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Association of the tetraspanin CD151 with the laminin-binding integrins 3ß1, 6ß1, 6ß4 and 7ß1 in cells in culture and in vivo

¹ Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
² Division of Pathology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
³ Division of Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

View larger version (59K): [in a new window]   Fig. 1. Immunohistochemical distribution of CD151 in kidney, skin and heart. Cryostat sections of human kidney (A), skin (B) and heart tissue (C) were stained by immunoperoxidase reaction with anti-CD151 (P48). Intense staining is seen in the glomeruli and the epithelia of kidney tubules (A), keratinocytes of the skin (B) and in vascular smooth (A,B) and cardiac muscle cells (C). Note that CD151 is localized on the basolateral surface of epithelia and at the sarcolemma of cardiac myocytes. It is also detected at costameres and intercalated discs (ID). v, vessel. Bars, 50 (A), 100 (B) and 10 µm (C).

View larger version (128K): [in a new window]   Fig. 2. Colocalization of CD151 with laminin receptors 3ß1 and dystroglycan in the glomerulus and with 6ß1, dystroglycan and the Lutheran molecule in tubular epithelial cells. Cryostat sections of human kidney were processed for double immunofluorescence confocal microscopy using P48 against CD151 (A,B), polyclonal antibodies against 3 (A,C,E,F), polyclonal (AP83) and monoclonal antibodies against dystroglycan (DG) (B,C, respectively), LU4F2 against the Lutheran blood group molecule (LU) (D), GoH3 against 6 (D), 6H1 against CD63 (E) and M38 against CD81 (F). Composite images were generated by superimposition of the green and red signals, with areas of overlap appearing as yellow. In the glomerulus, CD151 is co-distributed with laminin-receptors 3ß1 and DG but not with LU. The distribution pattern of 3ß1 is mainly restricted to the GBM side, where it completely overlaps with that of CD151 and partly with that of DG. CD151 is also present in the cytoplasm of cells and at cell surfaces other than the GBM side. Although prominently present in the cytoplasm of glomerular cells, CD63 and CD81 show hardly any overlap with 3ß1 at the GBM side. In tubular epithelial cells, CD151 is colocalized with 6ß1, DG (arrow) and LU. CD81 is only present at a low level, and CD63 is mainly present throughout the cytoplasm. Note the prominent staining with anti-LU in the subendothelial matrix and with 6ß1 present on the endothelial lining. Bar, 10 µm.

View larger version (136K): [in a new window]   Fig. 9. Immunohistochemical staining of smooth and striated muscle tissue with P48 and TS151R against CD151. Cryostat sections of human smooth (A,C) and striated muscle (B,D) were stained by immunoperoxidase reaction with P48 (A,C) or TS151R (B,D). Both types of muscle cells were strongly stained by P48. Intense staining of CD151 by TS151R was only seen in smooth muscle cells, no staining is found in skeletal muscle. n, nerve; v, vessel. Bar, 10 µm.

View larger version (59K): [in a new window]   Fig. 3. Colocalization of CD151 with the laminin receptor 7ß1 in smooth and skeletal muscle. Cryostat sections of human smooth and striated muscle were processed for double immunofluorescence confocal microscopy using P48 against CD151 (A) and polyclonal antibodies against 7B (B). The composite image (C) was generated by superimposition of the green and red signals, with areas of overlap appearing as yellow. The distribution pattern of CD151 and 7B completely overlap both at the sarcolemma of striated skeletal muscle (lower half) and on smooth muscle cells present in vessel walls (upper half). Note the prominent presence of CD151 in endothelial cells and the small amounts of CD151 in the stroma. Bar, 10 µm.

View larger version (57K): [in a new window]   Fig. 4. Association of CD151 with 3ß1 and 6ß1 in transfected K562 cells. Transfected K562 cells stably expressing 3ß1 or 6ß1 were 125I-surface-labeled, extracted with 1% CHAPS and lysates were subjected to immunoprecipitation with antibodies. Antibodies used were the anti-integrin antibodies J143 against the 3- and J8H against the 6 subunit and the anti-tetraspanin antibodies MEM62 against CD9, MEM53 against CD53, 6H1 against CD63, M38 against CD81, C33 against CD82 and 5C11 against CD151. Both 3ß1 and 6ß1 are present in immunoprecipitates prepared with antibodies against CD63, CD81 and CD151. However, the amount of integrin that is coprecipitated with CD151 is much greater than with the other tetraspanins, reflecting the stability and selectivity of the CD151-integrin binding. The formation of multimolecular complexes is reflected by the additional presence of CD81 (TM4) precipitated by antibodies against CD63 and CD151.

View larger version (27K): [in a new window]   Fig. 5. CD151 is strongly associated with both splice variants (X1 and X2) of 7Bß1 in transfected K562 cells. K562 cells stably expressing the 7ß1 extracellular splice variant X1 or X2 were lysed in 1% CHAPS (C) or 1% Nonidet P-40 (N). Lysates were subjected to immunoprecipitation with mAb P48 against CD151 (upper panel) or with polyclonal antibodies against 7B (lower panel). The presence of 7B or CD151 in the immune complexes was detected by immunoblotting with anti-7B or anti-CD151 (mAb 8C3) antibodies. No immunoreactive bands corresponding to 7B were detected in control (ctrl) immunoprecipitates (upper panel). As a further control (ctrl), the presence of CD151 in total lysates is shown (lower panel). Note that 7B is coprecipitated with CD151 and vice versa, and that this is independent of the extracellular splice variant or the lysis buffer used.

View larger version (70K): [in a new window]   Fig. 6. The absence of cleavage of the integrin 6 subunit on the association with CD151 in transfected K562 cells has no effect. Transfected K562 cells, stably expressing 6ARGGRß1 (uncleaved 6A), were 125I-surface-labeled, lysed in 1% CHAPS and immunoprecipitated with anti-integrin subunit or anti-tetraspanin antibodies. Antibodies used for immunoprecipitation were J8H directed against 6, Sam-1 against 5, TS2/16 against ß1, M38 against CD81 and 5C11 against CD151. Note that 6ARGGRß1 is coprecipitated with CD151 and to a lesser extent with CD81. The integrin 5ß1 is not coprecipitated with tetraspanins; the typical smear around 150 kDa representing 5 is not detected in the precipitates obtained with antibodies against these molecules.

View larger version (12K): [in a new window]   Fig. 7. Monoclonal antibody TS151R does not recognize CD151 when it is bound to 3ß1 or 7ß1. Transfected K562 cells stably expressing 3ß1 (left), 7ß1 (middle) or Vß1 (right) were lysed in 1% Nonidet P-40 and subjected to immunoprecipitation with the mAbs P48 or TS151R, followed by immunoblotting with the indicated polyclonal antibodies against 3, 7 or V. Immunoblot analysis of the lysates (LY) show the level of expression of each integrin.

View larger version (122K): [in a new window]   Fig. 8. Reactivity of CD151 with antibody TS151R is cell type specific. Cryostat sections of human kidney, skin and heart muscle were processed for double immunofluorescence confocal microscopy using TS151R (A,G,J) and P48 (D) against CD151, GoH3 against 6 (E), 439-9B against ß4 (H) and polyclonal antibodies against 3 (B) or 7 (K). The composite images (C,F,I,L) were generated by superimposition of the green and red signals, with areas of overlap appearing as yellow. TS151R against CD151 reacts with CD151 in tubular epithelial cells but hardly reacts in glomerular cells (A-C), keratinocytes (G-I) and cardiomyocytes (J-L). P48 against CD151 is used in the skin for comparison and shows a prominent staining especially at the dermo-epidermal junction (D-F). Bar, 10 µm.

View larger version (24K): [in a new window]   Fig. 10. Expression of laminin-binding integrins on transfected K562 cells increases CD151 cell surface expression. Flow cytometric analysis of wild-type K562 cells endogenously expressing 5ß1 and transfected K562 also expressing Vß1, 3ß1, 6ß1, 6ß4, 7X1ß1 or 7X2ß1. Solid lines in the left panels indicate staining with anti-integrin antibodies: anti-5 mAb Sam-1 (K562 cells), anti-V mAb 13C2 (K562-V), anti-3 mAb J143 (K562-3), anti-6 mAb GoH3 (K562-6), anti-ß4 mAb 439-9B (K562-6ß4) and anti-7 mAb CA25 (K562-7BX1 and K562-7BX2). Staining of cells with anti-CD151 antibodies is shown in the right panels. Solid lines represent staining of cells with mAb TS151R and broken lines with mAb P48. A negative control (dotted line) with secondary goat anti-mouse or rat IgG alone is shown in each panel. R represents the ratio of the mean fluorescence intensity of cells stained with mAb P48 and with secondary antibody alone. A three- to sixfold increase of CD151 expression is observed in transfected cells that ectopically express laminin-binding integrins. The TS151R epitope is masked in all cells expressing one of the laminin-binding integrins.

View larger version (27K): [in a new window]   Fig. 11. No complexes are formed between dystroglycan or the Lutheran blood group molecule and CD151-3ß1 complexes in human podocytes and proximal tubular epithelial cells in culture. Human podocytes (upper panels) and proximal tubular epithelial cells (PTEC) (lower panels) expressing the laminin receptors 3ß1, dystroglycan (DG) and Lutheran blood group antigens (LU) were lysed in 1% CHAPS and precipitated with polyclonal antibodies against 3 or DG (AP83) and mAbs against CD151 (P48) or LU (LU4F2). The immunoprecipitates were analyzed by immunoblotting for the presence of 3 and DG using specific antibodies. Note that DG is not coprecipitated with 3ß1 or CD151 and that 3ß1 is not seen in the precipitates obtained by antibodies against LU or DG. 3L, 3 light chain; ß-DG, ß-dystroglycan.