SITE-DIRECTED ESTROGEN-RECEPTOR ANTIBODIES STABILIZE 4-HYDROXYTAMOXIFEN LIGAND, BUT NOT ESTRADIOL, AND INDICATE LIGAND-SPECIFIC DIFFERENCESIN THE RECOGNITION OF ESTROGEN RESPONSE ELEMENT DNA IN-VITRO

Conformational differences between the I antiestrogen-liganded estroge n receptor and estradiol (E(2))-liganded estrogen receptor (ER) are th ought 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 conform ation, we quantitated the effect of site-directed ER-specific antibodi es raised against synthetic peptides corresponding to the DNA-binding domain of human ER on ER-ERE binding in vitro. Although 4-hydroxamoxif en-liganded-ER (4-OHT-ER) and E(2)-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 E(2)-ER binding to all ERE se quences tested. In contrast, the ERE binding stoichiometry of tamoxife n aziridine-liganded ER (TAz-ER) is identical to that of E(2)-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-speci fic polyclonal antibodies AT3A or AT3B prevented 4-OHT ligand dissocia tion, yielding an increase in specific 4-OHT-ER-ERE binding to a Ievel equal to that of E(2)-ER- or TAz-ER-ERE binding. However, higher amou nts of AT3A or AT3B inhibited specific ERE binding of both 4-OHT- and E(2)-ER. We conclude that differences in ER conformation when liganded with 4-OHT versus E(2) are revealed by these antibodies and that such differences in receptor conformation may influence subsequent interac tion of the receptor with other proteins necessary for transactivation .