Epidermal growth factor receptor binding is not a simple one-step process

The binding kinetics of ¹²⁵I-labeled mouse epidermal growth factor (EGF) to receptors on human fibroblast cells in monolayer culture were measured at 4 °C. Initial binding rates as a function of hormone concentration allowed estimation of simple two-state on-off rate constants of 1.2 x 10⁶ M^-1 s^-1 and 4.9 x 10^-3 s^-1, respectively. These two-state parameters gave inadequate computer fits to long term kinetic and equilibrium-binding data, suggesting that an additional process(es) was occurring. Nonlinear equilibrium Scatchard plots and transient 'pseudo-Scatchard' plots taken at pre-equilibrium times support the idea that at least one other process is occurring during receptor binding. ¹²⁵I-EGF-receptor dissociation kinetic plots were biphasic, yielding rate constants of 1.5 x 10^-2 s^-1 and 5.6 x 10^-5 s^-1 with the ratio of the two components changing with the time of initial incubation with ¹²⁵I-EGF. Application of a ternary complex model which assumed complexation of the bound receptor with a cell surface interaction molecule gave satisfactory fits to all data.