title: Re-localization of activated EGF receptor and its signal transducers to multivesicular compartments downstream of early endosomes in response to EGF.
abstract: The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction. However, accumulating data indicate that signal transduction from internalized cell surface receptors also occurs from endosomes. We show that a substantial fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGFR) and Shc, Grb2 and Cbl after internalization relocates from early endosomes to compartments which are negative for the early endosomes, recycling vesicle markers EEA1 and transferrin in EGF-stimulated cells. These compartments contained the multivesicular body and late endosome marker CD63, and the late endosome and lysosome marker LAMP-1, and showed a multivesicular morphology. Subcellular fractionation revealed that activated EGFR, adaptor proteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-positive fractions. Co-immunoprecipitations showed EGFR in complex with both Shc, Grb2 and Cbl. Treatment with the weak base chloroquine or inhibitors of lysosomal enzymes after EGF stimulation induced an accumulation of tyrosine-phosphorylated EGFR and Shc in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activation of ERK 1 and 2. These results suggest that EGFR signaling is not spatially restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.
authors.affiliation: Institute of Pathology, The National Hospital, University of Oslo, Norway. m.p.oksvold@labmed.uio.no
authors.forename: M P
authors.initials: MP
authors.lastname: Oksvold
authors.forename: E
authors.initials: E
authors.lastname: Skarpen
authors.forename: L
authors.initials: L
authors.lastname: Wierød
authors.forename: R E
authors.initials: RE
authors.lastname: Paulsen
authors.forename: H S
authors.initials: HS
authors.lastname: Huitfeldt
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD / Q000032:analysis / Q000378:metabolism; D000962:Antimalarials / Q000494:pharmacology; D002451:Cell Compartmentation / Q000502:physiology; D002738:Chloroquine / Q000494:pharmacology; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000032:analysis / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D006367:HeLa Cells; D006801:Humans; D019751:Intramolecular Transferases / Q000378:metabolism; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D008854:Microscopy, Electron; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D060149:Tetraspanin 30; D014443:Tyrosine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D033921:Vesicular Transport Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D000962:Antimalarials; C554586:CD63 protein, human; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D060149:Tetraspanin 30; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D014443:Tyrosine; D004815:Epidermal Growth Factor; D002738:Chloroquine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase; C067419:CBL protein, human