Regulation of epidermal growth factor receptor signaling by endocytosis and intracellular trafficking

Ligand activation of the epidermal growth factor receptor (EGFR) leads to i ts rapid internalization and eventual delivery to lysosomes. This process i s thought to be a mechanism to attenuate signaling, but signals could poten tially be generated after endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and select ively isolate internalized EGFR and associated molecules with the use of re versibly biotinylated anti-EGFR antibodies. In addition, we developed antib odies specific to tyrosine-phosphorylated EGFR. With the use of a combinati on of fluorescence imaging and affinity precipitation approaches, we evalua ted the state of EGFR activation and substrate association during trafficki ng in epithelial cells. We found that after internalization, EGFR remained active in the early endosomes. However, receptors were inactivated before d egradation, apparently due to ligand removal from endosomes. Adapter molecu les, such as Shc, were associated with EGFR both at the cell surface and wi thin endosomes. Some molecules, such as Grb2, were primarily found associat ed with surface EGFR, whereas others, such as Eps8, were found only with in tracellular receptors. During the inactivation phase, c-Cbl became EGFR ass ociated, consistent with its postulated role in receptor attenuation. We co nclude that the association of the EGFR with different proteins is compartm ent specific. In addition, ligand loss is the proximal cause of EGFR inacti vation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.