Applications for Green Fluorescent Protein in Cell Signaling

  • Paul Brennan
  • Emmanuelle Astoul
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 60)


The discovery of green fluorescent protein (GFP) as a naturally fluorescing protein and its subsequent modification to allow easy detection with standard fluorescent equipment such as the fluorescence-activated cell sorter (FACS) has revolutionized our ability to detect the presence of transfected proteins and to determine their location in whole cells and in organisms. GFP was found in the bioluminescent jellyfish Aequorea (1,2). It has since been modified to change its excitation and emision (3, 4, 5). Expression vectors are now supplied commercially that allow proteins to be tagged easily (6,7). Furthermore, it is a small protein, only 29 kDa, and the DNA encoding it can be easily modified using polymerase chain reaction to introduce any restriction sites for any subcloning work required.


Green Fluorescent Protein Cell Electroporation Cell Line RBL2H3 Green Fluorescent Protein Expression Vector Zeiss Laser Scan 
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  1. 1.
    Morin, J. G. and Hastings, J. W. (1971) Energy transfer in a bioluminescent system. J. Cell. Physiol. 77, 313–318.PubMedCrossRefGoogle Scholar
  2. 2.
    Shimomura, O., Johnson, F. H., and Saiga, Y. (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol. 59, 223–227.CrossRefGoogle Scholar
  3. 3.
    Cormack, B. P., Valdivia, R., and Falkow, S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.PubMedCrossRefGoogle Scholar
  4. 4.
    Heim, R., Cubitt, A. B., and Tsien, R. Y. (1995) Improved green fluorescence. Nature 373, 663–664.PubMedCrossRefGoogle Scholar
  5. 5.
    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.PubMedCrossRefGoogle Scholar
  6. 6.
    Clontech (1999) Living Colors User Manual, vol. PT2040-1, Clontech, Palo Alto, CA, pp. 16–17.Google Scholar
  7. 7.
    Clontech (1996) New Living Colors Vectors. CLONTECHniques XI, Clontech, Palo Alto, CA, pp. 20–23.Google Scholar
  8. 8.
    Turner, H., Gomez, M., McKenzie, E., Kirchem, A., Lennard, A., and Cantrel, L. D. A. (1998) Rac-1 regulates nuclear factor of activated T cells (NFAT) C1 nuclear translocation in response to Fcepsilon receptor type 1 stimulation of mast cells. J. Exp. Med. 188, 527–537.PubMedCrossRefGoogle Scholar
  9. 9.
    Sakai, N., Sasaki, K., Ikegaki, N., Shirai, Y., Ono, Y., and Saito, N. (1997) Direct visualization of the translocation of the gamma-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein. J. Cell Biol. 139, 1465–1476.PubMedCrossRefGoogle Scholar
  10. 10.
    Feng, X., Zhang, J., Barak, L. S., Meyer, T., Caron, M. G., and Hannun, Y. A. (1998) Visualization of dynamic trafficking of a protein kinase C betaII/green fluorescent protein conjugate reveals differences in G protein-coupled receptor activation and desensitization. J. Biol. Chem. 273, 10,755-10,762.Google Scholar
  11. 11.
    Meller, N., Liu, Y.-C., Collins, T. L., Bonnefoy-Berard, N., Baier, G., Isakov, N., et al. (1996) Direct interaction between protein kinase C theta (PKC theta) and 14–3–3 tau in T cells: 14–3–3 overexpression results in inhibition of PKC theta translocation and function. Mol. Cell. Biol. 16, 5782–5791.PubMedGoogle Scholar
  12. 12.
    Astoul, E., Watton, S.,and Cantrell, D. (1999) The dynamics of protein kinase B regulation during B cell antigen receptor engagement. J. Cell Biol. 145, 1511–1520.PubMedCrossRefGoogle Scholar
  13. 13.
    Watton, S. J. (1999) Akt/PKB localisation and 3′ phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction. Curr. Biol. 9, 433–436.PubMedCrossRefGoogle Scholar
  14. 14.
    Cheng, L., Fu, J., Tsukamoto, A., and Hawley, R. G. (1996) Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells. Nature Biotechnol. 14, 606–609.CrossRefGoogle Scholar
  15. 15.
    Brand. (1995) GFP in Drosophila. Trends Genet. 11, 324–325.Google Scholar
  16. 16.
    Ikawa, M., Kominami, K., Yoshimura, Y., Tanaka, K., Nishimune, Y., and Okabe, M. (1995) Green fluorescent protein as a marker in transgenic mice. Dev. Growth Differ. 37, 455–459.CrossRefGoogle Scholar
  17. 17.
    Matus, A. (1999) Introduction: GFP illuminates everything. Trends Cell Biol. 9,43.CrossRefGoogle Scholar
  18. 18.
    Genot, E., Cleverley, S., Henning, S., and Cantrell, D. (1996) Multiple p21ras effector pathways regulate nuclear factor of activated T cells. EMBO J. 15, 3923–3933.PubMedGoogle Scholar
  19. 19.
    Reif, K., Lucas, S., and Cantrell, D. (1997) A negative role for phosphatidylinositol 3-kinase in T cell antigen receptor function. Curr. Biol. 7, 285–293.PubMedCrossRefGoogle Scholar
  20. 20.
    Turner, H. and Cantrell, D. A. (1997) Distinct Ras effector pathways are involved in FcεR1 regulation of the transcriptional activity of Elk-1 and NFAT in mast cells. J. Exp. Med. 185, 43–57.PubMedCrossRefGoogle Scholar
  21. 21.
    Chen, C. A. and Okayama, H. (1988) Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques 6, 632–638.PubMedGoogle Scholar
  22. 22.
    Rosenthal, N. (1987) Identification of regulatory elements of cloned genes with functional assays. Methods Enzymol. 152, 704–709.PubMedCrossRefGoogle Scholar
  23. 23.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Paul Brennan
    • 1
  • Emmanuelle Astoul
    • 2
  1. 1.Department of MedicineUniversity of Wales College of MedicineWales, UK
  2. 2.Lymphocyte Activation Laboratory, Imperial Cancer Research FundLondonUK

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