Estrogen regulates target gene expression by binding to specific nuclear receptors that function as ligand-dependent transcription factors. Estrogen receptors (ERs) 1 contain two transcription activation domains, AF1 at the N terminus and AF2 in the C-terminal ligand-binding domain (1-3). Several proteins interact with AF2 in the presence of estrogen, some of which have the properties of transcriptional coactivators (4 -6).For instance, the p160 family of coactivators, which in humans includes the three proteins SRC1/N-CoA1 (7, 8), TIF2/GRIP1 (9, 10), and AIB1/ACTR/RAC3 (11-13), can interact with most nuclear receptors in a ligand-dependent manner and potentiate transcription of their target genes.The crystal structures of several nuclear receptor ligandbinding domains (LBDs) have now been determined (14 -19) and have revealed a striking conservation despite modest sequence homology (20). The LBD folds into a structure described as a sandwich of ␣-helices with a central hydrophobic ligandbinding pocket. In the presence of ligand, helices 3, 5, and 12 form a hydrophobic groove (21-24) important for interaction with the LXXLL motifs (9, 25-27) found in the p160 family members and also in other coactivators. However, the crystal structure of estrogen receptor ␣ (ER␣) revealed that helix 12 is repositioned in the presence of the antagonists tamoxifen (Tam) or raloxifene (Ral), thereby disrupting the surface of interaction with coactivators (16, 23). The side chain of these antiestrogens plays an important role in displacing helix 12. This suggests that amino acids of the ligand-binding domain that interact with the antiestrogen side chain play an important role in the transcriptionally inactive conformation of this domain. It has been suggested that integrity of aspartate 351, which forms a hydrogen bond with the tertiary amine present at the end of the side chains of Tam and Ral, is the key to the antiestrogenic character of these analogs (28). Indeed, a mutation of Asp-351 to tyrosine was isolated from an MCF7 tumor variant that was not inhibited but rather stimulated by Tam (29,30). Both Tam and Ral also behaved as agonists for expression of the estrogen target gene transforming growth factor-␣ in MDA-MB-231 cells stably transfected with this mutant of ER␣, while the full antiestrogen ICI182,780 remained inactive (28).Here we have introduced several mutations at position 351 and tested the functional consequences of these changes on ER␣ transactivation properties in the presence of estrogen and of antiestrogens. Our results demonstrate that Asp-351 can be mutagenized to Gly, Ala, or Val without diminishing the antagonist activity of antiestrogens in HeLa cells. However, we provide evidence for a stabilizing effect of Asp-351 on the active conformation of the wild-type ER LBD in the absence of hormones. EXPERIMENTAL PROCEDURESMaterials-Cell culture media and fetal bovine serum were purchased from Life Technologies, Inc. Estradiol, 4-hydroxytamoxifen (OHT), and ICI182,780 were purchased from Sigma. RU39,411 and RU...
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