Quantitative detection of the ligand-dependent interaction between the androgen receptor and the co-activator, Tif2, in live cells using two color, two photon fluorescence cross-correlation spectroscopy.
Two-photon, two-color fluorescence cross-correlation spectroscopy (TPTCFCCS) was used to directly detect ligand-dependent interaction between an eCFP-fusion of the androgen receptor (eCFP-AR) and an eYFP fusion of the nuclear receptor co-activator, Tif2 (eYFP-Tif2) in live cells. As expected, these two proteins were co-localized in the nucleus in the presence of ligand. Analysis of the cross-correlation amplitude revealed that AR was on average 81% bound to Tif2 in the presence of agonist, whereas the fractional complex formation decreased to 56% in the presence of antagonist. Residual AR-Tif2 interaction in presence of antagonist is likely mediated by its ligand-independent activation function. These studies demonstrate that using TPTCFCCS it is possible to quantify ligand-dependent interaction of nuclear receptors with co-regulator partners in live cells, making possible a vast array of structure-function studies for these important transcriptional regulators.
Rosales
Tilman
T
Optical Spectroscopy - Section, LBC, NHLBI, NIH, Bethesda, MD 20892-1412, USA.
Georget
Virginie
V
Malide
Daniela
D
Smirnov
Aleksandr
A
Xu
Jianhua
J
Combs
Christian
C
Knutson
Jay R
JR
Nicolas
Jean-Claude
JC
Royer
Catherine A
CA
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D008024:Ligands; D036641:Microscopy, Fluorescence, Multiphoton; D051958:Nuclear Receptor Coactivator 2 / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011944:Receptors, Androgen / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D008024:Ligands; C498001:NCOA2 protein, human; D051958:Nuclear Receptor Coactivator 2; D011944:Receptors, Androgen