Real-time studies of the interactions between epidermal growth factor and its receptor during endocytic trafficking

The interactions of growth factors with cell, surface receptors regulate fu ndamental cell processes, such as growth, differentiation and transformatio n. Understanding the nature of these interactions at the molecular level is of fundamental importance in cell biology. This Is not only from the point of view of basic science, but also because of the repercussions such knowl edge might have in understanding the mode of action of drugs in cells. Rece ptor mediated endocytosis has been implicated in the downregulation of the mitogenic signal. However, no data are thus far available on how growth fac tor/receptor interactions might control endocytic trafficking. Here we show that information on modes of binding and receptor conformational changes c an be obtained using time-resolved fluorescence methods. We have found that fluorescent probes bound to epidermal growth factor (EGF) show dynamic flu orescence quenching when EGF is bound to internalising EGF receptors (EGFR) . We propose that this dynamic quenching takes place because EGF-bound prob es interact with tryptophan residues in the extracellular domain of the EGF -EGFR complex. Real-time accumulation of fluorescent decays has also allowe d us to follow the time course of a conformational change in EGFR occurring during endocytosis, and correlate this information with endosomal traffick ing and EGFR recycling.