The relationship between EGF binding and receptor activation was analyzed for each binding site of EGF. For this purpose, conformational change of the cytoplasmic domain of EGFR after tyrosine phosphorylation was detected using a specific monoclonal antibody (22). The Fab' fragment of the antibody was labelled with a fluorophore Alexa488 (Alexa488-Fab'). To introduce Alexa488-Fab' into the cytoplasm, semi-intact cells perforated by streptolysin O (SLO) were used (6, 23). After a pulse stimulation of the semi-intact cells with EGF conjugated with another fluorophore, tetramethyl rhodamine (Rh-EGF), the cells were equilibrated with Alexa488-Fab'. Then ATP was added to start the reaction.
Binding of Rh-EGF and Alexa488-Fab' to the plasma membrane was simultaneously visualized in single molecules (Fig. 3A). Superimposition of the images of Rh-EGF over the images of Alexa488-Fab' demonstrated that there were three categories of fluorescent spots: spots containing only EGF, both EGF and Fab', and only Fab'. The first category represents EGF /EGFR complexes that failed to be activated or stained by Fab'. The second was the site of receptor activation induced by binding of EGF. The third represents activated EGFR without binding of EGF. Dissociation of EGF from EGFR was not observed in this experiment (up to 30 min after addition of ATP). In addition, binding of Alexa488-Fab' was specific for the activation of EGFR since the binding was strongly dependent on the presence of ATP and no binding was seen on the membrane of cells known to be EGFR-null. Therefore, the presence of the third category indicates an indirect mechanisms of EGFR activation in the cells stimulated with EGF. Activation of EGFR is believed to start in 2:2 complexes between EGF and EGFR. It is possible, however, that EGFR molecules activated in 2:2 complexes exchanged the dimerization pair to activate other unliganded EGFR. Once activated, unliganded EGFR may be able to activate other unliganded molecules. Reorganization of dimers is thought to be the mechanism of heterodimerization between EGFR families (21). Structural fluctuation of EGFR dimer (Fig. 2B) and lateral mobility of EGFR along the plasma membrane seems to be important for this process. Propagation of EGF signals (24) most likely uses this process.
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