1 Department of Molecular and Cellular Biology, Baylor
College of Medicine, Houston, Texas 77030,
2 Department of Cell and Structural Biology, University
of Illinois, Urbana-Champaign, Illinois 61801.
* Corresponding author. Mailing address: Department of
Molecular and Cellular Biology, Baylor College of Medicine, One BaylorPlaza,
Houston, TX 77030. Phone: (713) 798-8952. Fax: (713) 798-8005.
E-mail: mancini@bcm.tmc.edu.
Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor a (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-tagged lacrepressor-ER chimera (CFP-LacER) was used in live cells to discretelyimmobilize 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.
1. "A Steroid Receptor Coactivator, SRA, Functions as an RNA and is present in a SRC-1 Complex".
2. "Activation of DNA Transcription within Repressed Chromatin by Nuclear RNA Species".