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Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.
Under normal conditions Grb2 is localized throughout the entire cell. Upon stimulation of a growth factor receptor it translocates to the plasma membrane and is subsequently internalized into endosomes as shown in Figure 4. The expression of a cell surface receptor capable of binding Grb2 is sufficient to induce this translocation. In a typical screening experiment, no change in GFP-Grb2 localization is observed. However, when a protein is expressed that recruits Grb2 to a sub-cellular site, there is a change in localization that can be easily visualized by fluorescence microscopy. For instance, expression of the EGFR results in relocalization of GFP-Grb2 to endosome-like structures (Figure 4). Thus, when applying a genome-wide library, it can be expected that novel Grb2-binding cell surface proteins can be identified.
This method is not limited to the detection of cell surface proteins. For instance, Dynamin2 induces a change in sub-cellular localization of GFP-Grb2 displaying endosome-like recruitment (Figure 5). Dynamin2 binds to the C-terminal SH3 domain of Grb2 and is involved in endocytosis of this complex9,10. Thus, this approach enables the identification of general protein binding complexes and is not limited to the identification of interaction partners for the SH2 domain.
Several different types of translocation can be expected such as localization to the plasma membrane, to endosomes, other cytoplasmic vesicular structures or to the nucleus. Therefore, it is recommended that all images are also inspected by eye. However, when screening large sets of cDNAs an automated image analysis algorithm is more practical. In this case, a combination of algorithms is desirable, such as spot detection to identify endosomes, cytoplasm/nucleus detection to identify nuclear shuttling and general morphological algorithms to identify cellular shape changes. The use of these different phenotypic detection methods has been discussed in more detail elsewhere11.
Figure 1. Overall scheme of the experiment. The experiment details a complete high-content screening workflow starting with the amplification and preparation of the cDNA library, transfection of cDNA vectors plus reporter constructs, image acquisition and image analysis using the free open software IMAGEJ.
Figure 2. Configuration of the Tecan Deck. (1) On the left-hand side, five 100 ml troughs need to be filled with buffers 1 (40 ml), 2 (40 ml), 3 (50 ml), AW (60 ml) and A4 (100 ml). Trough A4 will need to be re-filled once during the procedure. (2) The vacuum manifold is located on position 14 in this example. (3) The deepwell plate with bacterial pellets is located on position 30, next to the elution buffer trough with 25 ml Elution buffer. (4) Disposable tips for the 8-channel head need to be loaded in the tip racks on the left side and tips for the multichannel head need to be filled on position 40. The Tecan FreedomEvo liquid handler that is used in this experiment is equipped with 500 μl syringes. Click here to view larger figure.
Figure 3. Automation of image acquisition on the Opera LX. A) Select the Configuration tab (1). Activate the Laser lines required for the experiment (2). Select the cameras for image acquisition (3). Select the plate type and objective lens for the experiment (4). B) Select the microscope tab (1). Define the light source and exposure time for exposure 1 (2,3). Focus on one well and take height (4). C) Select the Experiment Definition tab (1). Drag and drop exposure, skewcrop, reference, layout and sublayout files (2). Drag and drop the experiment file (3). D) Select Automatic Experiment (1). Drag and drop experiment file (2). Allocate appropriate barcode (3). Start image acquisition by clicking on the start button (4). Click here to view larger figure.
Figure 4. GFP-Grb2 translocation by cDNA expression. GFP-Grb2 relocates from a predominant cytoplasmic localization to endosome-like structures upon overexpression of a transmembrane growth factor receptor and subsequently internalizes to endosomes. On the left, microscopy images of COS M6 cells are shown that were transiently transfected with GFP-Grb2 (top) or GFP-Grb2 plus EGFR (bottom). Co-expression of the EGFR results in relocation of GFP-Grb2 to endosome-like structures.
Figure 5. Example of cDNA-induced translocation of GFP-Grb2. The GTPase Dynamin2, which is involved in Grb2-mediated endocytosis, relocates GFP-Grb2 to endosome-like structures (circled). In this experiment, GFP-Grb2 was co-transfected with DNM2 into HEK293T cells. Cells were stained with Hoechst 33342 after 24 hr and images were acquired on the Opera LX. One field of view of the green (GFP) channel and the UV (blue; Hoechst 33342) is displayed.
Expression cloning is a powerful tool that has been used in the past to identify novel cellular components such as virus receptors and blood cell antigens12. Here, we describe a method to facilitate the identification of novel putative signal transduction receptors that bind to Grb2.
There are a few critical steps in the protocol.
The Grb2 translocation assay has been used by other groups to identify small molecule inhibitors of EGFR kinase activation6. In that case, the EGFR is specifically activated with ligand causing recruitment of Grb2 to the plasma membrane. Disruption of this interaction can then be investigated using small molecule compounds. Similarly, it can be envisaged that siRNA screens can be employed to identify endogenous genes involved in EGFR-Grb2 signaling or other Grb2-binding growth factor receptors such as c-KIT or the erythropoietin receptor. Thus, there are multiple potential applications for this technique. An analogous approach could be applied to GFP-tagged reporter systems for other adapter molecules such as Shc, Gab or IRS.
One major advantage of using this cell-based assay in mammalian cells is that it enables the identification of physiologically relevant interactions. The assay is a readout for protein complex formation, but more importantly, the relevant interactions are monitored at the correct sub-cellular site. In this regard, this technique overcomes artifacts from other protein-protein interaction methods such as yeast-two-hybrid or in vitro assays. It should be noted though, that indirect interactions may also result in Grb2 recruitment. Similarly, the transcriptional up-regulation of binding partners may be induced by cDNA expression. To distinguish between these possibilities, it is necessary to perform the appropriate secondary assays to distinguish between direct and indirect binding effects.
In conclusion, the GFP-adapter molecule translocation assay promises high potential for genome-wide screening and drug discovery applications.
No conflicts of interest declared.
This work was supported by the Medical Research Council and the Marie-Curie International Reintegration Grant scheme (to JKV).