Ligand-mediated assembly and real-time cellular dynamics of estrogen receptor alpha-coactivator complexes in living cells.
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.
Stenoien
D L
DL
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Nye
A C
AC
Mancini
M G
MG
Patel
K
K
Dutertre
M
M
O'Malley
B W
BW
Smith
C L
CL
Belmont
A S
AS
Mancini
M A
MA
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
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