Skip to main content
SpringerLink
Log in

Application of Green Fluorescent Protein to the Study of Dynamic Protein-Protein Interactions and Subcellular Trafficking of Steroid Receptors

  • Protocol
Steroid Receptor Methods

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 176))

  • 507 Accesses

Abstract

The green fluorescent protein (GFP) from the jellyfish, Aequoria victoria, converts blue light to green fluorescence when expressed in intact cells and transgenic animals, and has proven to be a powerful tool for biological and medical research. This chapter describes the application of spectrally distinguishable variants of GFP to the investigation of steroid hormone receptor action. Topics that are covered include the design of GFP-receptor chimeras, the expression of GFP-fusion proteins in cells in culture, the detection of the GFP-tagged receptors in living and fixed cells, and the use of GFP-variants to study the colocalization and interaction of steroid receptors and other proteins. Specifically, the authors describe the application of GFP-tagged steroid receptors to assess issues in receptor trafficking and receptor interaction with coactivator proteins. The latter approach employs fluorescence resonance energy transfer (FRET), a technique that effectively permits a 100-fold enhancement beyond the inherent resolving power of the light microscope.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ward, W. W., Cody, C. W., Hart, R. C., and Cormier, M. J. (1980) Spectrophotometric identity of the energy transfer chromophores in Renilla and Aequorea green-fluorescent proteins. Photochem. Photobiol. 31, 611–615.

    Article  CAS  Google Scholar 

  2. Presley, J. F., Cole, N. B., Schroer, T. A., Hirschberg, K., Zaal, K.J., and Lippincott-Schwartz, J. (1997) ER-to-Golgi transport visualized in living cells. Nature 389, 81–85.

    Article  PubMed  CAS  Google Scholar 

  3. Aubin, J. E. (1979) Autofluorescence of viable cultured mammalian cells. J. Histochem. Cytochem. 27, 36–43.

    PubMed  CAS  Google Scholar 

  4. Heim, R., Cubitt, A. B., and Tsien, R. Y. (1995) Improved green fluorescence. Nature 373, 663–664.

    Article  PubMed  CAS  Google Scholar 

  5. Cormack, B. P., Valdivia, R. H., and Falkow, S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.

    Article  PubMed  CAS  Google Scholar 

  6. Crameri, A., Whitehorn, E. A., Tate, E., and Stemmer, W. P. (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnol. 14, 315–319.

    Article  CAS  Google Scholar 

  7. Heim, R., Prasher, D. C., and Tsien, R. Y. (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91, 12,501–12,504.

    Article  PubMed  CAS  Google Scholar 

  8. Heim, R. and Tsien, R. Y. (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178–182.

    Article  PubMed  CAS  Google Scholar 

  9. Ellenberg, J., Lippincott-Schwartz J., and Presley, J. F. (1998) Two-color green fluorescent protein time-lapse imaging. Biotechniques 25, 838–842.

    PubMed  CAS  Google Scholar 

  10. Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887.

    Article  PubMed  CAS  Google Scholar 

  11. Dopf, J. and Horagan, T. M. (1996) Deletion mapping of Aequorea victoria green fluorescent protein. Gene 173, 39–44.

    Article  PubMed  CAS  Google Scholar 

  12. Lim, C. S., Baumann, C. T., Htun, H., Xian, W., Irie, M., Smith, C. L., and Hager, G. L. (1999) Differential localization and activity of the A-and B-forms of the human progesterone receptor using green fluorescent protein chimeras. Mol. Endocrinol. 13, 366–375.

    Article  PubMed  CAS  Google Scholar 

  13. Carey, K. L., Richards, S. A., Lounsbury, K. M., and Macara, I. G. (1996) Evidence using a green fluorescent protein-glucocorticoid receptor chimera that the Ran TC4 GTPase mediates an essential function independent of nuclear protein import. J. Cell Biol. 133, 985–996.

    Article  PubMed  CAS  Google Scholar 

  14. Htun, H., Barsony, J., Renyi, I., Gould, D. L., and Hager, G. L. (1996) Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera. Proc. Natl. Acad. Sci. USA 93, 4845–4850.

    Article  PubMed  CAS  Google Scholar 

  15. Galigniana, M. D., Scruggs, J. L., Herrington, J., Welsh, M. J., Carter-Su, C., Housley, P. R., and Pratt, W. B. (1998) Heat shock protein 90-dependent (geldanamycin-inhibited) movement of the glucocorticoid receptor through the cytoplasm to the nucleus requires intact cytoskeleton. Mol. Endocrinol. 12, 1903–1913.

    Article  PubMed  CAS  Google Scholar 

  16. Okamoto, K., Tanaka, H., Ogawa, H., Makino, Y., Eguchi, H., Hayashi, S., et al. (1999) Redox-dependent regulation of nuclear import of the glucocorticoid receptor. J. Biol. Chem. 274, 10,363–10,371.

    Article  PubMed  CAS  Google Scholar 

  17. Galigniana, M. D., Housley, P. R., DeFranco, D. B., and Pratt, W. B. (1999) Inhibition of glucocorticoid receptor nucleocytoplasmic shuttling by okadaic acid requires intact cytoskeleton. J. Biol. Chem. 274, 16,222–16,227.

    Article  PubMed  CAS  Google Scholar 

  18. Nishi, M., Takenaka, N., Morita, N., Ito, T., Ozawa, H., and Kawata, M. (1999) Real-time imaging of glucocorticoid receptor dynamics in living neurons and glial cells in comparison with non-neural cells. Eur. J. Neurosci. 11, 1927–1936.

    Article  PubMed  CAS  Google Scholar 

  19. Strubing, C. and Clapham, D. E. (1999) Active nuclear import and export is independent of lumenal Ca2+ stores in intact mammalian cells. J. Gen. Physiol. 113, 239–248.

    Article  PubMed  CAS  Google Scholar 

  20. Day, R. N. (1998) Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy. Mol. Endocrinol. 12, 1410–1419.

    Article  PubMed  CAS  Google Scholar 

  21. Day, R. N., Nordeen, S. K., and Wan, Y. (1999) Visualizing protein-protein interactions in the nucleus of the living cell. Mol. Endocrinol. 13, 517–526.

    Article  PubMed  CAS  Google Scholar 

  22. Htun, H., Holth, L. T., Walker, D., Davie, J. R., and Hager, G. L. (1999) Direct visualization of the human estrogen receptor alpha reveals a role for ligand in the nuclear distribution of the receptor. Mol. Biol. Cell 10, 471–486.

    PubMed  CAS  Google Scholar 

  23. Georget, V., Lobaccaro, J.M., Terouanne, B., Mangeat, P., Nicolas, J. C., and 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  PubMed  CAS  Google Scholar 

  24. Georget, V., Terouanne, B., Lumbroso, S., Nicolas, J. C., and Sultan, C. (1998) Trafficking of androgen receptor mutants fused to green fluorescent protein: a new investigation of partial androgen insensitivity syndrome. J. Clin. Endocrinol. Metab. 83, 3597–3603.

    Article  PubMed  CAS  Google Scholar 

  25. Fejes-Toth, G., Pearce, D., and Naray-Fejes-Toth, A. (1998) Subcellular localization of mineralocorticoid receptors in living cells: effects of receptor agonists and antagonists. Proc. Natl. Acad. Sci. USA 95, 2973–2978.

    Article  PubMed  CAS  Google Scholar 

  26. Zhu, X. G., Hanover, J. A., Hager, G. L., and Cheng, S.Y. (1998) Hormoneinduced translocation of thyroid hormone receptors in living cells visualized using a receptor green fluorescent protein chimera. J. Biol. Chem. 273, 27,058–27,063.

    Article  PubMed  CAS  Google Scholar 

  27. Racz, A. and Barsony, J. (1999) Hormone-dependent translocation of vitamin D receptors is linked to transactivation. J. Biol. Chem. 274, 19352–19360.

    Article  PubMed  CAS  Google Scholar 

  28. Lee, C. H., Chinpaisal, C., and Wei, L. N. (1998) Cloning and characterization of mouse RIP 140, a corepressor for nuclear orphan receptor TR2. Mol. Cell. Biol. 18, 6745–6755.

    PubMed  CAS  Google Scholar 

  29. Wan, Y., Coxe, K. K., Thackray, V. G., Housley, P. R., and Nordeen, S. K. (2001) Separable features of the ligand-binding domain determine the differential subcellular localization and ligand-binding specificity of glucocorticoid receptor and progesterone receptor. Mol. Endocrinol. 15, 17–31.

    Article  PubMed  CAS  Google Scholar 

  30. Liu, J. and DeFranco, D. B. (1999) Chromatin recycling of glucocorticoid receptors: implications for multiple roles of heat shock protein 90. Mol. Endocrinol. 13, 355–365.

    Article  PubMed  CAS  Google Scholar 

  31. Tyagi, R. K., Amazit, L., Lescop, P., Milgrom, E., and Guiochon-Mantel, A. (1998) Mechanisms of progesterone receptor export from nuclei: role of nuclear localization signal, nuclear export signal, and ran guanosine triphosphate. Mol. Endocrinol. 12, 1684–1695.

    Article  PubMed  CAS  Google Scholar 

  32. Hache, R. J. G., Tse, R., Reich, T., Savory, J.G.A., and Lefebvre, Y. A. (1999) Nucleocytoplasmic trafficking of steroid-free glucocorticoid receptor. J. Biol. Chem. 274, 1432–1439.

    Article  PubMed  CAS  Google Scholar 

  33. McNally, J. G., Muller, W. G., Walker, D., Wolford, R., and Hager, G. L. (2000) The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science 287, 1262–1265.

    Article  PubMed  CAS  Google Scholar 

  34. Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A., and Tsien, R. Y. (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20, 448–455.

    Article  PubMed  CAS  Google Scholar 

  35. Mitra, R. D., Silva, C. M., and Youvan, D. C. (1996) Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein Gene 173, 13–17.

    Article  PubMed  CAS  Google Scholar 

  36. Pollok, B. A. and Heim, R. (1999) Using GFP in FRET-based applications. Trends Cell Biol. 9, 57–60.

    Article  PubMed  CAS  Google Scholar 

  37. Gordon, G.W., Berry, G., Liang, X. H., Levine, B., and Herman, B. (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74, 2702–2713.

    Article  PubMed  CAS  Google Scholar 

  38. Mahajan, N. and Herman, B. (1997) Alterations in the molecular interaction of Bcl-2 and Bax during apoptosis assessed using fluorescence resonance energy transfer (FRET) microscopy and green fluorescent protein (GFP-Bax and blue fluorescent protein (BFP)-Bcl-2 expressing proteins, in Proceedings of Microscopy and Microanalysis (Baily, G. W., Dimlich, R. V. W., Alexander, K.B., McCarthy, J. J., and Pretlow, T. P., eds.), vol. 3, Springer-Verlag, New York pp.135–136.

    Google Scholar 

  39. Periasamy, A. and Day, R. N. (1998) FRET imaging of PIT-1 protein interactions in living cells. J. Biomed. Optics 3, 154–160.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, CO

    Steven K. Nordeen & Yihong Wan

  2. Department of Pharmacology and Physiology, University of South Carolina School of Medicine, Columbia, SC

    Paul R. Housley

  3. Departments of Medicine and Cell Biology, National Science Foundation Center for Biological Timing, University of Virginia Health Sciences Center, Charlottesville, VA

    Richard N. Day

Authors
  1. Steven K. Nordeen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paul R. Housley
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yihong Wan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Richard N. Day
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Blueprint Technologies, Englewood, CO

    Benjamin A. Lieberman

Rights and permissions

Reprints and permissions

Copyright information

© 2001 Humana Press Inc.

About this protocol

Cite this protocol

Nordeen, S.K., Housley, P.R., Wan, Y., Day, R.N. (2001). Application of Green Fluorescent Protein to the Study of Dynamic Protein-Protein Interactions and Subcellular Trafficking of Steroid Receptors. In: Lieberman, B.A. (eds) Steroid Receptor Methods. Methods in Molecular Biology™, vol 176. Humana Press. https://doi.org/10.1385/1-59259-115-9:179

Download citation

  • DOI: https://doi.org/10.1385/1-59259-115-9:179

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-754-0

  • Online ISBN: 978-1-59259-115-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation