Site-directed estrogen receptor antibodies stabilize 4-hydroxytamoxifen ligand, but not estradiol, and indicate ligand-specific differences in the recognition of estrogen response element DNA in vitro.
Conformational differences between type I antiestrogen-liganded estrogen receptor and estradiol (E2)-liganded estrogen receptor (ER) are thought to be responsible for differentiating agonist versus antagonist ER activity at individual genes. To examine the impact of ER ligand on estrogen-response element (ERE) binding kinetics and receptor conformation, we quantitated the effect of site-directed, ER-specific antibodies raised against synthetic peptides corresponding to the DNA-binding domain of human ER on ER-ERE binding in vitro. Although 4-hydroxytamoxifen-liganded-ER (4-OHT-ER) and E2-ER bind a consensus ERE with equal high affinity, the stoichiometry of 4-OHT-ER-ERE binding at saturation is approximately 50% lower than that of E2-ER binding to all ERE sequences tested. In contrast, the ERE binding stoichiometry of tamoxifen aziridine-liganded ER (TAz-ER) is identical to that of E2-ER: one receptor dimer bound per ERE. The difference in binding stoichiometry is caused by dissociation of one molecule of 4-OHT from the ER as the dimeric receptor binds DNA. Addition of low concentrations of ER-specific polyclonal antibodies AT3A or AT3B prevented 4-OHT ligand dissociation, yielding an increase in specific 4-OHT-ER-ERE binding to a level equal to that of E2-ER- or TAz-ER-ERE binding. However, higher amounts of AT3A or AT3B inhibited specific ERE binding of both 4-OHT- and E2-ER. We conclude that differences in ER conformation when liganded with 4-OHT versus E2 are revealed by these antibodies and that such differences in receptor conformation may influence subsequent interaction of the receptor with other proteins necessary for transactivation.
Klinge
C M
CM
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642, USA.
Traish
A M
AM
Driscoll
M D
MD
Hilf
R
R
Bambara
R A
RA
D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D004958:Estradiol / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D011960:Receptors, Estrogen / Q000235:genetics / Q000276:immunology*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism
D008024:Ligands; D011960:Receptors, Estrogen; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol; D004247:DNA