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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

Biochemical characterization of mutant epidermal growth factor receptor (EGFR). (A). wtEGFR and mutant EGFR were translated into 35S-cysteine labelled proteins in a cell free expression system and labeled proteins detected by autoradiography following SDS-PAGE; (B). NR6 cells expressing wtEGFR or mutant EGFR were stained with secondary antibody alone (green), isotype control antibody (orange), mAb528 (red) or mAb806 (blue) and subjected to Fluorescence Activated Cell Sorting (FACS) analysis. The representative profiles for each cell line are shown; (C). wtEGFR or mutant EGFR cells were grown under serum free conditions, lysed and IP'd using mAb528. Western blot analysis of total EGFR using a mAb806 probe (upper panel) or a C-terminal EGFR probe (lower panel) are shown. A431 cells that overexpress the EGFR were included as a positive control.
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f1-cancers-03-02032: Biochemical characterization of mutant epidermal growth factor receptor (EGFR). (A). wtEGFR and mutant EGFR were translated into 35S-cysteine labelled proteins in a cell free expression system and labeled proteins detected by autoradiography following SDS-PAGE; (B). NR6 cells expressing wtEGFR or mutant EGFR were stained with secondary antibody alone (green), isotype control antibody (orange), mAb528 (red) or mAb806 (blue) and subjected to Fluorescence Activated Cell Sorting (FACS) analysis. The representative profiles for each cell line are shown; (C). wtEGFR or mutant EGFR cells were grown under serum free conditions, lysed and IP'd using mAb528. Western blot analysis of total EGFR using a mAb806 probe (upper panel) or a C-terminal EGFR probe (lower panel) are shown. A431 cells that overexpress the EGFR were included as a positive control.

Mentions: The cell-free translation of mutant EGFR demonstrated that Mw and expression levels of the proteins were similar to the wtEGFR, indicating that the mutations did not affect translation efficiency (Figure 1A). Both the wtEGFR and mutant EGFR were expressed on the cell surface of NR6 cells as determined by Fluorescence Activated Cell Sorting (FACS) (Figure 1B). Since the mAb528 antibody is highly conformation dependant, its ability to recognize the mutants suggests that they are folded correctly. The mAb806 recognized a portion of the overexpressed wtEGFR as expected (Figure 1B), but a greater shift was evident for wtEGFR and A289V than the R324L or E330K mutants, suggesting that the latter two mutations may affect the binding of the mAb806. This was also observed in our Western analyses (Figure 1C) where mAb806 displayed reduced binding especially to the R324L mutation relative to wtEGFR (upper panel). Binding of a C-terminal EGFR antibody was unchanged (lower panel) when comparing the fully glycosylated upper EGFR band. Thus the R324L and E330K mutations are expressed on the cell surface and have a minor, but reproducible, impact on mAb806 binding.


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)

Biochemical characterization of mutant epidermal growth factor receptor (EGFR). (A). wtEGFR and mutant EGFR were translated into 35S-cysteine labelled proteins in a cell free expression system and labeled proteins detected by autoradiography following SDS-PAGE; (B). NR6 cells expressing wtEGFR or mutant EGFR were stained with secondary antibody alone (green), isotype control antibody (orange), mAb528 (red) or mAb806 (blue) and subjected to Fluorescence Activated Cell Sorting (FACS) analysis. The representative profiles for each cell line are shown; (C). wtEGFR or mutant EGFR cells were grown under serum free conditions, lysed and IP'd using mAb528. Western blot analysis of total EGFR using a mAb806 probe (upper panel) or a C-terminal EGFR probe (lower panel) are shown. A431 cells that overexpress the EGFR were included as a positive control.
© Copyright Policy
Related In: Results  -  Collection

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

f1-cancers-03-02032: Biochemical characterization of mutant epidermal growth factor receptor (EGFR). (A). wtEGFR and mutant EGFR were translated into 35S-cysteine labelled proteins in a cell free expression system and labeled proteins detected by autoradiography following SDS-PAGE; (B). NR6 cells expressing wtEGFR or mutant EGFR were stained with secondary antibody alone (green), isotype control antibody (orange), mAb528 (red) or mAb806 (blue) and subjected to Fluorescence Activated Cell Sorting (FACS) analysis. The representative profiles for each cell line are shown; (C). wtEGFR or mutant EGFR cells were grown under serum free conditions, lysed and IP'd using mAb528. Western blot analysis of total EGFR using a mAb806 probe (upper panel) or a C-terminal EGFR probe (lower panel) are shown. A431 cells that overexpress the EGFR were included as a positive control.
Mentions: The cell-free translation of mutant EGFR demonstrated that Mw and expression levels of the proteins were similar to the wtEGFR, indicating that the mutations did not affect translation efficiency (Figure 1A). Both the wtEGFR and mutant EGFR were expressed on the cell surface of NR6 cells as determined by Fluorescence Activated Cell Sorting (FACS) (Figure 1B). Since the mAb528 antibody is highly conformation dependant, its ability to recognize the mutants suggests that they are folded correctly. The mAb806 recognized a portion of the overexpressed wtEGFR as expected (Figure 1B), but a greater shift was evident for wtEGFR and A289V than the R324L or E330K mutants, suggesting that the latter two mutations may affect the binding of the mAb806. This was also observed in our Western analyses (Figure 1C) where mAb806 displayed reduced binding especially to the R324L mutation relative to wtEGFR (upper panel). Binding of a C-terminal EGFR antibody was unchanged (lower panel) when comparing the fully glycosylated upper EGFR band. Thus the R324L and E330K mutations are expressed on the cell surface and have a minor, but reproducible, impact on mAb806 binding.

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