Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Epidermal growth factor receptor dimerization monitored in live cells

Nature Biotechnology volume 18, pages 218–222 (2000)Cite this article

Abstract

We present a method for monitoring receptor dimerization at the membrane of live cells. Chimeric proteins containing the epidermal growth factor (EGF) receptor extracellular and transmembrane domains fused to weakly complementing β-galactosidase (β-gal) deletion mutants were expressed in cells in culture. Treatment of the cells with EGF-like compounds for as little as 15 s resulted in chimeric receptor dimerization detectable as β-gal enzymatic activity. The dose response of chimeric receptors was ligand specific. β-galactosidase complementation was reversible upon removal of ligand and could be reinduced. Antibodies that block ligand binding inhibited receptor dimerization and β-gal complementation. These results demonstrate that β-gal complementation provides a rapid, simple, and sensitive assay for protein interactions and for detecting and monitoring the kinetics of receptor dimerization.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: EGF receptor dimerization monitored using β-gal complementation.
Figure 2: Chimeric receptor expression compared to A431 cells.
Figure 3: Kinetics of EGF receptor dimerization.
Figure 4: Antibodies which inhibit ligand binding inhibit dimerization.

Similar content being viewed by others

References

  1. Schlessinger, J. & Ullrich, A. Growth factor signaling by receptor tyrosine kinases. Neuron 9, 383–391 (1992).

    Article  CAS  Google Scholar 

  2. Ullrich, A. & Schlessinger, J. Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203–212 (1990).

    Article  CAS  Google Scholar 

  3. Weiss, F.U., Daub, H. & Ullrich, A. Novel mechanisms of RTK signal generation. Curr. Opin. Genet. Dev. 7, 80–86 (1997).

    Article  CAS  Google Scholar 

  4. Fields, S. & Song, O. A novel genetic system to detect protein-protein interactions. Nature 340, 245–246 (1989).

    Article  CAS  Google Scholar 

  5. Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    Article  CAS  Google Scholar 

  6. Rossi, F., Charlton, C.A. & Blau, H.M. Monitoring protein–protein interactions in intact eukaryotic cells by β-galactosidase complementation. Proc. Natl. Acad. Sci. USA 94, 8405–8410 (1997).

    Article  CAS  Google Scholar 

  7. Pelletier, J.N., Campbell-Valois, F.X. & Michnick, S.W. Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc. Natl. Acad. Sci. USA 95, 12141–12146 (1998).

    Article  CAS  Google Scholar 

  8. Ullmann, A., Perrin, D., Jacob, F. & Monod, J. Identification par complémentation in vitro et purification d'un segment de la β-galactosidase d'Escherichia coli. J. Mol. Biol. 12, 918–923 (1965).

    Article  CAS  Google Scholar 

  9. Mohler, W.A. & Blau, H.M. Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells. Proc. Natl. Acad. Sci. USA 93, 12423–12427 (1996).

    Article  CAS  Google Scholar 

  10. Rossi, F.M.V., Blakely, B.T., Charlton, C.A. & Blau, H.M. in Chimeric proteins (eds Emr, S.D. & Thorner, J.) in press (Academic Press, San Diego, CA; 2000).

    Google Scholar 

  11. Rossi, F.M.V., Blakely, B.T. & Blau, H.M. Interaction blues: protein interactions monitored live in mammalian cells by β-galactosidase complementation. Trends Cell Biol. 10, 119–122 (2000).

    Article  CAS  Google Scholar 

  12. Livneh, E. et al. Reconstitution of human epidermal growth factor receptors and its deletion mutants in cultured hamster cells. J. Biol. Chem. 261, 12490–12497 (1986).

    CAS  PubMed  Google Scholar 

  13. Gadella, T.W., Jr. & Jovin, T.M. Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation. J. Cell Biol. 129, 1543–1558 (1995).

    Article  CAS  Google Scholar 

  14. Nagata, S. Apoptosis by death factor. Cell 88, 355–365 (1997).

    Article  CAS  Google Scholar 

  15. Haigler, H., Ash, J.F., Singer, S.J. & Cohen, S. Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431. Proc. Natl. Acad. Sci. USA 75, 3317–3321 (1978).

    Article  CAS  Google Scholar 

  16. Stoscheck, C.M. & Carpenter, G. Characterization of the metabolic turnover of epidermal growth factor receptor protein in A-431 cells. J. Cell Physiol. 120, 296–302 (1984).

    Article  CAS  Google Scholar 

  17. Cowley, G.P., Smith, J.A. & Gusterson, B.A. Increased EGF receptors on human squamous carcinoma cell lines. Br. J. Cancer 53, 223–229 (1986).

    Article  CAS  Google Scholar 

  18. Felder, S., LaVin, J., Ullrich, A. & Schlessinger, J. Kinetics of binding, endocytosis, and recycling of EGF receptor mutants. J. Cell Biol. 117, 203–212 (1992).

    Article  CAS  Google Scholar 

  19. Kiyokawa, N., Lee, E.K., Karunagaran, D., Lin, S.Y. & Hung, M.C. Mitosis-specific negative regulation of epidermal growth factor receptor, triggered by a decrease in ligand binding and dimerization, can be overcome by overexpression of receptor. J. Biol. Chem. 272, 18656–18665 (1997).

    Article  CAS  Google Scholar 

  20. Beerli, R.R. & Hynes, N.E. Epidermal growth factor-related peptides activate distinct subsets of ErbB receptors and differ in their biological activities. J. Biol. Chem. 271, 6071–6076 (1996).

    Article  CAS  Google Scholar 

  21. Gill, G.N. et al. Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity. J. Biol. Chem. 259, 7755–7760 (1984).

    CAS  PubMed  Google Scholar 

  22. Winkler, M.E., O'Connor, L., Winget, M. & Fendly, B. Epidermal growth factor and transforming growth factor alpha bind differently to the epidermal growth factor receptor. Biochemistry 28, 6373–6378 (1989).

    Article  CAS  Google Scholar 

  23. Carraway, K.L. & Cerione, R.A. Inhibition of epidermal growth factor receptor aggregation by an antibody directed against the epidermal growth factor receptor extracellular domain. J. Biol. Chem. 268, 23860–23867 (1993).

    CAS  PubMed  Google Scholar 

  24. Yarden, Y. & Schlessinger, J. Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor. Biochemistry 26, 1443–1451 (1987).

    Article  CAS  Google Scholar 

  25. Cochet, C. et al. Demonstration of epidermal growth factor-induced receptor dimerization in living cells using a chemical covalent cross-linking agent. J. Biol. Chem. 263, 3290–3295 (1988).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Remy, I., Wilson, I.A. & Michnick, S.W. Erythropoietin receptor activation by a ligand-induced conformation change. Science 283, 990–993 (1999).

    Article  CAS  Google Scholar 

  28. Zabin, I. β-Galactosidase α-complementation. A model of protein–protein interaction. Mol. Cell. Biochem. 49, 87–96 (1982).

    Article  CAS  Google Scholar 

  29. Langley, K.E. & Zabin, I. β-Galactosidase α complementation: properties of the complemented enzyme and mechanism of the complementation reaction. Biochemistry 15, 4866–4875 (1976).

    Article  CAS  Google Scholar 

  30. Pegram, M. et al. Inhibitory effects of combinations of HER-2/neu antibody and chemotherapeutic agents used for treatment of human breast cancers. Oncogene 18, 2241–2251 (1999).

    Article  CAS  Google Scholar 

  31. Fiering, S.N. et al. Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs. Cytometry 12, 291–301 (1991).

    Article  CAS  Google Scholar 

  32. Nolan, G.P., Fiering, S., Nicolas, J.F. & Herzenberg, L.A. Fluorescence-activated cell analysis and sorting of viable mammalian cells based on β-D-galactosidase activity after transduction of Escherichia coli lacZ. Proc. Natl. Acad. Sci. USA 85, 2603–2607 (1988).

    Article  CAS  Google Scholar 

  33. Alberti, S., Parks, D.R. & Herzenberg, L.A. A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry 8, 114–119 (1987).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Xiao-Jia Chang, Carol Zuschlag, Carol Charlton, Joyce Tung (funded by Howard Hughes Medical Institute undergraduate research fellowship), and Patricia Wong (funded by the Stanford Medical School Alumni Scholarship) for technical assistance. F.M.V.R. was funded by a fellowship from the Human Frontier Science Program. This research was supported by National Institute of Health grants to H.M.B.

Author information

Author notes

  1. Bruce T. Blakely and Fabio M. V. Rossi: The first two authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, 94305-5332, CA

    Bruce T. Blakely, Fabio M. V. Rossi, Angeles Estelles & Helen M. Blau

  2. PE Biosystems, Advanced Discovery Sciences, Bedford, 01730, MA

    Bonnie Tillotson & Michelle Palmer

Authors
  1. Bruce T. Blakely
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fabio M. V. Rossi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bonnie Tillotson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michelle Palmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Angeles Estelles
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Helen M. Blau
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Helen M. Blau.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Blakely, B., Rossi, F., Tillotson, B. et al. Epidermal growth factor receptor dimerization monitored in live cells. Nat Biotechnol 18, 218–222 (2000). https://doi.org/10.1038/72686

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/72686

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing