Real-time kinetics of ligand/cell surface receptor interactions in living cells: Binding of epidermal growth factor to the epidermal growth factor receptor

We describe a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. Whil e most mammalian cell lines cannot survive the shear forces associated with turbulent stopped-flow mixing, we determined that a murine hematopoietic p recursor cell line, 32D, is capable of surviving rapid mixing using flow ra tes as great as 4.0 mL/s, allowing rapid processes to be quantitated with d ead times as short as 10 ms. 32D cells do not express any endogenous epider mal growth factor (EGF) receptor or other ErbB family members and were used to establish monoclonal cell lines stably expressing the EGF receptor. Ass ociation of fluorescein-labeled H22Y-murine EGF (F-EGF) to receptor-express ing 32D cells was observed by measuring time-dependent changes in fluoresce nce anisotropy following rapid mixing. Dissociation of F-EGF from EGF-recep tor-expressing 32D cells was measured both by chase experiments using unlab eled 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. D ata from a series of association and dissociation experiments, performed at various concentrations of F-EGF in the nanomolar range and at multiple cel l densities, were simultaneously analyzed using global analysis techniques and fit to a two independent receptor-class model. Our analysis is consiste nt with the presence of two distinct receptor populations having associatio n 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 expose d to nanomolar concentrations of ligand, ligand capture and release may fun ction as the first line of regulation of the EGF receptor-induced signal tr ansduction cascade.