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.

FIGURE 1.

Disulfide cross-linking of EGFR cysteine mutants in Ba/F3 transfectants treated with or without EGFR-specific kinase inhibitor, gefitinib. The cells were treated with 10 μm gefitinib or Me₂SO control for 45 min. The lysates were subjected to nonreducing SDS 5% PAGE and Western blotting with protein C antibody to detect EGFR protein. a, cysteine substitutions in the domain II (D279C/H280A) and domain IV (Y602C) interfaces in the crystal structure of liganded dimer. b, linker residues. c and d, TM domain residues. e, comparison of disulfide cross-linking induced by gefitinib, EGF, and gefitinib in combination with EGF. EGF-induced cross-linking data were published previously (9). MW, molecular mass; D, dimeric; M, monomeric.

FIGURE 2.

Two distinct classes of kinase inhibitors have different effect on EGFR dimerization. a and b, Ba/F3 stable transfectants expressing EGFR cysteine mutant A623C (a) or N615C (b) were incubated with 10 μm gefitinib, erlotinib, lapatinib, PD168393, HKI-272, or Me₂SO (DMSO) for 45 min and treated with or without 100 nm EGF for 5 min. The lysates were subjected to nonreducing SDS 5% PAGE and Western blotting with protein C antibody (upper panel) or phosphotyrosine antibody 4G10 (lower panel). D, dimeric; M, monomeric.

FIGURE 3.

Disruption of the asymmetric kinase domain interface by mutations abolishes kinase inhibitor-induced EGFR dimerization. a, Ba/F3 transfectants expressing WT or mutant EGFR were treated with or without EGF. The lysates were subjected to reducing SDS 7.5% PAGE and Western blotting with protein C antibody (upper panel) or phosphotyrosine antibody 4G10 (lower panel). b, cells were incubated with 10 μm gefitinib or erlotinib and treated with or without EGF. The lysates were subjected to nonreducing SDS 5% PAGE and Western blotting with protein C antibody. c, quantitation of cross-linking in b. d, Ba/F3 transfectants expressing WT (upper panel) or mutant V924R (lower panel) were treated with kinase inhibitors with or without EGF as in b. Surface cross-linking was carried by incubation with 1 mm BS³ for 1 h at 4 °C. The lysates were subjected to reducing SDS 5% PAGE and Western blotting with protein C antibody. D, dimeric; M, monomeric.

FIGURE 4.

Gel filtration chromatograms of detergent-solubilized, purified EGFR proteins. The purified EGFR truncation mutants indicated in panels a–c, with any additional mutations shown above each trace (0.7 μm), were treated with or without kinase inhibitors (200 μm), EGF (20 μm), or cetuximab Fab (40 μm) for 5 min on ice for 5 min and subjected to gel filtration on a Superose 6 HR column equilibrated with running buffer containing 0.2 mm dodecylmaltoside and 0.2 mm DTT. The inset in b shows Western blots with protein C antibody of fractions from the upper trace, demonstrating that EGFR is present only in the dimer peak, and not in the second peak. The arrows mark positions of dimeric (D), monomeric (M), and Fab complex material (Fab). Traces of Fab complexes are truncated so that only the beginning, rising portion of the Fab peak is shown, which is dashed.

FIGURE 5.

Kinase inhibitor promotes formation of the asymmetric kinase dimer with ectodomain conformation distinct from liganded dimer. a, representative class averages of unliganded, monomeric EGFR Δ998. b, representative class averages of EGF-bound, dimeric EGFR Δ998, in which symmetric ectodomain dimers are linked to asymmetric kinase dimers (subpanels 1–3) or symmetric kinase dimers (subpanels 4–6). c, representative class averages of PD168393-bound EGFR Δ998 with asymmetric kinase dimer (subpanels 1–3) or less interpretable densities (subpanels 4–6). d, representative class averages of PD168393-bound EGFR ΔectoΔ998 with asymmetric kinase dimer (subpanels 1–3) or symmetric kinase dimer (subpanel 4). e, representative class averages of EGFR(de2–7)Δ998 in complex with cetuximab Fab. f, representative class averages of EGFR Δ998 with PD168393 in complex with cetuximab Fab. Scale bars, 10 nm.

FIGURE 6.

Correlation of EM densities with crystal structures. Representative class averages are shown in the first row of each panel, with masked areas in the second row (labeled a), best correlating projections with crystal structures and cross-correlation scores in the third row (labeled b), and ribbon diagrams in the same orientation but at larger scale in the bottom row (labeled c). The cross-correlations in a and b are with the asymmetric kinase dimer from (10) (Protein Data Bank code 2GS6). Cross-correlations in c are with the Fab and EGFR domain III moieties (residues 311–503) from the crystal structure of cetuximab Fab bound to EGFR (25). In c, each monomeric unit was separately masked and cross-correlated. Using the orientation established for the Fab and domain III, the entire EGFR ectodomain Fab complex for each monomer is shown, preserving their relative spatial orientations, in the bottom panel of c. Scale bars, 10 nm.