The Laminin α2-Chain Short Arm Mediates Cell Adhesion through Both the α1β1 and α2β1 Integrins*

Laminin-2, a heterotrimer composed of α2, β1, and γ1 subunits, is the primary laminin isoform found in muscle and peripheral nerve and is essential for the development and stability of basement membranes in these tissues. Expression of a domain VI-truncated laminin α2-chain results in muscle degeneration and peripheral nerve dysmyelination in the dy 2Jdystrophic mouse. We have expressed amino-terminal domains VI through IVb of the laminin α2-chain, as well as its laminin-1 α1-chain counterpart, to identify candidate cell-interactive functions of this critical region. Using integrin-specific antibodies, recognition sites for the α1β1 and α2β1 integrins were identified in the short arms of both laminin α1- and α2-chain isoforms. Comparisons with a β-α chimeric short arm protein possessing β1-chain domain VI further localized these activities to α-chain domain VI. In addition, we found that the laminin α2-chain short arm supported neurite outgrowth independent of other laminin-2 subunits. A heparin/heparan sulfate binding activity was also localized to this region of the laminin α2 subunit. These data provide the first evidence that domain VI of the laminin α2-chain mediates interactions with cell surface receptors and suggest that these integrin and heparin binding sites, alone or in concert, may play an important role in muscle and peripheral nerve function.

Laminin-2, a heterotrimer composed of ␣2, ␤1, and ␥1 subunits, is the primary laminin isoform found in muscle and peripheral nerve and is essential for the development and stability of basement membranes in these tissues. Expression of a domain VI-truncated laminin ␣2-chain results in muscle degeneration and peripheral nerve dysmyelination in the dy 2J dystrophic mouse. We have expressed amino-terminal domains VI through IVb of the laminin ␣2-chain, as well as its laminin-1 ␣1-chain counterpart, to identify candidate cell-interactive functions of this critical region. Using integrin-specific antibodies, recognition sites for the ␣1␤1 and ␣2␤1 integrins were identified in the short arms of both laminin ␣1and ␣2-chain isoforms. Comparisons with a ␤-␣ chimeric short arm protein possessing ␤1-chain domain VI further localized these activities to ␣-chain domain VI. In addition, we found that the laminin ␣2-chain short arm supported neurite outgrowth independent of other laminin-2 subunits. A heparin/heparan sulfate binding activity was also localized to this region of the laminin ␣2 subunit. These data provide the first evidence that domain VI of the laminin ␣2-chain mediates interactions with cell surface receptors and suggest that these integrin and heparin binding sites, alone or in concert, may play an important role in muscle and peripheral nerve function.
Members of the laminin family of glycoproteins are thought to be important for the development and stability of basement membranes, both through architecture-forming interactions with other laminins and matrix components and through recognition of cell signaling molecules such as integrins. EHS 1 laminin, or laminin-1, is currently the best understood constituent of this family (reviewed in Refs. [1][2][3][4]. Laminin-1 is composed of three unique polypeptide chains, ␣1, ␤1, and ␥1, that form a large ϳ800-kDa heterotrimer composed of three short arms and one long arm. All other known laminin isoforms adopt a similar ␣-␤-␥ trimeric composition but assemble using varying combinations of unique ␣, ␤, and ␥ isoforms. Laminins 2 and 4, previously referred to as merosin, both contain the ␣2-chain subunit (5,6). The ␣2-chain subunit is the primary laminin ␣-chain found in skeletal muscle and peripheral nerve basement membranes, but its specific interactions are currently less well understood than those of ␣1-chain of laminin-1. The importance of laminins 2 and 4 in these tissues, particularly muscle, is much clearer; mutations that result in the absence of the ␣2-chain cause autosomal recessive congenital muscular dystrophies, both in humans and in dy mice (7)(8)(9)(10).
Several cell-interactive functions have been assigned to the carboxyl-terminal long arm of laminins 2 and 4, including ␣3␤1, ␣6␤1, ␣7␤1, and ␣6␤4 integrin recognition. (11)(12)(13)(14). It has also been shown that the G-domain at the carboxyl terminus of the laminin ␣2-chain provides a linkage between the extracellular matrix and the dystrophin-associated glycoprotein complex through its interaction with ␣-dystroglycan (see Refs. 15 and 16;reviewed in Ref. 17). The repertoire of known laminin ␣2-chain functions have thus far all been associated with its long arm; however, attention has recently focused on possible function(s) of the short arm following the identification of muscular dystrophies in which the amino-terminal short arm of the ␣2-chain is truncated or otherwise mutated (18 -21). In the dy 2J mouse, muscular dystrophy develops as the result of a truncation in ␣2-chain domain VI within the context of an otherwise functional heterotrimeric laminin molecule 2 (18,19). This mutation furthermore leads to peripheral nerve defects, also seen in the allelic dy mouse where the laminin ␣2 subunit is completely absent (7,8). In contrast to the long arm functions, little is currently known about the ␣2-chain short arm and its potential role as a ligand for cell surface receptors.
In this study, we have expressed laminin short arm ␣2-chain and ␣1-chain proteins to identify and characterize cell recognition functions specific for these amino-terminal domains. We report here that the ␣2-chain of laminin contains two distinct integrin binding sites within its amino-terminal domain, recognizing both the ␣1␤1 and ␣2␤1 integrins. These cell recognition sites are conserved in domain VI of the laminin ␣1-chain isoform, as reported previously in the case of ␣1␤1 integrin recognition (22)(23)(24)(25). Furthermore, we show that the ␣2-chain short arm contains heparin binding sites that may mediate interactions with cell surface proteoglycans or other charged glycosaminoglycans. These shared integrin recognition sites and heparin-binding activities may act alone or in concert with ␣-chain domain VI polymer-forming sites 3 (25,26), modulating cell activity and matrix architecture.

MATERIALS AND METHODS
Expression Constructs-pCIS, a mammalian expression vector containing the CMV promoter, was used to express all described recombinant laminin proteins (27). Mouse laminin ␣1-chain construct ␣1(VI-IVb)Ј, encoding domains VI, V, IVb and approximately 1 ⁄3 of domain IIIb, was described previously (25). A human laminin ␣2-chain partial cDNA (nucleotides 13-6927) in Bluescript KS was generously provided by Dr. Ulla Wewer (University of Copenhagen, Denmark) and Dr. Eva Engvall (Burnham Institute, La Jolla, CA). A stop codon was inserted at base 2642 in domain IIIb of the ␣2-chain cDNA by blunt end ligation of oligonucleotide dGCTTAATTAATTAAGC at an AhdI site. Construct ␣2(VI-IVb)Ј was generated by cloning a 2.7-kilobase pair SpeI-PacI fragment corresponding to the 5Ј-end of the ␣2 cDNA into pCIS at an XbaI site in the polylinker region. To generate chimeric laminin ␤1-␣1 construct ␤1(VI)␣1(V-IVb)Ј, a complete SpeI digest and a partial BsmI digest was used to purify a fragment containing ␣1(VI-IVb)Ј in pCIS missing 1952 bases at the 5Ј-end. A SpeI-ScaI fragment from the 5Ј-end of the mouse ␤1-chain cDNA was inserted into this region using a linker fragment generated by polymerase chain reaction. The linker piece extended from the ScaI site through the BsmI site and was generated using mouse ␤1 cDNA as a template with the following primers: 5Ј-T-CCGAGAGAAGTACTATTACGCTGTTTATGATATGGTGGTTCGAG-GGAACTGCATTTGCTACGGC-3Ј (sense) and 5Ј-TTGTGACAGTTGC-ATTCC-3Ј (antisense).
Purification of Recombinant Proteins-Human 293 cells (ATCC CRL 1573) were transfected by the calcium phosphate precipitation method as described by Chen and Okayama (28). DNA constructs were cotransfected with pSV2pac, a puromycin resistance plasmid kindly provided by Dr. Roswitha Nischt (University of Cologne, Germany). Antibioticresistant clones were obtained following several weeks of selection using 1 g/ml puromycin. Conditioned medium from resistant colonies was screened in Western blots for the presence of recombinant laminin proteins using antibodies specific for the corresponding laminins. Colonies that expressed high levels of protein were expanded and grown for 48 h in serum-free medium (DMEM-F12, Life Technologies, Inc.). Collected medium was placed on ice, adjusted to 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2 mM EDTA, and centrifuged in the cold for 30 min at 10,000 rpm to clear debris. Proteins ␣1(VI-IVb)Ј and ␣2(VI-IVb)Ј were precipitated in a 40% ammonium sulfate solution slowly stirred overnight on ice, followed by centrifugation at 20,000 rpm at 4°C for 30 min. Precipitate was then resuspended in 20 ml of 50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, and 0.5 mM PMSF and dialyzed overnight at 4°C against the same buffer. Next, protein was bound to an HPLC heparin-5PW affinity column (Toso-Haas) under the same buffer conditions and eluted with a 0 -1.0 M linear NaCl gradient. A peak that eluted between 0.16 and 0.19 M NaCl was pooled and then dialyzed overnight at 4°C against 50 mM Tris-HCl, pH 8.5, 0.5 mM EDTA, and 0.5 mM PMSF. Dialyzed protein was then bound to a DEAE-5PW anion exchange column (Toso-Haas) and eluted with a 0 -0.5 M linear NaCl gradient. Fractions containing purified protein were pooled, concentrated by Aquacide (Calbiochem), and dialyzed into 50 mM Tris-HCl, pH 7.4, 90 mM NaCl, 0.1 mM EDTA, 0.5 mM PMSF. Protein ␤1(VI)␣1(V-IVb)Ј did not bind heparin affinity beads and was purified as follows. Conditioned medium was precipitated with 45% ammonium sulfate and processed as described above, except that protein was dialyzed against 50 mM Tris, pH 8.5, 0.5 mM EDTA, 0.5 mM PMSF. Protein was then bound to a DEAE-5PW column and eluted with a 0 -0.5 M linear NaCl gradient. Fractions containing ␤1(VI)␣1(V-IVb)Ј were pooled and dialyzed overnight at 4°C against 20 mM Tris, pH 7.4, 0.2 mM EDTA, 0.5 mM PMSF. Protein was then adjusted to 1.5 M ammonium sulfate and bound to a phenyl-Superose hydrophobic interaction column (Toso-Haas) under the same buffer and salt conditions. Protein was eluted from the column using a 1.5-0 M decreasing linear ammonium sulfate gradient. Fractions containing ␤1(VI)␣1(V-IVb)Ј were pooled and concentrated to 0.5 ml and passed through a Superose 6 (Pharmacia) gel filtration column. Fractions containing purified protein were pooled, concentrated, and dialyzed against 50 mM Tris-HCl, pH 7.4, 90 mM NaCl, 0.1 mM EDTA, 0.5 mM PMSF.
Preparation of Laminins from Tissue-Mouse laminin-1 was extracted from lathyritic EHS tumor and purified as described by Yurchenco and Cheng (26). A preparation containing laminin isoforms with the ␣2-chain subunit (a mixture of laminins 2 and 4) was purified from human placenta using a modification of the procedure developed by (29). Briefly, homogenized human placentas were digested with bacterial collagenase (Worthington), followed by extraction with EDTAcontaining buffer at 0°C. The laminin-2/4 mixture was then purified from extracted material using ion exchange, gel filtration, and heparin affinity chromatography.
Cell Adhesion-HT1080 human fibrosarcoma cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum (Gemini) in a 5% CO 2 humidified atmosphere at 37°C. PC12 rat pheochromocytoma cells were also maintained in these conditions, except that Dulbecco's modified Eagle's medium was supplemented with 10% horse serum and 5% fetal bovine serum. Both cell types were prepared for cell adhesion studies by overnight incubation in 10 Ci/ml [methyl-3 H]thymidine (specific activity 85 Ci/mmol (Amersham Corp.)). Protein-coated wells were prepared by incubating proteins in 50 mM Tris-HCl, pH 7.4, 90 mM NaCl, 1 mM CaCl 2 buffer at 4°C overnight in half-area 96-well tissue culture plates (Costar). The plates were then washed three times with PBS containing 0.5% heat-inactivated bovine serum albumin and blocked for 2 h at 37°C in the same buffer. Labeled cells were detached with 0.1% trypsin and 0.5 mM EDTA, washed once in Dulbecco's modified Eagle's medium containing 0.5% heat-inactivated bovine serum albumin, and resuspended in the same medium at approximately 6 ϫ 10 5 cells/ml. For studies using integrin-blocking antibodies, cell suspensions were preincubated in the presence of the appropriate antibodies for 30 min at 37°C. Fifty l of cell suspension was added to each protein-coated well and incubated at 37°C for 70 min. Plates were then washed three times with PBS to remove unattached cells, followed by treatment with 2% SDS for 1 h to solubilize remaining cells. Solubilized cell suspensions were added to scintillation mixture (Ecoscint A, National Diagnostics), and radioactivity was determined using a model LS6000IC scintillation counter (Beckman Instruments). One hundred percent adhesion was defined as radioactivity of the total added cell suspension. Error bars represent S.E.
Antibodies-Antibodies against human integrin subunits were used at 10 g/ml in cell adhesion studies unless otherwise stated. Mouse monoclonal antibodies against the ␣1 and ␣2 subunits of human ␤1 integrins were purchased from Upstate Biotechnologies Inc. Mouse monoclonal antibody against the ␣3-chain of the human ␣3␤1 integrin was purchased from Chemicon. Monoclonal rat anti-human ␣6 integrin subunit (GoH3) was generously provided by Dr. Arnoud Sonnenberg (Netherlands Cancer Institute, Amsterdam). Monoclonal antibody 3A3, a mouse IgG specific for the ␣1 subunit of rat ␣1␤1 integrin (23), was generously provided by Dr. Sal Carbonetto (McGill University, Montreal). Ascites fluid containing monoclonal 3A3 was diluted at 1:50 in cell adhesion studies.

Expression and Characterization of Laminin ␣-Chain Short
Arm Proteins-Previous studies investigating the role of laminin-2 in cell adhesion and signaling have used the entire threechain ϳ800-kDa molecule (11)(12)(13)(14)(31)(32)(33)(34). Consequently, the interaction of whole laminin-2 with many different integrins has been reported, including ␣3␤1, ␣6␤1, ␣6␤4, and ␣7␤1 (11)(12)(13)(14). In this study, we sought to identify cell-interactive functions found exclusively in the laminin ␣2-chain amino-terminal region as well as to draw comparisons to those found in the analogous region of laminin-1. To analyze this region in isolation, we used a mouse laminin ␣1-chain short arm protein ␣1(VI-IVb)Ј, described and characterized previously (25), as a model to design an analogous ␣2-chain protein. Human recombinant ␣2(VI-IVb)Ј was expressed in 293 cells transfected with a mammalian expression vector (pCIS) containing the 5Ј-region of the ␣2-chain cDNA. This protein begins at the ␣2-chain amino terminus and extends through domains VI, V, and IVb, ending about a third of the way into domain IIIb. ␤1(VI)␣1(V-IVb)Ј, in which domain VI from ␣1(VI-IVb)Ј was replaced by domain VI of the laminin ␤1-chain, was assembled from mouse laminin ␤1-chain and ␣1-chain cDNAs, and was also expressed in 293 cells using the pCIS vector. This chimeric molecule was chosen to evaluate the cell recognition function of the ␣-chain in the absence of its domain VI for two reasons. First, a swap between corresponding domains, rather than a domain deletion, was most likely to maintain the proper folding found to be crucial for other laminin activities (24,35,36). Second, the short arm of the ␤1-chain does not interact with any of the cells used in this study (25,37).
Morphology and domain structure of the three short arm proteins was compared in glycerol rotary shadows (Fig. 2). Electron microscopy of platinum/carbon replicas revealed that all three molecules have a dumbbell-like appearance with a short tail visible in some species. These observations fit the predicted domain structure of the laminin short arm region, in which globular domains VI and IVb are separated by the rodlike EGF repeats of domain V. The stub-like extension seen in some molecules represents the one-third of domain IIIb present at the carboxyl terminus of these proteins.
Heparin Binds Laminin ␣1and ␣2-Chain Short Arm Proteins through Domain VI-Studies using proteolytic fragments derived from laminin-1 have shown that both G-domain and domain VI of the laminin ␣1-chain contain distinct heparin binding activities (25,(37)(38). HPLC heparin affinity chromatography was used to evaluate the laminin ␣2 subunit short arm, as well as the ␤1-␣1 laminin short arm chimeric protein, for the ability to interact with heparin. Laminin ␣2-chain short arm protein ␣2(VI-IVb)Ј bound heparin and was eluted from the column by 0.16 M NaCl, showing a reduced relative affinity compared with ␣1(VI-IVb)Ј, which required 0.19 M NaCl (Fig.  3). Chimeric protein ␤1(VI)␣1(V-IVb), lacking an ␣-chain domain VI, did not bind heparin at all. Both ␣-domain VI-containing proteins showed lower relative affinity than ␣1-chain G-domain fragment E3, which eluted at 0.27 M NaCl.
HT1080 Human Fibrosarcoma Cells Adhere to the ␣1and ␣2-Chain Short Arms of Laminin Isoforms-Previous studies have shown that HT1080 cells interact with the carboxyl-terminal long arm of laminin-1 through the ␣6␤1 integrin (36,39). However, the same adhesion-blocking antibodies used to map the ␣6␤1 integrin recognition site to this region are unable to block adhesion to full-size laminin-1, implying that other ␤1class integrins participate in HT1080 adhesion to laminin-1, possibly recognizing the short arm region. HT1080 adhesion to laminin-1, a mixture of laminins 2 and 4 (both ␣2-chain-containing laminins), and their respective ␣-chain short arm proteins, ␣1(VI-IVb)Ј and ␣2(VI-IVb)Ј, was assayed over a range of substrate concentrations (Fig. 4). The cells adhered to full-size laminin isoforms and to ␣1(VI-IVb)Ј and ␣2(VI-IVb)Ј; however, they did not adhere to chimeric protein ␤1(VI)␣1(V-IVb)Ј. From these observations we concluded that laminins 1, 2, and 4 contained additional cell recognition site(s) present in their ␣-chain short arm regions. Furthermore, this activity could be localized to the amino-terminal globule, domain VI. Integrins ␣2␤1, ␣3␤1, and ␣6␤1 Mediate Adhesion of HT1080 Cells to Laminins 1, 2, and 4 -A battery of integrin-blocking antibodies was used to assess which integrins were involved in the adhesion of HT1080 cells to laminin substrates. Cells were preincubated with integrin-blocking antibodies, alone or in combination, and then assayed for adhesion to full-size laminin substrates (Fig. 5). Although ␣6␤1 integrin blocking antibodies could partially inhibit HT1080 adhesion to laminin 1, a combination of ␣2␤1 and ␣6␤1 integrin-blocking antibodies was required to completely block adhesion (Fig. 5A), indicating that both receptors participate. Additionally, a combination of ␣3␤1 and ␣6␤1 integrin-blocking antibodies was more effective at blocking adhesion than ␣6␤1 integrin-blocking antibodies alone. Since adhesion could be completely inhibited without inclusion of the ␣3␤1 integrin-blocking antibodies, we suggest that an interaction between ␣3␤1 integrin and laminin-1 is either not required in this cell type or only occurs when additional ␤1-class integrins are occupied.
The ␣1␤1 Integrin Mediates Adhesion to Domain VI of Laminin ␣1and ␣2-Chains-PC12 rat pheochromocytoma cells were used to determine whether the ␣2-chain short arm of laminin isoforms 2 and 4 possessed a recognition site for the ␣1␤1 integrin, a function previously localized to the analogous region of the laminin ␣1-chain (25). Laminin short arm protein ␣2(VI-IVb)Ј was found to support adhesion of PC12 cells similar to ␣1(VI-IVb)Ј (Fig. 7), and adhesion to both substrates was completely blocked by 3A3, a monoclonal antibody specific for the rat ␣1 integrin subunit. Chimeric protein ␤1(V)␣1(V-IVb)Ј did not support adhesion of PC12 cells, demonstrating that the recognition site for the ␣1␤1 integrin is located in domain VI, a finding previously suggested using domain-specific blocking antibodies against laminin-1 (25).
Neurite Outgrowth of PC12 Cells Is Supported by the ␣2-Chain Short Arm of Laminin Isoforms 2 and 4 -The ␣2-chain short arm of laminin was evaluated for its ability to support neurite outgrowth of nerve growth factor-primed PC12 cells (Fig. 8). Cells were maintained overnight on dishes coated with either EHS laminin-1, human placental laminin-2/4, ␣1(VI-IVb)Ј, ␣2(VI-IVb)Ј, or ␤1-chain proteolytic fragment E4 (short  ␣2␤1, ␣3␤1, and ␣6␤1. A, adhesion to laminin-1 in the presence of integrin-blocking antibodies. HT1080 cells were plated onto wells coated with 5 g/ml laminin-1. Monoclonal antibodies specific for integrins ␣1␤1, ␣2␤1, ␣3␤1, and ␣6␤1 were included, in various combinations, each at a concentration of 10 g/ml. Rat IgG at 10 g/ml was included as a negative control. The combination of ␣2␤1 and ␣6␤1 integrin-blocking antibodies completely inhibited adhesion to laminin-1, whereas anti-␣3␤1 had a mild effect, and anti-␣1␤1 had no effect. Although ␣2␤1 integrin-blocking antibodies alone had no effect and ␣6␤1 integrin-blocking antibodies alone had a small effect, the complete blockade seen in the presence of both antibodies demonstrates the requirement for both ␣2␤1 and ␣6␤1 integrins in mediating HT1080 adhesion to laminin-1. B, adhesion to laminin-2/4 in the presence of integrin-blocking antibodies. Conditions were the same as in A except that substrate was 5 g/ml laminin-2 and -4 mixture. A combination of ␣2␤1, ␣3␤1, and ␣6␤1 integrin-blocking antibodies was required to block adhesion, and again anti-␣1␤1 had no effect. arm domains VI and V). After 24 h, long branching neurites were seen on all substrates, with the exception of E4. DISCUSSION In the present study, two independent integrin recognition sites, ␣1␤1 and ␣2␤1, have been identified in domain VI of the laminin ␣2-chain (Fig. 9). In addition, a heparin binding activity was identified in this domain, suggesting that this domain also interacts with either cell surface or matrix-bound glycosaminoglycans. The ␣1␤1 and ␣2␤1 integrin binding sites are the first cell recognition activities to be assigned to this functionally rich region of skeletal muscle laminin isoforms 2 and 4. We have also shown that these recognition sites are found in domain VI of the laminin-1 ␣-chain. In addition, the short arm regions of laminin ␣1and ␣2-chains mediate neurite outgrowth of PC12 rat pheochromocytoma cells independently of long arm receptor contributions.

. HT1080 cell adhesion to laminin isoforms is mediated by integrins
To assign these functions to domain VI, we compared the binding activities of ␣1and ␣2-chain short arm proteins with a chimera in which domain VI was replaced by domain VI of ␤1.
Domain VI of the ␤1-chain was chosen as an ideal control for cell adhesion studies, since a proteolytic fragment containing this domain was previously unable to support adhesion of the cell types used here (25,37). Native morphology appeared to be maintained in all three short arm molecules, appearing as similar dumbbell-shaped molecules representing globular domains VI and IVb separated by a rod-like domain V.
The ability of the ␣2-chain short arm to support heparin interactions was assessed using HPLC heparin affinity chromatography. We found that the short arm of the ␣2 subunit isoform bound heparin, although its relative affinity was less than that of its ␣1-chain counterpart. Both short arm proteins had a lower relative affinity than carboxyl-terminal G-domain fragment E3, which has been shown to mediate a heparinsensitive interaction with dystroglycan, a molecule that provides a linkage between the dystrophin-glycoprotein complex and the extracellular matrix in skeletal muscle (15,16). Arginine and lysine are two basic amino acids thought to be essential in mediating electrostatic interactions with the negatively charged sulfate and carboxylic acid groups of heparin (40). Domains VI of the laminin ␣1and ␣2-chains have net positive charges of ϩ9 and ϩ2, respectively. It is interesting to note that domain VI of the dystrophic dy 2J mouse ␣2-chain contains an internal deletion that results in a net loss of basic residues, reducing the net charge to zero (19). It is possible that loss of these positively charged residues could disrupt a crucial interaction with cell surface molecules containing negatively charged groups such as heparan sulfate or polysialic acid.
We found that a combination of antibodies against the ␣2␤1 and ␣6␤1 integrins completely blocked adhesion of HT1080 cells on a substrate of laminin-1. These results demonstrate that the ␣2␤1 and ␣6␤1 integrins are required in order for HT1080 cells to mediate adhesion to laminin-1. However, since a combination of ␣3␤1 and ␣6␤1 integrin-blocking antibodies was more effective than ␣6␤1 blocking antibodies alone, we suggest that laminin-1 may weakly associate with the ␣3␤1 integrin as long as additional ␤1-class integrin sites are occupied. Despite these observations, the ␣3␤1 integrin was clearly unable to support adhesion to laminin-1 in the absence of ␣2␤1 and ␣6␤1 interactions. In contrast, laminin isoforms 2 and 4 were found to utilize the ␣2␤1, ␣6␤1, and ␣3␤1 integrins in mediating adhesion to HT1080 cells, with a combination of antibodies against these three integrins causing the most significant decrease in adhesion. These observations may address the discrepancy apparent in the finding that although the ␣3␤1 integrin has been described as a receptor for laminin-1 (41), K562 cells transfected with the ␣3␤1 integrin were unable to adhere to EHS laminin-1, whereas adhesion was detected on human placental laminins (12). Also consistent with our observations are studies involving sensory neurons of the dorsal root ganglion, where the ␣3␤1 integrin is utilized more efficiently on a laminin-2/4 substrate than on an EHS laminin substrate (45).
We next examined whether the ␣2␤1 integrin interacted with the laminin ␣1and ␣2-chain short arm proteins. We found both ␣1and ␣2-chain proteins mediated adhesion to HT1080 cells, whereas our chimeric ␣-chain protein containing ␤1-chain domain VI did not. Furthermore, adhesion on both substrates was completely blocked in the presence of ␣2␤1 integrin-specific antibodies. The ␣2␤1 integrin was first described as a receptor for laminin in studies using human umbilical endothelial cells (46). This integrin has since been shown to participate in epithelial tissue processes such as wound repair, inflammation, regulation of keratinocyte proliferation, and establishment of polarity (reviewed in Ref. 47). Several studies examining the role of extracellular matrix proteins in these processes in vivo support an interaction between both laminin-1 and laminin-2 with the ␣2␤1 integrin. The ␣2␤1 integrin exhibits a highly restricted distribution during epithelial branching morphogenesis of the developing lung, appearing only at the branching tips (48), and this distribution is spatially and temporally correlated with the expression of laminin-1 and collagen IV at sites of rapid matrix deposition. Branching morphogenesis of mammary epithelial cells grown on a mixture of laminin-1 and collagen IV has also been shown to be dependent on expression of the ␣2␤1 integrin (49). Expression of the ␣2␤1 integrin in developing and adult peripheral nerves and Schwann cells, tissues where ␣2-chain-containing laminins are the predominant laminin isoforms, has been observed in several studies (50 -52).
Earlier studies have shown that ␣1␤1 integrin-mediated cell attachment occurs via the short arms of laminin-1 (22)(23)(24), specifically through domain VI of the ␣1-chain (25). Data shown here demonstrate that the ␣2-chain short arm of laminin isoforms 2 and 4 also mediated cell adhesion through the ␣1␤1 integrin, as well as supported neurite outgrowth. PC12 rat pheochromocytoma cells adhered and spread on dishes coated with laminin short arm ␣2-chain protein but were inhibited in the presence of antibodies specific for the ␣1 integrin subunit. Nerve growth factor-primed PC12 cells extended long, branching neurites when grown on ␣2-chain short arm protein, appearing similar to cells grown on full-size laminin-1, -2, or -4. Analysis of the developing avian embryo has shown that the ␣1␤1 integrin is expressed in the central nervous system, sympathetic and spinal sensory ganglia, capillary endothelium, and skeletal, smooth, and cardiac muscle; although in the adult, expression becomes restricted to muscle and endothelium (53,54). Studies have shown that the ␣1␤1 integrin mediates neural crest cell migration on laminin-1 (55-57), and we suggest that the ␣1␤1 integrin may mediate cell migration or adhesion during the development of tissues where laminin-2 and -4 isoforms play a prominent role, such as muscle or peripheral nerve (5,58). Analysis of mice engineered to lack the ␣1 integrin subunit do not exhibit gross muscle or nerve defects, showing only subtle disturbances in cell adhesion and migration (59); however, the ␣2␤1 integrin has an overlapping pattern of expression and ligand binding, and its presence may prevent such defects from occurring. Targeted disruption of the gene encoding the ␣2 integrin subunit as well as the generation of mice lacking both ␣1 and ␣2 integrin subunits are needed to more definitively assess the in vivo role of these integrinlaminin short arm interactions.
␣2-chain assembles with its ␤ and ␥ subunit partners into laminin-2 or -4 heterotrimers, 4 is secreted by cells and appears to be localized to the extracellular matrix (18,19). The removal of domain VI from an otherwise functional laminin molecule nonetheless leads to progressive muscle degeneration characteristic of human congenital muscular dystrophies. At least four functions can now be assigned to this critical ␣2-chain domain: ␣1␤1 and ␣2␤1 integrin recognition, heparin binding, and polymer formation (described as being analogous to the laminin-1 assembly mechanism). 3 We also note that heparin and integrin binding sites in laminin are all found at extreme ends of the molecule in domains VI and G, suggesting a functional basis for this spatial clustering. The transmembrane heparan sulfate proteoglycan syndecan, along with the ␣5␤1 integrin, has been proposed to function as a co-receptor for the matrix protein fibronectin (60). The co-localization of binding sites in laminin suggests that a similar pairing of integrin binding and cell surface proteoglycan binding may be involved in laminin-mediated cell attachment and signaling. The phenotype seen in the dy 2J mouse indicates that one or more of these domain VI activities may be required to maintain normal muscle stability.
Peripheral nerve function is also disrupted in the dy 2J dystrophic mouse, where dysmyelination results in large bundles of naked axons (61). The spinal roots show fewer Schwann cells than normal at birth, and many of the cells that are present do not differentiate. However, these uncommitted cells are capable of differentiation when transplanted into a normal environment (62). It may be that an intact laminin ␣2-chain domain VI is needed either for proper Schwann cell migration during development or to provide a differentiation cue to uncommitted precursor cells. It remains unclear whether the ␣2-chain domain VI integrin binding sites described here are involved in this process; however, the identification of these sites represents a significant step forward in our understanding of laminin ␣2-chain function and its requirement in muscle and peripheral nerve development and stability.