title: Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells.
abstract: In order to better understand the structural requirements for effective high affinity binding of estrogens and antiestrogens by the human estrogen receptor (ER), a comparative study was undertaken in which we examined: 1) native ER from the MCF-7 ER-positive human breast cancer cell line; 2) full length ER expressed in yeast; 3) the ER hormone binding domain (amino acid residues 302-595) expressed in yeast; 4) a bacterially expressed protein A fusion product encoding a truncated ER (amino acid residues 240-595); and 5) a synthetic peptide encompassing amino acids 510-551 of the ER. The binding parameters studied included affinity, kinetics, structural specificity for ligands, and stability. Full length ER expressed in yeast was very similar to the MCF-7 ER in its affinity [dissociation constant (Kd), 0.35 +/- 0.05 nM], dissociation rate (t1/2, 3-4 h at 25 C), and structural specificity for both reversible and covalently attaching affinity ligands. While the truncated ER expressed in yeast was similar to MCF-7 ER in its specificity of ligand binding, it showed a slightly reduced affinity for estradiol (Kd, 1.00 +/- 0.17 nM). The bacterially expressed ER also had a lower affinity for estradiol (Kd, 1.49 +/- 0.16 nM), which may be due in part to an increase in the dissociation rate (t1/2, 0.5 h at 25 C). The attachment of covalent affinity ligands and structural specificity for a variety of reversible ligands was comparable in the bacterially expressed ER to that observed for the receptors expressed in MCF-7 cells and yeast.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Physiology and Biophysics, University of Illinois, Urbana.
authors.forename: C H
authors.initials: CH
authors.lastname: Wooge
authors.forename: G M
authors.initials: GM
authors.lastname: Nilsson
authors.forename: A
authors.initials: A
authors.lastname: Heierson
authors.forename: D P
authors.initials: DP
authors.lastname: McDonnell
authors.forename: B S
authors.initials: BS
authors.lastname: Katzenellenbogen
mesh_terms: D001388:Aziridines / Q000378:metabolism; D001665:Binding Sites; D001943:Breast Neoplasms / Q000737:chemistry; D004926:Escherichia coli; D004958:Estradiol / Q000378:metabolism*; D006801:Humans; D007659:Ketones / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009376:Neoplasms, Hormone-Dependent / Q000737:chemistry; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D001388:Aziridines; D007659:Ketones; D008024:Ligands; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D013629:Tamoxifen; C052977:ketononestrol aziridine; D004958:Estradiol; C031859:tamoxifen aziridine