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.
Pearce
Sandra Timm
ST
Robert H. Lurie Comprehensive Cancer Center, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.
Liu
Hong
H
Jordan
V Craig
VC
D001224:Aspartic Acid / Q000737:chemistry*; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D000077267:Fulvestrant; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism* / Q000502:physiology*; D013629:Tamoxifen / Q000494:pharmacology*; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D008024:Ligands; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D016211:Transforming Growth Factor alpha; D013629:Tamoxifen; D000077267:Fulvestrant; D001224:Aspartic Acid; D004958:Estradiol; D008156:Luciferases