PMC

CD82 links HSPG-activated PKC, ligand-bound EGFR and c-Cbl. The model depicts the role of CD82 in early events of EGFR activation by heparin-binding domain containing ligand (e.g., HB-EGF). A, in absence of CD82 EGFR is not recruited to the tetraspanin-enriched microdomains. HB-EGF binding leads to robust phosphorylation of the receptor, recruitment of c-Cbl, and further ubiquitylation. B, when CD82 is expressed, a subset of EGFR associated with the tetraspanin is recruited to TERM where it is placed in close proximity to syndecans. Stimulation with HB-EGF leads to activation of PKC recruited to TERM by syndecans and CD82. PKC attenuates EGFR signaling by affecting c-Cbl recruitment to this subset of receptors. Additionally, active PKC negatively regulates activity of E3 ligase by serine/threonine phosphorylation.

CD82 modulates PKC-dependent phosphorylation of EGFR and c-Cbl in HB-EGF-stimulated cells. A, phosphorylation of c-Cbl on Tyr⁷⁷⁴ was studied by Western blotting (WB) with phosphospecific anti-Cbl antibody in cells (HB2 and HB2/CD82) stimulated with HB-EGF. Total c-Cbl and actin were used as control for loading. B, serine phosphorylation of c-Cbl in HB-EGF-stimulated cells (HB2 and HB2/CD82) was studied by immunoprecipitation (IP) with anti-c-Cbl mAb followed by Western blotting with anti-phosphoserine polyclonal antibody. The same membrane was redeveloped with anti-c-Cbl polyclonal antibody to confirm that equal amount of protein was immunoprecipitated in each sample. In parallel experiments, cells were pretreated for 1.5 h with 5 μm (final concentration) of anti-PKC inhibitor Calphostin C (CalC) before stimulation with HB-EGF. The data presented are from one of three independent experiments. C, phosphorylation of EGFR on Thr⁶⁵⁴ was studied by Western blotting with phosphospecific anti-EGFR^Thr654 antibody (Millipore). The blot is a representative of three independent experiments. Quantification of three experiments is presented on the graph. The p value was determined in paired two-tailed t test.

Co-localization of internalized CD82 and EGFR after HB-EGF stimulation. HB2/CD82 and HB2/CD82ΔC cells were incubated with HB-EGF (25 ηg/ml) and anti-CD82 mAb (TS82b) at 4 °C for 1 h. After three washes, the cells were chased at 37 °C for the indicated time intervals. The cells were fixed, permeabilized, and co-stained with anti-EGFR mAb. Co-localization of CD82 and EGFR was assessed using isotype specific Alexa Fluor-conjugated goat anti-mouse antibodies. A, representative confocal images of cells following 60 min of chase are presented. Scale bar, 10 μm. B, quantification of co-localized EGFR and CD82 after internalization was calculated in an average 40–50 cells at each time point in each cell line after collection of z-sections. The data are presented as Pearson's coefficient. The values are means from three independent experiments. The p values were determined by paired two-tailed t test.

Ubiquitylation of EGFR is impaired in CD82-overexpressing cells. A, expression levels of CD82 and EGFR in HB2, HB2/CD82, and HB2/CD82ΔC cells were determined by Western blotting (WB) with anti-CD82 mAb (TS82b) or with anti-EGFR (Ab-15) mAb, respectively. WB with anti-actin mAb is a control for equal loading. B–D, HB2 and HB2/CD82 cells were serum-starved overnight and incubated with 15 ηg/ml EGF (B) or 20 ηg/ml HB-EGF (C) or 15 ηg/ml AR (D) for the indicated time intervals. After incubation, the cells were lysed in Triton X-100 (1%), EGFR was immunoprecipitated (IP) with anti-EGFR mAb (Ab-12; Neomarkers), and ubiquitylation (Ubiq) of the receptor was determined by Western blotting with anti-ubiquitin mAb (FK2; Enzo). The membranes were reblotted with anti-EGFR pAb (Cell Signaling). The results of one of three independent experiments are shown. Densitometric analysis was carried out on the films of equal exposure time using the ImageJ program.

Impairment of ubiquitylation of HB-EGF-stimulated EGFR is dependent on CD82 expression level and heparin-binding domain of the ligand. A, 2.5.2A/wt and 2.5.2A/shCD82 cells were serum-starved overnight and incubated with 25 ηg/ml HB-EGF for indicated time intervals. After incubation, the cells were lysed in Triton X-100 (1%), EGFR was immunoprecipitated (IP) with anti-EGFR mAb (Ab-12; Neomarkers), and ubiquitylation (ubiq) of the receptor was determined by Western blotting (WB) with anti-ubiquitin mAb (FK2; Enzo). The membranes were reblotted with anti-EGFR pAb (Cell Signaling). The results of one of three independent experiments are shown. B, expression levels of CD82 and EGFR in 2.5.2A wild type and CD82 knockdown cells were determined by Western blotting with the appropriate antibodies. Equal loading was controlled by WB with anti-actin mAb. C, HB2, HB2/CD82 cells were serum-starved overnight, incubated with 10 ηg/ml sΔΗΒ-EGF for indicated time intervals. Ubiquitylation of the receptor was determined by Western blotting with anti-ubiquitin mAb (FK2; Enzo) after immunoprecipitation as described above. The results of one of two independent experiments are shown. D, schematic structures of wild type and mutant forms of HB-EGF (adapted from Ref. ).

CD82 and recruitment of c-Cbl to HB-EGF-stimulated EGFR. A, serum-starved HB2 and HB2/CD82 cells were incubated with HB-EGF (15 ηg/ml) at 37 °C for indicated time intervals. Equal amounts of protein (∼10 μg) were loaded onto polyacrylamide gels, and kinetics of phosphorylation was determined by Western blotting (WB) with the appropriate antibodies. Blots were developed using HRPO-conjugated secondary antibody from Dako. The levels of total EGFR were also tested. The data presented are from one of three independent experiments. Quantification of this experiment is shown in a graph. Densitometry was carried out using ImageJ. B, interaction of c-Cbl with EGFR in HB-EGF-stimulated cells was studied by immunoprecipitation (IP) with anti-c-Cbl antibody. EGFR was detected by Western blotting with anti-EGFR polyclonal antibody as described under “Experimental Procedures.” The same membrane was redeveloped with anti-c-Cbl goat antibody. The results of a representative experiment are shown (three in total).

Distribution of CD82 after 5 and 60 min of chase with anti-CD82 mAb analyzed by electron microscopy. HB2/CD82wt and HB2/CD82ΔC were incubated with anti-CD82 mAb (TS82b) for 1 h and then with protein A gold 10 nm for 40 min at 4 °C and subsequently chased for 5 or 60 min at 37 °C. Then the cells were fixed and processed according to the protocol described under “Experimental Procedures.” A, B, and G, after 5 min of chase, CD82wt and CD82ΔC were distributed to the plasma membrane and in the uncoated invaginations of PM. C (main panel and inset), after 5 min of chase, internalized CD82wt was found in the uncoated structures (arrow) but not coated vesicles (arrowheads). D, after 5 min of chase, CD82wt was observed in rosette-like endosomal structures. E, after 60 min of chase, internalized CD82wt was mainly distributed to the intracellular multivesicular compartments (arrows) and found occasionally at the PM. F, after 60 min of chase, gold-labeled CD82wt was also found on extracellular vesicles (arrows). H–J, CD82ΔC after 60 min chase was found on internal (H and J) and on extracellular (I) vesicles. K, quantification of the distribution of gold particles (average 1500) between various membranous compartments after 5 and 60 min of chase with anti-CD82 mAb in HB2/CD82wt and HB2/CD82ΔC cells. Scale bar, 200 nm.

Mutation in C-terminal of CD82 changes intracellular trafficking of the tetraspanin. A, expression levels of CD82wt and CD82ΔC determined by Western blotting (WB). B, internalization rate of CD82 in HB2/CD82 and HB2/CD82ΔC cells was studied using microplate internalization assay as described under “Experimental Procedures.” Internalization of anti-CD82 mAb (IA4 or TS82b) was monitored (after 1 and 2 h of chase at 37 °C) with IRDye 800CW and Odyssey Infrared Imaging System (LI-COR Biosciences). The graph presents average of mean fluorescence values from three experiments (± S.D.) for each cell line at indicated time point. The values presented in the table are ratios at particular time points relative to values at zero time point. The p value for each time point was determined in paired two-tailed t test. C, representative confocal images following uptake of anti-CD82 mAb (IA4) after 1 h of chase in cells expressing CD82wt or CD82ΔC are shown. The cells were acid-washed, fixed, permeabilized, and co-stained with anti-CD63 (1B5) mAb (as described under “Experimental Procedures”). Co-localization of CD82 and CD63 was assessed using isotype specific Alexa Fluor-conjugated goat anti-mouse antibodies. Z-sections were taken at the interval of 0.30–0.35 μm. Scale bar, 10 μm.

Postendocytic trafficking of EGFR following HB-EGF stimulation is altered in the presence of CD82. A and B, HB2, HB2/CD82wt, and HB2/CD82ΔC cells plated on coverslips were serum-starved overnight and incubated with HB-EGF (25 ηg/ml) at 37 °C for indicated time intervals. The cells were fixed and labeled for EGFR (anti-EGFR mAb; Ab-15) and the early endosomal marker EEA1 (anti-EEA1 mAb; Transduction Labs) (as described under “Experimental Procedures”). A, Representative confocal images of the cells following 15 min of EGFR internalization. Bars, 10 μm. B, quantification of the amount of EGFR co-localizing with EEA1 in an average of 40–50 cells for each time point is presented as Pearson's coefficient (average of three experiments). The p values were determined by paired two-tailed t test. C, ubiquitylation (ubiq or Ubi) of EGFR in HB2 and HB2/CD82ΔC cells was determined by immunoprecipitation (IP) as described under “Experimental Procedures.” The results of one of three independent experiments are shown. D, time course of EGFR recycling in HB2 and HB2/CD82 cells following stimulation with HB-EGF. The cells were incubated on ice with the ligand, washed, and incubated at 37 °C for indicated time intervals. The amount of EGFR at the cell surface was determined by flow cytometry. The values presented are means (percentages) ± S.D. from four experiments. WB, Western blotting.