α4β1- and α6β1-integrins are functional receptors for midkine, a heparin-binding growth factor

Midkine is a heparin-binding growth factor that promotes the growth, survival, migration and differentiation of various target cells. So far, receptor-type protein tyrosine phosphatase ζ, low-density-lipoprotein-receptor-related protein and anaplastic lymphoma kinase have been identified as receptors for midkine. We found β1 integrin in midkine-binding proteins from 13-day-old mouse embryos. β1-Integrin bound to a midkine-agarose column and was eluted mostly with EDTA. Further study revealed that the α-subunits capable of binding to midkine were α4 and α6. Purified α4β1- and α6β1-integrins bound midkine. Anti-α4 antibody inhibited the midkine-dependent migration of osteoblastic cells, and anti-α6 antibody inhibited the midkine-dependent neurite outgrowth of embryonic neurons. After midkine treatment, tyrosine phosphorylation of paxillin, an integrin-associated molecule, was transiently increased in osteoblastic cells. Therefore, we concluded that α4β1- and α6β1-integrins are functional receptors for midkine. We observed that the low-density-lipoprotein-receptor-related-protein-6 ectodomain was immunoprecipitated with α6β1-integrin and α4β1-integrin. The low-density-lipoprotein-receptor-related-protein-6 ectodomain was also immunoprecipitated with receptor-type protein tyrosine phosphatase ζ. α4β1- and α6β1-Integrins are expected to co-operate with other midkine receptors, possibly in a multimolecular complex that contains other midkine receptors.

. We have recently identified MK as a key molecule in the inflammatory response (Horiba et al., 2000;Sato et al., 2001). In MKdeficient mice, leukocyte infiltration into the blood vessels and kidney after ischemic injury is suppressed, leading to the suppression of neointima formation and nephritis after ischemia (Horiba et al., 2000;Sato et al., 2001). In the deficient mice, antibody-induced arthritis and intraperitoneal adhesions after surgery are also suppressed (Inoh et al., 2004;Maruyama et al., 2004).
So far, receptor-type protein tyrosine phosphatase ζ (PTPζ) (Maeda et al., 1999), anaplastic lymphoma kinase (ALK) (Stoica et al., 2002) and low-density-lipoprotein (LDL) receptor-related protein (LRP)  have been proposed as the MK receptors. PTPζ is a chondroitin sulfate proteoglycan with an intracellular tyrosine phosphatase domain, an MK receptor for the migration of embryonic neurons (Maeda et al., 1999) and osteoblast-like cells (Qi et al., 2001), and involved in the survival of embryonic neurons (Sakaguchi et al., 2003). PTPζ is also a PTN receptor for the migration of embryonic neurons (Maeda et al., 1996;Maeda and Noda, 1998). ALK is a transmembrane tyrosine kinase (Iwahara et al., 1997) and has been reported to serve as a PTN/HB-GAM and MK receptor primarily in the promotion of cell growth (Stoica et al., 2001;Stoica et al., 2002).
LRP is a member of the LDL-receptor family and is a receptor of MK necessary for the survival of embryonic neurons . It also plays a role in the internalization of MK (Shibata et al., 2002). Although the LDL-receptor family primarily serves as endocytosis receptors, some members have been found as components of signaling receptors (Herz and Bock, 2002). Their role in the signaling of reelin (Howell et al., 1999;Trommsdorff et al., 1999) is of particular interest. Reelin is an extracellular-matrix (ECM) protein required for proper neuronal migration, and the reelin receptor is composed of three components: a member of the LDL-receptor family (VLDL receptor or apoE2 receptor) (Howell et al., 1999;Trommsdorff et al., 1999); cadherinrelated neural receptor (Senzaki et al., 1999); and α 3 β 1 -integrin (Dulabon et al., 2000). In addition, LRP5/6 function as components of the Wnt receptor complex (Tamai et al., 2000;Wehrli et al., 2000). LRP also associates with the plateletderived growth factor (PDGF) receptor, leading to a downregulation of receptor activity (Boucher et al., 2003).
In this communication, we provide evidence that α 4 β 1 -and α 6 β 1 -integrins bind directly to MK and are involved in MKdependent cell migration and neurite outgrowth. Furthermore, we propose that integrins and other MK receptors mentioned above co-operate upon MK signaling, as in the case of reelin receptors.

Materials
Recombinant human MK was expressed in yeast (Pichia pastoris GS115) and purified to homogeneity as described previously . MK-agarose was prepared by coupling 50 mg human MK to 10 ml CNBr-activated Sepharose 4B (Amersham Biosciences, NJ) as described previously . Laminin was purchased from BD Bioscience (Bedford, MA, USA) and laminin-agarose was prepared by coupling laminin to CNBractivated Sepharose 4B. Protein-A/agarose and Protein-G/agarose were from Amersham Biosciences. V-10 peptide (GPEILDVPST) and fibronectin active fragment (GRGDS) were products of Peptide Institute (Osaka, Japan). Protease inhibitor cocktail tablets were from Roche (Mannheim, Germany).
Purification of MK-binding proteins and sequence analysis MK-binding membrane proteins were isolated from about 130 g day-13 mouse embryos as described previously . They were subjected to sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) under reducing conditions, and their sequences were analysed using a 494A Protein Sequencer (Applied Biosystems, Foster City, CA) after in-gel trypsin digestion and peptide separation .

DNA constructs
The coding sequences of human β 1 -integrin (Argraves et al., 1987) and human α 6 -integrin (Tamura et al., 1990) were ligated into an expression vector pcDNA3.1 (Invitrogen Life Technologies, Carlsbad, CA, USA) with the HA tag sequence at the C-terminus. The coding sequence of human α 4 -integrin (Takada et al., 1989) was also ligated into pcDNA3.1 with the FLAG-tag sequence or Myc-tag sequence at the C-terminus. A cDNA encoding the extracellular domain of mouse LRP6 (Brown et al., 1998;Sakaguchi et al., 2003) was ligated into pcDNA3.1 with the FLAG-tag sequence at the C-terminus. The cDNA encoding rat PTPζ (Maeda et al., 1994) was inserted into pcDNA3.1.

Binding of integrins to MK
The COS-7 cells transfected with HA-tagged β 1 -integrin or α 6integrin or FLAG-tagged α 4 -integrin cDNA were cultured in DMEM with 10% FCS for 24 hours and lysed in buffer A (20 mM Tris-HCl, pH 7.5, 0.3% CHAPS {3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid}, 0.15 M NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , protease inhibitors). The cell lysate was applied to an MK-agarose column (0.2 ml) equilibrated with buffer A. After washing with buffer A, the column was eluted stepwise with 1 ml of buffer B (20 mM Tris-HCl, pH 7.5, 0.3% CHAPS, protease inhibitor) containing 0.2 M, 0.3 M, 0.4 M and 0.5 M NaCl with or without 20 mM EDTA. The eluate was subjected to SDS-PAGE using a 7% gel under reducing conditions, and the proteins were transferred to a PVDF membrane. The membrane was blocked with 5% nonfat milk in PBS and reacted with anti-HA or anti-FLAG antibody. Then, it was reacted with HRPconjugated anti-rat IgG or anti-mouse IgG, and bands were revealed using an ECL detection kit (Amersham Biotechnology). COS-7 cells transfected with α 4 -integrin/FLAG cDNA were cultured in DMEM supplemented with 10% FCS overnight. The medium was changed to DMEM without L-methionine, L-cysteine or FCS. After 2 hours, 40 µl (20 MBq) L-[ 35 S] methionine [PRO-MIX; L-[ 35 S] in vitro cell labeling mix (Amersham Biosciences)] was added and culture was continued for 40 minutes. Then, the cells were lysed in 1 ml buffer A and the lysate was applied to an anti-FLAG-agarose column (0.4 ml). Material eluted with 2 ml of buffer A containing FLAG peptide (100 µg ml -1 ) was applied to an MK-agarose column. MK-binding proteins were subjected to SDS-PAGE using a 7% gel under reducing conditions, and analysed using an image analyser BAS2000 (Fujifilm, Japan). COS-7 cells were also co-transfected with α 4 -integrin/FLAG and β 1 -integrin/HA, and methionine labeled in the same way. The cell lysate was applied to anti-HA/agarose column. Material eluted with buffer A containing 1 mg ml -1 HA-peptide was applied to an MK-agarose column and analysed as above.

Immunoprecipitation
After 24-48 hours of culture, the transfected cells were rinsed with PBS. All the following steps were done on ice or at 4°C using icecold buffer. The cells on a 60-mm cell culture plate were covered with 0.5 ml buffer A and scraped with a cell scraper. The cell lysate was transferred to a 1.5 ml microcentrifuge tube, incubated for 20 minutes at 4°C and centrifuged at 10,000 g for 10 minutes to remove cellular debris. The samples were pre-adsorbed with 30 µl Protein-A/agarose or Protein-G/agarose by rotating the sample tubes for 2 hours at 4°C. After centrifugation, 1-10 µl (0.5-5 µg) of primary antibody was added to 0.5 ml of the supernatant fluid and the mixture was incubated at 4°C on a rotating device for between 5 hours and overnight. After addition of 30 µl of Protein-A/agarose or Protein-G/agarose (for anti-HA antibody), the mixture was incubated for 2 hours. The pellet was collected by centrifugation at 3000 g for 3 minutes, washed three times with buffer A and resuspended in 30 µl of 2× electrophoresis sample buffer and subjected to SDS-PAGE and western blotting as mentioned above.
The level of expression of the tagged proteins in the transfected cells were estimated using the [ 35 S]-labeled cells; we determined the percentage of radioactivity immunoprecipitated by an anti-tag antibody to radioactivity precipitated by 10% trichloroacetic acid. The values were 0.03-0.12% for α 4, α 6 , β 1 and LRP6.
For analysis of paxillin phosphorylation, UMR106 cells were cultured without FCS for 8 hours and then incubated with DMEM containing MK for the indicated periods. Cells were lysed in 0.5 ml buffer A containing 0.5 mM sodium vanadate. The cell lysate was immunoprecipitated with anti-paxillin antibody and subjected to western blotting using anti-phosphotyrosine or anti-paxillin antibody.

Assays for cell migration and neurite outgrowth
The migration of UMR106 cells was assayed using Chemotaxicell (Kurabo, Osaka, Japan) with pores 8 µm in diameter, as described previously (Qi et al., 2001). The lower surface of the filter was coated with 20 µg ml -1 MK or poly-L-lysine in PBS and 600 µl 0.3% bovine serum albumin (BSA) in DMEM was placed in the lower chamber. UMR-106 cells (1×10 5 ) in 100 µl 0.3% BSA/DMEM were added to the upper chamber and incubated for 6 hours. For the inhibition assay, UMR-106 cells were preincubated at room temperature for 30 minutes with antibodies, peptides or IgG, and added to the upper chamber with the inhibitors. Statistical analysis was performed with Student's t test.
A grid assay of neurite outgrowth was performed as described previously (Kaneda et al., 1996), adopting the method described by Rauvala et al. (Rauvala et al., 1994). Neurite outgrowth on wells coated with 20 µg ml -1 MK was performed as described previously .

Flow cytometric analysis
The expression of α 4 and β 1 -integrin on UMR106 cells was analysed by flow cytometry. UMR cells were detached by incubation for 10 minutes at 37°C with non-enzymatic cell dissociation solution (Sigma). The cells (1×10 6 ) were washed with PBS and suspended in 1 ml PBS containing 1% BSA. The cell suspension was mixed with 10 µl rat anti-mouse β 1 subunit or mouse anti-rat CD49d, incubated at 4°C on a rotating device for 45 minutes and, after washing with PBS, stained with FITC-conjugated anti-rat IgG or Alexa-Fluor-488/goat anti-mouse IgG at 4°C for 45 minutes. Background fluorescence intensity was assessed in the absence of primary antibody. The expression of integrins were quantified using a Beckman Coulter Flow Cytometer (EPICS XL-2)

Results β 1 -Integrin binds to MK
To identify new components of the MK receptor, membrane proteins of 13-day mouse embryos were solubilized with 0.3% CHAPS and MK-binding glycoproteins were isolated by affinity chromatography on Ricinus communis agglutininagarose and MK-agarose. These proteins were separated by SDS-PAGE and subjected to trypsin digestion and protein sequence analysis . In the previous study, a 110-kDa protein that migrated between entactin and heat-shock protein 90-β was not identified . In the present study, it was identified as β 1 -integrin, because two peptides derived from the protein by trypsin digestion [LSENNIQTIF (amino acids 326-335) and WDTGENPIYK (amino acids 775-784)] were present in β 1integrin.
To examine the binding of β 1 -integrin to MK in more detail, we produced HA-tagged β 1 -integrin. The cell lysate of COS-7 cells transfected with HA-tagged β 1 -integrin cDNA was applied to an MK-agarose column. β 1 -Integrin bound to the column at a NaCl concentration of 0.15 M. Under conditions without EDTA, β 1 -integrin was mainly eluted by 0.3 M and 0.4 M NaCl, although a significant portion was also eluted by 0.5 M NaCl and part of it remained in the column and was eluted by adding EDTA (Fig. 1A). This strong binding to an MKagarose column had been observed in the LRP family Sakaguchi et al., 2003) but not in other MK-binding proteins such as PRP-8 (Takahashi et al., 2001) and NCAM (data not shown). The addition of Mn 2+ did not change the elution profile (data not shown). In the presence of EDTA, a large amount of β 1 -integrin was eluted without changing the NaCl concentration, and the remainder by 0.2 M and 0.3 M (Fig. 1A). Therefore, β 1 -integrin bound specifically to MK and the binding was cation dependent, although the incomplete elution by EDTA indicates that the MK/β 1 -integrin interaction is somewhat different from the usual interaction of integrins with proteins in ECM.
Based on these results, we transfected cDNAs of HA-tagged α 6 -integrin and FLAG-tagged α 4 -integrin into COS-7 cells. The lysate of COS-7 cells transfected with FLAG-tagged α 4 -integrin was applied to an MK-agarose column and the adsorbed material was eluted with increasing NaCl concentrations. α 4 -Integrin was mainly eluted with 0.3 M and 0.4 M NaCl, and part of it remained in the column, as in the case of β 1 -integrin (Fig. 1C). When the lysate of COS-7 cells transfected with HA-tagged α 6 -integrin was applied to MKagarose column and eluted with NaCl, it was also eluted with 0.3 M and 0.4 M NaCl, in a manner similar to β 1 -integrin or α 4 -integrin (Fig. 1D).
We estimated the proportion of integrins bound to the MK column relative to the total amount in the extract by quantifying the amount of HA-tagged or FLAG-tagged integrins by western blotting after SDS-PAGE. In the case of β 1 -integrin, 36% bound to the MK column. The values were 39% and 54% for α 4 -and α 6 -integrins, respectively. Cotransfection of β 1 -and α-subunit cDNAs did not change the result. As an example, when HA-tagged α 6 -subunit and HAtagged β 1 -subunit cDNAs were transfected, the amount of bound β 1 was 37% of total, and the amount of bound α 6 was 49% of total. The elution profile from the column was also unchanged.
To verify that α 4 -integrin was bound to MK without the help of other molecules, α 4 -integrin/FLAG was purified using an anti-FLAG-monoclonal-antibody/agarose column and eluted with 100 µg ml -1 FLAG peptide. We confirmed the purity of the affinity-purified α 4 -integrin by using the [ 35 S]-labeled preparation. Upon SDS-PAGE, the major band was about 130-140 kDa, which corresponds to the bands of both α 4 -and β 1subunits (Guan and Hynes, 1990) (Fig. 1E). A minor band of around 70 kDa corresponds to the C-terminal fragment of α 4integrin (Hemler et al., 1987). The major band was also detected, when FLAG-tagged α 4 -subunit and HA-tagged β 1subunit were co-expressed, and α 4 β 1 -integrin was purified by affinity chromatography on anti-HA-antibody/agarose (Fig.  1E). A slight difference of the size of integrin β 1 -subunit expressed in COS-7 cells was observed between Fig. 1A and Fig. 1E. We interpret this as showing that, upon overexpression for prolonged period as in Fig. 1A, less glycosylated β 1 -subunit is formed. The affinity-purified α 4 -integrin/FLAG also bound to the MK-agarose column (Fig. 1C) in the same manner as the unpurified one (Fig. 1C).
Because one ligand for α 6 β 1 -integrin is laminin, we also purified α 6 β 1 -integrin using a laminin-1/agarose column before application to the MK column. Although α 6 β 4 -integrin also interact with laminin-1, β 4 was not detected in COS-7 cells. The purified integrin bound to the MK column (Fig. 1D) in a manner indistinguishable from the unpurified one (Fig. 1D). Based on all these results, we concluded that MK bound specifically to α 4 β 1 -and α 6 β 1 -integrins.
α 4 β 1 -Integrin is involved in MK-induced migration of osteoblastic cells MK induces haptotactic migration of UMR-106 rat osteoblastic cells (Qi et al., 2001). Generally speaking, osteoblasts express α 2 -, α 3 -, α 4 -, α 5 -, α 6 -and β 1 -subunits Journal of Cell Science 117 (22) The lysate of COS-7 cells transfected with β 1 -HA was applied to the MK column and eluted with 0.5 M NaCl containing EDTA. The eluate was analysed by immunoblotting using anti-α 4 -integrin or anti-α 6 integrin antibodies. (C) Binding of α 4integrin to MK. The cell lysate of COS-7 cells transfected with FLAG-tagged α 4 -integrin-encoding cDNA was applied to the MK column before or after affinity purification using anti-FLAGantibody/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 α 6integrin to MK. The cell lysate of COS-7 cells transfected with HAtagged α 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 α 4 β 1 -integrin to MK. (Flag) COS-7 cells transfected with FLAG-tagged α 4 -integrin cDNA were labeled with [ 35 S]-methionine. After purification using anti-FLAGantibody/agarose, the radioactively labeled α 4 β 1 -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 α 4 -integrin and HA-tagged β 1 -integrin-encoding cDNAs. After purification using anti-HA-antibody/agarose, the radioactively labeled α 4 β 1 -integrin fraction, which was bound to the MK-agarose column, was analysed as in the case of 'FLAG'. (Nakayamada et al., 2003). Western-blot analysis indicated that UMR-106 cells strongly expressed α 4 β 1 -integrin. A weaker expression of the α 6 -subunit was also noted. Flow-cytometric analysis confirmed that α 4 β 1 -integrin was expressed on the surfaces of these cells ( Fig. 2A). It is known that α 4 β 1 -integrin governs the migration of leukocytes to inflammatory sites (Rose et al., 2001). To evaluate the potential contribution of α 4 β 1 -integrin in MK signaling, we examined whether or not the MK-induced migration of UMR106 cells is mediated by α 4 β 1 -integrin.
MK was coated on the lower surface of a filter at a concentration of 20 µg ml -1 and the cells were added to the upper chamber. Function-blocking anti-rat-α 4 -integrin antibody inhibited the cell migration in a dose-dependent manner, whereas mouse IgG did not (Fig. 2B). Anti-α 6 -integrin antibody was not inhibitory (data not shown). Furthermore, the V-10 peptide (GPEILDVPST), which contains the tripeptide of the α 4 -integrin-binding motif [leucine-asparatate-valine (Guan and Hynes, 1990)] also inhibited the migration in a concentration-dependent manner. An RGD peptide used as a control was not inhibitory. We confirmed that the V-10 peptide at 1 mg ml -1 concentration inhibited the binding of α 4 β 1integrin to an MK column to 44% compared with the binding in the absence of the inhibitor. These assays were performed by culturing cells for 6 hours. However, in the assay conducted by culturing cells for 3 hours, anti-α 4 -integrin antibody inhibited the migration in an identical manner; the antibody at the concentration of 100 µg ml -1 reduced the migration to 41% of the control. The result supports our conclusion that the adhesion is to MK and not to other proteins secreted by the cells. Therefore, we concluded that haptotactic migration of UMR-106 cells induced by MK was mediated by α 4 β 1integrin.
Anti-α 6 -integrin antibody inhibits MK-dependent neurite outgrowth When neurons from rat embryonic brains were cultured on the grid pattern of MK formed on culture plates, they attached and 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 400× 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. Fig. 3. Inhibition of MK-induced neurite outgrowth by anti-α 6integrin 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 electronmicroscopy grids. After blocking with 10 mg ml -1 BSA, brain cells (1×10 6 ) from mouse embryonic cerebral cortex were cultured without further addition (A) or with 0.0045% NaN 3 (B), 50 µg ml -1 anti-α 4 -integrin antibody (C) or anti-α 6 -integrin antibody (D) at 37°C under an atmosphere containing 5% CO 2 . (E) Effects of antiα 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 400× 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.
extended their neurites along the MK tracks (Kaneda et al., 1996) (Fig. 3A). To examine whether α 6 -integrin is involved in neurite outgrowth induced by MK, mouse embryonic brains were cultured with anti-α 6 -integrin antibody. Indeed, anti-α 6integrin antibody inhibited the attachment and neurite outgrowth of neurons on the MK-coated substratum (Fig. 3D), whereas anti-α 4 -integrin antibody or 0.0045% NaN 3 (which is present in the anti-α 6 -integrin antibody preparation), or mouse IgG did not have this effect (Fig. 3B,C, H.M. and T.M., unpublished). We also performed quantitative analysis of the effect of antiα 6 antibody on neurite outgrowth. In the grid assay mentioned above, individual neurons are difficult to observe. Thus, we just plated brain cells on wells coated with MK. Anti-α 6 antibody dramatically suppressed the number of cells with extended neurites and increased the number of cells without neurites (Fig. 3E).

MK stimulates tyrosine phosphorylation of paxillin
To further examine the physiological significance of MKintegrin interactions, we investigated whether phosphorylation of integrin-associated molecules is changed after stimulation with MK. Consequently, we found that 200-500 ng ml -1 MK transiently increased the tyrosine phosphorylation of paxillin in UMR-106 cells (Fig. 4A). The maximum response was found at 5 minutes after the addition of MK (Fig. 4B). The increase in the phosphorylation was two-to threefold compared with untreated cells. Anti-α 4 antibody suppressed the increased phosphorylation (Fig. 4C). This finding supports the proposal that the binding of MK to α 4 β 1 -integrins in these cells delivers an intracellular signal necessary for the stimulation of cell migration.
α 6 β 1 -Integrin is co-immunoprecipitated with LRP6 and PTPζ LRP is an important component of the receptor complex for MK . Therefore, we were interested in clarifying whether LRP forms a complex with α 4 β 1 -or α 6 β 1integrin. Because LRP has molecular mass of 600 kDa, it is hard to express by cDNA transfection. We previously found that LRP6, which is a component of the Wnt receptor complex (Tamai et al., 2000;Wehrli et al., 2000), binds to MK with similar affinity (Sakaguchi et al., 2003). Therefore, we decided to investigate this point by transfection of a cDNA for the LRP6 ectodomain. The ectodomain was used to avoid a possible non-specific association of transmembrane proteins. COS-7 cells were transfected with cDNAs of HA-tagged β 1 (β 1 -HA) and FLAG-tagged LRP6 ectodomain (LRP6-FLAG) together, LRP6-FLAG alone or β 1 -HA alone. Then, LRP6-FLAG was precipitated with antibody directed against the FLAG epitope, and the immunoprecipitates were probed for the presence of β 1 -HA by immunoblotting. As a result, β 1 -HA could be detected only in the precipitates from cotransfected cells (Fig. 5A). When β 1 -HA was precipitated with anti-HA antibody and the immunoprecipitates were probed for the presence of LRP6-FLAG by immunoblotting, it was detected only in the precipitates from co-transfected cells (Fig. 5B).
Taken together, these results suggested that the LRP6 ectodomain formed a complex with α 6 β 1 -integrin. The LRP6 ectodomain also appears to form a complex with α 4 β 1 -integrin, but further analysis is required to obtain a definitive conclusion. We also questioned whether the LRP6 ectodomain is co-immunoprecipitated with PTPζ. PTPζ has been identified as a receptor of MK in the MK-dependent migration of UMR-106 osteoblastic cells and embryonic neurons (Maeda et al., 1999;Qi et al., 2001). COS-7 was transfected with cDNAs of PTPζ and LRP-FLAG. Then, PTPζ was precipitated with anti-PTPζ antibody and the immunoprecipitates were probed for the presence of LRP6-FLAG by immunoblotting. A 180-kDa band of LRP6-FLAG could be detected only in anti-PTPζ precipitates from co-transfected COS-7 cells (Fig. 6A). When LRP6-FLAG was precipitated with anti-FLAG antibody and the immunoprecipitates were probed for PTPζ by immunoblotting, a broad band of PTPζ could be detected only in the precipitates from co-transfected cells (Fig. 6B).
As described here, we suggested that the LRP6 ectodomain formed a complex with α 6 β 1 -integrin and also with PTPζ. It is likely that LRP also forms a complex with α 6 β 1 -integrin. Although the distribution of LRP6 is restricted, LRP is expressed in a variety of cells (Herz and Bock, 2002). Thus, we can infer that α 6 β 1 -integrin and probably α 4 β 1 -integrin form a complex with PTPζ when they are co-expressed.
To test this possibility, COS-7 cells were transfected with cDNAs of β 1 -HA and PTPζ, β 1 -HA alone or PTPζ alone. After precipitation with anti-PTPζ antibody, the immunoprecipitates were probed for the presence of β 1integrin by immunoblotting using anti-HA antibody. Indeed, β 1 -HA could be detected only in the precipitates from co-transfected cells (Fig. 7A). When β 1 -HA was precipitated with anti-HA antibody and the immunoprecipitates were probed for the presence of PTPζ by immunoblotting using anti-PTPζ antibody, a broad band of PTPζ could be detected only in precipitates from co-transfected cells (Fig. 7B). We also examined the possible association of α 6 -integrin or α 4integrin with PTPζ and obtained the same results ( Fig. 7C-F). Although a possible non-specific association through the transmembrane domain could not be completely ruled out in this experiment, the result further supports the proposed presence of a large complex consisting of PTPζ, LRP or LRP6 and α 4 β 1 -or α 6 β 1 -integrin. By transfection with cDNAs of α 6 -HA, LRP6-FLAG and PTPζ, we also obtained a result supporting the presence of a complex containing all the three molecules. Thus, the immunoprecipitate pulled down with anti-HA antibody contained both LRP6-FLAG and PTPζ (Fig. 6C).
Finally, we investigated the proportion of a coimmunoprecipitated tagged molecule to the total tagged molecule expressed in the cells; the value was obtained by western blot analysis of a tagged molecule in the immunoprecipitate and in cell extract. α 6 -HA co-precipitated with LRP6-FLAG was 7.9% of total α 6 -HA, and LRP-FLAG co-precipitated with α 6 -HA was 6.9% of total LRP-FLAG.
PTPζ co-precipitated with α 6 -HA was 4.2% of total PTPζ. PTPζ co-precipitated with LRP6-FLAG was 10% of total PTPζ. β 1 -HA co-precipitated with LRP6-FLAG was 0.64% of total β 1 -HA, and LRP-FLAG co-precipitated with β 1 -HA was 0.40% of total LRP6-FLAG. PTPζ co-precipitated with β 1 -HA was 0.41% of total PTPζ. Thus, co-precipitation efficiency of β 1 -HA was low and, in other cases, the value was in the range 4-10%. It is likely that the β 1 -subunit plays an important role in complex formation and the HA tag hinders the process.
When 200 ng ml -1 of MK was added to the culture medium, the proportion of tagged molecules in the co-precipitates of α 6 -HA and LRP6-FLAG did not increase significantly (data not shown). However, the proportion of β 1 -HA co-precipitated with LRP6-FLAG increased about fourfold (Fig. 5G). When the duration of transfection was shortened to lower the expression of α 6 -HA and LRP6-FLAG, α 6 -HA co-precipitated with LRP6-FLAG was 2.8% of total α 6 -HA. On this occasion, 200 ng ml -1 of MK increased the co-precipitation of α 6 -HA about threefold (Fig. 5H). We concluded that exogenous MK increased the efficiency of co-precipitation when the degree of co-precipitation was low in the absence of MK.

Discussion
We have provided evidence that MK specifically binds to α 4 β 1 -and α 6 β 1 -integrins. The ligands of α 4 β 1 -integrin are fibronectin, a major component of the ECM, and VCAM, a member of the immunoglobulin superfamily. α 4 β 1 -Integrin recognizes LDV in the alternatively spliced III CS domain of fibronectin (Guan and Hynes, 1990;Kleiman and Mosher, 2002). α 4 β 1 -Integrin plays important roles in cell migrationit governs lymphocyte migration (Rose et al., 2001), is involved in recruitment of neutrophils to inflammatory sites (Burns et al., 2001;Henderson et al., 2001) and is essential for the migration of epicardial progenitor cells to the surface of the heart to form the epicardium (Sengbusch et al., 2002). MK also promotes the migration of neutrophils, macrophages, UMR-106 osteoblastic cells and neurons (Takada et al., 1997;Maeda et al., 1999;Horiba et al., 2000;Qi et al., 2001). Therefore, we postulated that MK-induced migration of UMR-106 cells might be mediated by α 4 β 1 -integrin. Indeed, a function-blocking monoclonal antibody against α 4integrin inhibited MK-induced migration of UMR-106 cells in a concentration-dependent manner. Furthermore, we found that an LDV-containing decapeptide of fibronectin (also called CSI), but not an RGD peptide, inhibited the migration of these cells.
We also found that, in UMR-106 osteoblastic cells, soluble MK transiently increased the tyrosine phosphorylation of paxillin, which is an adapter molecule that directly binds to the cytoplasmic portion of α 4 -integrins (Turner, 2000;Schaller, 2001;Rose et al., 2002). The phosphorylation of paxillin at a tyrosine residue is known to promote cell migration through downstream systems involving Crk-II (Petit et al., 2000) or by suppressing RhoA (Tsubouchi et al., 2002). It should also be mentioned that substratumbound MK, but not soluble MK stimulates migration of UMR-106 cells (Qi et al., 2001), whereas both substratum-bound and soluble MK stimulate migration of macrophages (Horiba et al., 2000). It is likely that, in addition to phosphorylation of paxillin, another intracellular signal is required for migration of UMR-106 cells, and that the signal is delivered by substratum-bound MK. One ligand of α 6 β 1 -integrin is laminin, another component of the ECM. Laminin is one of the most potent promoters of neurite outgrowth of neuronal cells in culture, and α 6 β 1 -integrin is implicated in neuronal adhesion and neurite outgrowth on laminin (DeCurtis and Reichardt, 1993). In embryonic neurons, MK promotes neurite outgrowth and migration, and also has anti-apoptotic activity (Muramatsu et al., 1991;Muramatsu et al., 1993;Michikawa et al., 1993;Owada et al., 1999). These effects on embryonic neurons appear to be at least partially mediated by α 6 -integrin, because MK-Journal of Cell Science 117 (22) Fig. 7. α 4 β 1 -or α 6 β 1 -Integrin co-precipitates with PTPζ. COS-7 cells were co-transfected with PTPζ and β 1 -HA (A,B), PTPζ and α 6 -HA (C,D) or PTPζ and α 4 -FLAG (E,F). Single transfections were also done. After precipitation with anti-PTPζ antibody (A,C,E), anti-HA antibody (B,D) or anti-FLAG antibody (F), the immunoprecipitates were probed for the presence of β 1 -HA (A), α 6 -HA (C), α 4 -FLAG (E) or PTPζ (B,D,F) by immunoblotting using appropriate antibodies.