Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis

Abstract

Phosphorylation of the cytoplasmic tyrosine residues of the epidermal growth factor receptor (EGFR) upon binding of EGF induces recognition of various intracellular signaling molecules, including Grb2. Here, the reaction kinetics between EGFR and Grb2 was analyzed by visualizing single molecules of Grb2 conjugated to the fluorophore Cy3 (Cy3-Grb2). The plasma membrane fraction was purified from human epithelial carcinoma A431 cells after stimulation with EGF and attached to coverslips. Unitary events of association and dissociation of Cy3-Grb2 on the EGFR in the membrane fraction were observed at different concentrations of Grb2 (0.1-100 nM). The dissociation kinetics could be explained by using a multiple-exponential function with a major (>90%) dissociation rate of 8 s ؊1 and a few minor components, suggesting the presence of multiple bound states. In contrast, the association kinetics could be described by a stretched exponential function, suggesting the presence of multiple reaction channels from many unbound substates. Transitions between the unbound substates were also suggested. Unexpectedly, the rate of association was not proportional to the Grb2 concentration: an increase in Cy3-Grb2 concentration by a factor of 10 induced an increase in the reaction frequency approximately by a factor of three. This effect can compensate for fluctuation of the signal transduction from EGFR to Grb2 caused by variations in the expression level of Grb2 in living cells.EGFR ͉ signal transduction ͉ tyrosine phosphorylation

Non-Markovian properties and multiscale hidden Markovian network buried in single molecule time series

“…These time constants were much longer than those of the ON-times. 16 Simultaneous binding or release of multiple spots at the same position on the glass surface was hardly detected, suggesting that EGFR exists in the monomer form. The SM time series are symbolized as a bound state by symbol "1" (fluorescent) and an unbound state by "0" (nonfluorescent) by introducing an appropriate threshold.…”

“…By using the scheme, we scrutinize the SSN constructed for discrete time series of association and dissociation kinetics between EGFR and Grb2. 16 We show that our analytic formula accurately reproduces the autocorrelation function, and that the underlying SSNs change their properties as a function of timescale. It is also found that one of the states constituting the network serves to reset the memory of the process.…”

“…The plasma membrane fraction from epithelial carcinoma A431cells was immobilized to the coverslip, and Grb2 labeled with the fluorophore Cy3 were added into the solution. Morimatsu et al 16 observed "intermittent pulses" repeatedly at the same positions on the glass surface by Electron Bombardment (EB) CCD camera equipped with a Micro Channel plate (MCP) image intensifier. The pulse arises from binding and release processes of the Cy3-Grb2 with EGFR localized at the cytoplasmic side of the membrane fragments.…”

“…III, we apply our SSN analysis combined with the SSM to analyze the fluorescence intensity time trace of the binding and unbinding processes between EGFR and Grb2 in an in vitro reconstituted system. 16 The conclusion and future perspectives will be given in Sec. IV.…”

“…Single-molecule (SM) spectroscopy provides a means to uncover the complex mechanism of molecular machinery at single molecule level that one cannot address in bulk measurements. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] For example, it was found in an in vitro reconstituted system that the recognition (i.e., dissociation and association) kinetics between epidermal growth factor receptor (EGFR) on the plasma membrane and its adaptor protein Ash/Grb2 (Grb2) show non-exponential kinetics, and association kinetics depends nonlinearly on the Grb2 concentration, suggesting the existence of molecular memory in the signaling process. 16 Such innovative experimental developments of synthetic approaches using intact plasma membrane fractions a) Email: tamiki@es.hokudai.ac.jp have offered new challenges in theoretical modeling of the underlying mechanism of molecular machinery.…”