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A similar technique can be used to detect recognition between EGFR and cytoplasmic proteins. Grb2 is one of the cytoplasmic SH2 proteins that recognize activated EGFR. Grb2 works as an adapter protein to combine activated EGFR and mSos1, a guanine nucleotide exchange protein of a small G-protein Ras (16). Grb2 recognizes EGFR directly or indirectly mediated by Shc. The kinetic parameters of association and dissociation reactions between Grb2 and EGFR have not been revealed in conditions where the structure of the biomembrane remains.
To observe the recognition between EGFR and Grb2 on the plasma membrane, the basal membrane of cells was prepared using the lysis squirting method. Recombinant Grb2 was purified and conjugated with Cy3 at the N-terminus (Cy3-Grb2). Gy5-EGF, Cy3-Grb2, and ATP were added to the basal membrane fraction under a TIR-FM. At the same position of EGF-binding, repeated attachments and detachments of Cy3-Grb2 were observed in single molecules (Fig. 3B). Statistical analysis of off times and on times between EGFR and Grb2 will provide the dissociation and association rate constants, respectively. Determination of these rate constants gives important information for understanding the mechanism of EGF signaling.
Intracellular calcium response induced by EGF
EGF induces several global responses to change properties of cells such as reorganization of the cytoskeleton, cell movements, or new gene expression.
Elevation of intracellular Ca2+ concentration is one of these cellular responses at the early stage of EGF signaling. Activation of EGFR in turn activates a cytoplasmic enzyme, PLC, to produce IP3 from membrane lipids. IP3 is the second messenger that induces calcium release from the endoplasmic reticulum. A pulse of Rh-EGF was added to HeLa cells to visualize binding of EGF and to induce the calcium response (Fig. 4). Changes of the intracellular calcium concentration were monitored using a fluorescent calcium indicator Fluo-4. Binding of Rh-EGF was observed in single-molecules over the entire surface of the cells by focusing up and down (5). The number of EGF molecules bound on each cell was counted and the elevation of the Ca2+ concentration in the same cells was measured. Surprisingly, most cells respond to the binding of only a few hundred EGF molecules, even though HeLa cells are known to express several ten thousand EGFR molecules. Amplification and propagation mechanisms of the activation of EGFR (Fig. 3A) may be responsible for this remarkably high sensitivity.
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