Characterization of Nuclear Receptor Ligands by Multiplexed Peptide Interactions

Marie A. Iannone1

1 GlaxoSmithKline, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.6
DOI:  10.1002/0471142956.cy1306s35
Online Posting Date:  February, 2006
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This unit describes a method to evaluate the effect that small molecules have on the binding interactions of a nuclear receptor protein with a series of peptides. The multiplexed microsphere‐based system employs peptide‐coupled microsphere populations that are fluorescently unique and thereby identifiable by flow cytometric analysis. Up to 100 different peptide–nuclear receptor interactions may be analyzed in a single well of a 96‐well microtiter plate. This approach allows rapid and sensitive characterization of nuclear receptor ligands based on nuclear receptor protein–peptide interaction profiles. Since nuclear receptor binding interactions are dynamically related to protein conformation, the approach allows rapid evaluation of nuclear receptor ligands that may impart unique protein structure. The no‐wash format and the high surface density of the microsphere‐coupled interaction partner offer a moderately high‐throughput system to examine low‐ to high‐affinity interactions with excellent sensitivity. This approach, although described for nuclear receptors, may also be applied to other types of molecular interactions.

Keywords: Microsphere; flow cytometry; nuclear receptor; coactivator; corepressor; cofactor; agonist; antagonist; ligand; ligand binding domain; protein conformation

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Table of Contents

  • Basic Protocol 1: Multiplexed Binding Assay
  • Support Protocol 1: Coupling of Biotinylated Peptides to Lumavidin‐Coated Microspheres
  • Support Protocol 2: Coupling of Biotinylated Nuclear Receptor LBD to Streptavidin‐Alexa fluor 532
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Multiplexed Binding Assay

  • Peptide‐coupled microsphere suspension (see protocol 2)
  • PBS‐TBN/DTT (see recipe)
  • DMSO with or without ligand
  • Alexa Fluor 532–labeled NR LBD (see protocol 3)
  • 96‐well flat‐bottom assay plates, non‐binding surface, non‐sterile (Corning)
  • Multichannel pipet
  • LX‐100 flow cytometer with x‐y plate sampler for automated sampling from 96‐well microtiter plates (Luminex Corporation)

Support Protocol 1: Coupling of Biotinylated Peptides to Lumavidin‐Coated Microspheres

  • Phosphate buffered saline without calcium or magnesium (PBS; appendix 2A)
  • xMAP LumAvidin‐coated polystyrene microsphere populations (5.6‐µm diameter; Luminex Corporation); store at 4°C in the dark
  • PBS‐TBN/DTT (see recipe)
  • Lyophilized biotinylated peptide (Synpep or American Peptide)
  • DMSO
  • 5 mM D‐biotin (see recipe)
  • 96‐well filter‐bottom plates, MultiScreen BV 1.2‐µm, clear, non‐sterile (Millipore)
  • MultiScreen vacuum manifold for 96‐well plates (Millipore)

Support Protocol 2: Coupling of Biotinylated Nuclear Receptor LBD to Streptavidin‐Alexa fluor 532

  • Biotinylated nuclear receptor ligand binding domain (NR LBD; non‐biotinylated receptors are commercially available from Invitrogen, Active Motif, Jena Bioscience, Protein One, as well as other manufacturers)
  • 16 µM streptavidin–Alexa Fluor 532 conjugate (see recipe)
  • 5 mM free D‐biotin (see recipe)
  • PBS‐TBN/DTT (see recipe)
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Literature Cited

Literature Cited
   Brzozowski, A.M., Pike, A.C.W., Dauter, Z., Hubbard, R.E., Bonn, T., Engström, O., Ohman, L., Greene, G.L., Gustafsson. J.‐A., and Carlquist, M. 1997. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753‐758.
   Glickman, J.F., Wu, X., Mercuri, R., Illy, C., Bowen, B.R., He, Y., and Sills, M. 2002. A comparison of ALPHAScreen, TR‐FRET, and TRF as assay methods for FXR nuclear receptors. J. Biomol. Screen. 7:3‐10.
   Iannone, M.A., Consler, T.G., Pearce, K.H., Stimmel, J.B., Parks, D.J., and Gray, J.G. 2001. Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres. Cytometry 44:326‐337.
   Iannone, M.A., Simmons, C.A., Kadwell, S.H., Svoboda, D.L., Vanderwall, D.E., Deng, S.‐J., Consler, T.G., Shearin, J., Gray, J.G. and Pearce, K.H. 2004. Correlation between in vitro peptide binding profiles and cellular activities for estrogen receptor modulating compounds. Mol. Endocrinol. 18:1064‐1081.
   Lee, J.W., Lee, Y.C., Na, S.‐Y., Jung, D.‐J. and Lee, S.‐K. 2001. Transcriptional coregulators of the nuclear receptor superfamily: Coactivators and corepressors. Cell. Molec. Life Sci. 58:287‐297.
   Lonard, D.M. and O'Malley, B.W. 2005. Expanding functional diversity of the coactivators. Trends Biochem. Sci. 30:126‐132.
   Myszka, D.G. 2000. Kinetic, equilibrium, and thermodynamic analysis of macromolecular interactions with BIACORE. Methods Enzymol. 323:325‐340.
   Nettles, K.W. and Greene, G.L. 2005. Ligand control of coregulator recruitment to nuclear receptors. Annu. Rev. Physiol. 67:309‐333.
   Nilsson, M., Dahlman‐Wright, K., and Gustafsson, J.A. 2004. Nuclear receptors in disease: The oestrogen receptors. Essays Biochem. 40:157‐167.
   Norris, J.D., Paige, L.A., Christensen, D.J., Chang, C.Y., Huacani, M.R., Fan, D., Hamilton, P.T., Fowlkes, D.M., and McDonnell, D.P. 1999. Peptide antagonists of the human estrogen receptor. Science 285:744‐746.
   Northrop, J.P., Nguyen, D., Piplani, S., Olivan, S.E., Kwan, S.T., Go, N.F., Hart, C.P., and Schatz, P.J. 2000. Selection of estrogen receptor beta‐ and thyroid hormone receptor beta‐specific coactivator‐mimetic peptides using recombinant peptide libraries. Mol. Endocrinol. 14:605‐622.
   Novac, N. and Heinzel, T. 2005. Nuclear receptors: Overview and classification. Curr. Drug Targets ‐ Inflammation & Allergy 3:335‐346.
   Paige, L.A., Christensen, D.J., Gron, H., Norris, J.D. Gottlin, E.B., Padilla, K.M., Chang, C.Y., Ballas, L.M., Hamilton, P.T., McDonnell, D.P., and Fowlkes, D.M. 1999. Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta. Proc. Natl. Acad. Sci. U.S.A. 96:3999‐4004.
   Parker, G.J., Law, T.L., Lenoch, F.J., and Bolger, R.E. 2000. Development of high throughput screening assays using fluorescence polarization: Nuclear receptor‐ligand‐binding and kinase/ phosphatase assays. J. Biomol. Screen. 5:77‐88.
   Pearce, K.H., Iannone, M.A., Simmons, C.A., and Gray, J.G. 2004. Discovery of novel nuclear receptor modulating ligands: An integral role for peptide interaction profiling. Drug Discov. Today 9:741‐751.
   Savkur, R.S., Bramlett, K.S., Clawson, D., and Burris, T.P. 2004. Pharmacology of nuclear receptor‐coregulator recognition. Vitam. Horm. 68:145‐183.
   Schulman, I.G. and Heyman, R.A. 2004. The flip side: Identifying small molecule regulators of nuclear receptors. Chem. Biol. 11:639‐646.
   Shiau, A.K., Barstad, D., Loria, P.M., Cheng, L., Kushner, P.J., Agard, D.A., and Greene, G.L. 1998. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927‐937.
   Sidhu, S.S., Lowman, H.B., Cunningham, B.C., and Wells, J.A. 2000. Phage display for selection of novel binding peptides. Methods Enzymol. 328:333‐363.
   Sladek, R. and Giguere, V. 2000. Orphan nuclear receptors: An emerging family of metabolic regulators. Adv. Pharmacol. 47:23‐87.
   Spencer, T.E., Jenster, G., Burcin, M.M., Allis, C.D., Zhou, J., Mizzen, C.A., McKenna, N.J., Onate, S.A., Tsai, S.Y., Tsai, M.J., and O'Malley, B.W. 1997. Steroid receptor coactivator‐1 is a histone acetyl‐transferase. Nature 389:194‐198.
   Zhou, G., Cummings, R., Hermes, J., and Moller, D.E. 2001. Use of homogeneous time‐resolved fluorescence energy transfer in the measurement of nuclear receptor activation. Methods 25:54‐61.
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