Identification of Cell-binding Sites on the Laminin alpha 5 N-terminal Domain by Site-directed Mutagenesis*

Peter K. Nielsen and Yoshihiko YamadaDagger

From the Molecular Biology Section, Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370

Received for publication, September 25, 2000, and in revised form, November 13, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The newly discovered laminin alpha 5 chain is a multidomain, extracellular matrix protein implicated in various biological functions such as the development of blood vessels and nerves. The N-terminal globular domain of the laminin alpha  chains has an important role for biological activities through interactions with cell surface receptors. In this study, we identified residues that are critical for cell binding within the laminin alpha 5 N-terminal globular domain VI (~270 residues) using site-directed mutagenesis and synthetic peptides. A recombinant protein of domain VI and the first four epidermal growth factor-like repeats of domain V, generated in a mammalian expression system, was highly active for HT-1080 cell binding, while a recombinant protein consisting of only the epidermal growth factor-like repeats showed no cell binding. By competition analysis with synthetic peptides for cell binding, we identified two sequences: S2, 123GQVFHVAYVLIKF135 and S6, 225RDFTKATNIRLRFLR239, within domain VI that inhibited cell binding to domain VI. Alanine substitution mutagenesis indicated that four residues (Tyr130, Arg225, Lys229, and Arg239) within these two sequences are crucial for cell binding. Real-time heparin-binding kinetics of the domain VI mutants analyzed by surface plasmon resonance indicated that Arg239 of S6 was critical for both heparin and cell binding. In addition, cell binding to domain VI was inhibited by heparin/heparan sulfate, which suggests an overlap of cell and heparin-binding sites. Furthermore, inhibition studies using integrin subunit monoclonal antibodies showed that integrin alpha 3beta 1 was a major receptor for domain VI binding. Our results provide evidence that two sites spaced about 90 residues apart within the laminin alpha 5 chain N-terminal globular domain VI are critical for cell surface receptor binding.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Laminins are heterotrimeric basement membrane proteins that exert multiple biological functions through interactions with other matrix molecules and cell surface receptors. The regulation of these interactions is critical to many biological processes, including cell adhesion, migration, angiogenesis, tumor progression, and neurite outgrowth (for review, see Ref. 1). The laminin family contains at least 11 chains (five alpha , three beta , and three gamma  chains). The newly discovered alpha 5 chain is the most widely expressed member of the laminin alpha  chain family (2). Mutant mice that lack the alpha 5 chain have defects in neural tube closure, digit septation, placentation, and glomerulogenesis, suggesting that the alpha 5 chain has multifunctional roles in tissue development (3, 4). The laminin alpha 5 chain associates with the gamma 1 chain and either beta 1 or beta 2 chains to form laminin-10 and laminin-11, respectively (2). Laminin-10/11 is a ligand for several cell surface receptors, including alpha -dystroglycan (5), alpha 3beta 1, alpha 6beta 1, and alpha 6beta 4 integrins (6, 7). Laminin-11 has been shown to inhibit neurite outgrowth in vitro, while other laminin isoforms promote neurite outgrowth, suggesting a unique role of the alpha 5 chain in neural development (see Refs. 8 and 9, for review, see Ref. 10). Interestingly, SV2, a transmembrane keratan sulfate proteoglycan found in synaptic vesicles, has recently been proposed to act as a laminin alpha 5 receptor, suggesting a role of the laminin alpha 5 chain in nerve regeneration (11).

The N-terminal globular domain (domain VI, ~250-270 residues) is specific for laminin and is the most conserved (~60% sequence identity) among the laminin domains (12). Studies with function-blocking antibodies and cell binding assays have indicated that domain VI of both the laminin alpha 1 and alpha 2 chains contains binding sites for the alpha 1beta 1 and alpha 2beta 1 integrins (13-15). Furthermore, domain VI is also capable of binding heparin and heparan sulfate chains of perlecan (13-15). In addition to binding functions for cell surface receptor and for matrix proteins, domain VI is also essential for the self-assembly of laminins (13, 16, 18). Previous studies showed that the synthetic peptide RQVFQVAYIIIKA (A-13), derived from domain VI of the laminin alpha 1 chain, binds beta 1 subunit-containing integrins (19). The active core sequence (VAYI) of this peptide is conserved in the alpha 5 chain, but the functional importance of this site within domain VI has not yet been examined.

In the present work, we studied cell binding functions of mouse laminin alpha 5 domain VI using site-directed mutagenesis and synthetic peptides. We found that two sites, spaced by ~90 amino acids, are required for cell binding. We also identified four residues within the two sites that are essential for the binding. In addition, we demonstrated that an arginine residue of one of the sites is critical for both heparin and cell binding. Our findings suggest that the protein conformation surrounding these sites is important for cell binding through integrin and heparan sulfate-containing cell surface receptors.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Expression Vectors and Site-directed Mutagenesis-- Mouse kidney cDNA was used as a template in polymerase chain reaction to amplify sequences encoding domain VI and the first four EGF1-like repeats of domain V of the laminin alpha 1 and alpha 5 chain. Polymerase chain reaction was performed with PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) using the following primers: 1, GAGAGAAAGCTTCGCACTCCCGGGGGCGATGGC; 2, GAGAGACTCGAGAGTGGCAGCTAGGCCCGTGGAATC; 3, GAGAGAAAGCTTCAGCAGAGAGGCTTGTTCCCTGC; 4, GAGAGACTCGAGAGGAGCAGCCCTCGGGGTTTCG. Primers 1 and 2 were used for laminin alpha 5 domain VI (Arg1-His514), and primers 3 and 4 for the laminin alpha 1 domain VI (Gln1-Ser484). For polymerase chain reaction amplification of the EGF-like repeats of laminin alpha 5 domain V (Arg273-His514), primers 2 and 5, GAGAGAAAGCTTCGCTGTGTCTGTCATGGCCACG, were used. In addition to the coding sequences, these primers contained either a HindIII or a XhoI restriction site. The polymerase chain reaction products were digested with HindIII and XhoI and ligated into the expression vector pSecTag2/Hygro B (Invitrogen, Carlsbad, CA). The resulting expression vectors encode the Igkappa chain leader sequence, a laminin domain, and a c-myc epitope followed by a hexahistidine affinity tag sequence. Site-directed mutagenesis was performed using the Quickchange kit method (Stratagene, La Jolla, CA) and the N-terminal globular domain VI and the first four EGF-like repeats of domain V of the mouse laminin alpha 5 chain, which has been cloned into pBluescript II SK(+) (Stratagene). The following laminin alpha 5 residues were individually substituted with alanine: Tyr130, Arg225, Lys229, Arg234, Arg236, and Arg239. All expression constructions and mutations were verified by DNA sequencing.

Expression and Purification of Recombinant Proteins-- The expression vectors were transiently transfected into monkey kidney COS-7 cells (CRL-1651, ATCC) using FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, IN). The cells were maintained at 37 °C in a humid atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Generation of secreted recombinant His-tagged laminin domains was confirmed by Western blotting of serum-free conditioned medium using an anti-His(C-terminal)-horseradish peroxidase monoclonal antibody (Invitrogen). For recombinant production, conditioned medium (Dulbecco's modified Eagle's medium containing 1% fetal calf serum) was collected 7 days after transfection. The medium was cleared of detached cells and debris through centrifugation, and 1 M Tris-HCl, pH 8.0, and 5 M NaCl were added to final concentrations of 50 mM and 0.5 M, respectively. In addition, 0.2 M phenylmethanesulfonyl fluoride and 3 M imidazole, pH 6.0, were added to concentrations of 1 and 10 mM, respectively. Nickel-charged agarose resin (Probond, Invitrogen) was equilibrated with 50 mM Tris-HCl, 0.3 M NaCl, 10 mM imidazole, pH 8.0, and incubated with the conditioned medium. After incubation at 4 °C for 1 h, the resin was transferred to a column and washed with 50 mM Tris-HCl, 0.3 M NaCl, 20 mM imidazole, pH 8.0. The His-tagged proteins were eluted with 50 mM Tris-HCl, 0.3 M NaCl, 250 mM imidazole, pH 8.0. Purified proteins were dialyzed against phosphate-buffered saline and quantified using a BCA protein assay kit with bovine serum albumin as a standard (Pierce, Rockford, IL). Purity was determined by SDS-polyacrylamide gel electrophoresis followed by colloidal Coomassie G-250 Blue (GelCode Blue, Pierce, Rockford, IL) staining and judged to >95%.

Single-site substitutions may result in a change of the overall fold of domain VI. Gel filtration analysis of mutants, including Y130A and R239A, were therefore performed in 0.05 M phosphate buffer with 0.15 M NaCl, pH 7.0, using a Superose 12 HR 10/30 column on an ÄKTA EXPLORER design system (Amersham Pharmacia Biotech). The majority of the mutants and the wild-type domain VI eluted at a volume corresponding to a molecular weight of ~65,000 (data not shown). The results indicate that these molecules are monomers without mis-folding.

Synthetic Peptides-- Peptide synthesis was performed on ABI model 433A peptide synthesizers at the Facility for Biotechnology Resources (U. S. Food and Drug Administration). All the peptides were prepared with a C-terminal amide group. The peptides were purified by reverse-phase high performance liquid chromatography and characterized by mass spectrometry.

Real-time Heparin Binding Kinetics of Recombinant Proteins Measured by Surface Plasmon Resonance-- Biotinylated heparin (Celsus Laboratories, Inc., Cincinnati, OH) at 40 µg/ml in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, including 0.005% surfactant P20) was immobilized on a streptavidin-coated sensor chip (Sensor Chip SA, BIAcore, Inc., Piscataway, NJ) at 10 µl/min for 4 min to an immobilization level of 300 resonance units. In the affinity measurements, recombinant proteins at different concentrations (50-200 nM) were injected on the heparin-coated surface at 30 µl/min in the running buffer at 25 °C, and the binding and dissociation were registered (2 min each) in a BIAcoreTM 1000 instrument (BIAcore, Inc.). The streptavidin-heparin surface was regenerated at the end of each run by two successive injections of 30 µl of 20 mM NaOH containing 1 M NaCl. In control experiments with the same concentrations of recombinant proteins, but with a blank streptavidin sensor chip, no binding was seen. The sensorgrams obtained were analyzed by nonlinear least square curve fitting using BIAevaluation 2.1 software assuming single-site association and dissociation models.

Cell Binding Assays-- HT-1080 fibrosarcoma cells (CCL-121, ATCC) were detached with 0.05% (w/v) trypsin, 0.02% (w/v) EDTA in PBS, washed with Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin, resuspended to a concentration of 3 × 105 cells/ml, and incubated at 37 °C for 30 min. For evaluation of the effects of synthetic peptides or monoclonal antibodies against integrin subunits, cells were incubated with peptides or antibodies at 37 °C for 30 min. Function-blocking monoclonal antibodies against integrin alpha 1 (FB12), alpha 2 (P1E6), alpha 3 (P1B5), alpha 3 (ASC-1), alpha 4 (P1H4), alpha 5 (P1D6), alpha 6 (NKI-GoH3), alpha V (P3G8), and beta 4 (ASC-3) were purchased from Chemicon International, Inc., Temecula, CA. Anti-integrin alpha 2 (A2-IIE10) was from Upstate Biotechnology, Lake Placid, NY, and anti-integrin beta 1 (mAb13) was a gift from Dr. K. Yamada, National Institutes of Health. Assays were performed in 96-well round-bottom microtiter plates (Immulon-2HB, Dynex Technologies, Inc., Chantilly, VA). Wells were coated for 1 h at room temperature with 50 µl of recombinant proteins or laminin-10/11 (Life Technologies, Inc., catalog number 12163-010, Life Technologies) diluted with Dulbecco's PBS (Dulbecco's PBS) and then blocked for 1 h at room temperature with 200 µl of 1% heat-denatured bovine serum albumin. After washing (Dulbecco's PBS), cells (100 µl) were added and incubated for 60 min at 37 °C in a humidified atmosphere of 5% (v/v) CO2. Wells were washed gently twice with Dulbecco's PBS and stained for 10 min with 0.2% (w/v) crystal violet (Sigma) in 20% (v/v) methanol. After washing with H2O, cells were dissolved in 10% SDS (w/v), and the absorbance at 600 nm was measured. Each sample was assayed in triplicate, and attachment to bovine serum albumin was subtracted from all measurements.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Cell Binding Activity of Recombinant Laminin alpha 5 N-terminal Globular Domain VI-- Constructs were generated for expression of mouse laminin alpha 5 domain VI and the first four EGF-like repeats of domain V in mammalian cells (Fig. 1). In addition, a similar construct was also generated for laminin alpha 1 domain VI, which corresponds to that described previously (15). The secreted recombinant proteins were purified by Ni-agarose chromatography from the conditioned medium of transfected COS-7 cells with a yield of 1-2 µg/ml medium. The purity of the protein preparations was more than 95% as judged from Coomassie Blue-stained gels after SDS-polyacrylamide gel electrophoresis (data not shown). Recombinant fragments containing domains VI through IV of the laminin alpha 1 and alpha 2 chains have previously been shown to promote binding of HT-1080 fibrosarcoma cells (13, 14). This cell line was therefore used to analyze the adhesive properties of the recombinant laminin alpha 5 domain VI. HT-1080 cells showed strong binding to domain VI, while the recombinant domain V, consisting of the EGF-like repeats, showed no cell binding, demonstrating that the cell binding activities reside in domain VI (Fig. 1).



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Fig. 1.   Expression of recombinant laminin alpha 5 domain VI. Schematic representation of the laminin alpha 5 chain constructs used for mammalian expression. The N-terminal globular domain VI and the first four EFG-like repeats of domain V are indicated by VI and E1-4, respectively. The positions of four sequences (S1, S2, S6, and S7), two of which overlap, that inhibited HT-1080 cell binding to laminin alpha 5 domain VI are indicated. Cell binding activity of recombinant laminin alpha 5 domain VI (A5VI) and V (A5V) is shown. HT-1080 cells were added to wells coated with the laminin domains (10 µg/ml). Cell attachment was quantified by crystal violet staining and is expressed as percentage of domain VI (A5VI).

Effect of Synthetic Peptides on Cell Binding to Laminin alpha 5 Domain VI-- To localize potential cell-binding sites on laminin alpha 5 domain VI, we screened synthetic peptides for their inhibitory effect on HT-1080 cell binding to domain VI. We synthesized 15 peptides that corresponded to possible integrin recognition sequences (for review, see Ref. 20) within laminin alpha 5 domain VI and to active sequences previously identified in laminin alpha 1 domain VI (Table I). Four peptides (S1, S2, S6, and S7) were found to inhibit HT-1080 cell binding to domain VI (Fig. 2), whereas the other peptides showed no or only small effects on the cell-domain interaction. A GRGDS peptide, which is reported to block the function of various integrins, had no effect on cell binding. The sequences of peptides S1 and S2 overlap with four residues, but only S2 showed strong activity when tested in a direct cell binding assay (data not shown). The S6 and S7 peptides overlap by eight residues and showed no activity when tested for direct cell binding (data not shown). None of the peptides inhibited cell binding to laminin-10/11, suggesting that other cell-binding sites are available on the intact molecule (data not shown). Taken together, these results suggest that two sequences: S2, 123GQVFHVAYVLIKF135 and S6, 225RDFTKATNIRLRFLR239, separated by ~90 residues, directly interact with cell surface receptors within domain VI.


                              
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Table I
Synthetic peptides of the laminin alpha 5 N-terminal domain VI
The underlined sequences compete with HT-1080 cell-domain VI interactions.



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Fig. 2.   Effects of synthetic peptides on cell/domain VI interactions. HT-1080 cells were incubated for 30 min at 37 °C with synthetic peptides (100 µg/ml) and added to wells coated with laminin alpha 5 domain VI (10 µg/ml). After incubation for 1 h, cell binding was quantified. Values are expressed as percentage of control without peptide (C) and are the mean of three different experiments. Duplicate experiments gave similar results.

Cell Binding Activity of Laminin alpha 5 Domain VI Mutants-- We next examined the functional role of these two sites within domain VI by alanine substitution mutagenesis. Our laboratory has previously identified several synthetic peptides active for cell binding within domain VI of the mouse laminin alpha 1 chain (19). The S2 peptide corresponds to the highly active peptide A-13 (RQVFQVAYIIIKA) of laminin alpha 1 domain VI. Deletion analysis revealed that the VAYI sequence within A-13 is critical for high cell binding activity (19). The active core sequence VAYI is conserved in the laminin alpha 5 chain with the exception of a substitution of Ile with Val (Fig. 3). To test the importance of the tyrosine in the VAYI site, we generated a single substitution mutant by replacing tyrosine with alanine (Y130A). This showed that Tyr130 plays a significant role for cell binding, as the mutant domain VI with Y130A (A5VI-Y130A) reduced cell binding to ~25% of that of wild-type domain VI (Fig. 4).



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Fig. 3.   Alignment of sequences of laminin domain VI corresponding to synthetic peptides active for competition of cell/domain VI interactions. Alignment of domain VI of laminin alpha 1, alpha 2, alpha 3B, and alpha 5 chains. All sequences are mouse. The sequences shown correspond to the two regions active for cell binding within domain VI defined by using synthetic peptides. The residues in bold within the S2 and S6 sites of the laminin alpha 5 chain were replaced with Ala residues by site-directed mutagenesis of laminin alpha 5 domain VI (A5VI) to generate A5VI-Y130A (S2 site), A5VI-R225A (S6 site), A5VI-K229A (S6 site), A5VI-R234A (S6 site), A5VI-R236A (S6 site), and A5VI-R239A (S6 site). Numbers represent residues of the laminin alpha 5 chain.



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Fig. 4.   Cell binding to laminin alpha 5 domain VI mutants. Wells were coated with laminin alpha 5 domain VI mutants (10 µg/ml). HT-1080 cells were added, and crystal violet staining was used to assess the number of attached cells after 1 h. Data are given as percentage of cell binding to the wild-type laminin alpha 5 domain VI (A5VI). Each value represents the mean of three separate determinations ± S.D. Duplicate experiments gave similar results.

The S6 peptide overlaps with two previously reported active synthetic peptides (A-24 and A-25) derived from the laminin alpha 1 chain (19). The S6 sequence is characterized by five basic residues (four Arg and one Lys). Notable, three of the four Arg residues are conserved in domain VI of all the laminin alpha  chains (Fig. 3). To analyze the contribution of the individual Arg/Lys residues within the S6 site to cell binding, we generated five single substitution mutants by replacing Arg/Lys residues with Ala (R225A, K229A, R234A, R236A, and R239A). All the mutants were expressed at a level similar to wild-type domain VI, suggesting that the mutations did not cause unstable synthesis of domain VI due to unfolding of the proteins (data not shown). Cell binding to A5VI-K229A was very poor, while neither A5VI-R234A nor A5VI-R236A had a significant effect on cell binding (Fig. 4). R225A and R239A also showed a significant reduction in cell binding, by ~60% of the wild type (Fig. 4). These data indicate that four residues, Tyr130, Arg225, Lys229, and Arg239, within these two regions are crucial for cell binding.

Kinetic Analysis of Interactions between Heparin and Laminin alpha 5 Domain VI Mutants using Surface Plasmon Resonance-- Since heparin binding activity has been localized to domain VI of the laminin alpha 1 and alpha 2 chains (13-15), cell binding to recombinant laminin alpha 5 domain VI was examined in the presence of heparin and other heparin-like glycosaminoglycans. Heparin and heparan sulfate inhibited HT-1080 cell binding to domain VI, whereas keratan sulfate was much less inhibitory (Fig. 5). These results indicate that the heparin-binding site overlaps with the cell-binding sites.



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Fig. 5.   Effects of heparin/heparan sulfate and EDTA on cell binding to laminin alpha 5 domain VI. HT-1080 binding assays were performed on control without inhibitors (C), or in the presence of heparin (HP), heparan sulfate (HS), keratan sulfate (KS), and 5 mM EDTA. Each value represents the mean of three separate determinations ± S.D. Duplicate experiments gave similar results.

To examine the relationships between the heparin and cell-binding site, the binding kinetics between immobilized biotinylated heparin and the laminin alpha 5 domain VI mutants were measured directly by real-time biomolecular interaction analysis using surface plasmon resonance on a BIAcoreTM system. Similar equilibrium dissociation constants (Kd = 9-16 nM) were obtained for the interactions of wild-type domain VI of the laminin alpha 5 chain and five of the mutants with heparin (Table II). Less than a 2-fold difference was observed for the kinetic binding constants between wild-type domain VI and five of the mutants including Y130A, R225A, K229A, R234A, and R236A. Statistically significant differences in kinetic constants derived from BIAcore experiments are generally considered to be at least 5-10-fold. This demonstrates that the heparin binding activity was unchanged, indicating that the structural integrity of domain VI was maintained in these mutants and that these positions were not part of the heparin-binding site. In contrast, no binding of the R239A mutant to heparin was observed even at a high protein concentration (200 nM). Mutation of Arg239 was also found to be critical for cell binding, reducing activity by ~60% compared with wild-type domain VI (see above). Consistent with the cell binding results, no heparin binding was observed for a recombinant protein consisting of the first four EGF-like repeats of domain V. These results demonstrate that the heparin-binding site is located within domain VI. Interestingly, the binding affinity of domain VI of the laminin alpha 1 chain to heparin was about 4-fold weaker (Kd = 45 nM), demonstrating differences in affinity for heparin between the laminin domain VI isoforms. Taken together, these results indicate that Arg239 within the S6 site is critical for both heparin and cell binding.


                              
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Table II
Effect of mutagenesis on laminin alpha 5 domain VI-heparin interaction
Kinectic constants were obtained by surface plasmon resonance analysis of real-time heparin-binding interactions of laminin alpha 5 domain VI mutants, domain V, and laminin alpha 1 domain VI. Recombinant domains were analyzed at several concentrations and the kinectic constants represent average values of 2-3 determinations.

Effect of Integrin Monoclonal Antibodies on Cell Binding to Laminin alpha 5 Domain VI-- Cell binding to laminin alpha 5 and alpha 1 domain VI and to laminins containing either the alpha 5 chain (laminin-10/11) or the alpha 1 chain (laminin-1) was examined in the presence of different anti-integrin antibodies to identify integrin receptors involved in domain VI binding. The beta 1 integrin antibody demonstrated partial inhibition of HT-1080 cell binding to laminin-10/11 but strong inhibition of cell binding to laminin-1 (Fig. 6). The monoclonal antibody against integrin alpha 6 inhibited cell binding to laminin-1 but not laminin-10/11, while a monoclonal antibody against integrin alpha 3 showed a weak inhibitory effect on cell binding to laminin-10/11. Other antibodies against integrin subunits, including beta 4, alpha 1, alpha 2, alpha 4, alpha 5, and alpha V, had no effect on cell binding. Cell binding to both laminin-1 and laminin-10/11 was dependent on divalent cations, since it was abolished by 5 mM EDTA (Fig. 6), which supports the role of integrins or alpha -dystroglycan as receptors. These results suggest that different integrin receptors mediate HT-1080 cell binding to laminin-1 and laminin-10/11 and that the alpha 3beta 1 integrin binds laminin-10/11, agreeing with previous reports (6, 7).



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Fig. 6.   Inhibition of cell binding by anti-integrin antibodies and EDTA to laminin-10/11 and laminin-1. Cell binding assays using HT-1080 cells were performed on controls without inhibitors (control), or in the presence of rat preimmune IgG (10 µg/ml) (IgG), function-blocking anti-integrin monoclonal antibodies (10 µg/ml), and EDTA (5 mM). Wells were coated with 2 µg/ml laminin-10/11 or laminin-1. The results are expressed as percent binding of cells without inhibitors. Each value represents the mean of three separate determinations ± S.D. Duplicate experiments gave similar results.

The monoclonal antibody against the beta 1 integrin subunit inhibited HT-1080 binding to recombinant domain VI of both laminin alpha 5 and alpha 1 (Fig. 7, A and B). These results show that beta 1 subunit containing integrins is critical for cell binding to domain VI of both chains. Furthermore, 5 mM EDTA was also found to inhibit cell binding, supporting cation and integrin dependence of the interaction (Fig. 5). We then tested several monoclonal antibodies against integrin alpha  subunits to identify partners for the beta 1 integrins. Anti-integrin alpha 3 antibodies strongly inhibited HT-1080 cell binding to laminin alpha 5 and alpha 1 domain VI (Fig. 7, A and B). Monoclonal antibodies against alpha 1, alpha 5, and alpha V had no or only small effects on cell binding, indicating that these integrins were not major mediators of cell binding to laminin alpha 5 and alpha 1 domain VI (Fig. 7, A and B). HT-1080 cell binding to laminin alpha 5 and alpha 1 domain VI was reduced to about 30-50% by anti-integrin alpha 2 and alpha 4 antibodies. Integrin alpha 6 antibody also weakly reduced cell binding to laminin alpha 5 domain VI but not to laminin alpha 1 domain VI (Fig. 7B). Taken together, these results identify alpha 3beta 1 integrin as a major receptor to alpha 5 domain VI.



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Fig. 7.   Inhibition of cell binding by anti-integrin antibodies to laminin alpha 1 and alpha 5 domain VI. Cell binding assays were carried out without inhibitor (control) or in the presence of function-blocking anti-integrin antibodies (10 µg/ml). Wells were coated with 10 µg/ml recombinant domain VI of the laminin alpha 1 (panel A) or alpha 5 chain (panel B). Each value represents the mean of three separate determinations ± S.D. Duplicate experiments gave similar results.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The newly discovered laminin alpha 5 chain has been implicated in various biological activities, such as angiogenesis and nerve regeneration. Previously, we identified a cell-binding site within the C-terminal G domain of the mouse laminin alpha 5 chain using recombinant proteins and synthetic peptides (21). In the present study, we identified cell-binding sites on the laminin alpha 5 N-terminal globular domain VI.

Recombinant proteins of laminin alpha 5 domain VI and the first four EGF-like repeats of domain V were generated by mammalian expression. The recombinant protein was highly active for HT-1080 fibrosarcoma cell binding, while a recombinant protein consisting of only the EGF-like repeats showed no binding, indicating that domain VI contributed to the cell-binding site. This result is similar to previous reports from studies on recombinant fragments consisting of domains VI through IV, of the laminin alpha 1 and alpha 2 chains (13-15). In addition, recombinant laminin alpha 5 domain VI was also found to bind other cell lines including mouse B16-F10 melanoma and mesangial cells, while weak binding was observed for a human submandibular gland cell line, indicating cell type-specific interaction of this domain (data not shown).

To localize cell-binding sites on laminin alpha 5 domain VI, we screened domain VI-derived synthetic peptides for their effect on HT-1080 cell/domain VI interactions. Four peptides: S1, 115EVNVTLDLGQVFH127; S2, 123GQVFHVAYVLIKF135; S6, 225RDFTKATNIRLRFLR239; and S7, 232NIRLRFLRTNTL243, inhibited cell binding. These peptides corresponded to sequences in the laminin alpha 1 chain (A-12, A-13, A-24, and A-25) previously reported to be highly active for cell binding (19). The S1 and S2 peptides overlap, as do S6 and S7, thus the results indicate that at least two sequences, 123GQVFHVAYVLIKF135 and 225RDFTKATNIRLRFLR239, spaced by ~90 residues, directly interact with cell surface receptors within domain VI (Fig. 3). Alanine mutagenesis of recombinant domain VI identified four positions within these two sequences as critically involved in cell binding: Tyr130, Arg225, Lys229, and Arg239. A comparison with the sequences of domain VI of the laminin alpha 1, alpha 2, and alpha 3B chains reveals that Tyr130 and Arg239 are conserved, while Arg225 and Lys229 are conserved between the alpha 3 and alpha 5 chains, indicating that the position of the cell-binding sites varies. These differences may be important for regulation and specificity of receptor interactions within domain VI. The site-directed mutagenesis data also demonstrate that the two sites together contribute to a cell binding epitope and imply that these cell-binding sites are highly dependent on the conformation of domain VI. The importance of the three-dimensional structure of cell-binding sites has also been shown for other integrin ligands, including fibronectin and vascular cell adhesion molecule-1, where multiple contacts, involving several different ligand peptide segments, are formed between ligand and receptor (22, 23).

Kinetic data obtained here by surface plasmon resonance analysis for six single-site alanine substitution mutants of laminin alpha 5 domain VI demonstrate that one position (Arg239) within the cell-binding site is also critical for heparin interactions. Cell binding to domain VI was sensitive to inhibition by heparin/heparan sulfate, demonstrating overlap of cell and heparin-binding sites. Interestingly, the binding constants for binding to heparin show a 4-fold difference between domain VI of the laminin alpha 5 and alpha 1 chain, with the alpha 5 domain demonstrating highest affinity. Strong heparin binding affinity suggests the potential to bind heparan sulfate containing matrix molecules or cell surface receptors (21, 24, 25); accordingly, these molecules may interact mainly with the basic residues within the S6 site. Our results suggest that heparan sulfate-containing cell surface receptor interaction is required for efficient cell binding to laminin alpha 5 domain VI. This is in accordance with previous studies, which indicate that heparan sulfate-containing cell surface receptors can function as co-receptors for integrins and that these co-receptors are essential for cell binding to some ligands, including the heparin III domain of fibronectin and the angiogenic inducer Cyr61 (17, 26). The heparan sulfate-containing cell surface receptors may include syndecan-1 or alpha -dystroglycan; the latter has been shown to bind laminin-10/11 and domain VI of the laminin alpha 1 chain (8, 24). The role of these interactions may be important for cell type-specific binding or signaling (17, 26). The heparin-binding site on laminin alpha 5 domain VI may also function as a binding site for other matrix molecules, since it has been reported that laminin alpha 1 domain VI binds to heparan sulfate chains of perlecan (24). Accordingly, binding through the heparin-binding site may be a mechanism for the regulation of interactions with cells and matrix assembly.

Several integrins have previously been implicated as receptors for laminin-10/11, including alpha 3beta 1, alpha 6beta 1, and alpha 6beta 4 (7). In this study, we demonstrate that domain VI of the laminin alpha 5 chain is a binding site for integrins alpha 3beta 1, alpha 2beta 1, alpha 4beta 1, and alpha 6beta 1. HT-1080 cell binding was completely blocked by anti-integrin alpha 3 or beta 1 antibodies, while function-blocking antibodies against the alpha 2, alpha 4, and alpha 6 integrins showed a weaker effect, suggesting that the alpha 3beta 1 integrin is a major mediator of cell binding. Small or no effects were observed with antibodies against alpha 1, alpha 5, and alpha V integrins. Comparison with a recombinant protein of laminin alpha 1 domain VI showed similar integrin specificity except for alpha 6, where no effect was observed for laminin alpha 1 domain VI. The inhibition results are in agreement with the reported integrin specificity of recombinant fragments of domains VI through IV, of the laminin alpha 1 and alpha 2 chains (13, 14). The previous studies used the same cell line as here, but only results using anti-integrin alpha 1 and alpha 2 antibodies were reported. Our results using various monoclonal antibodies suggest that several integrins bind domain VI of the laminin alpha 5 and alpha 1 chains and that these receptors bind similar recognition sites within domain VI.

In conclusion, the present data represent the first mapping of sites within the N-terminal globular domain VI of the mouse laminin alpha 5 chain responsible for cell binding. Our results suggest that heparan sulfate-containing receptors and integrins recognize domain VI. We found that two sequences, spaced by ~90 residues within laminin alpha 5 domain VI, are critical for cell surface receptor binding and that at least four residues within these two regions together form a binding site(s) critical for receptor binding.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Bldg. 30/Rm. 405, NIDCR, National Institutes of Health, 30 Convent Dr. MSC 4370, Bethesda, MD 20892-4370. Tel.: 301-496-2111; Fax: 301-402-0897; E-mail: yoshi.yamada@nih.gov.

Published, JBC Papers in Press, November 29, 2000, DOI 10.1074/jbc.M008743200


    ABBREVIATIONS

The abbreviations used are: EGF, epidermal growth factor; PBS, phosphate-buffered saline.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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