Ligand-receptor interactions in the membrane of cultured cells monitored by fluorescence correlation spectroscopy

We investigated the specific binding of epidermal growth factor (EGF) to it s membrane-bound receptors in cultured cells. The specificity of the bindin g was attested by the consistent displacement of bound rhodamine-labeled EG F (Rh-EGF) following addition of 1000-fold molar excess of unlabeled EGF. t he binding specificity of EGF was further confirmed when vascular EGF was u nable to displace Rh-EGF binding, demonstrating no cross-reaction, Evidence for the specific interactions was verified by an equilibrium saturation bi nding experiment. EGF binding to the cell membranes is saturated at nancmol ar concentration. The Scatchard plots show a binding process with K-ass of 1.5 x 10(9) M-1. The dissociation kinetics follow a single exponential func tion characteristic for a relatively slow dissociation process with K-diss = 2.9 x 10(-4) s(-1). The appearance of two binding complexes through the d istribution of diffusion times may suggest that these are representatives o f two different forms or subtypes of EGF receptors. This study is of pharma ceutical significance as it provides evidence that fluorescence correlation spectroscopy can be used as a rapid technique for studying ligand-receptor interactions in cell cultures. This is a step forward toward large-scale s creening in cell cultures.