Anti-estrogen stimulation is highly sensitive to mutations at K206. (A) Results of a HeLa cell transfection in which the phenotype of ERα and ERα.K206A are compared with other ERα.K206 mutants. Note that ERα.K206A shows superactivity in the presence of E2 and little or no activity in the presence of anti-estrogens. Other K206 mutations, with the exception of K206 mutation to a like amino acid (K206R), convert anti-estrogen stimulation to repression despite the lack of E2-associated superactivity. (B) Alterations in ERα steady state levels do not account for altered phenotypes. HeLa cells were transfected with 10 μg of each expression vector, induced with appropriate ligands for 24 hrs and processed for Western blotting. The panels show individual exposures of autoradiograms of Western blots of HeLa cell extracts transfected with individual ERα mutants or DBD-LBD and its K206A equivalent.