The canonical description of transmembrane receptor function is initial binding of ligand, followed by initiation of intracellular signaling and then internalization en route to degradation or recycling to the cell surface. It is known that low concentrations of extracellular ligand leads to a higher proportion of receptor that is recycled, and that non-canonical mechanisms of receptor activation, including phosphorylation by the kinase p38, can induce internalization and recycling. However, no

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... connections have been made between these pathways; i.e., it has yet to be established what happens to unbound receptors following stimulation with ligand. We herein demonstrate that a minimal level of activation of EGFR tyrosine kinase by low levels of ligand is sufficient to fully activate downstream MAPK pathways, with most of the remaining unbound EGFR molecules being efficiently phosphorylated at intracellular serine/threonine residues by activated MAPKs. This non-canonical, p38-mediated phosphorylation of the C-tail of EGFR, near Ser1015, induces the clathrin-mediated endocytosis of the unliganded EGFR monomers, which occurs slightly later than the canonical endocytosis of ligand-bound EGFR dimers via tyrosine autophosphorylation. EGFR endocytosed via the non-canonical pathway is largely recycled back to the plasma membrane as functional receptors, whereas p38-independent populations are mainly sorted for lysosomal degradation. Moreover, ligand concentrations balance these endocytic trafficking pathways. These results demonstrate that ligand-activated EGFR signaling controls unliganded receptors through feedback phosphorylation, identifying a dual-mode regulation of the endocytic trafficking dynamics of EGFR. Epidermal growth factor receptor (EGFR), one of the most characterized receptor tyrosine kinases (RTKs), regulates many cellular functions, including survival, proliferation, and differentiation. The aberrant activation of EGFR by overexpression or activating mutations is a major mechanism underlying the pathogenesis of human cancers, including colorectal and lung cancers, and participates in acquired resistance to anti-cancer agents (1-4). Ligand-bound EGFR proteins form an asymmetric homodimer on the plasma membrane, which is followed by the activation of its tyrosine kinase. Activated EGFR is then rapidly internalized via clathrin-mediated endocytosis and clathrin-independent endocytosis. Sequential sorting to several vesicular transport systems, including early endosomes, late endosomes, multivesicular bodies (MVBs), and recycling endosomes, http://www.jbc.org/cgi/