Published: Vol 7, Iss 17, Sep 5, 2017 DOI: 10.21769/BioProtoc.2528 Views: 7960
Reviewed by: Ralph Bottcherilgen MenderTomas Aparicio
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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).
Keywords: Grb2Background
Signal transduction by growth factor receptors is essential for cells to maintain proper function and thus requires tight control. Signal transduction by growth factor receptors is initiated by binding of an external ligand (e.g., Epidermal Growth Factor, EGF) to a transmembrane receptor such as the Epidermal Growth Factor Receptor (EGFR) and activation of downstream signaling cascades (Yao et al., 2015). A key regulator of EGFR-signaling is Growth Factor Receptor-bound protein 2 (Grb2), which is composed of an internal SH2 (Src homology 2) domain flanked by two SH3 domains. Grb2 binds to activated growth factor receptors at phosphorylated tyrosine residues through its SH2 domain, thus coupling receptor activation to SOS-Ras-MAPK (Mitogen-activated protein kinase) signaling cascades. The composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along multiple pathways. Mutations in signaling pathways frequently lead to the development of cancer. Hence, in order to better understand how aberrant signaling can lead to disease, it is important to identify novel signaling molecules in growth factor signaling. To accomplish this, we used the previously established microscopy-based GFP-Grb2 translocation assay that monitors the translocation of cytosolic GFP-tagged Grb2 to subcellular compartments upon expression of a cDNA library (Figure 1). We used this technique to identify novel proteins that can lead to translocation of GFP-Grb2 when overexpressed and in a second stage tested whether these proteins play a role in EGFR-signaling (Petschnigg et al., 2017). Examples that lead to punctate structures include TACC3, a novel EGFR-interactor, and AMPH (Figure 3). TACC3 led to induction of large GFP-Grb2 puncta, whereas AMPH results in formation of multiple small spots (Figure 3), pointing at potentially different mechanisms of those proteins in EGFR-signaling. In our recent study, we further characterized TACC3 and showed that TACC3 specifically binds to oncogenic EGFR variants and showed that TACC3 enhances EGFR-stability at the cell surface and increases EGFR-mediated signaling. The GFP-Grb2 assay can be expanded to multiple more applications. A siRNA/shRNA or CRISPR library could be co-expressed with GFP-Grb2 and translocation subsequently observed following EGF stimulation. As EGF stimulation would sequester GFP-Grb2 to endosomal structures and the plasma membrane, translocation from there upon siRNA/shRNA knockdown or CRISPR knockout could point at possible factors that ablate EGFR-Grb2 interactions and signaling. In a similar way, small molecule compound screens could be done to test for drugs that can specifically disrupt EGFR-Grb2 interactions upon EGF-stimulation (Figure 1). Other options could be to use mutated Grb2-variants that fail to bind to EGFR or other binding partners, thus the assay can give insight into which gene (when overexpressed or knock-downed) or which drug has an influence on specific binding domains of Grb2.
Figure 1. Schematic overview of the Grb2 translocation assay. Under non-stimulated or basal conditions, GFP-tagged Grb2 is mostly found in the cytosol (A), but can be recruited to localized interaction partners such as activated or endocytosed Epidermal Growth Factor Receptor, EGFR. Expression of a cDNA expression plasmid can lead to relocalization of GFP-Grb2 to the plasma membrane, endosomal structures or other sub-cellular locations by binding to Grb2 interaction partners or activation of Grb2-dependent cellular pathways (B). Stimulation with EGF recruits Grb2 to phosphotylated EGFR (C). Small molecule compounds or si/shRNA libraries can help identify genes or drugs that can disrupt Grb2 binding to the EGFR, impairing the recruitment to the receptor and thus predominant cytosolic localization of GFP-Grb2 (D). Since Grb2 can interact with other growth factor receptors as well, the assay can be adapted to monitor interaction/recruitment to other receptors as well.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Fluorescence images were analysed using the freely available software ImageJ. For our study, structures were analysed individually and different punctate structures were assessed by eye. However, for a high-throughput screen, structures can furthermore be quantified using a custom-made image analysis ImageJ-pipeline. For more details see as an example (Ketteler et al., 2017). Alternatively, other software can be used such as CellProfiler can be used. A typical high-content analysis image processing workflow consists of noise reduction, segmentation, and feature extraction steps. Typically, the original nuclear channel of a sample cell is smoothened with a median filter, segmented with k-means clustering algorithm and the objects are selected and quantified (Ketteler and Kriston-Vizi, 2016).
Notes
Recipes
Acknowledgments
This work was supported by the UK Medical Research Council core funding to the MRC-UCL University Unit (MC_EX_G0800785) and a BBSRC New Investigator Award to RK (BB/JO/5881/1). JP was supported by an EC-Marie Curie International Incoming Fellowship (FP7-PEOPLE-2013-IIF). This protocol has been adapted from (Petschnigg et al., 2017).
References
Article Information
Copyright
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Petschnigg, J. and Ketteler, R. (2017). GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling. Bio-protocol 7(17): e2528. DOI: 10.21769/BioProtoc.2528.
Category
Cancer Biology > Cancer biochemistry > Protein
Cell Biology > Cell staining > Protein
Cell Biology > Cell signaling > Intracellular Signaling
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