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Glioma Specific Extracellular Missense Mutations in the First Cysteine Rich Region of Epidermal Growth Factor Receptor (EGFR) Initiate Ligand Independent Activation.

Ymer SI, Greenall SA, Cvrljevic A, Cao DX, Donoghue JF, Epa VC, Scott AM, Adams TE, Johns TG - Cancers (Basel) (2011)

Bottom Line: We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors.Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation.Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR.

View Article: PubMed Central - PubMed

Affiliation: Oncogenic Signaling Laboratory, Monash Institute of Medical Research, Monash University, Clayton, VIC 3168, Australia. terry.johns@med.monash.edu.au.

ABSTRACT
The epidermal growth factor receptor (EGFR) is overexpressed or mutated in glioma. Recently, a series of missense mutations in the extracellular domain (ECD) of EGFR were reported in glioma patients. Some of these mutations clustered within a cysteine-rich region of the EGFR targeted by the therapeutic antibody mAb806. This region is only exposed when EGFR activates and appears to locally misfold during activation. We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors. Both mutants were autophosphorylated in the absence of ligand and enhanced cell survival and anchorage-independent and xenograft growth. The ECD truncation that produces the de2-7EGFR (or EGFRvIII), the most common EGFR mutation in glioma, generates a free cysteine in this same region. Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation. Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR. These ECD mutations confirm that the cysteine-rich region of EGFR around the mAb806 epitope has a significant role in receptor activation.

No MeSH data available.


Related in: MedlinePlus

(A). Alignment of amino acids 295-337 of the immature EGFR protein with amino acids 1-46 of the mature de2-7EGFR protein. The disulfide bonds (black lines), mAb806 epitope and position of two EGFR ECD mutations studied in this paper (red) are shown. A 267 amino acid truncation removes the C295 from the EGFR structure and results in a free C16 residue in the mature de2-7EGFR protein (pink) as well as a unique 6 amino acid N-terminal sequence (green); (B). Three dimensional structural model of module 7 incorporating the R324L mutation. R324 lies within the module 7 loop spanning amino acids 311-326 in Domain II of EGFR. The R324L mutation will abolish salt bridges (dashed lines) with E317 and E319 which stabilize the loop, leading to greater loop flexibility and significant strain on the C311-C326 bond (yellow). Analysis utilized the structure of ligand bound EGFR extracellular domain described previously [3]. The figure was prepared with PyMol v.1.2r2 software (Schrodinger, LLC).
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f8-cancers-03-02032: (A). Alignment of amino acids 295-337 of the immature EGFR protein with amino acids 1-46 of the mature de2-7EGFR protein. The disulfide bonds (black lines), mAb806 epitope and position of two EGFR ECD mutations studied in this paper (red) are shown. A 267 amino acid truncation removes the C295 from the EGFR structure and results in a free C16 residue in the mature de2-7EGFR protein (pink) as well as a unique 6 amino acid N-terminal sequence (green); (B). Three dimensional structural model of module 7 incorporating the R324L mutation. R324 lies within the module 7 loop spanning amino acids 311-326 in Domain II of EGFR. The R324L mutation will abolish salt bridges (dashed lines) with E317 and E319 which stabilize the loop, leading to greater loop flexibility and significant strain on the C311-C326 bond (yellow). Analysis utilized the structure of ligand bound EGFR extracellular domain described previously [3]. The figure was prepared with PyMol v.1.2r2 software (Schrodinger, LLC).

Mentions: Unfortunately, while we were able to show that the missense mutations were present as dimers using the techniques described here (data not shown), we were unable to definitively determine their nature because of their low abundance and the presence of alternative inactive dimers as previously reported [23]. Also, the ability to mutate the exact cysteine residue in the de2-7EGFR made the definitive experiments relatively straight-forward in this receptor. Obviously this approach is not possible in full-length receptors. We have postulated that the region around mAb806 must be locally misfolded during receptor activation [10]. As noted, the R324L mutation is found within the mAb806 epitope and the E330K mutation is in the adjacent cysteine loop (Figure 8A). Our results show that these mutations cause conformational changes that enhance receptor activation in the absence of ligand. The R324L mutation abolishes two salt bridges with adjacent E317 and E319 residues. These salt bridges appear to stabilize the C311-C326 disulfide loop (Figure 8B). Their removal should increase the flexibility and dynamic behavior of the loop and place strain on the C311-C326 disulfide bond. This would make the bond more susceptible to breaking and possibly prevent its formation, leaving a free cysteine for intermolecular bonding as for the de2-7EGFR. Indeed, the cysteine at 311 is only a few amino acids away from the free cysteine in de2-7 EGFR which is cysteine 307 (or C16 in the de2-7EGFR). Molecular modeling has suggested that a G336R ECD mutation found in zebra fish EGFR also disrupts a nearby disulphide bond resulting in the formation of disulphide linked homodimers [24]. Taken together these results suggest a general mechanism of activation, whereby mutations in this cysteine rich region lead to disruption of disulfide bonds which become available for stabilizing dimer interactions.


Glioma Specific Extracellular Missense Mutations in the First Cysteine Rich Region of Epidermal Growth Factor Receptor (EGFR) Initiate Ligand Independent Activation.

Ymer SI, Greenall SA, Cvrljevic A, Cao DX, Donoghue JF, Epa VC, Scott AM, Adams TE, Johns TG - Cancers (Basel) (2011)

(A). Alignment of amino acids 295-337 of the immature EGFR protein with amino acids 1-46 of the mature de2-7EGFR protein. The disulfide bonds (black lines), mAb806 epitope and position of two EGFR ECD mutations studied in this paper (red) are shown. A 267 amino acid truncation removes the C295 from the EGFR structure and results in a free C16 residue in the mature de2-7EGFR protein (pink) as well as a unique 6 amino acid N-terminal sequence (green); (B). Three dimensional structural model of module 7 incorporating the R324L mutation. R324 lies within the module 7 loop spanning amino acids 311-326 in Domain II of EGFR. The R324L mutation will abolish salt bridges (dashed lines) with E317 and E319 which stabilize the loop, leading to greater loop flexibility and significant strain on the C311-C326 bond (yellow). Analysis utilized the structure of ligand bound EGFR extracellular domain described previously [3]. The figure was prepared with PyMol v.1.2r2 software (Schrodinger, LLC).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757403&req=5

f8-cancers-03-02032: (A). Alignment of amino acids 295-337 of the immature EGFR protein with amino acids 1-46 of the mature de2-7EGFR protein. The disulfide bonds (black lines), mAb806 epitope and position of two EGFR ECD mutations studied in this paper (red) are shown. A 267 amino acid truncation removes the C295 from the EGFR structure and results in a free C16 residue in the mature de2-7EGFR protein (pink) as well as a unique 6 amino acid N-terminal sequence (green); (B). Three dimensional structural model of module 7 incorporating the R324L mutation. R324 lies within the module 7 loop spanning amino acids 311-326 in Domain II of EGFR. The R324L mutation will abolish salt bridges (dashed lines) with E317 and E319 which stabilize the loop, leading to greater loop flexibility and significant strain on the C311-C326 bond (yellow). Analysis utilized the structure of ligand bound EGFR extracellular domain described previously [3]. The figure was prepared with PyMol v.1.2r2 software (Schrodinger, LLC).
Mentions: Unfortunately, while we were able to show that the missense mutations were present as dimers using the techniques described here (data not shown), we were unable to definitively determine their nature because of their low abundance and the presence of alternative inactive dimers as previously reported [23]. Also, the ability to mutate the exact cysteine residue in the de2-7EGFR made the definitive experiments relatively straight-forward in this receptor. Obviously this approach is not possible in full-length receptors. We have postulated that the region around mAb806 must be locally misfolded during receptor activation [10]. As noted, the R324L mutation is found within the mAb806 epitope and the E330K mutation is in the adjacent cysteine loop (Figure 8A). Our results show that these mutations cause conformational changes that enhance receptor activation in the absence of ligand. The R324L mutation abolishes two salt bridges with adjacent E317 and E319 residues. These salt bridges appear to stabilize the C311-C326 disulfide loop (Figure 8B). Their removal should increase the flexibility and dynamic behavior of the loop and place strain on the C311-C326 disulfide bond. This would make the bond more susceptible to breaking and possibly prevent its formation, leaving a free cysteine for intermolecular bonding as for the de2-7EGFR. Indeed, the cysteine at 311 is only a few amino acids away from the free cysteine in de2-7 EGFR which is cysteine 307 (or C16 in the de2-7EGFR). Molecular modeling has suggested that a G336R ECD mutation found in zebra fish EGFR also disrupts a nearby disulphide bond resulting in the formation of disulphide linked homodimers [24]. Taken together these results suggest a general mechanism of activation, whereby mutations in this cysteine rich region lead to disruption of disulfide bonds which become available for stabilizing dimer interactions.

Bottom Line: We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors.Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation.Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR.

View Article: PubMed Central - PubMed

Affiliation: Oncogenic Signaling Laboratory, Monash Institute of Medical Research, Monash University, Clayton, VIC 3168, Australia. terry.johns@med.monash.edu.au.

ABSTRACT
The epidermal growth factor receptor (EGFR) is overexpressed or mutated in glioma. Recently, a series of missense mutations in the extracellular domain (ECD) of EGFR were reported in glioma patients. Some of these mutations clustered within a cysteine-rich region of the EGFR targeted by the therapeutic antibody mAb806. This region is only exposed when EGFR activates and appears to locally misfold during activation. We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors. Both mutants were autophosphorylated in the absence of ligand and enhanced cell survival and anchorage-independent and xenograft growth. The ECD truncation that produces the de2-7EGFR (or EGFRvIII), the most common EGFR mutation in glioma, generates a free cysteine in this same region. Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation. Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR. These ECD mutations confirm that the cysteine-rich region of EGFR around the mAb806 epitope has a significant role in receptor activation.

No MeSH data available.


Related in: MedlinePlus