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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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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.

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Reference

  • Bacia K, Majoul IV, Schwille P (2002) Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis. Biophys J 83:1184–1193

    Article  Google Scholar 

  • Bacia K, Kim SA, Schwille P (2006) Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3:83–89

    Article  Google Scholar 

  • Baek SH, Ohgi KA, Nelson CA, Welsbie D, Chen C, Sawyers CL, Rose DW, Rosenfeld MG (2006) Ligand-specific allosteric regulation of coactivator functions of androgen receptor in prostate cancer cells. Proc Natl Acad Sci USA 103(9):3100–3105

    Article  ADS  Google Scholar 

  • Baudendistel N, Muller G, Waldeck W, Angel P, Langowski J (2005) Two-hybrid fluorescence cross-correlation spectroscopy detects protein-protein interactions in vivo. Chemphyschem 6:984–990

    Article  Google Scholar 

  • Berland K, Shen G (2003) Excitation saturation in two-photon fluorescence correlation spectroscopy. Appl Opt 42:5566–5576

    ADS  Google Scholar 

  • Black BE, Paschal BM (2004) Intranuclear organization and function of the androgen receptor. Trends Endocrinol Metab 15:411–417

    Article  Google Scholar 

  • Burnstein KL (2005) Regulation of androgen receptor levels: implications for prostate cancer progression and therapy. J Cell Biochem 95:657–669

    Article  Google Scholar 

  • Chang C, Chen YT, Yeh SD, Xu Q, Wang RS, Guillou F, Lardy H, Yeh S (2004) Infertility with defective spermatogenesis and hypotestosteronemia in male mice lacking the androgen receptor in Sertoli cells. Proc Natl Acad Sci USA 101:6876–6881

    Article  ADS  Google Scholar 

  • Chen Y, Muller JD, So PT, Gratton E (1999) The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 77:553–567

    Google Scholar 

  • Chen Y, Wei LN, Muller JD (2003) Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy. Proc Natl Acad Sci USA 100:15492–15497

    Article  ADS  Google Scholar 

  • Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, Rosenfeld MG, Sawyers CL (2004) Molecular determinants of resistance to antiandrogen therapy. Nat Med 10:33–39

    Article  Google Scholar 

  • Chen Y, Tekmen M, Hillesheim L, Skinner J, Wu B, Muller JD (2005a) Dual-color photon-counting histogram. Biophys J 88:2177–2192

    Article  Google Scholar 

  • Chen Y, Wei LN, Muller JD (2005b) Unraveling protein-protein interactions in living cells with fluorescence fluctuation brightness analysis. Biophys J 88:4366–4377

    Article  Google Scholar 

  • Culig Z, Comuzzi B, Steiner H, Bartsch G, Hobisch A (2004) Expression and function of androgen receptor coactivators in prostate cancer. J Steroid Biochem Mol Biol 92:265–271

    Article  Google Scholar 

  • d’Ancona FC, Debruyne FM (2005) Endocrine approaches in the therapy of prostate carcinoma. Hum Reprod Update 11:309–317

    Article  Google 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 101:1327–1332

    Article  ADS  Google Scholar 

  • Edwards J, Bartlett JM (2005a) The androgen receptor and signal-transduction pathways in hormone-refractory prostate cancer. Part 1. Modifications to the androgen receptor. BJU Int 95:1320–1326

    Article  Google Scholar 

  • Edwards J, Bartlett JM (2005b) The androgen receptor and signal-transduction pathways in hormone-refractory prostate cancer. Part 2. Androgen-receptor cofactors and bypass pathways. BJU Int 95:1327–1335

    Article  Google Scholar 

  • Georget V, Lobaccaro JM, Terouanne B, Mangeat P, Nicolas JC, Sultan C (1997) Trafficking of the androgen receptor in living cells with fused green fluorescent protein-androgen receptor. Mol Cell Endocrinol 129:17–26

    Article  Google Scholar 

  • Georget V, Bourguet W, Lumbroso S, Makni S, Sultan C, Nicolas JC (2005) Glutamic acid 709 substitutions highlight the importance of the interaction between androgen receptor helices H3 and H12 for androgen and antiandrogen actions. Mol Endocrinol 20(4):724–734

    Article  Google Scholar 

  • Haustein E, Schwille P (2004) Single-molecule spectroscopic methods. Curr Opin Struct Biol 14:531–540

    Article  Google Scholar 

  • Heinze KG, Koltermann A, Schwille P (2000) Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis. Proc Natl Acad Sci USA 97:10377–10382

    Article  ADS  Google Scholar 

  • Karvonen U, Janne OA, Palvimo JJ (2002) Pure antiandrogens disrupt the recruitment of coactivator GRIP1 to colocalize with androgen receptor in nuclei. FEBS Lett 523:43–47

    Article  Google Scholar 

  • Kettling U, Koltermann A, Schwille P, Eigen M (1998) Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc Natl Acad Sci USA 95:1416–1420

    Article  ADS  Google Scholar 

  • Kim SA, Heinze KG, Waxham MN, Schwille P (2004) Intracellular calmodulin availability accessed with two-photon cross-correlation. Proc Natl Acad Sci USA 101:105–110

    Article  ADS  Google Scholar 

  • Kim SA, Heinze KG, Bacia K, Waxham MN, Schwille P (2005) Two-photon cross correlation analysis of intracellular reactions with variable stoichiometry. Biophys J 88:4319–4336

    Article  Google Scholar 

  • Larson DR, Gosse JA, Holowka DA, Baird BA, Webb WW (2005) Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells. J Cell Biol 171:527–536

    Article  Google Scholar 

  • Lonard DM, O’Malley BW (2005) Expanding functional diversity of the coactivators. Trends Biochem Sci 30:126–132

    Article  Google Scholar 

  • Nettles KW, Greene GL (2005) Ligand control of coregulator recruitment to nuclear receptors. Annu Rev Physiol 67:309–333

    Article  Google Scholar 

  • Powell SM, Christiaens V, Voulgaraki D, Waxman J, Claessens F, Bevan CL (2004) Mechanisms of androgen receptor signalling via steroid receptor coactivator-1 in prostate. Endocr Relat Cancer 11:117–130

    Article  Google Scholar 

  • Saito K, Wada I, Tamura M, Kinjo M (2004) Direct detection of caspase-3 activation in single live cells by cross-correlation analysis. Biochem Biophys Res Commun 324:849–854

    Article  Google Scholar 

  • Smith CL, O’Malley BW (2004) Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocr Rev 25:45–71

    Article  Google Scholar 

  • Thews E, Gerken M, Eckert R, Zapfel J, Tietz C, Wrachtrup J (2005) Cross talk free fluorescence cross-correlation spectroscopy in live cells. Biophys J 89:2069–2076

    Article  Google Scholar 

  • Tyagi RK, Lavrovsky Y, Ahn SC, Song CS, Chatterjee B, Roy AK (2000) Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells. Mol Endocrinol 14:1162–1174

    Article  Google Scholar 

  • Whitaker HC, Hanrahan S, Totty N, Gamble SC, Waxman J, Cato AC, Hurst HC, Bevan CL (2004) Androgen receptor is targeted to distinct subcellular compartments in response to different therapeutic antiandrogens. Clin Cancer Res 10:7392–7401

    Article  Google Scholar 

  • Wu RC, Smith CL, O’Malley BW (2005) Transcriptional regulation by steroid receptor coactivator phosphorylation. Endocr Rev 26:393–399

    Article  Google Scholar 

  • Xu J, Li Q (2003) Review of the in vivo functions of the p160 steroid receptor coactivator family. Mol Endocrinol 17:1681–1692

    Article  Google Scholar 

  • Zhu P, Baek SH, Bourk EM, Ohgi KA, Garcia-Bassets I, Sanjo H, Akira S, Kotol PF, Glass CK, Rosenfeld MG, Rose DW (2006) Macrophage/cancer cell interactions mediate hormone resistance by a nuclear receptor derepression pathway. Cell 124:615–629

    Article  Google Scholar 

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Acknowledgments

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.

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Authors and Affiliations

  1. Optical Spectroscopy - Section, LBC, NHLBI, NIH, Bethesda, MD, 20892-1412, USA

    Tilman Rosales, Aleksandr Smirnov, Jianhua Xu & Jay R. Knutson

  2. INSERM U540, Montpellier, France

    Virginie Georget & Jean-Claude Nicolas

  3. NHLBI Light Microscopy Core Facility, National Institutes of Health, Bethesda, MD, USA

    Daniela Malide & Christian Combs

  4. INSERM U554, Montpellier, France

    Catherine A. Royer

Authors
  1. Tilman Rosales
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  2. Virginie Georget
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  3. Daniela Malide
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  4. Aleksandr Smirnov
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  5. Jianhua Xu
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  6. Christian Combs
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  7. Jay R. Knutson
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  8. Jean-Claude Nicolas
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  9. Catherine A. Royer
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Correspondence to Catherine A. Royer.

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Rosales, T., Georget, V., Malide, D. et al. 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. Eur Biophys J 36, 153–161 (2007). https://doi.org/10.1007/s00249-006-0095-1

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  • DOI: https://doi.org/10.1007/s00249-006-0095-1

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