title: Silencing and reactivation of the selective estrogen receptor modulator-estrogen receptor alpha complex.
abstract: 4-Hydroxytamoxifen (4-OHT), a selective estrogen receptor modulator, is an agonist at a transforming growth factor-alpha (TGF-alpha) target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with wild-type human ERalpha. In contrast, raloxifene (Ral) is a complete antiestrogen silencing activation function (AF) 1 and AF2 in this system. A natural mutation D351YERalpha enhances 4-OHT agonist activity and changes Ral-like compounds from antagonists to partial agonists. We reasoned that: either the conformation of the Ral-D351YERalpha is altered, thereby reactivating AF2 in the ligand binding domain, or the change at amino acid 351 allosterically reactivates AF1 in the Ral-D351YERalpha complex. Unlike the estradiol-ERalpha complex, agonist activity of 4-OHT and raloxifene through ERalpha and D351YERalpha were not attributed to coactivator (such as SRC-1, AIB1) binding to the ligand binding domain. We conclude that the classic AF2 is not responsible for the agonist activities of 4-OHT-ERalpha, 4-OHT-D351YERalpha, and Ral-D351YERalpha. To address the role of AF1, stable transfectants of ERalpha or D351YERalpha with an AF1 deletion (D351deltaAF1, D351YdeltaAF1) were generated in MDA-MB-231 cells. Additionally, D538A/E542A/D545A triple mutations within helix 12 (D351-3m, D351Y3m) or the COOH-terminal 537 deletion (D351delta537) were tested. The agonist activities of 4-OHT and raloxifene were lost in these stable transfectants, but antiestrogenic action was retained. The reactivation of an estrogen-like property of the Ral-ERalpha complex through AF1 with the D351Y mutation illustrates a novel allosteric mechanism for the selective estrogen receptor modulator ERalpha complex.
authors.affiliation: Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611, USA.
authors.forename: H
authors.initials: H
authors.lastname: Liu
authors.forename: E S
authors.initials: ES
authors.lastname: Lee
authors.forename: A
authors.initials: A
authors.lastname: Deb Los Reyes
authors.forename: J W
authors.initials: JW
authors.lastname: Zapf
authors.forename: V C
authors.initials: VC
authors.lastname: Jordan
mesh_terms: D000494:Allosteric Regulation; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics; D047628:Estrogen Receptor alpha; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D011487:Protein Conformation; D020849:Raloxifene Hydrochloride / Q000378:metabolism / Q000494:pharmacology*; D011960:Receptors, Estrogen / Q000819:agonists / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020845:Selective Estrogen Receptor Modulators / Q000378:metabolism / Q000494:pharmacology*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D014407:Tumor Cells, Cultured
chemical_list: D047628:Estrogen Receptor alpha; D011960:Receptors, Estrogen; D020845:Selective Estrogen Receptor Modulators; D016211:Transforming Growth Factor alpha; D013629:Tamoxifen; C016601:afimoxifene; D020849:Raloxifene Hydrochloride