Limits...
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

The R324L and E330K mutants demonstrate enhanced transforming activity in anchorage independent growth assays. Transgenic NR6 cells were plated in an agarose matrix for 20 days and stained with MTT. (A) Colonies were counted and data graphed as the total colonies per well ± S.E. A random 10–20% sampling of the total numbers was analyzed for the percentage of cells (B) over 120 μm or (C) over 150 μm in size ± S.E.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3757403&req=5

f5-cancers-03-02032: The R324L and E330K mutants demonstrate enhanced transforming activity in anchorage independent growth assays. Transgenic NR6 cells were plated in an agarose matrix for 20 days and stained with MTT. (A) Colonies were counted and data graphed as the total colonies per well ± S.E. A random 10–20% sampling of the total numbers was analyzed for the percentage of cells (B) over 120 μm or (C) over 150 μm in size ± S.E.

Mentions: The ability of the transgenic NR6 cells to form colonies in anchorage-free conditions was examined (Figure 5). All of the cells tested formed colonies, with the R324L mutant forming significantly more colonies than the other cell lines (P = 0.009; Figure 5). The E330K mutant did not form significantly more colonies than wtEGFR-expressing cells. When the data was grouped into the percentage of colonies greater than 120 μm (Figure 5B) and 150 μm (Figure 5C), it was clear that both the mutant EGFR-expressing cells formed colonies that were significantly larger in size than wtEGFR. Thus, the ECD EGFR mutations significantly enhanced in vitro survival in serum free conditions and soft agarose when compared with wtEGFR.


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)

The R324L and E330K mutants demonstrate enhanced transforming activity in anchorage independent growth assays. Transgenic NR6 cells were plated in an agarose matrix for 20 days and stained with MTT. (A) Colonies were counted and data graphed as the total colonies per well ± S.E. A random 10–20% sampling of the total numbers was analyzed for the percentage of cells (B) over 120 μm or (C) over 150 μm in size ± S.E.
© Copyright Policy
Related In: Results  -  Collection

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

f5-cancers-03-02032: The R324L and E330K mutants demonstrate enhanced transforming activity in anchorage independent growth assays. Transgenic NR6 cells were plated in an agarose matrix for 20 days and stained with MTT. (A) Colonies were counted and data graphed as the total colonies per well ± S.E. A random 10–20% sampling of the total numbers was analyzed for the percentage of cells (B) over 120 μm or (C) over 150 μm in size ± S.E.
Mentions: The ability of the transgenic NR6 cells to form colonies in anchorage-free conditions was examined (Figure 5). All of the cells tested formed colonies, with the R324L mutant forming significantly more colonies than the other cell lines (P = 0.009; Figure 5). The E330K mutant did not form significantly more colonies than wtEGFR-expressing cells. When the data was grouped into the percentage of colonies greater than 120 μm (Figure 5B) and 150 μm (Figure 5C), it was clear that both the mutant EGFR-expressing cells formed colonies that were significantly larger in size than wtEGFR. Thus, the ECD EGFR mutations significantly enhanced in vitro survival in serum free conditions and soft agarose when compared with wtEGFR.

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