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First published online 17 October 2006
doi: 10.1242/jcs.03228

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TfR2 localizes in lipid raft domains and is released in exosomes to activate signal transduction along the MAPK pathway

¹ Department of Hematology, Oncology and Molecular Medicine,, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
² Department of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
³ Laboratory of Immunogenetics, Department of Genetics, Biology, and Biochemistry, University of Turin Medical School, Via Santena 19, 10125 Turin, Italy

View larger version (42K): [in a new window]   Fig. 1. Association of TfR2 with lipid rafts/caveolae by density gradient centrifugation. (A) K562 cav, K562 wt and HepG2 cells were lysed in Triton X-100 and subjected to sucrose gradient centrifugation. Aliquots of fractions collected from the top of the gradient were analyzed by western blotting with the antibodies against TfR2, CD81, TfR1 and caveolin-1. Equal protein amounts for each fraction were resolved by SDS-PAGE. (B) HepG2 cells were serum-starved overnight and subjected to sucrose gradient centrifugation (top panels). In a second set of experiments, serum-starved HepG2 cells were pre-treated with human holotransferrin (4 hours at 37°C), before sucrose gradient and western blot analysis.

View larger version (41K): [in a new window]   Fig. 2. Effect of Triton X-100 treatment and cholesterol depletion on TfR2 and TfR1 cell membrane expression. K562 cells were labeled with anti-TfR1, anti-TfR2 or anti-CD81 mAbs (left, middle and right panels, respectively) and the fluorescence was measured before (C) and after (TX-100) 5 minutes of detergent treatment. Depletion of plasma membrane cholesterol was achieved by incubation with methyl-ß-cyclodextrin (MBCD) before antibody labeling and TX-100 treatment.

View larger version (62K): [in a new window]   Fig. 3. Association between TfR2 and caveolin-1 in LDTI membranes as shown by immunofluorescence and immunoprecipitation experiments. (A) HepG2 cells were plated on coverslips and left to adhere overnight. Cells were stained with a mouse mAb directed against TfR2, followed by a Texas-Red-conjugated goat anti-mouse IgG. Cells were then fixed with paraformaldehyde (PFA, top panels) or with methanol/acetone (bottom panels), permeabilized and stained with a rabbit polyclonal anti-caveolin-1, followed by a FITC-conjugated anti-rabbit IgG. (B) HepG2 cells were stained with anti-TfR2 (red), then fixed (PFA) and labeled with a FITC-conjugated anti-CD81 mAb. (C) HepG2 cells were labeled with anti-TfR1 mAb, followed by a Texas-Red-conjugated goat anti-mouse IgG. Cells were then fixed and permeabilized (methanol/acetone, saponin) and stained with a rabbit polyclonal anti-caveolin-1, followed by a FITC-conjugated anti-rabbit IgG. (D) HepG2 cells were stained using the indicated primary mAbs, followed by a FITC-conjugated goat anti-mouse IgG. Where indicated, stained cells were exposed to Triton X-100 (TX-100) before analysis. Nuclei were counterstained using Syto59. (E) LDTI domains prepared from biotinylated HepG2 cells were immunoprecipitated with anti-TfR2 mAb. Top panel shows detection of immunoprecipitated (IP) and total (TOT) LDTI by anti-TfR2 or by streptavidin-horseradish peroxidase conjugated (strept). Bottom panel shows detection of IP and total LDTI by anti-caveolin 1 antibody. Only externally exposed biotinylated proteins were detected by streptavidin-HRP. As a control, an irrelevant antibody was used for immunoprecipitation (IgG). Positions of molecular size markers in kDa are indicated on the left. (F) Whole cell extracts prepared from biotinylated HepG2 cells were immunoprecipitated with a mouse mAb directed against TfR1. Total lysate (TOT) and immunoprecipitated fraction (IP) were detected by streptavidin-HRP-conjugated (strept) anti-TfR1 (top panel) and by anti-caveolin-1 (bottom panel). As a control, an irrelevant antibody was used for immunoprecipitation (IgG).

View larger version (27K): [in a new window]   Fig. 4. Surface and intracellular distribution of TfR1 and TfR2. (A) K562 cells were incubated at 4°C for 1 hour with either PBS (untreated cells, NT) or PBS containing trypsin (trypsinized cells, Tryps). For flow cytometry analysis, labeling with anti-TfR1 and anti-TfR2 was performed on intact cells and on fixed and permeabilized cells (fix-perm). (B-D) Aliquots of untreated (NT) and trypsinized (Tryps) K562 (B,D) or HepG2 (C) cells (4°C or 37°C) were lysed and total cellular lysates were resolved by SDS-PAGE and immunoblotted against TfR1, TfR2 and caveolin-1. Blots were stripped and reprobed for actin to ensure equal protein loading and transfer.

View larger version (55K): [in a new window]   Fig. 5. Subcellular fractionation of K562 cells. K562 cells were broken by nitrogen cavitation and the resulting lysate was clarified by centrifugation. The postnuclear supernatant was layered over the top of a sucrose density gradient and centrifuged (30,000 rpm, 2 hours). The four fractions and the pellet (fraction 5) were collected from the top of the tube and characterized by western blot using antibodies directed against TfR2, TfR1, Rab5, Rab11, Lamp-2 and Golgin-97.

View larger version (38K): [in a new window]   Fig. 6. Analysis of exosomes isolated from K562 cav cell culture media. (A) 20 µg cell debris obtained by centrifugation of culture medium from biotinylated-K562 cav cells was subjected to a 40%-5% continuous sucrose gradient in the absence of detergent, then analyzed by streptavidin and TfR2 immunoblotting. Positions of molecular size markers in kDa are indicated on the right. (B) 20 µg exosomes obtained as described in Materials and Methods were biotinylated then layered on a 30% to 10% continuous sucrose gradient and analyzed by streptavidin and TfR2 immunoblotting. In both cases equal volumes of fractions 1-11 were loaded. Sucrose densities were obtained for each fraction by refractometry. (C) Left panel, 3 µg of plasma membrane (PM) and 3 µg of LDTI prepared from biotinylated K562 cav cells or 1 µg of biotinylated exosomes (EXO) from K562 cav cells were analyzed by streptavidin immunoblotting. Note the differential pattern of biotinylated proteins displayed as exosomes, LDTI and PM. Right panel, equal protein amounts of plasma membrane (PM), LDTI and exosomes (EXO) prepared from K562 cav cells were analyzed by western blotting for CD45 and TfR1 expression.

View larger version (74K): [in a new window]   Fig. 7. Biochemical characterization of exosomes derived from K562 and HepG2 cells. Equal protein amounts of exosomes (EXO) and LDTI (left panel) or total cellular lysates (right panel) obtained from K562 wt, K562 cav and HepG2 cells were resolved by SDS-PAGE and immunoblotted with the use of antibodies for TfR2, CD81, TfR1, caveolin-1, the raft-marker Lyn, the exosomal-lysosomal marker Lamp-2, and actin to demonstrate equivalent protein loading and transfer.

View larger version (42K): [in a new window]   Fig. 8. Association between CD81 and TfR2 in LDTI membranes and exosomes. (A) 40 µg of exosomes (EXO) and 10 µg of LDTI purified from K562 cav cells were immunoprecipitated with an anti-CD81 mAb, resolved by SDS-PAGE under non-reducing conditions and immunoblotted against TfR2 and CD81. As a control of antibody specificity, non-immune serum (IgG) was used to immunoprecipitate the lysates. (B) Exosomes prepared from K562 cav cell culture medium were incubated with beads coated with anti-TfR2 mAb in the absence of detergents; the purified TfR2+ exosomes (IP with TfR2) were then lysed and immunoblotted against TfR2, TfR1 and CD81. (C) K562-derived exosomes were immunoprecipitated with anti-CD81 mAb (IP with CD81) and stained with mAbs for TfR2, TfR1 and CD81. One aliquot of total exosomal lysate (TOT) was loaded as a control.

View larger version (52K): [in a new window]   Fig. 9. Activation of ERK1/2 and p38 MAP kinases. (A) Top panel, K562 cells were serum-starved, treated with anti-TfR2 mAb G/14C2, with human transferrin (h-Tf) or with anti-TfR1 mAb for the indicated times and subjected to immunoblotting with anti-phospho-ERK1/2 Ab (pERK1/2). Middle panel: in control experiments, serum-starved K562 cells were exposed to anti-TfR1 Ab (5 minutes, 37°C), bovine serum albumin (BSA; 30 minutes, 37°C), bovine transferrin (b-Tf; 30 µM, 30 minutes, 37°C) and phorbol 12-myristate 13-acetate (PMA; 5 minutes, 37°C). Bottom panel, dose-response curve of ERK1/2 phosphorylation over a physiological and sub-physiological range of h-Tf and b-Tf concentrations. Blots were stripped and reprobed for ERK1/2 to ensure equivalent loading and transfer. (B) A TfR2-negative subclone of K562 cell line was serum-starved, treated with h-Tf (30 µM; 30 minutes, 37°C) and PMA (5 minutes, 37°C) and immunoblotted for TfR1, TfR2, pERK1/2 and actin. (C) Top panel, comparison of the level of ERK1/2 phosphorylation in K562 wt and caveolin-1 transfected K562 cells (K562 cav) exposed to anti-TfR2 (5 minutes, 37°C), h-Tf (30 µM; 30 minutes, 37°C) and PMA (5 minutes, 37°C). Bottom panel, serum-starved K562 cells were treated with anti-TfR2 or h-Tf and used for western analysis to assess the phosphorylation of p38 MAP kinase; blots were stripped and reprobed for tubulin to ensure equivalent loading and transfer. For each treatment, the level of ERK1/2 phosphorylation was quantified by densitometry with the Quantity One program (Bio-Rad) and reported in arbitrary units. Error bars show the range of values obtained in three independent experiments.

View larger version (39K): [in a new window]   Fig. 10. Effect of cholesterol depletion and repletion on TfR2-mediated ERK1/2 activation. Top panel, serum-starved K562 cells were incubated with or without 10 mM MBCD for 15 minutes at 37°C before treatment with anti-TfR2 mAb or PMA for 5 minutes at 37°C. One aliquot of MBCD-treated cells was incubated with 1 mM water soluble cholesterol. Cells were then collected and lysed. Equal aliquots of total cell lysates from untreated (NT), MBCD-treated (MBCD), and MBCD and cholesterol-treated (MBCD+cholest) cells were resolved by SDS-PAGE and immunoblotted against phospho-ERK1/2. Blots were stripped and reprobed with anti-tubulin Ab to ensure equal protein loading and transfer. Bottom panel, three experiments of this type were evaluated by densitometry to quantify the relative abundance of pERK1/2 induced in response to anti-TfR2 and PMA, normalized with respect to tubulin content and expressed as a percentage of the control (C). Error bars correspond to the range of values obtained in three independent experiments.

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