand, an in vitro interaction methodology using time-resolved fluorescence resonance energy transfer (TR–FRET) was developed using glutathione S-transferase (GST)-tagged ERRγ ligand-binding domain (LBD), a terbium-labeled anti-GST antibody, a fluorescein-labeled peptide containing sequences derived from coregulator proteins, and various ligands. An initial screen of these coregulator peptides bearing the coactivator LXXLL motif, the corepressor LXXI/HIXXXI/L motif, or other interaction motifs from natural coactivator sequences or random phage display peptides indicated that the peptides PGC1α, D22, and SRC1-4, known as class III coregulators, interacted most strongly with ERRγ in the absence of ligand. Given its assay window and biological relevance in energy metabolism and obesity, further studies were conducted with PGC1α. Fluorescein-labeled PGC1α peptide was displaced from the ERRγ LBD in the presence of increasing concentrations of 4-OHT and tamoxifen, but DES was less effective in PGC1α displacement. The statistical parameter Z′ factor that measures the robustness of the assay was greater than 0.8 for displacement of PGC1α from ERRγ LBD in the presence of saturating 4-OHT over an assay incubation time of 1–6 h, indicating an excellent assay. These findings also suggest that binding of 4-OHT, tamoxifen, or DES to ERRγ results in differential affinity of coregulators for ERRγ due to unique ligand-induced conformations.