EGFR Biosensor Cell Line

Human EGFR Live Cell Fluorescent Biosensor Assay

Sigma® Life Science offers a novel, robust live cell assay for measuring endogenous human EGFR activity using a fluorescent GFP-tagged EGFR-specific SH2 domain protein. This modified A549 cell line provides an innovative and sensitive research tool for studying the molecular mechanism and kinetics of endogenous receptor activation and internalization for EGFR (ERBB1).

Benefits of EGFR Biosensor Cell Line
Applications to Your Research
Live Cell Imaging of the Human EGFR Biosensor
Poster

Benefits of EGFR Biosensor Cell Line

• Live cell assays to study EGFR kinetics
• Specific to EGFR activity
• Robust and rapid assays with high sensitivity
• Economical for rapid cell processing and data analysis
• Adaptable to multiple HCS platforms including 384-well plates
• Stable expression of GFP-tagged biosensor
• Endogenous EGFR expression and activity

Applications to Your Research

• Live high content screening for both wild-type or mutant EGFR activity
• Live cell imaging to follow receptor kinetics of activation and internalization
• Ideal for high throughput screens

Live Cell Imaging of the Human EGFR Biosensor

The fluorescent EGFR Biosensor is stably expressed in A549 human lung carcinoma cells and is ideal for real-time live cell imaging of ligand binding and receptor activation and internalization. Activity is easily quantified by image analysis to count cytoplasmic granules following receptor internalization. These cells also provide a rapid and sensitive method for high throughput screening of drug libraries to identify compounds that modulate EGFR activity – both activators or inhibitors.

Figure 1. EGFR Biosensor Utilized in A549 Live Cell Assay – A. The structure of the EGFR biosensor construct consisting of the TagGFP green fluorescent protein and two tandem SH2 domains from adapter protein Grb2. B. EGFR activation and internalization in A549 cells. The EGFR biosensor plasmid was transfected into the A549 cell line, the transfected cells treated with 100 ng/mL EGF to activate EGFR and then the internalization process was observed in real time by fluorescence microscopy. The increasing internalization of the biosensor-EGFR complex over time can be quantitated using granularity analysis.

The EGFR Biosensor assay is highly selective for ligands that bind to ERBB1. EGF stimulation activates the receptor-biosensor interaction as initially observed by clustering of the biosensor at the cell surface within minutes of treatment. This is followed by receptor internalization which appears as dense fluorescent granules in the cytoplasm of the cell. Receptor activation can be blocked by specific inhibitors of ERBB1 (e.g., AG1478) and shows no activity for ligands specific for HER2 and HER 3 (e.g., heregulin-1β).

Figure 2. Specificity of A549 EGFR Biosensor Activity – EGF (100 ng/mL) stimulated EGFR (ERBB1) internalization while Tyrphostin AG1478 (1 µM), a selective inhibitor of EGFR, blocked receptor translocation to the plasma membrane and internalization. Hepatocyte growth factor (HGF, 100 ng/mL), a ligand specific for HGFR has low affinity for EGFR and showed much less activity than EGF. Bar = 10 µm.

Poster

• Genetically Encoded, SH2 Domain-Based Fluorescent Reporters of Endogenous Receptor Tyrosine Kinase Activity in Living Cells(2.3 Mb PDF)

Publication

• Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells.

For more information on these or any of our modified cell lines please contact us.

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