STABILITY OF THE LIGAND ESTROGEN-RECEPTOR INTERACTION DEPENDS ON ESTROGEN RESPONSE ELEMENT FLANKING SEQUENCES AND CELLULAR FACTORS

To determine whether accessory proteins mediate the ligand- and DNA se quence-dependent specificity of estrogen receptor (ER) interaction wit h DNA, the binding of partly purified vs highly purified bovine ER to various estrogen response elements (EREs) was measured in the presence of different ER ligands. Partly purified estradiol-liganded ER (E(2)- ER) binds cooperatively to stereoaligned tandem EREs flanked by natura lly occurring AT-rich sequences, with a stoichiometry of one E(2)-ER d imer per ERE. In contrast, highly purified E(2)-ER binds with a 10-fol d lower affinity and non-cooperatively to EREs flanked by the AT-rich region. Moreover, the binding stoichiometry of highly purified E(2)-ER was 0.5 E(2)-ER dimer, or one monomer per ERE, independent of the ERE flanking sequence. Interestingly, the binding of ER liganded with the antiestrogen 4-hydroxytamoxifen (4-OHT-ER) was non-cooperative with a n apparent stoichiometry of 0.5 4-OHT-ER dimer per ERE, regardless of ER purity or ERE flanking sequence. We recently showed that when 4-OHT -ER binds DNA, one molecule of 4-OHT dissociates from the dimeric 4-OH T-ER-ERE complex, accounting for the reduced apparent binding stoichio metry. In contrast, ER covalently bound by tamoxifen aziridine (TAz) g ave an ERE binding stoichiometry of one TAz-ER dimer per ERE, and TAz- ER binds cooperatively to multiple AT-rich EREs, regardless of the pur ity of the receptor. We have obtained evidence that purification of ER removes an accessory protein(s) that interacts with ER in a sequence- and/or DNA conformational-dependent manner, resulting in stabilizatio n of E(2), but not 4-OHT, in the ligand binding domain when the recept or binds to DNA. We postulate that retention of ligand by ER maintains the receptor in a conformation necessary to achieve high-affinity, co operative ERE binding. Copyright (C) 1996 Elsevier Science Ltd.