Estrogen receptor interactions and dynamics monitored in live cells by fluorescence cross-correlation spectroscopy.
Quantitative characterization of protein interactions in live cells remains one of the most important challenges in modern biology. In the present work we have used two-photon, two-color, fluorescence cross-correlation spectroscopy (FCCS) in transiently transfected COS-7 cells to measure the concentrations and interactions of estrogen receptor (ER) subtypes alpha and beta with one of their transcriptional coactivator proteins, TIF2, as well as heterodimerization between the two ER subtypes. Using this approach in a systematic fashion, we observed a strong ligand-dependent modulation of receptor-coactivator complexation, as well as strong protein concentration dependence for complex formation in the absence of ligand. These quantitative values for protein and complex concentrations provide the first estimates for the ER-TIF2 K(d) for the full-length proteins and in a cellular context (agonist, < approximately 6 nM; antagonist, > approximately 3 microM; unliganded, approximately 200 nM). Coexpression of the two ER subtypes revealed substantial receptor heterodimer formation. They also provide, for the first time, estimated homo- and heterodimerization constants found to be similar and in the low nanomolar range. These results underscore the importance of receptor and coregulator expression levels and stability in the tissue-dependent modulation of receptor function under normal and pathological conditions.
Savatier
Julien
J
Centre de Biochimie Structurale, INSERM U554, and CNRS UMR5048, Université Montpellier 1 and 2, Montpellier F-34090, France.
Jalaguier
Stéphan
S
Ferguson
Matthew L
ML
Cavaillès
Vincent
V
Royer
Catherine A
CA
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D011960:Receptors, Estrogen / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D036341:Intercellular Signaling Peptides and Proteins; D011960:Receptors, Estrogen