Allosteric silencing of activating function 1 in the 4-hydroxytamoxifen estrogen receptor complex is induced by substituting glycine for aspartate atamino acid 351

The active metabolite of tamoxifen, 4-hydroxytamoxifen (4-OHT), is used in the laboratory for mechanistic studies of antiestrogen action. This compoun d binds to the estrogen receptor alpha (ER) and silences activating functio n 2 (AF2) in the ligand binding domain, but activating function 1 (AF1) at the other end of the ER remains constitutive and is considered to be ligand independent. Amino acid D351 in the ligand binding domain appears to be cr itical for interactions with the antiestrogenic side chain of antiestrogens . We have devised an assay to evaluate the biological activity of 351 mutan t ERs and antiestrogens at the transforming growth factor alpha (TGF alpha) gene in situ (J.I. MacGregor Schafer et al,, Cancer Res., 59: 4308-4313, 1 999), The substitution of glycine for aspartate at position 351 results in the conversion of the 4-OHT:ER complex from estrogen-iike to completely ant iestrogenic, In cells stably expressing D351G ER, the ER retains responsive ness to estradiol (E-2) and also retains antiestrogenic responsiveness to b oth raloxifene and ICI 182,780, The relative binding affinity of E-2 for D3 51G ER (0.77 +/- 0.17 x 10(-9) M) is comparable with wild-type ER (0.42 +/- 0.08 x 10(-9) M). In addition, the D351G ER retains the ability to bind SR C-1 in the presence of E-2, thus D351G ER AF2 activity has not been comprom ised. We also used a cell line stably expressing an ER with a triple mutati on in helix 12 (D538A, E542A, and D545A) that ablated AF2 activity, which r esulted in decreased effects of E-2, suggesting that both AF1 and AF2 activ ity are required for maximal estrogen activity in MDA-MB-231 cells. Interes tingly, the triple mutation also completely reduced the estrogen-like actio ns of 4-OHT, We propose that a specific mutation at amino acid 351 can allo sterically silence AF1 in the 4-OHT:ER complex by either preventing the bin ding of coactivators or encouraging the binding of a corepressor molecule. We suggest that the 4-OHT specific site responsible for estrogen-like actio ns can be referred to as AF2b. This binding site would consist of at least four carboxylic acids at amino acids 351 and 538, 542 and 545 in helix 12 t o permit coactivator docking for gene activation. The AF2b site is distinct from AF2 for E-2 action. Further studies will provide insight into the est rogen-like actions of tamoxifen in select tissues and breast tumors and ide ntify a significant mechanism of drug resistance to tamoxifen.