The juxtamembrane region of the EGF receptor functions as an activation domain

Abstract

In several growth factor receptors, the intracellular juxtamembrane (JM) region participates in autoinhibitory interactions that must be disrupted for tyrosine kinase activation. Using alanine scanning mutagenesis and crystallographic approaches, we define a domain within the JM region of the epidermal growth factor receptor (EGFR) that instead plays an activating--rather than autoinhibitory--role. Mutations in the C-terminal 19 residues of the EGFR JM region abolish EGFR activation. In a crystal structure of an asymmetric dimer of the tyrosine kinase domain, the JM region of an acceptor monomer makes extensive contacts with the C lobe of a donor monomer, thus stabilizing the dimer. We describe how an uncharacterized lung cancer mutation in this JM activation domain (V665M) constitutively activates EGFR by augmenting its capacity to act as an acceptor in the asymmetric dimer. This JM mutant promotes cellular transformation by EGFR in vitro and is tumorigenic in a xenograft assay.

Figure 1. Effect of Scanning Alanine Mutagenesis of the JM Region on Tyrosine Phosphorylation

A. Schematic of full-length EGFR, with the transmembrane (TM), tyrosine kinase domain (TKD), and carboxy-terminal (CT) regions marked. The intracellular domain (ICD) construct includes the JM, TKD, and CT regions. An alignment of JM region sequences across the human ErbB receptor family is shown, with the putative JM activation domain (JMAD) indicated. B. Scanning alanine mutagenesis of the JM region using ICD-flag constructs. Cos-7 cells transiently expressing wild-type or mutated ICD-flag constructs were analyzed by western blotting and densitometry for both phosphotyrosine content and flag-epitope expression. The graph demonstrates the ratio of phosphotyrosine to flag expression for each construct relative to WT. Results represent the means of 2 experiments ± standard deviation. C. Effect of JM alanine substitutions on tyrosine autophosphorylation of full-length EGFR. NIH3T3 cells transiently expressing full-length EGFR with the indicated mutations were serum-starved and then treated (+) or not (−) with EGF for 10 min. Cell lysates were subjected to SDS-PAGE and immunoblotting, detecting phospho-EGFR with anti-EGFR-pY1173 (upper panel) and receptor levels with anti-EGFR (lower panel).

Figure 2. The Acceptor JM Region ‘Cradles’ the C-lobe of the Donor in an Asymmetric TKD Dimer

A. Cartoon representation of the crystal structure of EGFR^645-998 harboring an inactivating K721M mutation. The TKD forms a crystallographic dimer that closely resembles the asymmetric TKD dimer seen for protein that lacks the complete JM region (Zhang et al., 2006), shown in B. The ‘Acceptor’ TKD is colored green, and ‘Donor’ TKD yellow. The N-lobe and C-lobe of donor and acceptor molecules are labeled, as are key helices in the donor C-lobe. Helix αC in the acceptor N-lobe is also marked. The N-terminal part of the JM region of the acceptor ‘cradles’ the donor C-lobe. The short N-terminal α-helix of the acceptor (residues 654-663) projects away from the donor, and makes no direct contacts. B. Structure of the active asymmetric dimer of the wild-type EGFR TKD lacking residues 645-671 from the JM region (PDB ID 2GS6), from Zhang et al. (2006). Features labeled in A are also labeled for this structure. C. Detailed view of side-chains in the acceptor JM region (green) that make contact with the C-lobe of the donor TKD. The orientation for this figure is shown in the cartoon representation in the inset, and the region illustrated is boxed. All side-chains present in the crystal structure from T654-R681 are shown. Those side-chains colored light grey could be replaced by alanine with no effect on ICD activity in Figure 1B (including T669). Side-chains colored green could not be replaced by alanine without significant loss of activity. Residue labels boxed in green correspond to the N-terminal half of the JM activation domain, where alanine substitutions have the greatest effect. Note that the N-terminal helix of the acceptor structure makes no direct contact with the donor.

Figure 3. Effects of mutations at JM phosphorylation sites

A. Effect of a phosphomimetic mutation at T669 on ICD tyrosine Phosphorylation. The indicated flag-tagged ICD constructs were transiently expressed in Cos-7 cells. Lysates were subjected to SDS-PAGE and immunoblotting as in Figure 1C, but using anti-Flag to normalize for ICD expression levels. B. Regulation of constitutively active EGFR-L834R by a JM loss-of-function mutant. 293 cells transiently expressing indicated ICD constructs were lysed and subjected to SDS-PAGE and immunoblotted with anti-EGFR-pY-1173 and anti-Flag.

Figure 4. Effects of Clinically-Observed JM Mutations on EGFR Activity

A. The noted clinically-observed JM mutations were introduced into the Flag-tagged EGFR ICD. The resulting constructs were transiently expressed in Cos-7 cells, and ICD autophosphorylation was assessed as described in Figure 1. B. and C. The V665M, L668P, and P670L mutations were also introduced into full-length EGFR, and the influence on EGF-dependent (+) and EGF-independent (−) EGFR autophosphorylation was assessed as described for Figure 1C. The K721R inactivating mutation was included as a negative control (C).

Figure 5. EGFR-V665M is transforming in NIH3T3 cells

A. NIH3T3 cells stably expressing the indicated wild-type or mutated forms of EGFR (or pBabe-puro vector only) were seeded at a density of 8×10³ cells per well in a standard colony forming assay (see Experimental Procedures). Cells were incubated for 3 weeks in the absence or presence of 50ng/ml EGF, and colonies were then counted. The results are presented as the means of 3 experiments ± standard deviation. To control for possible differences in expression level of the mutants, NIH3T3 stably expressing each mutant were lysed and analyzed by SDS-PAGE and immunoblotting with anti-EGFR. B. Upper panel: Example of tumors isolated from nude mice 3 months after s.c. injection with NIH3T3 cells stably expressing wild-type or V665M EGFR or the pBabe-puro vector. Lower panel: Volumes of tumors derived from the cells indicated above were evaluated 3 months after s.c. injection. Open circles represent single mice with a total of 4 mice per cell type analyzed.

Figure 6. The V665M Mutation must be present in the Acceptor Molecule to Activate EGFR

ICD constructs containing mutations that limit them to functioning only as a donor (I682Q) or acceptor (V924R) in the asymmetric TKD dimer were used to assess the mechanism by which the V665M mutation activates EGFR. The noted combinations of ICD constructs were coexpressed in 293 cells as indicated, and lysates were subjected to SDS-PAGE and immunoblotting for phospho-EGFR (with anti-EGFR-pY1173) and for protein levels with anti-Myc, and anti-Flag as appropriate. Autophosphorylation levels are greater than wild-type (lane 2) only when the V665M mutation is present in the acceptor TKD (lanes 9 and 10).

Figure 7. Mechanism of Activating JM Mutations

A. Contacts between the acceptor JM domain and donor C-lobe are shown with the V665 side-chain represented as space-filling spheres. The V665 side-chain fails to fill a cavity on the donor C-lobe formed by the side-chains of Q788, Y789 and Q825. However, as shown in the right-hand panel, a methionine (red) at position 665 – modeled for this figure - would completely fill the cavity, increasing van der Waal’s contacts between acceptor and donor. B. Similarly, the L679F mutation is likely to promote interactions between the JM region (green) and the donor C-lobe, as indicated by modeling a phenylalanine at this position.