Abstract
The epidermal growth factor receptor (EGFR) is an essential receptor tyrosine kinase (RTK) that regulates cell proliferation and differentiation, and its abnormal activation contributes to a variety of human cancers. EGFR is activated by the binding of growth factors, such as epidermal growth factor (EGF), resulting in the formation of an asymmetric dimer in which one EGFR molecule allosterically activates the other molecule. This chapter provides a detailed protocol to purify and characterize full-length EGFR bound with EGF. This protocol facilitates the expression and preparation of full-length EGFR in transiently transfected mammalian cell culture. A method is also presented for quantitative evaluation of EGFR enzyme activity.
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References
Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141:1117–1134
Lynch TJ, Bell DW, Sordella R et al (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129–2139
Paez JG, Janne PA, Lee JC et al (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304:1497–1500
Rich JN, Rasheed BK, Yan H (2004) EGFR mutations and sensitivity to gefitinib. N Engl J Med 351:1260–1261, author reply 1260-1261
Wang Z, Longo PA, Tarrant MK et al (2011) Mechanistic insights into the activation of oncogenic forms of EGF receptor. Nat Struct Mol Biol 18:1388–1393
Yun CH, Boggon TJ, Li Y et al (2007) Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 11:217–227
Jura N, Endres NF, Engel K et al (2009) Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment. Cell 137:1293–1307
Liu P, Cleveland TE 4th, Bouyain S et al (2012) A single ligand is sufficient to activate EGFR dimers. Proc Natl Acad Sci U S A 109:10861–10866
Ogiso H, Ishitani R, Nureki O et al (2002) Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell 110:775–787
Zhang X, Gureasko J, Shen K et al (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125:1137–1149
Qiu C, Tarrant MK, Boronina T et al (2009) In vitro enzymatic characterization of near full length EGFR in activated and inhibited states. Biochemistry 48:6624–6632
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Wang, Z., Candelora, C. (2017). In Vitro Enzyme Kinetics Analysis of EGFR. In: Jimenez, G. (eds) ERK Signaling. Methods in Molecular Biology, vol 1487. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6424-6_2
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DOI: https://doi.org/10.1007/978-1-4939-6424-6_2
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6422-2
Online ISBN: 978-1-4939-6424-6
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