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.
Highlights
Small molecule antagonists to the kinase domain induce ectodomain dimerization
Reagents and Cell Lines—The kinase inhibitors gefitinib and erlotinib were from LC Laboratories (Woburn, MA); lapatinib was from Enzo Life Sciences (Farmingdale, NY); and PD168393 was from Calbiochem (Billerica, MA)
The pattern of disulfide cross-linking in the linker and TM regions induced by gefitinib was identical to that induced by EGF [9] and by the combination of gefitinib and EGF (Fig. 1e)
Summary
Results: EGF and kinase inhibitors that stabilize the asymmetric kinase domain dimer induce identical transmembrane domain interfaces as shown by cross-linking but distinct ectodomain conformations as shown by EM. 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. Small molecule kinase antagonists were found to shift the equilibrium between these three different states of association of the kinase domain These results are consistent with loose linkage between the EGFR ectodomain and intracellular domain [9]. Recent studies have shown that association of kinase domains across a specific, asymmetric dimerization interface activates kinase activity and that different classes of kinase antagonists favor the active-like conformation seen with asymmetric dimer interface or an inactive-like conformation seen with distinct symmetric dimer interfaces in crystal lattices or a monomeric state seen in EM (7, 18 –21). One dimeric kinase domain conformation can couple to different ectodomain conformations, further demonstrating complexity in coupling between the ectodomain and intracellular domains in EGFR transmembrane signaling
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