3.2.1. Mutations in the Extracellular Domain
Mutations in the extracellular domain of EGFR are frequently observed in GBM, in which the amplification of the EGFR
gene is particularly prominent [112
]. This gene amplification often accompanies deletions or insertions. The EGFR type I mutant, EGFRvI, lacks most of the extracellular domain, and is tumorigenic (Figure 1
]. EGFRvII has an in-frame deletion of exons 14 and 15, which encode amino acid residues 545–627 (521–603 in mature EGFR) of Subdomain IV [114
]. The constitutive phosphorylation of EGFRvII at a level similar to that of wild-type EGFR stimulated with EGF was observed in serum starved cells, demonstrating ligand-independent activation [115
]. The region deleted in EGFRvII encodes the “tethering arm,” which interacts with the “β-hairpin” of Subdomain II to form the inactive “tethered” conformation of the extracellular domain. Therefore, the EGFRvII deletion forces the mutant receptor to become the active extended form. EGFRvIII (also known as de2–7 EGFR or D2–7EGFR) has an in-frame deletion of exons 2–7, which encode amino acid resides 30–297 (6–273 in mature EGFR) encompassing Subdomain I and two-thirds of Subdomain II [112
]. Despite the lack of ligand binding, EGFRvIII is constitutively phosphorylated, and confers high tumorigenic potential [116
]. Unlike ligand-activated wild-type EGFR, however, EGFRvIII also activates the c-Jun N-terminal kinase through PI3K, but cannot activate the signal transducers and activators of transcription 1 (STAT1) and STAT3 [98
]. EGFRvIII fails to be ubiquitinated, thus prolonging oncogenic signaling [119
How are these mutants with deletions in the EGFR extracellular domain constitutively phosphorylated? As described above, the pre-formed dimeric structure of EGFR is facilitated through interactions between the cytoplasmic domains, the transmembrane domains, and the extracellular JM regions. Therefore, EGFR mutants with deleted extracellular ligand-binding domains are able to adopt a dimeric form, and are likely to trans
-autophosphorylate. This suggests that the extracellular domains of the EGFR dimer impose a negative constraint on receptor activation that is relieved by EGF binding. In the absence of the N-terminal extracellular regions, therefore, the cytoplasmic domain dimer of the deletion mutants may be unstable, and the kinase domain dimer tends to adopt an asymmetric active configuration. However, this active configuration does not seem to be the same configuration as that of ligand-activated wild-type EGFR. Only limited numbers of substrate tyrosine residues seem to be phosphorylated [120
]. This may cause abnormal downstream signaling and the lack of ubiquitination. The extracellular deletions may also induce the rotation of the transmembrane domains, which then rearranges the kinase domain dimer for partial activation. Indeed, EGFRvIII spontaneously forms cysteine disulfide-bridged dimers that are phosphorylated in the absence of the ligand [121
]. The cysteine disulfide bridging of the EGFRvIII receptor in the dimeric form may induce the rotation of its transmembrane domains, resulting in the reorientation of the intracellular kinase domains from a symmetric inactive form to an asymmetric active configuration. However, the mechanism of activation of EGFRvIII must await determination of its structure.
Missense mutations (R108K, T263P, A289V and G598V; R84K, T239P, A265V, and G574V in mature EGFR) in the extracellular domain of EGFR were also found in GBM and glioblastoma cell lines, which conferred anchorage-independent growth and tumorigenicity upon NIH 3T3 cells [113
]. These mutant EGFR proteins are phosphorylated at basal levels, and are still responsive to the ligand. R108K and A289V occur at the interface between Subdomains I and II, and T263P occurs in Subdomain II just before the “β-hairpin” that contacts Subdomain IV in the inactive “tethered” structure (Figure 2
a). These mutations may cause conformational changes of the extracellular domain of EGFR, and may release the tether between Subdomains II and IV. The released “β-hairpin” of Subdomain II may interact eith each other to induce the ligand-independent basal phosphorylation of the EGFR missense mutants (Figure 2
b). The detailed mechanism of activation of the mutants must await determination of their structures.
Sequencing of EGFR in a large cohort of GBM patients has identified over 30 different missense mutations within the extracellular domain, including C620Y, C620W, C624F, C628Y, and C636Y (C596Y, C596W, C600F, C604Y, and C612Y in mature EGFR). All of these cysteines are present in Subdomain IV [113
]. These Subdomain IV mutations leave an unpaired cysteine residue available for the formation of an intermolecular disulfide bond. Unlike EGFRvIII, which spontaneously formed a disulfide-bridge between protomers, these mutants form interprotomer cysteine disulfide bridges upon ligand stimulation [121
]. Three of the four mutants (C620Y, C624F, and C628Y) exhibit elevated levels of basal-tyrosine phosphorylation, with C620Y having the highest level. In particular, cell lines expressing the C620Y or C624F mutant grew better in soft agar than the control cells expressing wild-type EGFR, suggesting that these mutations are oncogenic [121
]. An EGFR-ligand autocrine loop may initiate the activation of the mutant receptors, and may then induce disulfide bridge formation that in turn rotates the transmembrane domains to constitutively activate the mutant receptors. This proposed mechanism of activation of the mutants must await determination of their structures before and after ligand binding.
3.2.3. Mutations in the Kinase Domain
The G719S mutation (G695S in mature EGFR) also activates the kinase, presumably by the destabilization of the symmetric inactive configuration of the dimer [127
]. G719 contributes to interactions that hold helix αC in the inactive conformation [130
]. The G719S mutation may not be able to do this, and may allow the helix to adopt its active conformation in the asymmetric dimeric configuration. Substitutions of G719 to alanine or cysteine also occur in NSCLC [131
]. Structural studies of the mutants are required to test this mechanism of activation.
Deletion mutants in the intracellular domain have also been found in NSCLC, and various deletions within the range encompassing E746 to I759 (E722 to I735 in mature EGFR) have been identified [133
]. A deletion mutant, ∆E746–A750 (∆E722–A726 in mature EGFR), is constitutively phosphorylated in the absence of the ligand [134
]. Two other mutations are a deletion in which residues from L747 to P753 (L723–P729 in mature EGFR) are replaced with a single serine to give the ∆L747–P753insS mutant [133
], and the mutant ∆S752–I759 (∆S728–I735 in mature EGFR) in which residues 752–759 are deleted [133
]. When these mutant EGFR
genes were expressed in the murine hematopoietic Ba/F3 cell line, they all showed significantly higher basal (ligand-independent) tyrosine phosphorylation levels than wild-type receptors [128
]. The regions deleted from the N-terminus of helix αC in the mutants interact with the A-loop in inactive EGFR [64
]. This suggests that the removal of the region by deletion may activate EGFR by disrupting the autoinhibited kinase domain conformation through the release of the A-loop from the active site for substrate binding [128
]. This conformational change of the active site of EGFR may cause the transition from a symmetric inactive configuration to an asymmetric active form in the dimer. The detailed mechanism of activation of the mutants must await determination of their structures.
The R776H mutation (R752H in mature EGFR) is associated with lung cancer where there is no smoking history, and is found both in normal and tumor tissues [137
]. This mutant receptor is constitutively autophosphorylated in the absence of the ligand [137
]. The constitutive activation of the receptor relies on the intact interface in its asymmetric dimeric kinase configuration. The R776H mutant receptor preferentially adopts the “receiver” position when paired with wild-type EGFR [138
], providing support for the “superacceptor” hypothesis [139
]. This is similar to the L858R mutation described below.
G735S, G796S, and E804G mutations (G711S, G772S, and E780G in mature EGFR) found in prostate cancer are oncogenic, causing increased cell growth, transformation, and invasion [141
]. All of these mutant receptors demonstrate the increased phosphorylation of four tyrosine residues (Y869, Y1016, Y1092, and Y1197; Y845, Y992, Y1068, and Y1173 in mature EGFR) compared to wild-type EGFR in the absence of the ligand, indicating that the mutants are constitutively active. The G735S and G796S mutations were also found in thyroid cancer and squamous cell carcinoma of the head and neck, respectively [142
]. The G735 residue is located in the N-lobe, and this mutation is likely to cause a conformational change of the kinase domain for activation [142
]. G796 and E804 are located at the interface between two protomers of a symmetric inactive dimer, and seem to be involved in inactive dimer formation. The G796S and E804G mutations may destabilize the inactive dimer to form an asymmetric active configuration. It is necessary to test this proposed mechanism by observing the effects of mutagenesis of the residues and interacting residues on the autophosphorylation of the mutant receptors.
The kinase domain mutation L858R (L834R in mature EGFR) has been observed in 40–45% of mutations in NSCLC [144
]. An asymmetric configuration of the kinase domain dimer is required for the ligand-independent activity of NSCLC-associated EGFR kinase mutants [140
]. Crystal structural analysis indicates that the L858R mutation is likely to shift the equilibrium of the kinase domain dimer toward an active configuration by preventing key hydrophobic interactions between L858 and other residues in the N-lobe that lock the regulatory helix αC in the inactive position [128
]. This occurs by suppressing intrinsic disorder within the kinase domain N-lobe to allow the mutant kinase domain to act as a “superacceptor” within asymmetric kinase dimers [139
]. In this ligand-independent activation of EGFR due to L858R, “β-hairpin” interaction of the extracellular Subdomain II plays a critical role [54
]. Interestingly, live-cell FRET measurements reveal that the L858R kinase mutation alters the extracellular domain structure such that unliganded mutant EGFR adopts an extended (untethered) configuration. Therefore, the transition from a symmetric inactive configuration of the kinase dimer to an asymmetric active form affects configurations of the extracellular domains, causing the “β-hairpins” to interact. This inside-out transmembrane signaling may occur through a rotation of the transmembrane domains. To test this possibility, a structural study of the mutant is required.
The L861Q mutation (L837Q in mature EGFR) found in NSCLC is also in the A-loop of the kinase domain [122
]. A short α-helix in the A-loop that includes residues L858 and L861 interacts with helix αC in the inactive form of EGFR. Side chains of both L858 and L861 contribute to a hydrophobic core formed by residues from the helix αC, the A-loop, and elsewhere [64
]. The L861Q mutation could also disrupt the autoinhibitory interaction between the A-loop and helix αC of EGFR by destabilizing the set of hydrophobic interactions [128
]. Therefore, L861Q is likely to shift the equilibrium of the kinase domain toward an active conformation, which acts as a “superacceptor” within asymmetric kinase dimers.
An in-frame, tandem duplication of EGFR residues 688–1054 (664–1030 in mature EGFR) has been detected in GBM [113
]. This duplication region comprising exons 18–26 encodes the entire kinase domain, and the mutant designated “TKD-EGFR” displays chronically elevated basal autophosphorylation at five known phosphorylation sites [148
]. An EGFR kinase domain duplication has also been found in lung, brain, and other cancers [149
]. This mutant, EGFR-KDD, has a tandem, in-frame duplication of exons 18–25, which encode the entire EGFR kinase domain, and is constitutively active. Computational modeling suggests that EGFR-KDD can be activated by virtue of an asymmetric intramolecular dimeric structure, unlike the typical asymmetric, intermolecular dimeric structure between adjacent protomers [149
3.2.4. Mutations in the C-Terminal Tail Region
As described above, a large fraction of GBM cells display EGFR
gene amplification that correlates with receptor overexpression [102
], and also exhibit an EGFRvIII deletion mutation in the extracellular domain of the receptor [150
]. EGFR molecules with C-terminal deletions have also been found in GBM, and are collectively termed EGFRvIV. EGFRvIVa lacks exons 25–27 (from G983 to P1090; from G959 to P1066 in mature EGFR), and EGFRvIVb lacks exons 25 and 26 (from G983 to G1054; from G959 to G1030 in mature EGFR) [113
]. These two mutants have transforming and tumorigenic properties in the absence of the ligand, and show ligand-independent constitutive activation. The oncogenic function of these mutants depends upon the intrinsic kinase activity [153
]. Recent genomic studies in GBM and NSCLC patients have identified an additional class of oncogenic mutations caused by the deletion of C-terminal coding regions [103
]. Among them, mutants with a deletion of exon 25 (from G983 to L1038; from G959 to L1014 in mature EGFR) or both exons 25 and 26 (from G983 to G1054; from G959 to G1030 in mature EGFR, which is equivalent to EGFRvIVb above) are constitutively phosphorylated, and cause phosphorylation of downstream STAT3 when expressed in NIH 3T3 cells in the absence of the ligand [156
]. Exon 25 encodes 56 amino-acid residues from G983 to L1038 (from G959 to L1014 in mature EGFR), and encompasses the AP-2 helix and the electrostatic hook, which are involved in the stabilization of the symmetric inactive configuration of the kinase dimer [63
]. Therefore, it is likely that the deletion of exon 25 destabilizes the inactive kinase dimer, and shifts the equilibrium of the kinase domain toward an asymmetric active configuration in the dimeric structure. The detailed mechanism of activation of the mutants must await determination of their structures.