title: Mechanisms for kinase-mediated dimerization of the epidermal growth factor receptor.
abstract: We study a mechanism by which dimerization of the EGF receptor (EGFR) cytoplasmic domain is transmitted to the ectodomain. Therapeutic and other small molecule antagonists to the kinase domain that stabilize its active conformation, but not those that stabilize an inactive conformation, stabilize ectodomain dimerization. Inhibitor-induced dimerization requires an asymmetric kinase domain interface associated with activation. EGF and kinase inhibitors stimulate formation of identical dimer interfaces in the EGFR transmembrane domain, as shown by disulfide cross-linking. Disulfide cross-linking at an interface in domain IV in the ectodomain was also stimulated similarly; however, EGF but not inhibitors stimulated cross-linking in domain II. Inhibitors similarly induced noncovalent dimerization in nearly full-length, detergent-solubilized EGFR as shown by gel filtration. EGFR ectodomain deletion resulted in spontaneous dimerization, whereas deletion of exons 2-7, in which extracellular domains III and IV are retained, did not. In EM, kinase inhibitor-induced dimers lacked any well defined orientation between the ectodomain monomers. Fab of the therapeutic antibody cetuximab to domain III confirmed a variable position and orientation of this domain in inhibitor-induced dimers but suggested that the C termini of domain IV of the two monomers were in close proximity, consistent with dimerization in the transmembrane domains. The results provide insights into the relative energetics of intracellular and extracellular dimerization in EGFR and have significance for physiologic dimerization through the asymmetric kinase interface, bidirectional signal transmission in EGFR, and mechanism of action of therapeutics.
authors.affiliation: Immune Disease Institute, Children's Hospital Boston and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Chafen
authors.initials: C
authors.lastname: Lu
authors.forename: Li-Zhi
authors.initials: LZ
authors.lastname: Mi
authors.forename: Thomas
authors.initials: T
authors.lastname: Schürpf
authors.forename: Thomas
authors.initials: T
authors.lastname: Walz
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Springer
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000077156:Gefitinib; D006801:Humans; D008854:Microscopy, Electron; D009154:Mutation; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011799:Quinazolines / Q000494:pharmacology
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; C509204:PD168393; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D004815:Epidermal Growth Factor; D010770:Phosphotransferases; D066246:ErbB Receptors; D000077156:Gefitinib