Modulation of estrogen receptor alpha function and stability by tamoxifen and a critical amino acid (Asp-538) in helix 12.

Estrogen receptor alpha (ER) is a ligand-activated transcription factor implicated in breast cancer growth. Selective estrogen receptor modulators (SERMs), such as tamoxifen (4-OHT), bind to the ER and affect the position of helix 12, thereby influencing coregulator binding and ER transcriptional activation. Previous studies have shown that a triple mutation in helix 12 (3m; D538A/E542A/D545A) caused a change in ER stability and obliterated 4-OHT action (Liu, H., Lee, E. S., de los Reyes, A., Zapf, J. W., and Jordan, V. C. (2001) Cancer Res. 61, 3632-3639). Two approaches were taken to determine the role of individual mutants (D538A, L540Q, E542A, and D545A) on the activity and stability of the 4-OHT.ER complex. First, mutants were evaluated using transient transfection into ER-negative T47D:C4:2 cells with an ERE3-luciferase reporter, and second, transforming growth factor alpha (TGFalpha) mRNA was used as a gene target in situ for stable transfectants of MDA-MB-231 cells. Transcriptional activity occurred in the presence of estrogen in all of the mutants, although a decreased response was observed in the L540Q, 3m, and D538A cells. The 3m and D538A mutants lacked any estrogenic responsiveness to 4-OHT, whereas the other mutations retained estrogen-like activity with 4-OHT. Unlike the other mutants, the ER was degraded in the D538A mutant with 4-OHT treatment. However, increasing the protein levels of the mutant with the proteasome inhibitor MG132 did not restore the ability of 4-OHT to induce TGFalpha mRNA. We suggest that Asp-538 is a critical amino acid in helix 12 that not only reduces the estrogen-like actions of 4-OHT but also facilitates the degradation of the 4-OHT.D538A complex. These data further illustrate the complex role of specific surface amino acids in the modulation of the concentration and the estrogenicity of the 4-OHT.ER complex.