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J. Biol. Chem., Vol. 282, Issue 20, 14853-14860, May 18, 2007

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The LG1-3 Tandem of Laminin 5 Harbors the Binding Sites of Lutheran/Basal Cell Adhesion Molecule and 31/61 Integrins^*

Yamato Kikkawa^¶1, Takako Sasaki^||, Mai Tuyet Nguyen^||, Motoyoshi Nomizu^¶, Toshihiro Mitaka, and Jeffrey H. Miner^**

From the Renal Division, Department of Internal Medicine and ^**Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, Department of Pathophysiology, Cancer Research Institute, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan, ^¶Laboratory of Clinical Biochemistry, Tokyo University of Pharmacy and Life Science, Tokyo 192-0392, Japan, and ^||Max-Planck-Institut fur Biochemie, D-82152 Martinsried, Germany

Received for publication, December 21, 2006 , and in revised form, March 23, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The laminin-type globular (LG) domains of laminin chains have been implicated in various cellular interactions that are mediated through receptors such as integrins, -dystroglycan, syndecans, and the Lutheran blood group glycoprotein (Lu). Lu, an Ig superfamily transmembrane receptor specific for laminin 5, is also known as basal cell adhesion molecule (B-CAM). Although Lu/B-CAM binds to the LG domain of laminin 5, the binding site has not been precisely defined. To better delineate this binding site, we produced a series of recombinant laminin trimers containing modified chains, such that all or part of 5LG was replaced with analogous segments of human laminin 1LG. In solid phase binding assays using a soluble Lu (Lu-Fc) composed of the Lu extracellular domain and human IgG1 Fc, we found that Lu bound to Mr5G3, a recombinant laminin containing 5 domains LN through LG3 fused to human laminin 1LG4–5. However, Lu/B-CAM did not bind other recombinant laminins containing 5LG3 unless 5LG1–2 was also present. A recombinant 5LG1-3 tandem lacking the laminin coiled coil (LCC) domain did not reproduce the activity of Lu/B-CAM binding. Therefore, proper structure of the 5LG1-3 tandem with the LCC domain was essential for the binding of Lu/B-CAM to laminin 5. Our results also suggest that the binding site for Lu/B-CAM on laminin 5 may overlap with that of integrins 31 and 61.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Laminins are a family of heterotrimeric glycoproteins that assemble into basement membranes. The laminin-511 trimer (511) is a major isoform widely expressed in fetal and adult tissues. Many of the biological functions of laminin-511 are mediated through the 5 subunit. Mice lacking laminin 5 die during late embryogenesis with several developmental defects, including defects in neural tube formation, digit separation, placentation, glomerulogenesis, lung lobe separation, intestinal smooth muscle development, and tooth morphogenesis (1–6). A grafting experiment to bypass embryonic lethality has shown that laminin 5 is also required for hair follicle development (7). Deletion of 5 during lung development causes a delayed differentiation of distal epithelial cells and a reduced capillary density associated with a decrease in lung vascular endothelial growth factor production (8). Moreover, a hypomorphic mutation in laminin 5 causes polycystic kidney disease (9).

All five laminin chains have a laminin-type globular (LG)² domain at their COOH termini, which consists of five homologous domains (LG1–LG5). Molecular dissection of the laminin 5 LG domain in vivo revealed that the LG1-2 tandem harbors most of the functionality of the 5LG domain with regard to mediating developmental processes, whereas the 5LG3-5 tandem appears to be involved in glomerular filtration (5, 10).

The interaction of cells with laminins is mediated by various receptors, including integrins and non-integrin receptors (11). Laminin-511/521 (511/521) trimers are bound by several different receptors, including integrins 31, 61, and 64 (12, 13), -dystroglycan (14), and Lutheran/basal cell adhesion molecule (Lu/B-CAM) (15–17). Lu is a member of the immunoglobulin superfamily and has been studied primarily in the contexts of blood group antigens and sickle cell disease (18, 19). B-CAM, a splice variant of Lu, has the same NH₂-terminal extracellular domain as Lu, but it lacks the COOH-terminal 40 amino acids of the cytoplasmic tail, which carries an Src homology 3 binding motif and potential phosphorylation sites that could be involved in intracellular signaling pathways (20).

In previous studies, we have reported that laminin 5 (but not the other four laminin chains) is a ligand for Lu/B-CAM (17, 21). We have also reported that the Lu/B-CAM binding site maps within the 5LG domain and that the 5LG3 domain is essential for Lu/B-CAM binding to laminin 5. Similarly, several groups have attempted to identify binding sites for integrins, syndecans, and -dystroglycan within laminin chain LG domains (22, 23). Yu and Talts (24) reported that 5LG1-3 and 5LG4-5 recombinant fragments had cell-adhesive activity dependent on 31/61 integrins and -dystroglycan, respectively. Recently, Ido et al. (25) produced recombinant laminin trimers containing a modified 5 chain with serial deletions of LG1–5 and found that 5LG3 was essential for binding of the 31 and 61 integrins. These results lead to the conclusion that 5LG3 is critical for the binding of Lu/B-CAM and integrins 31 and 61. However, the precise structure of the binding sites for these receptors within 5LG remains unclear.

Here we produced a series of recombinant laminins to narrow the binding site of Lu/B-CAM within the laminin 5LG domain. Solid phase binding assays to these laminins were performed with a soluble recombinant Lu (Lu-Fc) composed of the extracellular domain of Lu fused to IgG1 Fc. Lu/B-CAM binding to laminin required the 5LG1-3 tandem, as did integrins 31 and 61 binding to laminin. Furthermore, we examined competition between Lu/B-CAM and 31/61 integrins for binding to 5LG1-3. Our results indicate that the binding site for Lu/B-CAM on 5LG1-3 overlaps with that of the 31/61 integrins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—Human embryonic kidney (HEK293) and human fibrosarcoma (HT1080) cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and Health Science Research Resources Bank (Osaka, Japan), respectively. Both cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum. K562 human erythroleukemia cells transfected with cDNA encoding human integrin 3A or 6A subunits were kindly provided by Dr. Arnoud Sonnenberg (The Netherlands Cancer Institute) and maintained in RPMI 1640 medium containing 10% fetal calf serum and 1 mg/ml G418 (Sigma) (26).

Antibodies and Proteins—Monoclonal antibody against human laminin 1LG4–5 (163DE4) (27) was a gift from Dr. Ismo Virtanen (University of Helsinki, Helsinki, Finland). Polyclonal antibody against laminin 5 domain LN/LEa has been described previously (28). Monoclonal antibodies against human Lu (BRIC108 and BRIC221) were purchased from Biogenesis (Kingston, NH) and Serotec (Oxford, UK), respectively. Monoclonal antibody against human integrin 3 (P1B5) was purchased from Chemicon International (Temecula, CA). Rat monoclonal antibody against human integrin 6 (GoH3) was purchased from Pharmingen. Anti-human integrin 1 (TS2/16) was purified on a protein G-Sepharose 4B column from the conditioned medium of hybridoma cells purchased from the ATCC. Human IgG1 Fc control protein was prepared as described previously (29).

Construction of Expression Vectors—Laminin chain expression vectors were prepared as follows. cDNA clones encoding full-length mouse laminin 5 with the FLAG sequence at the COOH terminus (generated in our laboratory) and human laminin 1 (provided by Dr. Karl Tryggvason, Stockholm, Sweden) were used to construct expression vectors encoding the chimeric laminin chains Mr51, Mr5G1, Mr5G2, Mr5G3, Mr5G13, and Mr5G23. PCR was used to introduce restriction sites at the appropriate locations and to seamlessly join amplified fragments with overlapping sequences by sequential PCR (30). For all PCR, Vent polymerase (New England Biolabs, Beverly, MA) was used according to the manufacturer's instructions. Preparation of Mr5, Mr51, Mr5G2, and Mr5G3 has been described previously (17). For generating Mr5G1, we took advantage of a unique AgeI site at the end of the sequence encoding 5LG1. To construct Mr5G1, human 1LG2–5 was amplified with sense (5'-AGGCGCGCCTCTAGAGCACCGGTTCCCAGAATGAAGACCCTTCC-3') and antisense (5'-AGGCGCGCCCGTACGTCAGGACTCGGTCCCAGGAC-3'). This segment was ligated to the cDNA encoding 5 domains LN through LG1. To construct Mr5G13, we used AgeI and SpeI sites in sequences encoding the 5LG domain. Human 1LG2 was amplified with sense (using the same sense primer to generate Mr5G1) and antisense (5'-AGTCATGGTGCGTCCCACTAGCAGGAAGCTAACACTCCGGAT-3'); mouse 5LG3 was amplified with sense (5'-ATCCGGAGTGTTAGCTTCCTGCTAGTGGGACGCACCATGACT-3') and antisense (5'-CCGACCACATGTCGGATGCGA-3'). These two products were mixed and subjected to PCR again for 20 cycles to join them. The amplified PCR product was inserted into the AgeI and SpeI sites of Mr5G3. To construct Mr5G23, we used the Mr51 cDNA to make a chimeric segment of 5LCC and 1LG1. The chimeric segment was amplified with sense (5'-GCCCTGAATGAGCTGGCATC-3') and antisense (5'-ACCGGTCTGGGAGCTTCCGAAGCACCCA-3'). The amplified PCR product was inserted into the MluI and AgeI sites of Mr5G3. The full-length mouse laminin 5 with the COOH-terminal FLAG tag and chimeric chains were then cloned into pcDNA3.1Zeo(+) (Invitrogen) and used to produce recombinant laminins. cDNA clones encoding mouse laminin 1 chains (provided by Dr. Albert E. Chung, University of Pittsburgh, Pittsburgh, PA) and 1 (provided by Dr. Peter D. Yurchenco, Robert Wood Johnson Medical School, Piscataway, NJ) were cloned into pcDNA3.1Neo(+) (Invitrogen) and pIREShyg2 (BD Biosciences Clontech), respectively.

The expression vector containing 5LG1-3 was prepared as follows. The DNA sequence encoding the LG1-3 domain of laminin 5 was amplified by reverse transcription-PCR using RNA from mouse embryonic endothelial cells and the primer pair sense (5'-GTCAGCTAGCCGCCAGCAAGGTCAAGG-3') and antisense (5'-GTCACTCGAGCTACCTTGAGGTCTCGATGAG-3'). The PCR product was inserted into the NheI-XhoI site of the episomal expression vector pCEP-Pu containing the BM-40 signal peptide (31).

An expression vector containing Lu-Fc was prepared as follows. A DNA fragment encoding human IgG1 Fc was prepared from the CD4-Ig vector (32) and inserted into the BamHI-XbaI sites of pcDNA3.1Neo(+). A full-length human Lu cDNA was purchased from Invitrogen and used as a template for PCR. DNA sequences encoding the extracellular domain of Lu/B-CAM was amplified using sense (5'-CGGGATCCGCCACCATGGAGCCCCCGGACGCACCG-3') and antisense (5'-CGGGATCCACTCCAGCCTGGGAGGTCTG-3'). The PCR product was inserted into the BamHI site of the above human IgG1 Fc expression vector.

Expression and Purification of Recombinant Proteins—HEK293 cells were transfected with the mouse laminin 1 expression vector using Lipofectamine 2000 (Invitrogen), and stable clones were selected using 400 µg/ml hygromycin (Invitrogen). All further cell culture and clonal expansion were carried out in the presence of the relevant antibiotics for positive selection. A clone highly expressing mouse laminin 1 was then transfected with the mouse laminin 1 expression vector, and stable clones were selected using 800 µg/ml G418 (Sigma). A clone highly expressing laminin 1 and 1 was transfected with mouse laminin 5 or chimeric chain expression vectors, and stable clones were selected using 400 µg/ml Zeocin (Invitrogen). The selected clones were grown to confluency in 24-well culture plates with DMEM containing 10% fetal bovine serum. The confluent cells were incubated in serum-free DMEM for four days. The conditioned media were harvested and clarified by centrifugation at 10,000 revolutions/min for 20 min. They were screened for the expression of recombinant laminins by immunoblotting with antibody against the laminin 5 domain LN/LEa. The clones showing high secretion were grown to confluency in culture dishes with DMEM containing 10% fetal bovine serum. The confluent cells were incubated in serum-free DMEM for four days. The conditioned media were harvested and clarified by sequential centrifugation at 500 revolutions/min for 10 min and 10,000 revolutions/min for 20 min. The collected media were precipitated with ammonium sulfate at 80% saturation. The resulting precipitates were collected by centrifugation at 10,000 revolutions/min for 30 min and then dissolved in and dialyzed against PBS(–). The 30-fold concentrated media were used for purification. The recombinant laminins were purified from the conditioned culture media by anti-FLAG or anti-human laminin 1LG4–5 monoclonal antibody coupled to agarose. The eluted fractions were pooled and dialyzed against 10 mM Tris-HCl, pH 7.5, 150 mM, 0.1% CHAPS, 1 mM EDTA, and 1% sucrose.

Human embryonic kidney EBNA-293 cells were transfected with the 5LG1-3 expression vector and the serum-free media were collected as described previously (31). The serum-free media were passed over a heparin-Sepharose column (2 x 25 cm) equilibrated in 50 mM Tris-HCl, pH 7.4, and eluted with 0–0.5 M NaCl gradient. The second purification was achieved on Superose 12 HR16/50 (GE Healthcare Biosciences) equilibrated with 0.2 M ammonium acetate. The pooled fraction was further purified on MonoQ HR5/5 (GE Healthcare Biosciences) equilibrated with 20 mM Tris-HCl, pH 8.0, and eluted with a linear gradient of 0–0.6 M NaCl.

HEK293 cells were transfected with Lu-Fc expression vector using Lipofectamine 2000 (Invitrogen), and stable clones were selected using 1 mg/ml G418 (Sigma). The conditioned media were prepared as described above. Recombinant protein was purified from the conditioned media by protein-A-Sepharose (GE Healthcare Biosciences). The eluted fractions were pooled and dialyzed against PBS(–).

Electrophoretic Analysis and Immunoblotting—SDS-PAGE was carried out on 5 or 7.5% gels under reducing or non-reducing conditions. Proteins were visualized with silver staining or Coomassie Brilliant Blue. For immunoblotting, sample proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Proteins on the membrane were reacted with first antibodies followed by incubation with secondary antibody conjugated with horseradish peroxidase (GE Healthcare Biosciences). Bound antibodies were visualized with ECL Western blotting detection reagents (GE Healthcare Biosciences).

Solid Phase Binding Assay—Solid phase binding assays were carried out with recombinant proteins coated onto the plastic surface of microtiter plates. Plates were blocked with PBS(–) containing 1% bovine serum albumin and incubated with Lu-Fc at 37 °C for 1 h. After washing with PBS(–), the bound Lu-Fc was detected with a biotinylated anti-human IgG1 Fc monoclonal antibody (Sigma). After further washing, the bound antibodies were detected by the addition of streptavidin-conjugated horseradish-peroxidase (GE Healthcare Biosciences) followed by the addition of 1 mg/ml o-phenylenediamine and 0.012% H₂O₂. The absorbance was measured at 450 nm with a Microplate Reader Model 550 (Bio-Rad).

Cell Adhesion Assays—Cell adhesion assays were performed as described previously (13). Briefly, 96-well microtiter plates (Nunc, Roskilde, Denmark) were incubated with recombinant laminins at 37 °C for 1 h and then blocked with PBS(–) containing 1% bovine serum albumin for another hour. K562 transfectants were suspended in serum-free RPMI 1640 medium at 4 x 10⁵ cells/ml and preincubated with or without 1 µg/ml anti-integrin 1 monoclonal antibody TS2/16 at room temperature for 10 min. 50 µl of cell suspension were added to the laminin-coated wells and incubated at 37 °C for 1 h. The attached cells were fixed with 4% formaldehyde, stained with Diff-Quik (International Regents Corp., Kobe, Japan), and counted under the microscope.

Cell adhesion inhibition assays were performed on the basis of the adhesion assay described above. HT1080 cells were suspended in serum-free DMEM at 4 x 10⁵ cells/ml and preincubated with 1 µg/ml anti-integrin 1 monoclonal antibody TS2/16 at room temperature for 10 min. The cells were further preincubated with 10 µg/ml monoclonal antibodies against different integrins and 20 µg/ml recombinant Lu-Fc protein at room temperature for 10 min. TS2/16-stimulated K562 transfectants were also preincubated with 20 µg/ml recombinant Lu-Fc protein at room temperature for 10 min. The preincubated cells were transferred to plates coated with 10 µg/ml of the recombinant laminins. After incubation at 37 °C for 20 min, the attached cells were stained and counted as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Production of Recombinant Laminins—In a previous study, we reported that 5LG3 is essential for Lu/B-CAM binding to laminin 5 (17). Although our results showed that the 5LG4-5 tandem is not required for Lu/B-CAM binding, whether or not 5LG1-2 is required remains an open question. To further define the Lu/B-CAM binding site on the laminin 5LG domain, we produced a series of recombinant laminin trimers containing chimeric 5 chains in which all or part of laminin 5LG was replaced with analogous segments of human laminin 1 (Fig. 1). Although the functional structure of laminins has in some cases been examined by producing deletion mutants (25, 33), the deletion approach often leads to disruptions in the tertiary structure. Therefore, we undertook a chimeric approach using laminin 5 and 1 chains. The human laminin 1LG domain does not bind to Lu/B-CAM, and it also bears an epitope in the 1LG4-5 tandem recognized by a monoclonal antibody.

View larger version (44K): [in this window] [in a new window]   FIGURE 1. Schematic diagrams of laminin 5 and chimeric laminin chains. Shown are the following: full-length mouse laminin 5 chain (Mr5), the chimeric construct encoding laminin 5 domains LN through LCC linked to the human laminin 1 LG domain (shaded, Mr51), and Mr5G1, Mr5G2, and Mr5G3, which contain laminin 5 domains LN through 5 LG1, LG2, and LG3 fused to the human laminin 1 LG2-5, LG3-5, and LG4-5 domains, respectively. LG2 and LG1 of Mr5G3 were replaced with analogous human laminin 1 segments (shaded, Mr5G13 and Mr5G23), respectively.

View larger version (61K): [in this window] [in a new window]   FIGURE 2. Purification of recombinant laminins in mammalian cells. HEK293 cells expressing laminin1 and1 were transfected with a laminin5 expression vector or chimeric laminin chains. Recombinant laminins containing Mr5 or a chimeric laminin chain were purified from the conditioned media of triple-transfected HEK293 cells using anti-FLAG or anti-human laminin 1 LG4–5 monoclonal antibodies coupled to agarose. Purified proteins were analyzed by 5% SDS-PAGE under reducing conditions and visualized by silver staining. The chains assembled with 1 and 1 chains. The position of reduced Engelbreth-Holm-Swarm laminin-111 (400 and 200 kDa) is indicated at the left.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Solid phase binding assays of Lu-Fc to immobilized recombinant laminins. A, Lu-Fc and Fc purified from conditioned medium of the transfectants were subjected to SDS-PAGE on a 7.5% gel under reducing conditions. Lu-Fc (lane 1) and Fc (lane 2) were stained with Coomassie Brilliant Blue. Molecular mass standards are indicated. B, 96-well microtiter plates were coated with recombinant laminins at 10 µg/ml. After blocking, the wells were incubated with 5 µg/ml Lu-Fc at 37 °C for 1 h. The recombinant Fc protein was used as the control. Bound Lu-Fc was detected with anti-human IgG1 Fc antibody. Each column represents the mean ± S.D. of triplicate assays. OD, optical density.

Solid phase binding assays were performed using the purified recombinant laminins and Lu-Fc. Consistent with our previous study, Lu-Fc bound to recombinant laminin trimers containing Mr5G3 but not Mr5G2 (Fig. 3B). The straightforward interpretation is that Lu/B-CAM binds to 5LG3. However, the other recombinant laminins containing 5LG3 (Mr5G13 and Mr5G23) did not bind Lu-Fc. These results indicate that Lu/B-CAM binding requires not only 5LG3 but also 5LG1–2. We conclude that the entire 5LG1-3 tandem structure is essential for Lu/B-CAM binding to laminin 5.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Lu-Fc binding activity of recombinant 5LG1–3. A, 5LG1-3 purified from conditioned media was subjected to SDS-PAGE on a 7.5% gel under non-reducing conditions and stained with Coomassie Brilliant Blue. Molecular mass standards are indicated. B, dose-response binding of Lu-Fc to recombinant laminin-511 (open circles) or 5LG1–3 only (open squares) coated in increasing concentrations on 96-well microtiter plates. Lu-Fc slightly bound to 5LG1–3 and only at high concentrations. Similar results were obtained in three independent experiments. OD, optical density.

The Binding of Lu/B-CAM and Integrins on the Laminin 5 LG Domain—Laminin 5 is also recognized by integrins 31, 61, 64, and v3 (13, 36). Ido et al. (25) report that 5LG3 was essential for the binding of 31/61 integrins. Recent advances have shown that the binding of integrin 61 requires not only 5LG3 but also 5LG1–2 (35). To confirm the binding sites for both 31 and 61 integrins, we examined the adhesion of K562 erythroleukemia cells transfected with integrin 3 or 6 to the recombinant laminins containing chimeric chains. The transfectants expressing integrin 31or 61 could bind to recombinant laminins containing Mr5G3 but not Mr5G2, indicating that 5LG3 is essential for the binding of 31/61 integrins (Fig. 5). Moreover these integrins could not bind to the other recombinant laminins containing 5LG3 but lacking the 5LG1-2 tandem, consistent with the reports cited above. Thus, the entire 5LG1-3 tandem was required for integrin binding to laminin 5, as it is for Lu/B-CAM binding.

That both the integrins and Lu/B-CAM bind to 5LG1–3 suggests that the binding of Lu/B-CAM to 5LG1–3 might compete with binding by 31/61 integrins. To test this possibility, cell adhesion inhibition assays in the presence of Lu-Fc were performed using human fibrosarcoma HT1080 cells stimulated with activating anti-integrin 1 monoclonal antibody TS2/16 (Fig. 6). The adhesion of unstimulated and TS2/16-stimulated HT1080 cells to laminin 5 was inhibited by a combination of anti-integrin 3 and 6 antibodies, in agreement with our previous study (13). Lu-Fc could partially inhibit the adhesion of unstimulated HT1080 cells to laminin 5, but the adhesion of TS2/16-stimulated cells was not inhibited any longer by Lu-Fc. The inhibitory effects of Lu-Fc were dependent on the activation state of integrins, suggesting that the binding of Lu/B-CAM on laminin 5 competed with that of 31/61 integrins. Furthermore, to determine whether the binding of Lu/B-CAM to laminin 5 occupied or obscured the binding site for 31/61 integrins, we performed adhesion assays using K562 cells transfected with integrin 3or 6 in the presence of Lu-Fc. Lu-Fc could efficiently inhibit the adhesion of the transfectants to laminin 5 (Fig. 7). Therefore, we conclude that the binding site for Lu/B-CAM on laminin 5 is close to that for 31/61 integrins.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Adhesion of K562 transfectants expressing integrin 31or 61 to recombinant laminins. K562 non-transfectants (K562) and transfectants expressing 31(3A/K562) or 61(6A/K562) were preincubated with or without the activating anti-integrin 1 antibody TS2/16. The unstimulated (white columns) or stimulated (gray columns) cells were added to 96-well microtiter plates coated with 10 µg/ml recombinant laminins and incubated at 37 °C for 1 h. Adherent cells were stained and counted as described under "Experimental Procedures." Each column represents the mean ± S.D. of triplicate assays.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Inhibitory effects of anti-integrin antibodies and Lu-Fc on adhesion of HT1080 human fibrosarcoma cells to laminin 5. Unstimulated (white columns) or TS2/16-stimulated (gray columns) HT1080 cells were preincubated with anti-integrin antibody, Lu-Fc, or Fc only and added to wells coated with 10 µg/ml recombinant laminin-511. Adherent cells were stained and counted as described under "Experimental Procedures." The number of cells adhering to laminin-511 in the absence of antibody (none) was taken as 100%. Each column represented the mean ± S.D. of triplicate assays.

View larger version (13K): [in this window] [in a new window]   FIGURE 7. Inhibitory effects of Lu-Fc on the adhesion of K562 transfectants expressing integrin 31or 61 to laminin 5 chain. K562 non-transfectants (K562) and transfectants expressing 31(3A/K562) or 61 (6A/K562) were incubated with anti-integrin 1 antibody TS2/16. The stimulated transfectants were incubated in wells coated with 10 µg/ml recombinant laminin-511 for 30 min at 37 °C in the absence (white columns) and presence of Lu-Fc (gray columns) or Fc (black columns). Each column represents the mean ± S.D. of triplicate assays.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Integrin 61 binding activity is known to be present in LG1–3 of both laminin-511 and laminin-111 (22, 35). Therefore, all chimeric chains, including either 1or 5LG1–3, should bind to integrin 61. However, because the binding of integrin 61 to laminin 1 is weaker than to 5 (13, 37), the chimeric chains carrying all or part of 1LG1–3 exhibited drastically reduced binding to integrin 61 (Fig. 5). As shown in the figure, the coating concentration of 10 µg/ml is enough for the adhesion of K562 cells expressing integrin 61 to 5LG1–3 but not to the modified 5 chains containing all or part of 1LG1–3. Recently, another group produced a similar series of recombinant laminins containing laminin 5 chains swapped with each of the 1LG1-3 domains (35). The altered 5 chain also reduced the binding affinity of integrin 61.

A recombinant human Lu extracellular domain fused with the Fc portion of human IgG1 (Lu-Fc) was produced in mammalian cells, as described in other studies (15, 38). The recombinant proteins dimerized at the Fc region. As expected, dimerized Lu-Fc bound to laminin 5 more effectively than did monomeric Lu (Sol-Lu) (17). Solid phase binding assays showed that recombinant laminin containing Mr5G3 but not Mr5G2 bound to Lu-Fc. In agreement with our previous study, the 5LG3 domain is definitely required for Lu/B-CAM binding. However, the other recombinant laminins containing 5LG3 (Mr5G13 and Mr5G23) were not bound by Lu-Fc, suggesting that the 5LG3 domain is necessary but not sufficient to impart binding activity. These results allowed us to conclude that the entire structure of the 5LG1-3 tandem is essential for Lu/B-CAM binding to laminin 5.

The LG1-3 tandem of laminin chains is predicted to form the shape of a cloverleaf in contact with the LCC domain (22). The binding site of Lu/B-CAM could be formed at the tertiary structure level. Moreover, because the recombinant isolated 5LG1-3 tandem did not bind to Lu-Fc, the LCC domain may be required for the proper folding of 5LG1–3 within the LG domain. In addition, the Lu-Fc binding activity of Mr5G3 was lower than that of the full-length 5 chain, suggesting that the 5LG4-5 tandem (replaced by 1LG4–5 in Mr5G3) is involved in Lu/B-CAM binding. Accumulating evidence indicates that LG4-5 tandems are involved in laminin binding to -dystroglycan and syndecan (22, 23). It is unlikely that the manner of Lu/B-CAM binding is similar to that of -dystroglycan and syndecans. Because structural analysis predicts that LG4–5 is spatially located over the cloverleaf of LG1–3 (22, 33), the possibility exists that 5LG4–5 regulates the binding affinity of Lu/B-CAM to 5LG1–3.

Integrin-mediated cell adhesion is one of the hallmarks of the biological functions of laminin LG domains. Many studies that have functionally dissected LG domains have addressed the regions of various laminin isoforms that are responsible for integrin-mediated cell adhesion (22, 24, 25). Recently, Ido et al. (35) reported that the entire 5LG1-3 tandem was essential for the binding of integrin 61. We also found that the binding sites for 31/61 integrins were within the 5LG1-3 tandem. In a recent study, we have reported that soluble recombinant Lu inhibits the adhesion of human corneal epithelial cells to laminin-511 as well as function blocking antibodies to 3 and 1, suggesting that the binding sites for Lu and integrin 31 overlap (39). Here, Lu-Fc inhibited 31/61 integrin-mediated cell adhesion to laminin 5. The most straightforward interpretation is that the binding site of Lu/B-CAM on 5LG1-3 overlaps with that of 31/61 integrins. Alternative explanations that cannot be ruled out are that 1) Lu/B-CAM binding causes a change in the structure of 5LG1–3 that prevents integrin binding or 2) steric hindrance prevents binding of both Lu/B-CAM and the integrin. We have also reported that Lu/B-CAM mediates the adhesion of human mesangial and endothelial cells to laminin 5 in collaboration with integrins containing the 1 subunit (5, 40). As Lu-Fc did not inhibit the adhesion of HT1080 cells activated with anti-integrin 1 monoclonal antibody, the activation status of integrins on cells may affect the binding of Lu/B-CAM to 5LG1–3.

Distribution studies in various tissues have shown that Lu/B-CAM and 31/61 integrins are localized at the basal surface of cells, facing a basement membrane containing laminin 5 (20, 41–43). We have also observed that Lu/B-CAM is co-localized with 31/61 integrins in some mouse tissues (21).³ Therefore, it is likely that Lu/B-CAM competitively interacts with 31/61 integrins for binding to the 5LG1–3 domain in vivo. Lu (but not B-CAM) contains the 40 amino acids of the cytoplasmic domain that carries the Src homology 3 binding motif and potential phosphorylation sites that could be involved in intracellular signaling pathways (20). Hines et al. (44) show that the physiologic stress mediator epinephrine, acting through the 2-adrenergic receptor, increased the adhesion of sickle red blood cells to laminin 5 via inside-out signals. Recently, it has been reported that cAMP signaling can promote adhesion to laminin 5 via Lu through two distinctive signaling pathways (45, 46). The activated binding of Lu to laminin 5 may modulate intra-cellular signaling from the 5 chain via 31/61 integrins.

This study provides new insights into both integrin- and non-integrin-mediated cell adhesion to basement membranes containing laminin 5. Although we have narrowed the binding site of Lu/B-CAM on the laminin 5LG domain to the LG1-3 tandem, in the future it will be important to analyze the binding site at the level of tertiary structure. Also, Lu-Fc may serve as an important tool to identify integrin binding sites on the 5LG1-3 tandem.

	FOOTNOTES

 
^* This work was supported, in part, by Grants 17590307 and 18060041 from the Ministry of Education, Sciences, Sports, and Culture, Japan (to Y. K.) and National Institutes of Health Grants R01DK064687 and R01GM060432 (to J. H. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

¹ To whom correspondence should be addressed: Tokyo University of Pharmacy and Life Science, School of Pharmacy, Laboratory of Clinical Biochemistry, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Tel.: 81-42-676-5670; Fax: 81-42-676-5669; E-mail: kikkawa{at}ps.toyaku.ac.jp.

² The abbreviations used are: LG, laminin-type globular; LCC, laminin coiled coil; Lu, Lutheran blood group glycoprotein; B-CAM, basal cell adhesion molecule; PBS(–), Ca²⁺ and Mg²⁺-free phosphate-buffered saline; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

³ Y. Kikkawa and J. H. Miner, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Dr. Arnoud Sonnenberg for providing K562 transfectants.

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