title: Subnuclear trafficking of estrogen receptor-alpha and steroid receptor coactivator-1.
abstract: We have analyzed ligand-dependent, subnuclear movements of the estrogen receptor-alpha (ERalpha) in terms of both spatial distribution and solubility partitioning. Using a transcriptionally active green fluorescent protein-ERalpha chimera (GFP-ERalpha), we find that 17beta-estradiol (E2) changes the normally diffuse nucleoplasmic pattern of GFP-ERalpha to a hyperspeckled distribution within 10-20 min. A similar reorganization occurs with the partial antagonist 4-hydroxytamoxifen; only a subtle effect was observed with the pure antagonist ICI 182,780. To examine the influence of ligand upon ERalpha association with nuclear structure, MCF-7 cells were extracted to reveal the nuclear matrix (NM). Addition of E2, 4-hydroxytamoxifen, or ICI 182,780 causes ERalpha to partition with the NM-bound fraction on a similar time course (10-20 min) as the spatial reorganization suggesting that the two events are related. To determine the effects of E2 on the redistribution and solubility of GFP-ERalpha, individual cells were directly examined during both hormone addition and NM extraction and showed that GFP-ERalpha movement and NM association were coincident. Colocalization experiments were performed with antibodies to identify sites of transcription (RNA pol Ilo) and splicing domains (SRm160). Using E2 treated MCF-7 cells, minor overlap was observed with transcription sites and a small amount of the total ERalpha pool. Experiments performed with bioluminescent derivatives of ERalpha and steroid receptor coactivator-1 (SRC-1) demonstrated both proteins colocalize to the same NM-bound foci in response to E2 but not the antagonists tested. Deletion mutagenesis and in situ analyses indicate intranuclear colocalization requires a central SRC-1 domain containing LXXLL motifs. Collectively, our data suggest that ERalpha transcription function is dependent upon dynamic early events including intranuclear rearrangement, NM association, and SRC-1 interactions.
authors.affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Stenoien
authors.forename: M G
authors.initials: MG
authors.lastname: Mancini
authors.forename: K
authors.initials: K
authors.lastname: Patel
authors.forename: E A
authors.initials: EA
authors.lastname: Allegretto
authors.forename: C L
authors.initials: CL
authors.lastname: Smith
authors.forename: M A
authors.initials: MA
authors.lastname: Mancini
mesh_terms: D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure*; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D000077267:Fulvestrant; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D051548:Histone Acetyltransferases; D006801:Humans; D007223:Infant; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D016296:Mutagenesis; D015530:Nuclear Matrix / Q000378:metabolism; D056920:Nuclear Receptor Coactivator 1; D011960:Receptors, Estrogen / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D047628:Estrogen Receptor alpha; D008164:Luminescent Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D013629:Tamoxifen; D049452:Green Fluorescent Proteins; C016601:afimoxifene; D000077267:Fulvestrant; D004958:Estradiol; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1