EGFR Redistribution Assay

• Designed to assay compounds for their ability to modulate internalization of EGFR
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Epidermal growth factor receptor (EGFR, ErbB-1) is a member of the ErbB receptor family, a subfamily of four closely related tyrosine kinases. The other members of the ErbB receptor family are HER2/c-neu (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4). EGFR is a ubiquitous single-transmembrane receptor, which in its inactive state is present as monomers in the cell membrane.

Upon activation by a ligand, the receptor dimerizes with another member of ErbB receptor family, e.g. another EGFR molecule or HER2. The homo- and heteromer ErbB receptor dimers have different ligands. EGFR dimers and EGFR/ErbB-2 heterodimers are activated by epidermal growth factor (EGF), transformation growth factor α (TGFα), and amphiregulin (AR). Binding of ligand to the receptor and subsequent dimerization leads to stimulation of the intrinsic tyrosine kinase activity of EGFR and activation of multiple downstream signaling pathways, including the MAP kinase pathway, the AKT pathway, and the STAT pathway. Following activation, the EGFR is internalized through clathrin coated pits and subsequently sorted to and degraded in lysosomes. The ubiquitin ligase c-Cbl targets the EGFR to endocytosis by tagging the receptor with multiple monoubiquitin molecules important for receptor down-regulation. The EGFR and HER2 are involved in cell proliferation and differentiation, and they are often over-expressed or amplified in cancers, making them important targets for cancer therapy.

Figure 1. Internalization of EGFR-EGFP in response to EGF. Cells were untreated (DMSO control, left panel), or treated with 10 ng/ml EGF for 30 min in the presence (right panel) or absence (center panel) of EGFR inhibitor. Arrows indicate the EGFR-EGFP internalization that is detected by the image analysis algorithm.

Figure 2. A) Concentration response of EGF in the EGFR Redistribution assay (n=8). Cells were treated with EGF for 30 min. Cells were then fixed and receptor internalization was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. The EC₅₀ of EGF is ~0.2 ng/ml. B) Concentration response of the EGFR inhibitor compound in the EGFR Redistribution assay (n=16). Cells were incubated with compound for 1 hr, followed by EGF treatment for 30 min. Cells were then fixed and receptor internalization was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. The EC₅₀ of the EGFR inhibitor is ~1 μM.