Supplementary results and discussion

Role of receptor internalization. Comparing the kinetics of activation of the different signaling molecules, it can be seen that the majority of the signal results from the receptors at the plasma membrane at the EGF concentration of 50 ng/ml. The phosphorylation and association of phosphorylated Shc to receptors, which are still at the surface, is so fast that Ras-GTP activation has already peaked and then decayed to <50% before phosphorylated endocytosed receptors accumulate in the endosomes to a significant extent. The internalized receptors could also contribute to Ras activation, in the range of <10% of the total signal. This implies that all the subsequent steps of the MAP kinase cascade exhibit kinetics very similar to the activation of plasma membrane receptors. Thus, the high initial velocity of (EGFEGFR)2* phosphorylation seems to be sufficient to dominate ERK activation (Fig.4 in the main text).