Epidermal growth factor and membrane trafficking. EGF receptor activation of endocytosis requires Rab5a.
Activated epidermal growth factor receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between signaling and endocytosis 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 phase and receptor mediated, by EGF was enhanced by Rab5a expression, but not by Rab5b, Rab5c, or Rab5a truncated at the NH(2) and/or COOH terminus. Dominant negative Rab5a (Rab5:N34) blocked EGF-stimulated receptor-mediated and fluid-phase endocytosis. EGF activation of Rab5a function was dependent on tyrosine residues in the COOH-terminal domain of the EGF receptor (EGFR). Removal of the entire COOH terminus by truncation (c'973 and c'991) abrogated ligand-induced Rab5a activation of endocytosis. A "kinase-dead" EGFR failed to stimulate Rab5a function. However, another EGF receptor mutant (c'1000), with the kinase domain intact and a single autophosphorylation site effectively signaled Rab5 activation. These results indicate that EGFR and Rab5a are linked via a cascade that results in the activation of Rab5a and that appears essential for internalization. The results point to an interdependent relationship between receptor activation, signal generation and endocytosis.
Barbieri
M A
MA
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Roberts
R L
RL
Gumusboga
A
A
Highfield
H
H
Alvarez-Dominguez
C
C
Wells
A
A
Stahl
P D
PD
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects*; D011992:Endosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts; D005799:Genes, Dominant / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D014162:Transfection; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D020033:Protein Isoforms; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D019000:Phosphotyrosine; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D066246:ErbB Receptors; D020696:rab5 GTP-Binding Proteins