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First published online 16 August 2005
doi: 10.1242/jcs.02520

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Evidence for the presence of a low-mass ß1 integrin on the cell surface

¹ Manitoba Centre for Proteomics and Systems Biology, Department of Internal Medicine, University of Manitoba, 715 McDermot Avenue, Winnipeg, MB, Canada R3E 3P4
² Rheumatic Diseases Research Laboratory, Department of Internal Medicine, University of Manitoba, 715 McDermot Avenue, Winnipeg, MB, Canada R3E 3P4
³ Time of Flight Laboratory, Department of Physics and Astronomy, University of Manitoba, Winnipeg, MB, Canada R3T 2N2
⁴ The Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK

View larger version (50K): [in a new window]   Fig. 1. (A) Differential expression of ß1 integrin epitopes on K562 Cells. Cells were grown in serum-free medium and either stained with JB1A, B3B11 or 12G10, or reacted with only the secondary antibody (BG). The cells were analysed by flow cytometry. The geometric means of the expression values for each category were BG 8. 27, 12G10 29. 97, B3B11 61. 95 and JB1A 72. 27. (B) Distribution of ß1 integrin species identified by B3B11 or 12G10. K562 cells were fixed in 4% paraformaldehyde, stained with the indicated antibodies and visualized with Cy3-conjugated goat anti-mouse immunoglobulin. The cells were stained with a polyclonal antibody 5 and stained with an Oregon Green goat anti-rabbit immunoglobulin. (C) 12G10-reactive ß1 does not codistribute with B3B11 reactive ß1. Cells were reacted with 12G10 and the bound integrins were cross-linked with Cy3-coupled goat anti-mouse immunoglobulin. The cells were fixed and blocked with mouse immunoglobulin to saturate the combining sites on the anti-mouse immunoglobulin. The cells were then reacted with biotin-labelled B3B11 and the bound antibody was detected with an FITC-labelled Avidin conjugate. 12G10 labelled integrins were clustered; in contrast B3B1ll integrins were diffusely distributed. The overlay of the two images shows that there was little or no colocalization of the integrins detected by these two antibodies.

View larger version (65K): [in a new window]   Fig. 2. Differential distribution of the integrin 5 chain and 12G10 reactive ß1 chains on the cell surface. K562 cells were incubated with B3B11 antibody (A-C) or 12G10 (D-F), washed, and a Cy3 (red)-conjugated anti-mouse immunoglobulin second antibody was applied to trigger aggregation of the antibody-reactive ß1 species. The cells were fixed and stained with a polyclonal anti-5, followed by Oregon Green-conjugated second antibody (A-F). A representative double stained cell was selected and photographed. (A) B3B11, (B) 5, (C) overlay of A and B. (D) 12G10, (E) 5, (F) overlay of D and E. (G-I) 5 integrins of K562 cells were cross-linked with polyclonal anti-5 and Oregon Green 488-conjugated secondary antibody. The cells were fixed and stained with 12G10 plus Cy3-conjugated second antibody. (G) 12G10, (H) 5, (I) overlay of G and I.

View larger version (33K): [in a new window]   Fig. 3. The chain expression pattern of K562 cells. Cells were surface labelled with the indicated chain-specific antibodies or with secondary antibody only (BG) and analysed by flow cytometry. 5 was the only one of the integrins detected in significant levels on these cells. The activities of each of these antibodies were confirmed on control cell lines that expressed the corresponding integrins.

View larger version (82K): [in a new window]   Fig. 4. (A) Distinct species of ß1 chain on the cell surface. Lanes 1-4: K562 were surface labelled with different antibodies to ß1 integrin (lane 1, control; 2, B3B11; 3, JB1A; 4, 12G10) and washed to remove free antibodies. The cells were lysed and the antibody-associated integrins were immunoprecipitated, separated by SDS-PAGE and immunoblotted with JB1A to detect total ß1. Lanes 5-7: total cell lysates from untreated cells were prepared and incubated with antibodies for immunoprecipitation (lanes 5, B3B11; 6, JB1A; 7, 12G10). The samples were analysed as described above. Note that the patterns of capture are different for the surface-labelled and the cell lysate-derived integrins demonstrating the specificity of the cell surface capture. Lanes 8-10: cells were grown in serum-free medium and surface labelled with 12G10, lane 8; 12G10 in the presence of 120 kDa fragment of fibronectin, lane 9; or surface labelled with 12G10, washed and exposed to the 120 kDa fragment of fibronectin, lane 10. The bound integrins were isolated by immunoprecipitation and processed as described above. (B) Lack of association between 5 and the 12G10 reactive ß1 subunits. Cell surface integrins were immunoprecipitated with antibodies to 5 with JBS5 or to ß1, with B3B11, JB1A or 12G10. The precipitates were separated by SDS-PAGE and probed by western blot analysis with anti-ß1, JB1A (upper panel) or a polyclonal anti-5 (lower panel).

View larger version (65K): [in a new window]   Fig. 5. Mass spectrometric analysis of ß1 integrin-associated proteins on the cell surface. Cells were surface labelled with antibody (lane 1, control; lane 2, B3B11; lane 3, 12G10). The protein complexes were isolated and separated in 8% SDS-PAGE gels under reducing conditions and stained with Coomassie Blue. The visible protein bands were cut out for in-gel digestion and mass spectrometric analysis. The identified proteins are labelled and marked with arrows.

View larger version (60K): [in a new window]   Fig. 6. (A) N-link glycosylation contributed to the mass loss. The surface integrins isolated with either B3B11 or 12G10 were either analysed directly (-) or treated (+) with PGNase F for 2 hours at 37°C. The integrins were then separated and analysed by western blotting with JB1A. The glycosylation resulted in a decrease in the molecular masses of the 12G10- and B3B11-reactive integrins to 90 kDa, indicating that the mass differences are related to N-glycosylation levels or patterns on the two integrin species.

View larger version (42K): [in a new window]   Fig. 7. Expression patterns of ß1 integrin chains on different cell lineages. Human cell lines of different lineages (Jurkat, T lymphoma; HT1080, fibrosarcoma; MDA-MB-231 breast cancer) were surface labelled with anti-integrin ß1 antibody B3B11 (lanes 1, 4, 7), JB1A (lanes 2, 5, 8) or 12G10 (lanes 3, 6, 9) and the immunoprecipitates were immunoblotted with JB1A. The 120 kDa integrin ß1 were identified in HT1080 and MDA cells by JB1A and 12G10, but not in Jurkat cells.

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