Fluorescence Anisotropy Microplate Assay to Investigate the Interaction of Full-Length Steroid Receptor Coactivator-1a with Steroid Receptors

  • Chen Zhang
  • Steven K. Nordeen
  • David J. Shapiro
Part of the Methods in Molecular Biology book series (MIMB, volume 977)


Estrogens, acting via estrogen receptor (ER) play key roles in growth, differentiation, and gene regulation in the reproductive, central nervous, and skeletal systems. ER-mediated gene transcription contributes to the development and spread of breast, uterine, and liver cancer. Steroid receptor coactivator-1a (SRC1a) belongs to the P160 family of coactivators, which is the best known of the many coactivators implicated in ER-mediated transactivation. Binding of full-length P160 coactivators to steroid receptors has been difficult to investigate in vitro. This chapter details how to investigate the interaction of SRC1a with ER using the fluorescence anisotropy/polarization microplate assay (FAMA).

Key words

Estrogen receptor Steroid receptor coactivator-1a Fluorescence anisotropy Microplate LxxLL motif 


  1. 1.
    Luconi M, Forti G, Baldi E (2002) Genomic and nongenomic effects of estrogens: molecular mechanisms of action and clinical implications for male reproduction. J Steroid Biochem Mol Biol 80:369–381PubMedCrossRefGoogle Scholar
  2. 2.
    Katzenellenbogen BS, Katzenellenbogen JA (2000) Estrogen receptor transcription and transactivation: estrogen receptor alpha and estrogen receptor beta: regulation by selective estrogen receptor modulators and importance in breast cancer. Breast Cancer Res 2:335–344PubMedCrossRefGoogle Scholar
  3. 3.
    Tsai MJ, O’Malley BW (1994) Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486PubMedCrossRefGoogle Scholar
  4. 4.
    Evans RM (1988) The steroid and thyroid hormone receptor superfamily. Science 240: 889–895PubMedCrossRefGoogle Scholar
  5. 5.
    Mosselman S, Polman J, Dijkema R (1996) ER beta: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53PubMedCrossRefGoogle Scholar
  6. 6.
    Carroll JS, Brown M (2006) Estrogen receptor target gene: an evolving concept. Mol Endocrinol 20:1707–1714PubMedCrossRefGoogle Scholar
  7. 7.
    Carroll JS, Liu XS, Brodsky AS, Li W, Meyer CA, Szary AJ, Eeckhoute J, Shao W, Hestermann EV, Geistlinger TR, Fox EA, Silver PA, Brown M (2005) Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1. Cell 122:33–43PubMedCrossRefGoogle Scholar
  8. 8.
    O’Lone R, Frith MC, Karlsson EK, Hansen U (2004) Genomic targets of nuclear estrogen receptors. Mol Endocrinol 18:1859–1875PubMedCrossRefGoogle Scholar
  9. 9.
    Klinge CM, Jernigan SC, Smith SL, Tyulmenkov VV, Kulakosky PC (2001) Estrogen response element sequence impacts the conformation and transcriptional activity of estrogen receptor alpha. Mol Cell Endocrinol 174:151–166PubMedCrossRefGoogle Scholar
  10. 10.
    Krieg SA, Krieg AJ, Shapiro DJ (2001) A unique downstream estrogen responsive unit mediates estrogen induction of proteinase inhibitor-9, a cellular inhibitor of IL-1beta- converting enzyme (caspase 1). Mol Endocrinol 15:1971–1982PubMedCrossRefGoogle Scholar
  11. 11.
    Glass CK, Rosenfeld MG (2000) The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14:121–141PubMedGoogle Scholar
  12. 12.
    Xu J, O’Malley BW (2002) Molecular mechanisms and cellular biology of the steroid receptor coactivator (SRC) family in steroid receptor function. Rev Endocr Metab Disord 3:185–192PubMedCrossRefGoogle Scholar
  13. 13.
    Spiegelman BM, Heinrich R (2004) Biological control through regulated transcriptional coactivators. Cell 119:157–167PubMedCrossRefGoogle Scholar
  14. 14.
    Kalkhoven E, Valentine JE, Heery DM, Parker MG (1998) Isoforms of steroid receptor co-activator 1 differ in their ability to potentiate transcription by the oestrogen receptor. EMBO J 17:232–243PubMedCrossRefGoogle Scholar
  15. 15.
    Wang SY, Ahn BS, Harris R, Nordeen SK, Shapiro DJ (2004) Fluorescence anisotropy microplate assay for analysis of steroid receptor-DNA interactions. Biotechniques 37:807-8, 810-7Google Scholar
  16. 16.
    Wang S, Zhang C, Nordeen SK, Shapiro DJ (2007) In vitro fluorescence anisotropy analysis of the interaction of full-length SRC1a with estrogen receptors alpha and beta supports an active displacement model for coregulator utilization. J Biol Chem 282:2765–2775PubMedCrossRefGoogle Scholar
  17. 17.
    Mao C, Patterson NM, Cherian MT, Aninye IO, Zhang C, Montoya JB, Cheng J, Putt KS, Hergenrother PJ, Wilson EM, Nardulli AM, Nordeen SK, Shapiro DJ (2008) A new small molecule inhibitor of estrogen receptor alpha binding to estrogen response elements blocks estrogen-dependent growth of cancer cells. J Biol Chem 283:12819–12830PubMedCrossRefGoogle Scholar
  18. 18.
    Kretzer NM, Cherian MT, Mao CJ, Aninye IO, Reynolds PD, Schiff R, Hergenrother PJ, Nordeen SK, Wilson EM, Shapiro DJ (2010) A noncompetitive small molecule inhibitor of estrogen-regulated gene expression and breast cancer cell growth that enhances proteasome-dependent degradation of estrogen receptor alpha. J Biol Chem 285:41863–41873PubMedCrossRefGoogle Scholar
  19. 19.
    Shapiro DJ, Mao CJ, Cherian MT (2011) Small molecule inhibitors as probes for estrogen and androgen receptor action. J Biol Chem 286:4043–4048PubMedCrossRefGoogle Scholar
  20. 20.
    Zhang CC, Krieg S, Shapiro DJ (1999) HMG-1 stimulates estrogen response element binding by estrogen receptor from stably transfected HeLa cells. Mol Endocrinol 13:632–643PubMedCrossRefGoogle Scholar
  21. 21.
    Thackray VG, Nordeen SK (2002) High-yield purification of functional, full-length steroid receptor coactivator 1 expressed in insect cells. Biotechniques 32(260):262–263Google Scholar
  22. 22.
    Bramlett KS, Wu Y, Burris TP (2001) Ligands specify coactivator nuclear receptor (NR) box affinity for estrogen receptor subtypes. Mol Endocrinol 15:909–922PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Chen Zhang
    • 1
  • Steven K. Nordeen
    • 2
  • David J. Shapiro
    • 1
  1. 1.Department of BiochemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  2. 2.University of Colorado at Denver and Health Sciences CenterAuroraUSA

Personalised recommendations