The interaction of epidermal growth factor with its receptor in A431 cell membranes: a stopped-flow fluorescence...

We describe a quantitative examination of the interaction of epidermal growth factor (EGF) with the EGF receptor using A431 cell membrane vesicles as a receptor source. Using T-format steady-state fluorescence anisotropy detection coupled with stopped-flow mixing, we measured the association and EGF-induced dissociation kinetics of fluorescein 5-isothiocyanate-labeled mEGF (FITC-EGF) with the EGF receptor over a wide range of FITC-EGF concentrations, membrane dilutions, and time scales (milliseconds to minutes). Fluorescence anisotropy-based equilibrium binding titrations were also performed. All studies utilized the same receptor preparation, ligand preparation, and detection system. The entire data surface (approximately 78,000 data points) was simultaneously analyzed using global analysis techniques with a variety of kinetic models. Our analysis identified with a high level of confidence receptor populations with two association rate constants (k(on) = 1.2 x 10(6) M-1 s-1, 7.2 x 10(6) M-1 s-1) and three dissociation rate constants (k(off0 = 0.95 x 10(-2) s-1, 0.13 x 10(-2) s-1, 0.32 x 10(-3) s-1), which reflect the presence of at least two distinct receptor populations in A431 cell membranes. Analysis of the kinetic data was found to be much more sensitive to the presence of multiple receptor populations than was the analysis of the equilibrium binding data.