title: 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 (E2-ER) binds EREs with a stoichiometry of one E2-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 E2-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 (Kd = 0.27 nM), comparable to that of E2-ER and 4-OHT-ER. In contrast to 4-OHT-ER, the ERE binding stoichiometry of TAz-ER was identical to that of E2-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]E2 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.
authors.affiliation: Department of Biochemistry, University of Rochester School of Medicine and Dentistry, NY 14642 USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Klinge
authors.forename: A M
authors.initials: AM
authors.lastname: Traish
authors.forename: R A
authors.initials: RA
authors.lastname: Bambara
authors.forename: R
authors.initials: R
authors.lastname: Hilf
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011960:Receptors, Estrogen / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D017186:Titrimetry / Q000379:methods; D014316:Tritium / Q000737:chemistry
chemical_list: D011960:Receptors, Estrogen; D013629:Tamoxifen; D014316:Tritium; C016601:afimoxifene; D004958:Estradiol; C031859:tamoxifen aziridine; D004247:DNA