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Published on: April 24, 2012
GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling
Julia Petschnigg1, Robin Ketteler1
1MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom.
Abstract:
High-content screening is a useful tool to understand complex cellular processes and to identify genes, proteins or small molecule compounds that modulate such pathways. High-content assays monitor the function of a protein or pathway by visualizing a change in an image-based readout, such as a change in the localization of a reporter protein. Examples of this can be the translocation of a fluorescently tagged protein from the cytoplasm to the nucleus or to the plasma membrane. One protein that is known to undergo such translocation is the Growth Factor Receptor-bound protein 2 (GRB2) that is recruited to the plasma membrane upon stimulation of a growth factor receptor and subsequently undergoes internalization. We have used GFP-tagged Grb2 previously to identify genes that are involved in EGFR signaling (Petschnigg et al., 2017). Ultimately, the assay can be adapted to cDNA expression cloning (Freeman et al., 2012) and can be used in early stage drug discovery to identify compounds that modulate or inhibit EGFR signaling and internalization (Antczak and Djaballah, 2016).
Insights
High-content screening uses image-based readouts to study cellular pathways. This method, utilizing Growth Factor Receptor-bound protein 2 (GRB2) translocation, aids in identifying genes and compounds affecting EGFR signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- High-content screening (HCS) is a powerful technique for analyzing complex cellular processes.
- Image-based readouts, such as protein localization changes, are key to HCS assays.
- Growth Factor Receptor-bound protein 2 (GRB2) exhibits translocation, making it a useful reporter.
Purpose of the Study:
- To leverage HCS with GRB2 translocation to understand EGFR signaling.
- To identify genes involved in EGFR signaling pathways.
- To apply HCS for early-stage drug discovery targeting EGFR signaling.
Main Methods:
- Utilizing a GFP-tagged GRB2 reporter protein.
- Monitoring GRB2 translocation via image-based readouts in response to growth factor receptor stimulation.
- Adapting the assay for cDNA expression cloning and compound screening.
Main Results:
- Previous studies successfully identified genes involved in EGFR signaling using GFP-tagged GRB2 (Petschnigg et al., 2017).
- The HCS assay is adaptable for cDNA expression cloning (Freeman et al., 2012).
- The assay can identify compounds modulating or inhibiting EGFR signaling and internalization (Antczak and Djaballah, 2016).
Conclusions:
- High-content screening with GRB2 translocation is effective for dissecting EGFR signaling.
- This approach facilitates the discovery of genes and small molecules impacting cellular pathways.
- The method holds significant potential for early-stage drug discovery and target identification.
