title: Ligand-mediated assembly and real-time cellular dynamics of estrogen receptor alpha-coactivator complexes in living cells.
abstract: Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor alpha (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-tagged lac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integrated lac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP-SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP-SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP-SRC-1, while antagonist additions diminish YFP-SRC-1-CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER-SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP-SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.
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: A C
authors.initials: AC
authors.lastname: Nye
authors.forename: M G
authors.initials: MG
authors.lastname: Mancini
authors.forename: K
authors.initials: K
authors.lastname: Patel
authors.forename: M
authors.initials: M
authors.lastname: Dutertre
authors.forename: B W
authors.initials: BW
authors.lastname: O'Malley
authors.forename: C L
authors.initials: CL
authors.lastname: Smith
authors.forename: A S
authors.initials: AS
authors.lastname: Belmont
authors.forename: M A
authors.initials: MA
authors.lastname: Mancini
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000494:pharmacology; D017930:Genes, Reporter; D051548:Histone Acetyltransferases; D007763:Lac Operon / Q000235:genetics*; D008024:Ligands; D008856:Microscopy, Fluorescence; D056920:Nuclear Receptor Coactivator 1; D011485:Protein Binding / Q000187:drug effects; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D047628:Estrogen Receptor alpha; D004967:Estrogens; D008024:Ligands; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D051548:Histone Acetyltransferases; D056920:Nuclear Receptor Coactivator 1