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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00793

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Tetraspanin CD82 regulates compartmentalisation and ligand-induced dimerization of EGFR

Cancer Research UK Institute for Cancer Studies, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

View larger version (54K): [in a new window]   Fig. 1. The role of CD82 in the dimerization of ErbB receptors. (A) Expression of ErbB receptors in HB2/ZEO and HB2/CD82 cells. Protein lysates (10 µg) were resolved in 8% SDS-PAGE, transferred to the nitrocellulose membrane and probed with the anti-EGFR (mAb-15), anti-ErbB2 (mAb-1) and anti-ErbB3 (C-17) Abs. (B) Cells were incubated with 125I-EGF (or 125I-TGF) at 4°C for 2 hours. The unbound ligand was removed and the cells were treated with 0.5 mM BS3 for 1 hour at 4°C. The complexes were purified as described in the Materials and Methods and resolved in 6% SDS-PAGE. The Abs used were: polyclonal 1005, anti-EGFR; mAb-11, anti-ErbB2; polyclonal C-17, anti-ErbB3. ErbB3 in the immunoprecipitates was probed with the mAb HER-3 Ab-6. (C) 0.5 µg of rs-CD82 was immunoprecipitated using a panel of anti-CD82 mAbs (lanes 2-5) or a negative control (187.1) mAb. 0.2 µg of purified rs-CD82 was used as a positive control (lane 1). The immunoprecipitates were resolved in 10% SDS-PAGE transferred to the membrane and probed with the anti-CD82 mAb (C33). In the additional experiments we found that denatured rs-CD82 is not recognised by any of the anti-CD82 mAbs (results are not shown). (D) The immunoprecipitation analysis was carried out as described in A except that cells were pre-incubated with the recombinant soluble (rs) proteins prior to (1 hour at 4°C) and during binding of 125IEGF. The experiments with HB2/ZEO and HB2/CD82 cells were done in parallel. Both gels were exposed for 14 days.

View larger version (57K): [in a new window]   Fig. 2. CD82 does not affect ligand-independent dimerization of EGFR and ErbB2. Cells were pre-treated with 0.5 mM BS3 for 1 hour at 4°C and then subjected to immunoprecipitation analysis as described in Materials and Methods. Immunoprecipitated proteins were resolved in 6% SDS-PAGE, transferred to the nitrocellulose membrane and probed with anti-EGFR (mAb-15) or anti-ErbB2 (mAb-1) Abs.

View larger version (26K): [in a new window]   Fig. 3. CD82 does not affect ligand-induced dimerization of ErbB3 with ErbB2. (A) Expression of ErbB receptors in MCF-7/ZEO and MCF-7/CD82 cells. Protein lysates (10 µg) were resolved in 8% SDS-PAGE, transferred to the nitrocellulose membrane and probed with the anti-ErbB2 (mAb-1) and anti-ErbB3 (C-17). (B) MCF-7/ZEO and MCF-7/CD82 cells were incubated with 125I-HRG at 4°C for 2 hours. Formation of ErbB2-ErbB3 dimers was analysed as described in Fig. 1B.

View larger version (35K): [in a new window]   Fig. 4. CD82 is associated with ErbB2 and ErbB3. (A) Expression of ErbB proteins in MCF-7/CD82, HB2/CD82 and T47D cells. Equal amounts of total protein lysates were resolved in 8% SDS-PAGE, transferred to the membrane and probed with antibodies to EGFR (mAb Ab-15), ErbB3 (C-17) or ErbB2 (Ab-1). (B) Cells were lysed in 1% Brij98 and the complexes were immunoprecipitated using specific antibodies: anti-ErbB3, mAb-4; anti-ErbB2, mAb-11; anti-CD82, C11; negative control, 187.1. The immunoprecipitated complexes were resolved in 8% SDS-PAGE, transferred to a nitrocellulose membrane and probed with Abs to ErbB3 (C-17) or ErbB2 (mAb-1) or CD82 (mAbs C33 and TS82b). (C) Stimulation with HRG does not affect the association of CD82 with ErbB2 and ErbB3. MCF-7/CD82 cells were stimulated with 50 ng/ml HRG for indicated times at 37°C. CD82-ErbB complexes were analysed as described above.

View larger version (64K): [in a new window]   Fig. 5. The role of CD82 in compartmentalisation of membrane proteins. Cells were lysed in ice-cold 1% Brij98/MES and the lysates were fractionated in 0.2-0.9 M gradient of sucrose as described in the Materials and Methods. Equal volumes of each fraction were resolved in 10% SDS-PAGE. Distribution of proteins in the gradient fractions was assessed by western blotting using specific antibodies: mAb-15 for EGFR, mAb-1 for ErbB2, mixture of mAbs C33 and TS82b for CD82; mAb 8G6 for EMMPRIN. Caveolin was detected using rabbit polyclonal antibody. The results of a representative (of three) experiment are shown.

View larger version (44K): [in a new window]   Fig. 6. CD82 causes redistribution of EGFR and GD1a. HB2/ZEO (A,C,E) and HB2/CD82 cells (B,D,F-J) were grown on glass coverslips for 48 hours. Cells were fixed with paraformaldehyde and indirect immunofluorescence staining was carried out using mAbs to EGFR (A,B), GD1a (C,D), FITC-conjugated cholera toxin (E,F). Double staining was carried out using a combination of anti-GD1a (GD1a-1) and anti-CD82 (C33) mAbs (G,H) or anti-GD1a (GD1a-1) and anti-EGFR (Ab-16) mAbs (I,J). Staining was visualised using FITC-conjugated goat anti-mouse IgG (A-D) or a combination of Texas Red-conjugated goat anti-IgG1 and Alexa Fluor 488-conjugated goat anti-IgG2a (G-J). H and J are digitally magnified highlighted areas of G and I, respectively (x6). Images were acquired using the Nikon Eclipse E600 microscope (Plan Apo 60xA/1.4 oil) and the Leica DC200 digital camera. The images were subsequently processed using the DC200 image processing programme.

View larger version (32K): [in a new window]   Fig. 7. CD82 causes redistribution of GD1a in MCF-7 cells. Cells were prepared for immunofluorescence staining as described in the legend to Fig. 6. (A) MCF-7/ZEO cells were stained with anti-GD1a mAb. Staining was visualised using FITC-conjugated goat anti-mouse IgG. (B,C) Double staining of MCF-7/CD82 cells was carried out using a combination of anti-GD1a (GD1a-1) and anti-CD82 (C12) mAbs (B and C, respectively). Staining was visualised using a combination of Texas Red-conjugated goat anti-IgG1 and Alexa Fluor 488-conjugated goat anti-IgG2a. (D) Digital superimposition of B and C.