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
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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.
KeywordsAndrogen Receptor Tif2 Casodex Androgen Receptor Function Selective Androgen Receptor Modulator
This work was supported by intra-mural funding to the investigators through the NIH and the INSERM. The authors would like to sincerely thank Dawn Walker, Gordon Hager, Cem Elbi and Bill DeGraff for their assistance in cell culture and transfections, as well as for stimulating discussions.
- Chen Y, Muller JD, So PT, Gratton E (1999) The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 77:553–567Google Scholar
- De Gendt K, Swinnen JV, Saunders PT, Schoonjans L, Dewerchin M, Devos A, Tan K, Atanassova N, Claessens F, Lecureuil C, Heyns W, Carmeliet P, Guillou F, Sharpe RM, Verhoeven G (2004) A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. Proc Natl Acad Sci USA 101:1327–1332CrossRefADSGoogle Scholar