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)-taggedlac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integratedlac 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.
ACKNOWLEDGMENTS
We thank A. Cooney, D. Moore, Z. D. Sharp, and J. Wong for many helpful discussions.
This work was supported by grants from the National Institutes of Health to M. A. Mancini (R01-DK55622), A. Belmont (R01-GM58460 and R01-GM42516), and C. L. Smith (RO1 DK53002); a National American Heart Association Scientist Development Award and funds from the Department of Cell Biology, Baylor College of Medicine to M. A. Mancini; an NIH postdoctoral fellowship (1F32DK09787) to D. L. Stenoien; and an NIH Cell and Molecular Biology traineeship (T32-GM07283) and Howard Hughes Medical Institute predoctoral fellowship to A. Nye.