title: Raloxifene and ICI182,780 increase estrogen receptor-alpha association with a nuclear compartment via overlapping sets of hydrophobic amino acids in activation function 2 helix 12.
abstract: The basis for the differential repressive effects of antiestrogens on transactivation by estrogen receptor-alpha (ERalpha) remains incompletely understood. Here, we show that the full antiestrogen ICI182,780 and, to a lesser extent, the selective ER modulator raloxifene (Ral), induce accumulation of exogenous ERalpha in a poorly soluble fraction in transiently transfected HepG2 or stably transfected MDA-MB231 cells and of endogenous receptor in MCF7 cells. ERalpha remained nuclear in HepG2 cells treated with either compound. Replacement of selected hydrophobic residues of ERalpha ligand-binding domain helix 12 (H12) enhanced receptor solubility in the presence of ICI182,780 or Ral. These mutations also increased transcriptional activity with Ral or ICI182,780 on reporter genes or on the endogenous estrogen target gene TFF1 in a manner requiring the integrity of the N-terminal AF-1 domain. The antiestrogen-specific effects of single mutations suggest that they affect receptor function by mechanisms other than a simple decrease in hydrophobicity of H12, possibly due to relief from local steric hindrance between these residues and the antiestrogen side chains. Fluorescence anisotropy experiments indicated an enhanced regional stabilization of mutant ligand-binding domains in the presence of antiestrogens. H12 mutations also prevent the increase in bioluminescence resonance energy transfer between ERalpha monomers induced by Ral or ICI182,780 and increase intranuclear receptor mobility in correlation with transcriptional activity in the presence of these antiestrogens. Our data indicate that ICI182,780 and Ral locally alter the ERalpha ligand binding structure via specific hydrophobic residues of H12 and decrease its transcriptional activity through tighter association with an insoluble nuclear structure.
authors.affiliation: Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, Québec, Canada.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Lupien
authors.forename: M
authors.initials: M
authors.lastname: Jeyakumar
authors.forename: Elise
authors.initials: E
authors.lastname: Hébert
authors.forename: Khalid
authors.initials: K
authors.lastname: Hilmi
authors.forename: David
authors.initials: D
authors.lastname: Cotnoir-White
authors.forename: Caroline
authors.initials: C
authors.lastname: Loch
authors.forename: Anick
authors.initials: A
authors.lastname: Auger
authors.forename: Guila
authors.initials: G
authors.lastname: Dayan
authors.forename: Geneviève-Anne
authors.initials: GA
authors.lastname: Pinard
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Wurtz
authors.forename: Dino
authors.initials: D
authors.lastname: Moras
authors.forename: John
authors.initials: J
authors.lastname: Katzenellenbogen
authors.forename: Sylvie
authors.initials: S
authors.lastname: Mader
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism; D000077267:Fulvestrant; D006801:Humans; D007930:Leucine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D020849:Raloxifene Hydrochloride / Q000494:pharmacology*; D012995:Solubility; D014158:Transcription, Genetic / Q000187:drug effects; D000071163:Trefoil Factor-1; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics
chemical_list: D000596:Amino Acids; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; C051838:TFF1 protein, human; D000071163:Trefoil Factor-1; D025521:Tumor Suppressor Proteins; D000077267:Fulvestrant; D020849:Raloxifene Hydrochloride; D004958:Estradiol; D007930:Leucine