|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
Research Article |
1 Laboratory of Cellular and Molecular Biology, Division of Basic Science,
National Cancer Institute, Bethesda, MD 20892-4255, USA
2 Cell Biology and Metabolism Branch, National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda, MD 20892,
USA
* Author for correspondence (e-mail: samelson{at}helix.nih.gov )
Accepted 28 January 2002
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.
Key words: Grb2, EGFR, SH3 domain, Endocytosis, GFP
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Tong, P. Taylor, S. M. Peterman, A. Prakash, and M. F. Moran Epidermal Growth Factor Receptor Phosphorylation Sites Ser991 and Tyr998 Are Implicated in the Regulation of Receptor Endocytosis and Phosphorylations at Ser1039 and Thr1041 Mol. Cell. Proteomics, September 1, 2009; 8(9): 2131 - 2144. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kawada, G. Upadhyay, S. Ferandon, S. Janarthanan, M. Hall, J.-P. Vilardaga, and V. Yajnik Cell Migration Is Regulated by Platelet-Derived Growth Factor Receptor Endocytosis Mol. Cell. Biol., August 15, 2009; 29(16): 4508 - 4518. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Leonard, A. Hayakawa, D. Lawe, D. Lambright, K. D. Bellve, C. Standley, L. M. Lifshitz, K. E. Fogarty, and S. Corvera Sorting of EGF and transferrin at the plasma membrane and by cargo-specific signaling to EEA1-enriched endosomes J. Cell Sci., October 15, 2008; 121(20): 3445 - 3458. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Chen and X. Zhuang Epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus PNAS, August 19, 2008; 105(33): 11790 - 11795. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Li, M. Lorinczi, K. Ireton, and L. A. Elferink Specific Grb2-mediated Interactions Regulate Clathrin-dependent Endocytosis of the cMet-tyrosine Kinase J. Biol. Chem., June 8, 2007; 282(23): 16764 - 16775. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Traub and G. L. Lukacs Decoding ubiquitin sorting signals for clathrin-dependent endocytosis by CLASPs J. Cell Sci., February 15, 2007; 120(4): 543 - 553. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Bonaccorsi, D. Nosi, M. Muratori, L. Formigli, G. Forti, and E. Baldi Altered endocytosis of epidermal growth factor receptor in androgen receptor positive prostate cancer cell lines J. Mol. Endocrinol., January 1, 2007; 38(1): 51 - 66. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Orth, E. W. Krueger, S. G. Weller, and M. A. McNiven A novel endocytic mechanism of epidermal growth factor receptor sequestration and internalization. Cancer Res., April 1, 2006; 66(7): 3603 - 3610. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Martin, J. M. Del Valle, I. Saborit, and P. Engel Identification of Grb2 As a Novel Binding Partner of the Signaling Lymphocytic Activation Molecule-Associated Protein Binding Receptor CD229 J. Immunol., May 15, 2005; 174(10): 5977 - 5986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhan, M. R. Tremblay, N. Melian, and S. Carbonetto Evidence That Dystroglycan Is Associated with Dynamin and Regulates Endocytosis J. Biol. Chem., May 6, 2005; 280(18): 18015 - 18024. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Huang and A. Sorkin Growth Factor Receptor Binding Protein 2-mediated Recruitment of the RING Domain of Cbl to the Epidermal Growth Factor Receptor Is Essential and Sufficient to Support Receptor Endocytosis Mol. Biol. Cell, March 1, 2005; 16(3): 1268 - 1281. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Du and S. R. Post Macrophage colony-stimulating factor differentially regulates low density lipoprotein and transferrin receptors J. Lipid Res., September 1, 2004; 45(9): 1733 - 1740. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Stang, F. D. Blystad, M. Kazazic, V. Bertelsen, T. Brodahl, C. Raiborg, H. Stenmark, and I. H. Madshus Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits Mol. Biol. Cell, August 1, 2004; 15(8): 3591 - 3604. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Crimeen-Irwin, S. Ellis, D. Christiansen, M. J. Ludford-Menting, J. Milland, M. Lanteri, B. E. Loveland, D. Gerlier, and S. M. Russell Ligand Binding Determines Whether CD46 Is Internalized by Clathrin-coated Pits or Macropinocytosis J. Biol. Chem., November 21, 2003; 278(47): 46927 - 46937. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Hinrichsen, J. Harborth, L. Andrees, K. Weber, and E. J. Ungewickell Effect of Clathrin Heavy Chain- and {alpha}-Adaptin-specific Small Inhibitory RNAs on Endocytic Accessory Proteins and Receptor Trafficking in HeLa Cells J. Biol. Chem., November 14, 2003; 278(46): 45160 - 45170. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Traub Sorting it out: AP-2 and alternate clathrin adaptors in endocytic cargo selection J. Cell Biol., October 27, 2003; 163(2): 203 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Huang, S.-O. Kim, J. Jiang, and S. J. Frank Growth Hormone-induced Phosphorylation of Epidermal Growth Factor (EGF) Receptor in 3T3-F442A Cells: MODULATION OF EGF-INDUCED TRAFFICKING AND SIGNALING J. Biol. Chem., May 23, 2003; 278(21): 18902 - 18913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Jiang, F. Huang, A. Marusyk, and A. Sorkin Grb2 Regulates Internalization of EGF Receptors through Clathrin-coated Pits Mol. Biol. Cell, March 1, 2003; 14(3): 858 - 870. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Baldassarre, A. Pompeo, G. Beznoussenko, C. Castaldi, S. Cortellino, M. A. McNiven, A. Luini, and R. Buccione Dynamin Participates in Focal Extracellular Matrix Degradation by Invasive Cells Mol. Biol. Cell, March 1, 2003; 14(3): 1074 - 1084. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Bunnell, D. I. Hong, J. R. Kardon, T. Yamazaki, C. J. McGlade, V. A. Barr, and L. E. Samelson T cell receptor ligation induces the formation of dynamically regulated signaling assemblies J. Cell Biol., September 29, 2002; 158(7): 1263 - 1275. [Abstract] [Full Text] [PDF]
|
|
