Fig. 2.

The DNA-binding mutant ERα(GSCKV) activates expression of a GRE-containing reporter gene. (A) Schematic diagram showing the ERE-Luc, GRE-Luc, and two chimeric reporter genes. (B) The amino acid sequence of the DBD of wild-type ERα (ERα−wt) and DNA-binding mutant ERα(GSCKV). The gray circles indicate the three amino acids that were mutated to enable binding to a GRE. (C) HepG2 cells cotransfected with 1 μg of the indicated reporter genes and 50 ng of expression vectors as indicated were treated with vehicle (Veh; 0.1% ethanol), 10 nM E2, or 10 nM CE for 24 hours. Data are presented as luciferase activity relative to their respective vehicle-treated control. (D) HepG2 cells were cotransfected with 1 μg of EGRE-Luc and ERα-wt (25 ng), ERα(GSCKV) (25 ng), or both [25 ng of ERα-wt + 25 ng ERα(GSCKV)] plasmids, and treated with vehicle or 10 nM CE for 24 hours. The luciferase activity of 10 nM CE-treated ERα-wt + ERα(GSCKV) cotransfected cells was set to 100. Values represent the average ± S.E.M.; (n = 3) for all experiments.