DISSOCIATION OF 4-HYDROXYTAMOXIFEN, BUT NOT ESTRADIOL OR TAMOXIFEN AZIRIDINE, FROM THE ESTROGEN-RECEPTOR AS THE RECEPTOR BINDS ESTROGEN RESPONSE ELEMENT DNA

Estradiol-liganded estrogen receptor (E(2)-ER) binds EREs with a stoic hiometry of one E(2)-ER dimer per estrogen response element (ERE). In contrast, although 4-hydroxytamoxifen (4-OHT)-liganded ER (4-OHT-ER) b inds EREs with high affinity, its saturation ERE binding capacity is c onsistently half that of E(2)-ER, giving an apparent stoichiometry of one 4-OHT-ER monomer per ERE. Here we show that one molecule of 4-OHT ligand dissociates from the ER dimer apparently during the process of binding to DNA. Under equilibrium conditions, the type I antiestrogen tamoxifen aziridine (TAz), covalently attached to ER (TAz-ER), binds a single ERE with high affinity (K-d = 0.27 nM), comparable to that of E(2)-ER and 4-OHT-ER. In contrast to 4-OHT-ER, the ERE binding stoichi ometry of TAz-ER was identical to that of E(2)-ER: one dimeric recepto r per ERE. By measuring [H-3]ligand that was initially bound to ER, a significant loss of [H-3]4-OHT from ER was detected after ERE binding, whereas all [H-3]E(2) or [H-3]TAz remained ER-bound. These results co nfirm that one molecule of 4-OHT ligand dissociates from the ER dimer as a consequence of ERE binding. Binding of 4-OHT and TAz are likely t o induce a conformation in ER dimers that alters their capacity for ge ne activation. Upon ER binding to DNA, this conformation reveals itsel f by allowing 4-OHT dissociation, and predictably would allow TAz diss ociation were it not bound covalently. (C) 1996 Elsevier Science Ltd.