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the monomeric and dimeric bindings of EGF (Figure 5.1C). Best
fit values of the
reaction parameters agreed very well with the results from direct observation
(Figure 5.2) and were consistent with previous studies. Since this is the simplest
model which takes into account both the presence of the reaction intermediate and is
consistent with experimentally obtained kinetic parameters, it was concluded that
this model is appropriate for the formation of signaling dimers of EGFR.
Our result can be
fitted to recent X-ray crystallographic studies by assuming
dynamic structural changes in EGFR. In crystal form, EGFR has two conformations,
a tethered and an extended state. Only the extended state is thought to bind
firmly to
EGFand formsignaling dimers [5]. Single-molecule experiments suggest amodel for
the formation of the EGFR signaling dimer as follows (Figure 5.3B). Most of the
receptor molecules are in the monomeric tethered state-like structure, which is the
slow binding site for EGF. However, a small percentage of receptor molecules form
predimers in an extended-like structure that binds with EGF more rapidly than the
monomer by a factor of about 100. EGFmolecules selectively bind to the predimers of
EGFR especially when their concentration is low. Binding of the
rst EGFmolecule to
the predimer induces an allosteric conformational change in the vacant binding site
to make binding of the second EGF even more rapid. In this model, signal
transduction of EGF is facilitated through receptor predimerization and positive
cooperative ligand binding.
5.4
Amplification and Propagation of EGFR Activation
Phosphorylation (activation) of EGFR after formation of the signaling dimers was
examined in single molecules [10]. Cells were incubated with Rh-EGF for 1min and
any unbound Rh-EGF was then washed out. After incubation for various periods of
time, cells were
fixed and activated EGFR was detected using the Fab
0
fragment of a
monoclonal antibody which recognizes the active conformation of the cytoplasmic
domain of EGFR after phosphorylation. The Fab
0
fragment was labeled with a green
fluorophore Alexa 488 (Alx-Fab
0
).
Rh-EGF and Alx-Fab
0
on the plasma membrane were visualized in single mole-
cules. Since, unfortunately, Rh-EGF dissociated from the cell surface during
fixation,
binding of Rh-EGF before the
fixation and Alx-Fab
0
after the
fixation were compared
between similarly treated but different cells. Both numbers of molecules and binding
sites of Alx-Fab
0
increased with time after stimulation with Rh-EGF. At the peak of
activation, the density of molecules and binding sites for Alx-Fab
0
were greater than
those for Rh-EGF by a factor of 3.0 and 2.3, respectively, i.e. the EGF signal was
ampli
ed during the activation process of EGFR.
A semi-intact cell technique [11] was used for the simultaneous imaging of Rh-EGF
and Alx-Fab
0
to study the process of ampli
cation. Semi-intact cells were prepared by
perforating the plasma membrane using the antibiotic streptolysin-O. Rh-EGF was
applied to the semi-intact cells. Since most of the cytoplasm leaked through the pores,
activation of EGFR did not take place at this stage. The cells were then washed and
