Epidermal growth factor and membrane trafficking: EGF receptor activation of endocytosis requires Rab5a

Activated epidermal growth factor receptors recruit various intracellular p roteins leading to signal generation and endocytic trafficking. Although ac tivated receptors are rapidly internalized into the endocytic compartment a nd subsequently degraded in lysosomes, the linkage between signaling and en docytosis is not well understood. Here we show that EGF stimulation of NR6 cells induces a specific, rapid and transient activation of Rab5a. EGF also enhanced translocation of the Rab5 effector, early endosomal autoantigen 1 (EEA1), from cytosol to membrane. The activation of endocytosis, fluid pha se and receptor mediated, by EGF was enhanced by Rab5a expression, but not by Rab5b, Rab5c, or Rab5a truncated at the NH2 and/or COOH terminus. Domina nt negative Rab5a (Rab5:N34) blocked EGF-stimulated receptor-mediated and f luid-phase endocytosis. EGF activation of Rab5a function was dependent on t yrosine residues in the COOH-terminal domain of the EGF receptor (EGFR). Re moval of the entire COOH terminus by truncation (c'973 and c'991) abrogated ligand-induced Rab5a activation of endocytosis. A "kinase-dead" EGFR faile d to stimulate Rab5a function. However, another EGF receptor mutant (c'1000 ), with the kinase domain intact and a single autophosphorylation site effe ctively signaled Rab5 activation. These results indicate that EGFR and Rab5 a are linked via a cascade that results in the activation of Rab5a and that appears essential for internalization. The results point to an interdepend ent relationship between receptor activation, signal generation and endocyt osis.