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.

Fig. 1.

Single-molecule observation of interactions between EGFR and Cy3–Grb2. (A and B) Cy3–Grb2 bound to the plasma membrane fraction with (A) and without (B) EGF stimulation. The fluorescence intensity of each spot represents the frequency of Cy3–Grb2 binding because images were accumulated during 1 min. (Scale bar: 5 μm.) (C) Repeated binding and release of Cy3–Grb2 were observed at a single site on the stimulated membrane fraction (see SI Movie 1). (D) Changes in the fluorescence intensity at single binding sites plotted against time. A histogram of the fluorescence intensity is shown on the right of the plot at 10 nM Cy3–Grb2. The averages and σ value are used to determine thresholds for the on and off states (see Materials and Methods). (E) The on- and off-time for each event were determined from the fluorescence intensity.

Fig. 2.

Dissociation kinetics. (A) Cumulative histograms of the on-times for each concentration of Grb2. The histograms were fitted with a single (green) and a sum of two (blue) or three (red) exponential functions. τ₁–τ₃ (in s) are the time constants in the fitting to the three-component multiple-exponential function. Numbers in parentheses are percentages of each fraction. (B) Distributions of the inverse of the average on-times at individual binding sites. The average of every binding site for each concentration of Grb2 is indicated by an arrowhead.

Fig. 3.

Association kinetics. (A) Distributions of the inverse of the average off-times at individual reaction sites divided by the Grb2 concentration. The average of every binding site for each concentration of Grb2 is indicated by an arrowhead. The left ends of the distributions were limited to avoid nonspecific bindings (see Materials and Methods). (B) Cumulative histograms of the off-times for each concentration of Grb2. The histograms were fitted with a single (green) and a sum of two (blue) or three (red) exponential functions. τ₁–τ₃ (in s) are the time constants in the fitting to multiple exponential functions. Numbers in parentheses are percentages of each fraction. τ₁–τ₃ at 100 nM Grb2 (10 nM Cy3–Grb2 mixed with 90 nM nonlabeled Grb2) are the normalized values assuming the same reaction rate for Cy3-labeled and nonlabeled Grb2.

Fig. 4.

Stretched exponential kinetics of the association events. Cumulative histograms of the off-times as shown in Fig. 3B were fitted with a stretched exponential function. Red lines are the results of fitting by floating both the time constant τ (in s) and the exponent α. Blue lines are the results of fitting with α fixed to 0.5 and floating the time constant τ′ (in s). τ and τ′ for 100 nM Grb2 are normalized values assuming the same reaction rate for Cy3-labeled and nonlabeled Grb2.

Fig. 5.

Distribution of the dissociation equilibrium constants. The ratio of the total off-times to the total on-times was calculated for individual reaction sites and multiplied by the Grb2 concentration. The average over every reaction site for each concentration of Grb2 is indicated by an arrowhead.