title: Role of Grb2 in EGF-stimulated EGFR internalization.
abstract: Grb2 is an adaptor molecule that couples membrane receptors such as the epidermal growth factor receptor (EGFR) to intracellular signaling pathways. To gain insight into the trafficking pathways followed by these molecules after activation by EGF, we visualized Grb2 and EGFR fused to GFP spectral variants in single live cells. In nonstimulated cells, Grb2-YFP was primarily localized diffusely in the cytoplasm, whereas EGFR-CFP was found on the plasma membrane and in endocytic structures localized in the perinuclear area. Within 1 minute of EGF stimulation, Grb2 redistributed to the plasma membrane where it bound EGFR-CFP in an SH2 dependent manner. The plasma membrane then began to dynamically ruffle, and Grb2-YFP and EGFR-CFP were found to internalize together in large macropinocytic structures. These structures were morphologically distinct from conventional, clathrin-derived endosomes and did not label with transferrin, AP-2 or clathrin heavy chain. Evidence that these structures did not require clathrin for internalization came from experiments showing that expression of the C-terminus of AP-180, which inhibited transferrin uptake, had no effect on EGF-induced internalization of EGFR. YFP-tagged Grb2 containing an inhibitory mutation in either N- or C-SH3 domain redistributed to the plasma membrane upon EGF stimulation, but the macropinocytic structures containing Grb2-YFP and EGFR-CFP did not translocate inward and appeared to remain tethered to the plasma membrane. This suggested that the Grb2 SH3 domain was responsible for coupling the membranes containing EGFR with downstream effectors involved in internalization of these membranes. Transferrin uptake was unaffected in the presence of all of the SH3 domain mutants, consistent with the EGF-stimulated EGFR internalization pathway being clathrin-independent. These results demonstrate a role for Grb2 in events associated with a macropinocytic internalization pathway for EGFR in activated cells.
authors.affiliation: Laboratory of Cellular and Molecular Biology, Division of Basic Science, National Cancer Institute, Bethesda, MD 20892-4255, USA.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Yamazaki
authors.forename: Kristien
authors.initials: K
authors.lastname: Zaal
authors.forename: Dale
authors.initials: D
authors.lastname: Hailey
authors.forename: John
authors.initials: J
authors.lastname: Presley
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lippincott-Schwartz
authors.forename: Lawrence E
authors.initials: LE
authors.lastname: Samelson
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002966:Clathrin / Q000378:metabolism; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D051380:GRB2 Adaptor Protein; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology*; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D033962:Adaptor Protein Complex 2; D048868:Adaptor Proteins, Signal Transducing; D002966:Clathrin; D051380:GRB2 Adaptor Protein; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D034281:Dynamins