Real-Time Kinetics of Ligand/Cell Surface Receptor Interactions in Living Cells:  Binding of Epidermal Growth Factor to the Epidermal Growth Factor Receptor

Abstract

We describe a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent stopped-flow mixing, we determined that a murine hematopoietic precursor cell line, 32D, is capable of surviving rapid mixing using flow rates as great as 4.0 mL/s, allowing rapid processes to be quantitated with dead times as short as 10 ms. 32D cells do not express any endogenous epidermal growth factor (EGF) receptor or other ErbB family members and were used to establish monoclonal cell lines stably expressing the EGF receptor. Association of fluorescein-labeled H22Y-murine EGF (F-EGF) to receptor-expressing 32D cells was observed by measuring time-dependent changes in fluorescence anisotropy following rapid mixing. Dissociation of F-EGF from EGF-receptor-expressing 32D cells was measured both by chase experiments using unlabeled mEGF and by experiments in which equilibrium was perturbed by dilution. Comparison of these dissociation experiments showed that little, if any, ligand-induced dissociation occurs in the chase dissociation experiments. Data from a series of association and dissociation experiments, performed at various concentrations of F-EGF in the nanomolar range and at multiple cell densities, were simultaneously analyzed using global analysis techniques and fit to a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations having association rate constants of k(on1) = 8.6 x 10(6) M(-1) s(-1) and k(on2) = 2.4 x 10(6) M(-1) s(-1) and dissociation rate constants of k(off1) = 0.17 x 10(-2) s(-1) and k(off2) = 0.21 x 10(-2) s(-1). The magnitudes of these parameters suggest that under physiological conditions, in which cells are transiently exposed to nanomolar concentrations of ligand, ligand capture and release may function as the first line of regulation of the EGF receptor-induced signal transduction cascade.

Single-molecule analysis of epidermal growth factor binding on the surface of living cells

“…The formation of signaling dimers from predimeric sites would be more efficient than association of two EGF/EGFR complexes, which is limited by diffusion along the plasma membrane, as predicted by Mayawala et al (2005) based on our previous results (Sako et al , 2000). The association rate constant to monomeric sites ( k 3 ) is consistent with the association rate constants between EGF and EGFR, as reported previously (Bellot et al , 1990; Berkers et al , 1991; Felder et al , 1992; Chung et al , 1997; Wilkinson et al , 2001). Our calculation suggests that approximately 98–99% of EGFRs exist as monomeric sites on the cell surface.…”

“…Bellot et al (1990), Berkers et al (1991), Felder et al (1992), Chung et al (1997) and Wilkinson et al (2001).…”

Pathogenic Activation of Receptor Tyrosine Kinases in Mammalian Membranes

“…50 The kinetics is described in terms of rates of reactions, proportional to rate constants and concentrations. The ratio of the rate constants of the forward and backward reactions are equilibrium constants, such as the deactivation constant K. Thus, there are similarities between the kinetic approach and the thermodynamic approach presented here, because .…”

An empirical Bayesian approach for model-based inference of cellular signaling networks

“…It may seem that an increase in the EGF binding rate may provide better agreement with the Moehren et al data. However, the rate constant for EGF binding was obtained from the literature [45] and is a factor of 10 5 slower than the other steps that lead to ErbB1 phosphorylation. Additional studies may be needed to rectify these seemingly conflicting observations.…”