Binding of EGF induces dimerization of EGFR, which in turn promotes tyrosine phosphorylation in the cytoplasmic domain of EGFR. Once phosphorylated on the tyrosine residues, EGFR is recognized by many cytoplasmic proteins containing SH2 or PTB domains. These are the early steps in signal transduction of EGF (16, 17). The process of EGFR dimerization has been visualized for single molecules of EGF/EGFR complexes (6). Cy3-EGF in solution could not be observed as fluorescent spots because of rapid Brownian movement. In addition, binding of Cy3-EGF on the cell surface was specific for EGFR (Fig. 1B). Thus the appearance of the fluorescent spots of Cy3-EGF on the cell surface means the formation of an EGF/EGFR complex. The binding ratio of EGF and EGFR is known to be 1:1. Therefore, dimerization of the EGF /EGFR complex can be detected as the formation of fluorescent spots that have twice the brightness of single Cy3-EGF spots. Two types of dimer formation have been visualized. In a few cases (10%), two fluorescent spots diffusing laterally along the plasma membrane collided and then moved together. However, in the majority of cases (90%), the fluorescence intensity of a spot increased suddenly by a factor of approximately two and then photobleached in two steps (Fig. 2A). This latter case is probably caused by the direct binding of EGF molecules in solution to an EGF-free EGFR located next to an EGF-bound EGFR, suggesting that dimers (or larger oligomers) of EGFR had been preformed. Other studies also suggest predimerization or preclustering of EGFR (18, 19).
Fluctuation of the EGFR dimers
Single-pair fluorescence resonance energy transfer (spFRET) (1, 20) has been used to examine dynamic conformational fluctuations of the dimers of EGF /EGFR complexes (6). A mixture of Cy3-EGF and Cy5-EGF was added to the cells. Exciting Cy3 with a 532-nm laser, fluorescence images at 580 nm (Cy3) and 670 nm (Cy5) were acquired simultaneously using dual-view optics. Anti-correlation of the fluorescence intensity changes of the same spot at the Cy3 and Cy5 channel have been visualized, indicating the occurrence of FRET (Fig. 2B). In some cases, the efficiency of FRET fluctuated with time, suggesting conformational fluctuations of the dimers in a time scale of seconds. It is highly likely that this dynamic fluctuation of the complexes was related to the reorganization of the dimers of EGF/EGFR complexes as proposed from biochemical studies (21).
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