title: Transactivation Function-2 of Estrogen Receptor α Contains Transactivation Function-1-regulating Element.
abstract: ERα has a ligand-dependent transactivation function in the ligand binding domain of ERα C terminus (AF-2) and a ligand-independent activation function in the N terminus (AF-1). It is still not fully understood how AF-1 and AF-2 activities are regulated cooperatively by ligands. To evaluate the AF-1 involvement in the estrogenic activities of various compounds, we analyzed these transactivation functions using AF-1-truncated and AF-2-mutated ERα mutants. AF-2 is composed of two domains with flexible and static regions. We used an AF-2 flexible region mutant and an AF-2 static region mutant. Both mutants have been reported as non-E2 responsive due to disruption of E2-mediated coactivator recruitment to the AF-2. The AF-2 mutants were not activated by agonists, but surprisingly antagonists and selective estrogen receptor modulators (SERMs) activated the AF-2 mutants. This antagonist reversal activity was derived from AF-1. Furthermore, we demonstrated that the AF-2 contains an AF-1 suppression function using C-terminal-truncated ERα mutants. From these findings we hypothesized that the mutation of AF-2 disrupted its ability to suppress AF-1, causing the antagonist reversal. To assess the AF-2-mediated AF-1 suppression, we analyzed the transcription activity of physically separated AF-1 and AF-2 using a novel hybrid reporter assay. We observed that the AF-1 activity was not suppressed by the physically separated AF-2. Furthermore, SERMs did not induce the AF-1-mediated activity from the separated mutant AF-2, which differed from the intact protein. These results imply that SERM activity is dependent on a conformational change of the full-length ERα molecule, which allows for AF-1 activation.
authors.affiliation: From the Receptor Biology Section, Reproductive and Developmental Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709.
authors.forename: Yukitomo
authors.initials: Y
authors.lastname: Arao
authors.affiliation: From the Receptor Biology Section, Reproductive and Developmental Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, Department of Pharmacology and Cancer Biology, Duke University, Medical Center, Durham, North Carolina 27710, and.
authors.forename: Laurel A
authors.initials: LA
authors.lastname: Coons
authors.affiliation: Department of Chemical Biology, GlaxoSmithKline, Research Triangle Park, North Carolina 27709.
authors.forename: William J
authors.initials: WJ
authors.lastname: Zuercher
authors.affiliation: From the Receptor Biology Section, Reproductive and Developmental Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, korach@niehs.nih.gov.
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Korach
mesh_terms: D000818:Animals; D052244:Endocrine Disruptors / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008024:Ligands; D051379:Mice; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048789:Phytoestrogens / Q000494:pharmacology; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D017384:Sequence Deletion; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D052244:Endocrine Disruptors; D047628:Estrogen Receptor alpha; D008024:Ligands; D050505:Mutant Proteins; D048789:Phytoestrogens; D011994:Recombinant Proteins; D020845:Selective Estrogen Receptor Modulators; D015534:Trans-Activators
keywords: agonist; antagonist; estrogen; estrogen receptor; protein structure; selected estrogen receptor modulators; steroid hormone; steroid hormone receptor