Growth Factors Internalized EGF-EGFR Complex Continues to Signal

Epidermal growth factor (EGF) binding to the epidermal growth factor receptor (EGFR) induces receptor dimerization and tyrosine autophosphorylation, initiating a complex cascade of events leading to cell proliferation. In addition to activating various signaling pathways, EGF binding triggers endocytosis of the EGF-EGFR complex. Most internalized EGF-EGFR is degraded in lysosomes; some, however, may contribute to the cellular response. Wang et al. used selective activation of endosomal EGFR to address the possible role of endosomal EGF-EGFR in intracellular signaling. After inactivation of EGFR with the EGFR tyrosine kinase inhibitor AG-1478, BT20 or MDCK cells were treated with EGF, to stimulate endocytosis, and with monensin, to inhibit EGFR recycling to the plasma membrane. Immunoblot analysis of cells fractionated through a sucrose-density gradient, as well as immunofluorescent analysis of fixed cells, demonstrated internalization of nonphosphorylated EGFR together with labeled EGF. Removal of AG-1478 and monensin led to reactivation of endosomal EGFR (phosphorylation) without its reappearance at the plasma membrane. Similar results were obtained without monensin by washing cells after AG-1478/EGF treatment. Western analysis of immunoprecipitates indicated that activated endosomal EGFR associated with growth receptor-binding protein 2, src homologous and collagen-like protein, and phospholipase C-1. Activation of endosomal EGFR led to phosphorylation of Ras and extracellular signal-regulated kinase 1 and 2, as well as Akt. Activated endosomal EGFR suppressed apoptosis in response to serum withdrawal. This work indicates that endocytosed EGFR does participate in intracellular signaling, and describes a system applicable to analyses of signaling by other endocytosed receptor tyrosine kinases.

Y. Wang, S. Pennock, X. Chen, Z. Wang, Endosomal signaling of epidermal growth factor receptor stimulates signal transduction pathways leading to cell survival. Mol. Cell. Biol. 22, 7279-7290 (2002). [Abstract] [Full Text]