title: Estrogen receptor α L543A,L544A mutation changes antagonists to agonists, correlating with the ligand binding domain dimerization associated with DNA binding activity.
abstract: A ligand-dependent nuclear transcription factor, ERα has two transactivating functional domains (AF), AF-1 and AF-2. AF-1 is localized in the N-terminal region, and AF-2 is distributed in the C-terminal ligand-binding domain (LBD) of the ERα protein. Helix 12 (H12) in the LBD is a component of the AF-2, and the configuration of H12 is ligand-inducible to an active or inactive form. We demonstrated previously that the ERα mutant (AF2ER) possessing L543A,L544A mutations in H12 disrupts AF-2 function and reverses antagonists such as fulvestrant/ICI182780 (ICI) or 4-hydoxytamoxifen (OHT) into agonists in the AF2ER knock-in mouse. Our previous in vitro studies suggested that the mode of AF2ER activation is similar to the partial agonist activity of OHT for WT-ERα. However, it is still unclear how antagonists activate ERα. To understand the molecular mechanism of antagonist reversal activity, we analyzed the correlation between the ICI-dependent estrogen-responsive element-mediated transcription activity of AF2ER and AF2ER-LBD dimerization activity. We report here that ICI-dependent AF2ER activation correlated with the activity of AF2ER-LBD homodimerization. Prevention of dimerization impaired the ICI-dependent ERE binding and transcription activity of AF2ER. The dislocation of H12 caused ICI-dependent LBD homodimerization involving the F-domain, the adjoining region of H12. Furthermore, F-domain truncation also strongly depressed the dimerization of WT-ERα-LBD with antagonists but not with E2. AF2ER activation levels with ICI, OHT, and raloxifene were parallel with the degree of AF2ER-LBD homodimerization, supporting a mechanism that antagonist-dependent LBD homodimerization involving the F-domain results in antagonist reversal activity of H12-mutated ERα.
authors.affiliation: From the Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 and.
authors.forename: Yukitomo
authors.initials: Y
authors.lastname: Arao
authors.affiliation: From the Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 and.
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Hamilton
authors.affiliation: From the Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 and; the Department of Pharmacology and Cancer Biology, Duke University, Medical Center, Durham, North Carolina 27710.
authors.forename: Laurel A
authors.initials: LA
authors.lastname: Coons
authors.affiliation: From the Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 and. Electronic address: korach@niehs.nih.gov.
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Korach
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D004247:DNA / Q000378:metabolism*; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000819:agonists* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004967:Estrogens / Q000494:pharmacology; D000077267:Fulvestrant; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D008024:Ligands; D051379:Mice; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D056920:Nuclear Receptor Coactivator 1 / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D059748:Proteolysis / Q000187:drug effects; D020218:Response Elements / Q000235:genetics; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D004967:Estrogens; D008024:Ligands; D050505:Mutant Proteins; D020845:Selective Estrogen Receptor Modulators; D000077267:Fulvestrant; D004958:Estradiol; D004247:DNA; D056920:Nuclear Receptor Coactivator 1
keywords: Antagonist Reversal; Dimerization; Estrogen; Estrogen Receptor; Fulvestrant; Mutant; Protein Domains; SERMs; Tamoxifen; Transcription