Silencing and Reactivation of the Selective Estrogen Receptor Modulator-Estrogen Receptor α Complex

4-Hydroxytamoxifen (4-OHT), a selective estrogen receptor modulator, is an agonist at a transforming growth factor-α (TGF-α) target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with wild-type human ERα. In contrast, raloxifene (Ral) is a complete antiestrogen silencing activation function (AF) 1 and AF2 in this system. A natural mutation D351YERα enhances 4-OHT agonist activity and changes Ral-like compounds from antagonists to partial agonists. We reasoned that: either the conformation of the Ral-D351YERα is altered, thereby reactivating AF2 in the ligand binding domain, or the change at amino acid 351 allosterically reactivates AF1 in the Ral-D351YERα complex. Unlike the estradiol-ERα complex, agonist activity of 4-OHT and raloxifene through ERα and D351YERα were not attributed to coactivator (such as SRC-1, AIB1) binding to the ligand binding domain. We conclude that the classic AF2 is not responsible for the agonist activities of 4-OHT-ERα, 4-OHT-D351YERα, and Ral-D351YERα. To address the role of AF1, stable transfectants of ERα or D351YERα with an AF1 deletion (D351ΔAF1, D351YΔAF1) were generated in MDA-MB-231 cells. Additionally, D538A/E542A/D545A triple mutations within helix 12 (D351–3m, D351Y3m) or the COOH-terminal 537 deletion (D351Δ537) were tested. The agonist activities of 4-OHT and raloxifene were lost in these stable transfectants, but antiestrogenic action was retained. The reactivation of an estrogen-like property of the Ral-ERα complex through AF1 with the D351Y mutation illustrates a novel allosteric mechanism for the selective estrogen receptor modulator ERα complex.