title: Breast cancer-derived M543V mutation in helix 12 of estrogen receptor alpha inverts response to estrogen and SERMs.
abstract: We have isolated from human breast cancers several mutations in the Helix 12 component of activation function 2 (AF-2) in the estrogen receptor alpha (ERalpha). We used a novel approach to detect changes in the hormone-binding domain of ERalpha, based on the evidence that antiestrogens, such as 4-hydroxytamoxifen (ZOHT) and ICI 182,780, block the function of ERalpha by binding and folding the AF-2 transcriptional domain in a way that inhibits its association with coactivator proteins. We have identified a Helix 12 mutation, M543V, which leads to greater ERalpha transcription with ZOHT and other antiestrogens (including 1,1-dichloro-2,2,3-triarylcyclopropanes, DTACs) than with 17-beta estradiol (E2). We also found an independent mutation at the same position, M543I, which did not show this inverted ligand phenotype. In comparison to further Helix 12 mutations made in vitro, it appears that relative hydrophobicity of the amino acid side chains on the inner face of Helix 12 is key to maintaining the transcriptionally active, agonist conformation with bound E2. This active conformation can be induced, resulting in increased transcription, by adding excess p160 coactivator AIB1 in transcriptional assays with E2-bound receptors, while the ZOHT-bound receptors were not further activated by AIB1. Other experiments show that the cross talk between ERalpha and AP-1 protein from AP-1-binding sites is not dependent on Helix 12 integrity. We show that two alleles containing a proline substitution in Helix 12 that inactivate AF-2 function of ERalpha at EREs have little negative effect on function through AP-1 elements, supporting a prominent role for the N-terminal AF-1 of ERalpha in AP-1/ERalpha transcriptional cross talk.
authors.affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA. mnichols@pitt.edu
authors.forename: Mark
authors.initials: M
authors.lastname: Nichols
authors.forename: Peng
authors.initials: P
authors.lastname: Cheng
authors.forename: Yue
authors.initials: Y
authors.lastname: Liu
authors.forename: Beatriz
authors.initials: B
authors.lastname: Kanterewicz
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Hershberger
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: McCarty
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D004967:Estrogens; D005260:Female; D000077267:Fulvestrant; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D009376:Neoplasms, Hormone-Dependent / Q000378:metabolism* / Q000473:pathology; D017354:Point Mutation; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000502:physiology; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection
chemical_list: C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D004967:Estrogens; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D020845:Selective Estrogen Receptor Modulators; D018808:Transcription Factor AP-1; D013629:Tamoxifen; C016601:afimoxifene; D000077267:Fulvestrant; D004958:Estradiol