In Vitro Enzymatic Characterization of Near Full Length EGFR in Activated and Inhibited States
journal contributionposted on 21.07.2009 by Chen Qiu, Mary K. Tarrant, Tatiana Boronina, Patti A. Longo, Jennifer M. Kavran, Robert N. Cole, Philip A. Cole, Daniel J. Leahy
Any type of content formally published in an academic journal, usually following a peer-review process.
The epidermal growth factor receptor (EGFR) is a single-pass transmembrane protein with an extracellular ligand-binding region and a cytoplasmic tyrosine kinase. Ligand binding activates the tyrosine kinase, which in turn initiates signaling cascades that influence cell proliferation and differentiation. EGFR activity is essential for normal development of many multicellular organisms, and inappropriate activation of EGFR is associated with multiple human cancers. Several drugs targeting EGFR activity are approved cancer therapies, and new EGFR-targeted therapies are being actively pursued. Much of what is known about EGFR structure and function is derived from studies of soluble receptor fragments. We report here an approach to producing an active, membrane-spanning form of EGFR of suitable purity, homogeneity, and quantity for structural and functional studies. We show that EGFR is capable of direct autophosphorylation of tyrosine 845, which is located on its kinase activation loop, and that the kinase activity of EGFR is ∼500-fold higher in the presence of EGF vs the inhibitory anti-EGFR antibody cetuximab. The potencies of the small molecule EGFR kinase inhibitors erlotinib and lapatinib for various forms of EGFR were measured, and the therapeutic and mechanistic implications of these results considered.