Ligand-mediated assembly and real-time cellular dynamics of estrogen receptor α-coactivator complexes in living cells

David L. Stenoien, Anne C. Nye, Maureen G. Mancini, Kavita Patel, Martin Dutertre, Bert W. O'Malley, Carolyn L. Smith, Andrew S. Belmont, Michael A. Mancini

Research output: Contribution to journalArticle

Abstract

Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor α (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.

Original languageEnglish (US)
Pages (from-to)4404-4412
Number of pages9
JournalMolecular and cellular biology
Volume21
Issue number13
DOIs
StatePublished - 2001

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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    Stenoien, D. L., Nye, A. C., Mancini, M. G., Patel, K., Dutertre, M., O'Malley, B. W., Smith, C. L., Belmont, A. S., & Mancini, M. A. (2001). Ligand-mediated assembly and real-time cellular dynamics of estrogen receptor α-coactivator complexes in living cells. Molecular and cellular biology, 21(13), 4404-4412. https://doi.org/10.1128/MCB.21.13.4404-4412.2001