Dissociation of 4-hydroxytamoxifen, but not estradiol or tamoxifen aziridine, from the estrogen receptor as the receptor binds estrogen response element DNA

Abstract

Estradiol-liganded estrogen receptor (E 2-ER) binds EREs with a stoichiometry of one E 2-ER dimer per estrogen response element (ERE). In contrast, although 4-hydroxytamoxifen (4-OHT)-liganded ER (4-OHT-ER) binds EREs with high affinity, its saturation ERE binding capacity is consistently 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 stoichiometry of TAz-ER was identical to that of E 2-ER: one dimeric receptor per ERE. By measuring [ 3H]ligand that was initially bound to ER, a significant loss of [ 3H]4-OHT from ER was detected after ERE binding, whereas all [ 3H]E 2 or [ 3H]TAz remained ER-bound. These results confirm 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 to induce a conformation in ER dimers that alters their capacity for gene activation. Upon ER binding to DNA, this conformation reveals itself by allowing 4-OHT dissociation, and predictably would allow TAz dissociation were it not bound covalently.