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Molecular and Cellular Biology, July 2001, p. 4404-4412, Vol. 21, No. 13
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.13.4404-4412.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Ligand-Mediated Assembly and Real-Time Cellular Dynamics of Estrogen Receptor alpha -Coactivator Complexes in Living Cells

David L. Stenoien,1 Anne C. Nye,2 Maureen G. Mancini,1 Kavita Patel,1 Martin Dutertre,1 Bert W. O'Malley,1 Carolyn L. Smith,1 Andrew S. Belmont,2 and Michael A. Mancini1,*

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030,1 and Department of Cell and Structural Biology, University of Illinois, Urbana-Champaign, Illinois 618012

Received 5 February 2001/Returned for modification 15 March 2001/Accepted 9 April 2001

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.

* Corresponding author. Mailing address: Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-8952. Fax: (713) 798-8005. E-mail: mancini{at}bcm.tmc.edu.

Molecular and Cellular Biology, July 2001, p. 4404-4412, Vol. 21, No. 13
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.13.4404-4412.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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