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Role of Grb2 in EGF-stimulated EGFR internalization

Tetsuo Yamazaki1, Kristien Zaal2, Dale Hailey2, John Presley2, Jennifer Lippincott-Schwartz2 and Lawrence E. Samelson*,1

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



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  		Fig. 1. Chimeric molecules containing Grb2 or EGFR. (A) YFP-tagged Grb2. Mutations in each chimeric molecule are indicated. GGGG; four-glycine linker. (B) C-terminally tagged EGFR. (C) Immunoblot of Grb2-YFP. Lysates of COS-7 cells transfected with either wild-type Grb2 or mutant Grb2 fused to YFP were analyzed by western blotting (lane 1, YFP alone; lane 2, wild-type Grb2-YFP; lane 3, SH2m-YFP; lane 4, NSH3m-YFP; lane 5, CSH3m-YFP; lane 6, NCSH3m-YFP). The blot was probed with anti-GFP antibody and reprobed with anti-Grb2 antibody. (D) Western blot of EGFR-CFP. Lysates of COS-7 cells expressing either CFP alone or EGFR-CFP were analyzed by western blotting. The blot was probed with anti-GFP antibody (lanes 1,2) and reprobed with anti-EGFR antibody (lanes 3,4). Arrows point to EGFR-CFP (upper band) and endogenous EGFR (lower band). Molecular weight marker proteins (kDa) are indicated at the left. (e) Grb2-YFP associates with EGFR in an EGF-dependent manner. Wild-type Grb2-YFP was immunoprecipitated by anti-GFP antibody from the lysate of A431 cells expressing wild-type Grb2-YFP either before or after EGF stimulation. The blot was probed with anti-GFP antibody (bottom) or anti-EGFR antibody (top). (F) Grb2-YFP constitutively associates with SOS, Cbl and dynamin II. GFP immunoprecipitates from lysates of A431 cells transfected with either YFP alone (lane 1) or wild-type Grb2-YFP (lane 2) were immunoblotted with either anti-SOS, anti-Cbl, anti-dynamin II or anti-GFP antibody.

 


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  		Fig. 2. EGF-induced redistribution of Grb2-YFP to the cell periphery in A431 cells. A431 cells were transfected with YFP fused to either wild-type Grb2, the SH2 domain mutant or the SH3 domain mutants of Grb2, as follows. (A,B) Wild-type Grb2-YFP, (C,D) SH2m-YFP, (E,F) NSH3m-YFP, (G,H) CSH3m-YFP and (I,J) NCSH3m-YFP. Images of live A431 cells in each column were taken prior to (A,C,E,G,I) and 1 minute after EGF stimulation (B,D,F,H,J) using confocal microscopy. Bars, 5 µm.

 


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  		Fig. 3. Internalization of EGFR-CFP with Grb2-YFP induced by EGF. Individual live COS-7 cells co-expressing wild-type Grb2-YFP and EGFR-CFP were monitored over time by confocal time-lapse imaging. Protein movement of Grb2-YFP (middle) and EGFR-CFP (left) upon EGF stimulation is shown. Arrows point to the characteristic internalized structures (see text).

 


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  		Fig. 4. Colocalization of Grb2-YFP with clathrin heavy chain, AP-2 and dynamin. COS-7 cells co-expressing EGFR-CFP with wild-type Grb2-YFP were stimulated. Cells were then immunostained with antibodies directed at clathrin heavy chain (CHC), AP-2 or dynamin, followed by a TRITC-conjugated secondary antibody (middle). Arrows point to co-localized structures.

 


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  		Fig. 5. Transferrin localization and dynamics in the presence of Grb2-YFP or NSH3m-YFP. (A) Transferrin uptake by COS-7 cells co-expressing EGFR-CFP with either wild-type Grb2-YFP (top) or NSH3m-YFP (bottom) was monitored by confocal time-lapse imaging. Images were collected 20 min after EGF stimulation following 40-min incubation with transferrin-TRITC. (B) Uptake and sequestration of biotinylated Tfn (B-Tfn) in COS-7 cells was measured by avidin inaccessibility (See Materials and Methods). Assays were performed in the absence (open symbols) or presence of EGF (closed symbols). Data shown are representative of three independent experiments.

 


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  		Fig. 6. Internalization of EGFR-CFP is not blocked by the inhibition of clathrin-mediated uptake. COS-7 cells were co-transfected with EGFR-CFP and the C-terminal domain of AP180 and serum starved. In nontransfected cells, transferrin localizes to the perinuclear region. In co-transfected cells, the C-terminal domain of AP180 inhibits clathrin-mediated uptake and transferrin fails to localize (left). When stimulated with EGF, co-transfected cells still take up EGFR-CFP in large structures (right). Images from a time-lapse series are presented. At time 0, EGF was added to a final concentration of 100 ng/ml. Confocal images were captured every 30 seconds. Arrows indicated a region of interest initially on the plasma membrane. The region is internalized and moves to the perinuclear region where it deforms the nuclear envelope.

 


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  		Fig. 7. Requirement of the intact N-terminal SH3 domain of Grb2 for EGFR internalization. Images of live COS-7 cells co-expressing NSH3m-YFP and EGFR-CFP were collected using confocal microscopy; NSH3m-YFP (left) and EGFR-CFP (middle). Shown at the bottom is a higher magnification view of the boxed region. Arrows point to large spherical structures associated with the plasma membrane (see text). Bar, 5 µm.

 


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  		Fig. 8. Colocalization of NSH3m-YFP with clathrin heavy chain, AP-2 and dynamin. COS-7 cells co-expressing EGFR-CFP with NSH3m-YFP were stimulated. Cells were then immunostained with antibodies directed at clathrin heavy chain (CHC), AP-2 or dynamin, followed by a TRITC-conjugated secondary antibody. Arrows point to co-localized structures.

 


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  		Fig. 9. EGFR internalization is blocked by NSH3m-YFP at the step of membrane fission. EGF-stimulated COS-7 cells co-expressing NSH3m-YFP and EGFR-CFP were fixed then either permeabilized (A,B) or left unpermeabilized (C,D). Cells were immunostained with an antibody against the extracellular domain of EGFR, followed by a TRITC-conjugated secondary antibody (B,D). Bar, 5 µm. (E) EGFR internalization in COS-7 cells overexpressing the indicated YFP chimeric molecules was measured by determining avidin inaccessibility. Assays were performed as described in Materials and Methods. Data shown are representative of three independent experiments.

 

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