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European Biophysics Journal

, Volume 36, Issue 2, pp 153–161 | Cite as

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

  • Tilman Rosales
  • Virginie Georget
  • Daniela Malide
  • Aleksandr Smirnov
  • Jianhua Xu
  • Christian Combs
  • Jay R. Knutson
  • Jean-Claude Nicolas
  • Catherine A. Royer
Biophysics Letter

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.

Keywords

Androgen Receptor Tif2 Casodex Androgen Receptor Function Selective Androgen Receptor Modulator 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

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.

Reference

  1. Bacia K, Majoul IV, Schwille P (2002) Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis. Biophys J 83:1184–1193CrossRefGoogle Scholar
  2. Bacia K, Kim SA, Schwille P (2006) Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3:83–89CrossRefGoogle Scholar
  3. 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):3100–3105CrossRefADSGoogle Scholar
  4. Baudendistel N, Muller G, Waldeck W, Angel P, Langowski J (2005) Two-hybrid fluorescence cross-correlation spectroscopy detects protein-protein interactions in vivo. Chemphyschem 6:984–990CrossRefGoogle Scholar
  5. Berland K, Shen G (2003) Excitation saturation in two-photon fluorescence correlation spectroscopy. Appl Opt 42:5566–5576ADSGoogle Scholar
  6. Black BE, Paschal BM (2004) Intranuclear organization and function of the androgen receptor. Trends Endocrinol Metab 15:411–417CrossRefGoogle Scholar
  7. Burnstein KL (2005) Regulation of androgen receptor levels: implications for prostate cancer progression and therapy. J Cell Biochem 95:657–669CrossRefGoogle Scholar
  8. 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 101:6876–6881CrossRefADSGoogle Scholar
  9. Chen Y, Muller JD, So PT, Gratton E (1999) The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 77:553–567Google Scholar
  10. Chen Y, Wei LN, Muller JD (2003) Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy. Proc Natl Acad Sci USA 100:15492–15497CrossRefADSGoogle Scholar
  11. 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 10:33–39CrossRefGoogle Scholar
  12. Chen Y, Tekmen M, Hillesheim L, Skinner J, Wu B, Muller JD (2005a) Dual-color photon-counting histogram. Biophys J 88:2177–2192CrossRefGoogle Scholar
  13. Chen Y, Wei LN, Muller JD (2005b) Unraveling protein-protein interactions in living cells with fluorescence fluctuation brightness analysis. Biophys J 88:4366–4377CrossRefGoogle Scholar
  14. 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 92:265–271CrossRefGoogle Scholar
  15. d’Ancona FC, Debruyne FM (2005) Endocrine approaches in the therapy of prostate carcinoma. Hum Reprod Update 11:309–317CrossRefGoogle Scholar
  16. 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
  17. 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 95:1320–1326CrossRefGoogle Scholar
  18. 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 95:1327–1335CrossRefGoogle Scholar
  19. 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 129:17–26CrossRefGoogle Scholar
  20. 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):724–734CrossRefGoogle Scholar
  21. Haustein E, Schwille P (2004) Single-molecule spectroscopic methods. Curr Opin Struct Biol 14:531–540CrossRefGoogle Scholar
  22. 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 97:10377–10382CrossRefADSGoogle Scholar
  23. Karvonen U, Janne OA, Palvimo JJ (2002) Pure antiandrogens disrupt the recruitment of coactivator GRIP1 to colocalize with androgen receptor in nuclei. FEBS Lett 523:43–47CrossRefGoogle Scholar
  24. 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 95:1416–1420CrossRefADSGoogle Scholar
  25. Kim SA, Heinze KG, Waxham MN, Schwille P (2004) Intracellular calmodulin availability accessed with two-photon cross-correlation. Proc Natl Acad Sci USA 101:105–110CrossRefADSGoogle Scholar
  26. Kim SA, Heinze KG, Bacia K, Waxham MN, Schwille P (2005) Two-photon cross correlation analysis of intracellular reactions with variable stoichiometry. Biophys J 88:4319–4336CrossRefGoogle Scholar
  27. 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 171:527–536CrossRefGoogle Scholar
  28. Lonard DM, O’Malley BW (2005) Expanding functional diversity of the coactivators. Trends Biochem Sci 30:126–132CrossRefGoogle Scholar
  29. Nettles KW, Greene GL (2005) Ligand control of coregulator recruitment to nuclear receptors. Annu Rev Physiol 67:309–333CrossRefGoogle Scholar
  30. 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 11:117–130CrossRefGoogle Scholar
  31. 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 324:849–854CrossRefGoogle Scholar
  32. Smith CL, O’Malley BW (2004) Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocr Rev 25:45–71CrossRefGoogle Scholar
  33. 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 89:2069–2076CrossRefGoogle Scholar
  34. 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 14:1162–1174CrossRefGoogle Scholar
  35. 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 10:7392–7401CrossRefGoogle Scholar
  36. Wu RC, Smith CL, O’Malley BW (2005) Transcriptional regulation by steroid receptor coactivator phosphorylation. Endocr Rev 26:393–399CrossRefGoogle Scholar
  37. Xu J, Li Q (2003) Review of the in vivo functions of the p160 steroid receptor coactivator family. Mol Endocrinol 17:1681–1692CrossRefGoogle Scholar
  38. 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 124:615–629CrossRefGoogle Scholar

Copyright information

© EBSA 2006

Authors and Affiliations

  • Tilman Rosales
    • 1
  • Virginie Georget
    • 2
  • Daniela Malide
    • 3
  • Aleksandr Smirnov
    • 1
  • Jianhua Xu
    • 1
  • Christian Combs
    • 3
  • Jay R. Knutson
    • 1
  • Jean-Claude Nicolas
    • 2
  • Catherine A. Royer
    • 4
  1. 1.Optical Spectroscopy - SectionLBC, NHLBI, NIHBethesdaUSA
  2. 2.INSERM U540MontpellierFrance
  3. 3.NHLBI Light Microscopy Core FacilityNational Institutes of HealthBethesdaUSA
  4. 4.INSERM U554MontpellierFrance

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