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
| Autor: | Benita S. Katzenellenbogen, Gisela M. Nilsson, Agneta Heierson, Donald P. McDonnell, Cynthia Harris Wooge |
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| Rok vydání: | 1992 |
| Předmět: |
Neoplasms
Hormone-Dependent Protein Conformation Recombinant Fusion Proteins Aziridines Saccharomyces cerevisiae Estrogen receptor Breast Neoplasms Peptide Ligands Endocrinology Escherichia coli Humans Molecular Biology chemistry.chemical_classification Binding Sites Estradiol biology General Medicine Ketones biology.organism_classification Peptide Fragments Yeast Neoplasm Proteins Amino acid Dissociation constant Kinetics Tamoxifen Receptors Estrogen Biochemistry chemistry biology.protein Protein A Protein Binding Binding domain |
| Zdroj: | Molecular Endocrinology. 6:861-869 |
| ISSN: | 1944-9917 0888-8809 |
| DOI: | 10.1210/mend.6.6.1495491 |
| Popis: | 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) |
| Databáze: | OpenAIRE |
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