FIGURE 5.
Determination of ligand potency for recruitment of SRC3 (ligand titration assay to determine RRP). In these assays, the recruitment of fluorescein-labeled SRC3 (100 nm) to the terbium-labeled LBDs of ERα or ERβ (1 nm) was evaluated as a function of increasing concentrations of different estrogenic compounds. The binding curves of control diffusion-enhanced FRET-corrected specific FRET values were normalized (with maximal tr-FRET values from 17β-E2 and different ligands set equal to 100%) and plotted against the log ligand concentrations, and the data were analyzed by nonlinear regression with an equation for the sigmoidal dose response (variable slope) in GraphPad Prism 4. Each experiment was repeated three times with replicates, and the results of a representative experiment are shown. The concentrations of each ligand that promoted 50% of maximal SRC3 recruitment (EC50) were obtained from the binding curves of three experiments and are expressed as mean EC50 ± S.D. in Table 3, and these values are also expressed relative to 17β-E2 as RRP values. Binding curves with various ligands are as follows: in the presence of 17β-E2, EE2, DMS, DES, and meso-Hex with ERα (A) and ERβ (B); in the presence of 17β-E2, E1, E3, Equ, Eqn, and Gen with ERα (C) and ERβ (D); and in the presence of 17β-E2, 17α-E2, and dl-Hex with ERα (E) and ERβ (F). Error bars, S.D.