Abstract
The androgen receptor (AR) is the central component of a dynamic conformational and interaction cascade initiated by androgenic hormones. AR function can be modified by cellular inputs not examined in test tube studies of AR action. Thus, there is a need to measure AR conformation and biochemistry directly within the cell where the intracellular locations, levels and availability of the hormone, AR, AR-interacting factors, DNA-binding sites, enzymes that modify those components of AR action, and factors that compete for the formation of functional AR–cofactor complexes may affect AR action. The dynamic nature of the AR functional cycle itself may introduce temporal fluctuations in factor status and location to affect AR output in the intact cell. This chapter focuses on the method of Förster resonance energy transfer which uniquely has the resolving power and ability to directly measure the conformation and biochemistry of AR signaling in living cells.
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References
Day, R.N., Davidson, M.W. (2009) The fluorescent protein palette: tools for cellular imaging. Chem. Soc. Rev. 38, 2887–2921.
Day, R.N., Schaufele, F. (2008) Fluorescent protein tools for studying protein dynamics in living cells. J. Biomed. Optics 13 (031202), 1–6.
Giepmans, B.N., Adams, S.R., Ellisman, M.H., Tsien, R.Y. (2006) The fluorescent toolbox for assessing protein location and function. Science 312, 217–224.
Shaner, N.C., Steinbach, P.A., Tsien, R.Y. (2005) A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909.
Jones, J.O., An, W.F., Diamond, M.I. (2009) AR Inhibitors identified by high-throughput microscopy detection of conformational change and subcellular localization. ACS Chem. Biol. 4, 199–208.
Schaufele, F., Carbonell, X., Guerbadot, M., Borngraeber, S., Chapman, M., Ma, A., Miner, J., Diamond, M. (2005) The structural basis of androgen receptor activation: intramolecular and intermolecular amino–carboxy interactions. Proc. Natl. Acad. Sci. USA 102, 9802–9807.
Heilemann, M. (2010) Fluorescence microscopy beyond the diffraction limit. J. Biotechnol. 149, 243–251.
Hell, S.W. (2007) Far-field optical nanoscopy. Science 316, 1153–1158.
Day, R.N., Schaufele, F. (2005) Imaging molecular interactions in living cells. Mol. Endocrinol. 19, 1675–1686.
Gordon, G.W., Berry, G., Liang, X.H., Levine, B., Herman, B. (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74, 2702–2713.
Vogel, S., Thaler, C., Koushik, S.V. (2006) Fanciful FRET. Sci STKE 2006 (331), re2.
Förster, T. (1948) Zwischenmolekulare energiewanderung und fluoreszenz. Ann. Phys. 6, 54–75.
Förster, T. (1959) Transfer mechanisms of electronic excitation. Discuss. Faraday Soc. 27, 1–17.
Stryer, L., Haugland. R.P. (1967) Energy transfer: a spectroscopic ruler. Proc. Natl. Acad. Sci. USA 58, 719–726.
Patterson, G.H., Piston, D.W., Barisas, B.G. (2000) Förster distances between green fluorescent protein pairs. Anal. Biochem. 284, 438–440.
van Royen, M.E., Cunha, S.M., Brink, M.C., Mattern, K.A., Nigg, A.L., Dubbink, H.J., Verschure, P.J., Trapman, J., Houtsmuller, A.B. (2007) Compartmentalization of androgen receptor protein–protein interactions in living cells. J. Cell. Biol. 177, 63–72.
Kofoed, E.M., Guerbadot, M., Schaufele, F. (2010) Structure, affinity, and availability of estrogen receptor complexes in the cellular environment. J. Biol. Chem. 285, 2428–2437.
Voss, T.C., Demarco, I.A., Day, R.N. (2005) Quantitative imaging of protein interactions in the cell nucleus. Biotechniques 38, 413–424.
Wallrabe, H., Periasamy, A. (2005) Imaging protein molecules using FRET and FLIM microscopy. Curr. Opin. Biotechnol. 16, 19–27.
Levitt, J.A., Matthews, D.R., Ameer-Beg, S.M., Suhling, K. (2009) Fluorescence lifetime and polarization-resolved imaging in cell biology. Curr. Opin. Biotechnol. 20, 28–36.
Piston, D.W., Rizzo, M.A. (2008) FRET by fluorescence polarization microscopy. Methods Cell. Biol. 85, 415–430.
Rizzo, M.A., Springer, G.H., Granada, B., Piston, D.W. (2004) An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449.
Nagai, T., Ibata, K., Park, E.S., Kubota, M., Mikoshiba, K., Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90.
Kofoed, E.M., Guerbadot, M., Schaufele, F. (2008) Dimerization between aequorea fluorescent proteins does not affect interaction between tagged estrogen receptors in living cells. J. Biomed. Opt. 13 (031207), 1–15.
Koushik, S.V., Chen, H., Thaler, C., Puhl, H.L., Vogel, S.S. (2006) Cerulean, Venus, and VenusY67C FRET reference standards. Biophys. J. 91, L99–L101.
Elangovan, M., Wallrabe, H., Chen, Y., Day, R.N., Barroso, M., Periasamy, A. (2003) Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. Methods 29, 58–73.
Chen, H., Puhl, H.L., Ikeda, S.R. (2007) Estimating protein-protein interaction affinity in living cells using quantitative Förster resonance energy transfer measurements. J. Biomed. Opt. 12 (054011), 1–9.
Hoppe, A., Christensen, K., Swanson, J.A. (2002) Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys. J. 83, 3652–3664.
Brewer, J.A., Sleckman, B.P., Swat, W., Muglia, L.J. (2002) Green fluorescent protein–glucocorticoid receptor knockin mice reveal dynamic receptor modulation during thymocyte development. J. Immunol. 169, 1309–1318.
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Schaufele, F. (2011). FRET Analysis of Androgen Receptor Structure and Biochemistry in Living Cells. In: Saatcioglu, F. (eds) Androgen Action. Methods in Molecular Biology, vol 776. Humana Press. https://doi.org/10.1007/978-1-61779-243-4_10
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DOI: https://doi.org/10.1007/978-1-61779-243-4_10
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