title: 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.
abstract: 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.
authors.affiliation: Optical Spectroscopy - Section, LBC, NHLBI, NIH, Bethesda, MD 20892-1412, USA.
authors.forename: Tilman
authors.initials: T
authors.lastname: Rosales
authors.forename: Virginie
authors.initials: V
authors.lastname: Georget
authors.forename: Daniela
authors.initials: D
authors.lastname: Malide
authors.forename: Aleksandr
authors.initials: A
authors.lastname: Smirnov
authors.forename: Jianhua
authors.initials: J
authors.lastname: Xu
authors.forename: Christian
authors.initials: C
authors.lastname: Combs
authors.forename: Jay R
authors.initials: JR
authors.lastname: Knutson
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Nicolas
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Royer
mesh_terms: 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*
chemical_list: D008024:Ligands; C498001:NCOA2 protein, human; D051958:Nuclear Receptor Coactivator 2; D011944:Receptors, Androgen