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First published online 5 October 2004
doi: 10.1242/jcs.01423

Journal of Cell Science 117, 5405-5415 (2004)
Published by The Company of Biologists 2004
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{alpha}4ß1- and {alpha}6ß1-integrins are functional receptors for midkine, a heparin-binding growth factor

Hisako Muramatsu1, Peng Zou1, Hiromichi Suzuki1, Yoshihiro Oda1, Guo-Yun Chen1, Nahoko Sakaguchi1, Sadatoshi Sakuma2, Nobuaki Maeda3,4, Masaharu Noda3, Yoshikazu Takada5 and Takashi Muramatsu1,*

1 Department of Biochemistry and Division of Disease Models, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
2 Cell Signals, 75-1 Ono-cho, Tsurumi-ku, Yokohama 230-0046, Japan
3 Division of Molecular Neurobiology, National Institute for Basic Biology, Myodaiji-cho, Okazaki 444-8585, Japan
4 Department of Developmental Neuroscience, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan
5 University of California Davis Medical Center, 4645 2nd Avenue, Sacramento, CA 95817, USA



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  		Fig. 1. {alpha}4ß1- and {alpha}6ß1-integrins bind to MK. (A) The binding of ß1-integrin to MK. The cell lysate of the COS-7 cells transfected with ß1-HA-encoding cDNA was applied to an MK-agarose column (0.2 ml) and bound proteins were eluted stepwise with 1 ml of buffer containing 0.15 M, 0.2 M, 0.3 M, 0.4 M and 0.5 M NaCl with or without 20 mM EDTA. A portion of the eluate was subjected to SDS-PAGE and analysed by western blotting using anti-HA antibody to detect ß1-integrin. (B) Identification of {alpha}-subunit of ß1-integrin capable of binding MK. The lysate of COS-7 cells transfected with ß1-HA was applied to the MK column and eluted with 0.5 M NaCl containing EDTA. The eluate was analysed by immunoblotting using anti-{alpha}4-integrin or anti-{alpha}6integrin antibodies. (C) Binding of {alpha}4-integrin to MK. The cell lysate of COS-7 cells transfected with FLAG-tagged {alpha}4-integrin-encoding cDNA was applied to the MK column before or after affinity purification using anti-FLAG-antibody/agarose, and bound proteins were eluted and analysed as in A, except that anti-FLAG antibody was used. (Before) MK column only. (After) MK column and FLAG column. (D) Binding of {alpha}6-integrin to MK. The cell lysate of COS-7 cells transfected with HA-tagged {alpha}6-integrin-encoding cDNA was applied to the MK column before or after affinity purification using laminin-agarose, and bound proteins were eluted and analysed as in A. (Before) MK column only. (After) MK column and FLAG column. (E) Binding of metabolically labeled {alpha}4ß1-integrin to MK. (Flag) COS-7 cells transfected with FLAG-tagged {alpha}4-integrin cDNA were labeled with [35S]-methionine. After purification using anti-FLAG-antibody/agarose, the radioactively labeled {alpha}4ß1-integrin fraction, which was bound to the MK-agarose column and was eluted by 0.5 M NaCl with 20 mM EDTA, and analysed by SDS-PAGE. (HA) COS-7 cells were transfected with FLAG-tagged {alpha}4-integrin and HA-tagged ß1-integrin-encoding cDNAs. After purification using anti-HA-antibody/agarose, the radioactively labeled {alpha}4ß1-integrin fraction, which was bound to the MK-agarose column, was analysed as in the case of `FLAG'.

 


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  		Fig. 2. {alpha}4ß1-Integrin is involved in MK-induced migration. (A) Expression of {alpha}4ß1-integrin on the surface of UMR-106 cells as revealed by flow cytometry. UMR cells were incubated with rat anti-mouse-ß1-integrin or mouse anti-rat-{alpha}4-integrin antibody, and stained with FITC/anti-rat-IgG or Alexa-Fluor 488-anti-mouse IgG. The negative controls without the primary antibody are indicated with light lines, whereas the signals from integrin staining are shown with a dark line. (B) Involvement of {alpha}4ß1-integrin in MK-induced migration. The lower surface of the Chemotaxicell filter was coated with MK or poly-L-lysine (PLL) and a migration assay using UMR-106 cells was performed. Ten fields at 400x magnification per filter were counted to determine the number of migrated cells (one field corresponds to 1/160th of the entire surface of the filter). The results are expressed as a ratio to the value obtained without the addition of reagents (PBS). The value is the mean ± s.e.m. (n=3). *, P<0.001 versus PBS or IgG. V10, the GPEILDVPST sequence; RGD, the GRGDS sequence.

 


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  		Fig. 3. Inhibition of MK-induced neurite outgrowth by anti-{alpha}6-integrin antibody. Plastic 24-well culture plates were coated with 10 µg ml–1 of MK for 2 hours at room temperature. Then, a grid pattern was made by the ultraviolet-inactivation technique using electron-microscopy grids. After blocking with 10 mg ml–1 BSA, brain cells (1x106) from mouse embryonic cerebral cortex were cultured without further addition (A) or with 0.0045% NaN3 (B), 50 µg ml–1 anti-{alpha}4-integrin antibody (C) or anti-{alpha}6-integrin antibody (D) at 37°C under an atmosphere containing 5% CO2. (E) Effects of anti-{alpha}6-integrin antibody on neurite outgrowth on wells coated with 20 µg ml–1 MK. The proportion of neurons with a defined neurite length was determined by enumerating cells in ten fields at 400x magnification. The average value obtained in three different wells is shown with the s.d. Phase-contrast photomicrographs were taken after culture for 48 hours using a Nikon DIAPHOTO and Olympus CCD camera C5530. Scale bar, 50 µm.

 


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  		Fig. 4. MK induces paxillin phosphorylation. UMR-106 cells were cultured for 8 hours in DMEM and stimulated with MK (0 ng ml–1, 50 ng ml–1, 200 ng ml–1, 500 ng ml–1 or 1000 ng ml–1) in DMEM for 5 minutes (A) or 200 ng ml–1 of MK for 0 minutes, 5 minutes, 10 minutes or 30 minutes (B). (C) Cells were incubated for 5 minutes without addition (C), with 200 ng ml–1 MK (MK) or 200 ng ml–1 MK plus 100 µg ml–1 anti-{alpha}4-integrin antibody (anti-{alpha}4 + MK). Paxillin was immunoprecipitated from cell lysate and analysed by western blotting using anti-phosphotyrosine antibody (Anti-P-Tyr) or anti-paxillin antibody (anti-pax). Relative densitometric densities are shown below the bands obtained by western blotting. In (C), three experiments were performed and a typical result was shown; the increased phosphorylation by MK was suppressed by anti-{alpha}4-integrin antibody to 36% of the value without the antibody.

 


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  		Fig. 5. {alpha}4ß1- or {alpha}6ß1-Integrin precipitates with LRP6. COS-7 cells were co-transfected with LRP6-FLAG and ß1-HA (A,B,G), LRP6-FLAG and {alpha}6-HA (C,D,H) or LRP6-FLAG and {alpha}4-Myc (E,F). Single transfections were also done. After precipitation with anti-FLAG antibody (A,C,E,G,H), anti-HA antibody (B,D) or anti-Myc antibody (F), the immunoprecipitates were probed for the presence of ß1-HA (A,G), {alpha}6-HA (C,H), {alpha}4-myc (E) or LRP6-FLAG (B,D,F) by immunoblotting. In (G), experiments were performed as in (A) except that cells were incubated with 200 ng ml–1 MK for appropriate period. In (H), experiments were performed as in (C) except that the time of transfection was reduced to 1.5 hours and that cells were incubated with 200 ng ml–1 MK for appropriate period.

 


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  		Fig. 6. LRP6 co-precipitates with PTP{zeta}. (A), (B) COS-7 cells were co-transfected with LRP6-FLAG and PTP{zeta}, or with each alone. After precipitation with anti-PTP{zeta} antibody (A) or anti-FLAG antibody (B), the immunoprecipitates were probed for the presence of LRP6-FLAG (A) or PTP{zeta} (B) by immunoblotting using anti-FLAG antibody (A) or anti-PTP{zeta} antibody (B). (C) COS-7 cells were co-transfected with LRP6-FLAG, PTP{zeta} and {alpha}6-HA. After precipitation with anti-HA antibody, the immunoprecipitate was probed for the presence of LRP6-FLAG or PTP{zeta}.

 


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  		Fig. 7. {alpha}4ß1- or {alpha}6ß1-Integrin co-precipitates with PTP{zeta}. COS-7 cells were co-transfected with PTP{zeta} and ß1-HA (A,B), PTP{zeta} and {alpha}6-HA (C,D) or PTP{zeta} and {alpha}4-FLAG (E,F). Single transfections were also done. After precipitation with anti-PTP{zeta} antibody (A,C,E), anti-HA antibody (B,D) or anti-FLAG antibody (F), the immunoprecipitates were probed for the presence of ß1-HA (A), {alpha}6-HA (C), {alpha}4-FLAG (E) or PTP{zeta} (B,D,F) by immunoblotting using appropriate antibodies.

 

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© The Company of Biologists Ltd 2004