Abstract
The epidermal growth factor (EGF) receptor possesses intrinsic protein-tyrosine kinase activity, and both overexpressed wild-type and mutated forms have been associated with many types of cancers. Therefore, understanding the mechanisms that modulate receptor activity and function is essential to the development of treatments for many of these cancers. However, to address this issue by either conventional or high-throughput screening methods requires the availability of large amounts of highly purified and active EGF receptor.
The technique described in this chapter utilizes immunoaffinity chromatography, which allows for the isolation of highly purified and active preparations of EGF receptor. By immobilizing an antibody that recognizes the ligand-binding domain of the receptor to Sepharose beads, the receptor can be eluted specifically from the antibody by the addition of EGF. This association establishes a unique interaction that ensures the isolation of a highly enriched preparation of EGF receptor. This protocol allows for the purification of large or small batches of receptor that retain their kinase activity. Additionally, this chapter reports on the subsequent steps necessary to characterize the receptor: kinase activity, mass, purity, and the ability of the receptor to undergo autophosphorylation.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lynch T. J., Bell D. W., Sordella R., et al. (2004) Activating mutations in theepidermal growth factor receptor underlying responsiveness of non-small-celllung cancer to gefitinib. N. Engl. J. Med. 350, 2129–2139.
Carpenter G. and Cohen S. (1990) Epidermal growth factor. J. Biol. Chem. 265, 7709–7712.
Bertics P. J. and Gill G. N. (1985) Self-phosphorylation enhances the proteintyrosinekinase activity of the epidermal growth factor receptor. J. Biol. Chem. 260, 14,642–14,647.
Buday L. and Downward J. (1993) Epidermal growth factor regulates p21rasthrough the formation of a complex of receptor, Grb2 adapter protein, and Sosnucleotide exchange factor. Cell 73, 611–620.
Wang X. J., Liao H. J., Chattopadhyay A., Carpenter G. (2001) EGF-dependenttranslocation of green fluorescent protein-tagged PLC-gamma1 to theplasma membrane and endosomes. Exp. Cell. Res. 267, 28–36.
Zhu G., Decker S.J., Saltiel A. R. (1992) Direct analysis of the binding ofSrc-homology 2 domains of phospholipase C to the activated epidermal growthfactor receptor. Proc. Natl. Acad. Sci. USA 89, 9559–9563.
Pao W., Miller V., Zakowski M., et al. (2004) EGF receptor gene mutations arecommon in lung cancers from “never smokers” and are associated with sensitivityof tumors to gefitinib and erlotinib. Proc. Natl. Acad. Sci. USA 101, 13,306–13,311.
Sordella R., Bell D. W., Haber D., Settleman J. (2004) Gefitinib-sensitizingEGFR mutations in lung cancer activate anti-apoptotic pathways. Science 305, 1163–1167.
Paez J. G., Janne P. A., Lee J. C., et al. (2004) EGFR mutations in lung cancer:correlation with clinical response to gefitinib therapy. Science 304, 1497–1500.
Weber W., Bertics P.J., Gill G. N. (1984) mmunoaffinity purification ofthe epidermal growth factor receptor. Stoichiometry of binding and kinetics ofself-phosphorylation. J. Biol. Chem. 259, 14,631–14,636.
Gronowski A. M. and Bertics P. J. (1993) Evidence for the potentiation of epidermalgrowth factor receptor tyrosine kinase activity by association with the detergent-insoluble cellular cytoskeleton: analysis of intact and carboxy-terminallytruncated receptors. Endocrinology 133, 2838–2846.
Beebe J. A., Wiepz G. J., Guadarrama A. G., Bertics P.J., Burke, T. J.(2003) A carboxyl-terminal mutation of the epidermal growth factor receptor alterstyrosine kinase activity and substrate specificity as measured by a fluorescencepolarization assay. J. Biol. Chem. 278, 26,810–26,816.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Wiepz, G.J., Guadaramma, A.G., Fulgham, D.L., Bertics, P.J. (2006). Purification and Assay of Kinase-Active EGF Receptor From Mammalian Cells by Immunoaffinity Chromatography. In: Patel, T.B., Bertics, P.J. (eds) Epidermal Growth Factor. Methods in Molecular Biology, vol 327. Humana Press. https://doi.org/10.1385/1-59745-012-X:25
Download citation
DOI: https://doi.org/10.1385/1-59745-012-X:25
Publisher Name: Humana Press
Print ISBN: 978-1-58829-421-0
Online ISBN: 978-1-59745-012-6
eBook Packages: Springer Protocols