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

Susie I Ymer,Timothy E Adams,Diana X Cao,Andrew M Scott,Anna Cvrljevic,V Chandana Epa,Terrance G Johns,Sameer A Greenall,Jacqui F Donoghue

doi:10.3390/cancers3022032

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.

Highlights

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTKs) that include ErbB2, ErbB3 and ErbB4 [1]
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)
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

Summary

Introduction

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTKs) that include ErbB2, ErbB3 and ErbB4 [1]. EGFR can bind at least seven ligands, of which the most widely studied is epidermal growth factor (EGF) [1]. Regulated activation of the EGFR requires high affinity ligand binding and involves homo- and/or hetero-dimerization with other. Dimerization is essential for activation of its intrinsic kinase activity and subsequent autophosphorylation [3,4,5,6]. These events activate a number of different downstream signaling pathways that regulate multiple cellular processes such as proliferation, differentiation and development. The EGFR is commonly overexpressed or mutated in many cancer types and its presence promotes tumor progression and survival

Results

Discussion

Conclusion