The muscle integrin binding protein (MIBP) interacts with alpha7beta1 integrin and regulates cell adhesion and laminin matrix deposition

Integrins are alphabeta transmembrane receptors that function in key cellular processes, including cell adhesion, differentiation, and extracellular matrix deposition through interactions with extracellular, membrane, and cytoplasmic proteins. We previously identified and cloned a muscle beta1 integrin cytoplasmic binding protein termed MIBP and found that the expression level of MIBP is critical in the decision-making process of terminal myogenic differentiation. We report here that MIBP interacts with the alpha7beta1 integrin but not the alpha5beta1 integrin in C2C12 myoblasts, suggesting an important role of integrin alpha chains in the regulation of the beta1-MIBP interaction. Furthermore, consistent with its selective binding activity toward the alpha7beta1 laminin receptor, we have found that overexpression of MIBP in C2C12 myoblasts resulted in a significant reduction of cell adhesion to laminin and inhibition of laminin matrix deposition. By contrast, neither cell adhesion to fibronectin nor fibronectin matrix deposition was significantly altered in cells overexpressing MIBP. Finally, we show that both the protein level and tyrosine phosphorylation of paxillin, a key signaling molecule involved in the cellular control of myogenic differentiation, are increased by MIBP. These results suggest that MIBP functions in the control of myogenic differentiation by regulating alpha7beta1 integrin-mediated cell interactions with laminin matrix and intracellular signaling through paxillin.     

Integrin alpha3 subunit participates in myoblast adhesion and fusion in vitro

Satellite cells are myogenic precursor cells, participating in growth, and regeneration of skeletal muscles. The proteins that play a role in myogenesis are integrins. In this report, we show that the integrin alpha3 subunit is expressed in quiescent satellite cells and activated myoblasts. We also find that in myoblasts the integrin alpha3 subunit is localized at cell-cell and cell-extracellular matrix contacts. We notice that increase in protein and mRNA encoding the integrin alpha3 subunit accompanies myoblast differentiation. Using double immunofluorescence and immunoprecipitation experiments, we demonstrate that the integrin alpha3 subunit co-localizes with actin, and binds the integrin beta1 subunit and ADAM12, suggesting that the complex alpha3beta1/ADAM12 is probably involved in myoblast fusion. Importantly, overexpression of the full-length integrin alpha3 subunit increases myoblast fusion whereas an antibody against its extracellular domain inhibits fusion. These data demonstrate that the integrin alpha3 subunit may contribute to satellite cell activation and then myoblast adhesion and fusion.     

Modulation of cell proliferation and differentiation through substrate-dependent changes in fibronectin conformation

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound alpha5 and beta1 integrin subunits but not alphav or beta3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of alpha5beta1 integrin bound to Fn, and differentiation was inhibited by anti-alpha5, but not anti-alphav, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications.     

Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin [beta] subunits.

Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins. Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin beta1A and beta1D subunits. RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues. Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (DeltaH1), were induced during in vitro differentiation of C2C12 mouse myoblasts. We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin beta subunits. The mere presence of the high-affinity binding site for beta1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts. Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers. When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes. Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region. These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.     

alpha 11beta 1 integrin recognizes the GFOGER sequence in interstitial collagens

The integrins alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1) are referred to as a collagen receptor subgroup of the integrin family. Recently, both alpha(1)beta(1) and alpha(2)beta(1) integrins have been shown to recognize triple-helical GFOGER (where single letter amino acid nomenclature is used, O = hydroxyproline) or GFOGER-like motifs found in collagens, despite their distinct binding specificity for various collagen subtypes. In the present study we have investigated the mechanism whereby the latest member in the integrin family, alpha(11)beta(1), recognizes collagens using C2C12 cells transfected with alpha(11) cDNA and the bacterially expressed recombinant alpha(11) I domain. The ligand binding properties of alpha(11)beta(1) were compared with those of alpha(2)beta(1). Mg(2+)-dependent alpha(11)beta(1) binding to type I collagen required micromolar Ca(2+) but was inhibited by 1 mm Ca(2+), whereas alpha(2)beta(1)-mediated binding was refractory to millimolar concentrations of Ca(2+). The bacterially expressed recombinant alpha(11) I domain preference for fibrillar collagens over collagens IV and VI was the same as the alpha(2) I domain. Despite the difference in Ca(2+) sensitivity, alpha(11)beta(1)-expressing cells and the alpha(11) I domain bound to helical GFOGER sequences in a manner similar to alpha(2)beta(1)-expressing cells and the alpha(2) I domain. Modeling of the alpha I domain-collagen peptide complexes could partially explain the observed preference of different I domains for certain GFOGER sequence variations. In summary, our data indicate that the GFOGER sequence in fibrillar collagens is a common recognition motif used by alpha(1)beta(1), alpha(2)beta(1), and also alpha(11)beta(1) integrins. Although alpha(10) and alpha(11) chains show the highest sequence identity, alpha(2) and alpha(11) are more similar with regard to collagen specificity. Future studies will reveal whether alpha(2)beta(1) and alpha(11)beta(1) integrins also show overlapping biological functions.     

None of the integrins known to be present on the mouse egg or to be ADAM receptors are essential for sperm-egg binding and fusion

Antibody inhibition and alpha6beta1 ligand binding experiments indicate that the egg integrin alpha6beta1 functions as a receptor for sperm during gamete fusion; yet, eggs null for the alpha6 integrin exhibit normal fertilization. Alternative integrins may be involved in sperm-egg binding and fusion and could compensate for the absence of alpha6beta1. Various beta1 integrins and alphav integrins are present on mouse eggs. Some of these integrins are also reported to be receptors for ADAMs, which are expressed on sperm. Using alpha3 integrin null eggs, we found that the alpha3beta1 integrin was not essential for sperm-egg binding and fusion. Oocyte-specific, beta1 integrin conditional knockout mice allowed us to obtain mature eggs lacking all beta1 integrins. We found that the beta1 integrin null eggs were fully functional in fertilization both in vivo and in vitro. Furthermore, neither anti-mouse beta3 integrin function-blocking monoclonal antibody (mAb) nor alphav integrin function-blocking mAb inhibited sperm binding to or fusion with beta1 integrin null eggs. Thus, function of beta3 or alphav integrins does not seem to be involved in compensating for the absence of beta1 integrins. These results indicate that none of the integrins known to be present on mouse eggs or to be ADAM receptors are essential for sperm-egg binding/fusion, and thus, egg integrins may not play the role in gamete fusion previously attributed to them.     

Cloning and characterization of a c-myc intron binding protein (MIBP1).

The cDNA for a c-myc intron 1 binding protein 1 (MIBP1) in the rat was isolated from lambda gt11 and lambda ZAPII cDNA libraries. Sequencing of the cDNA clones revealed a long ORF which encoded a putative protein of 2437 amino acid residues. This protein has two widely separated zinc finger regions, each of which carries C2H2 motifs. When expressed in E. coli as a fusion protein, part of the MIBP1 showed sequence-specific binding to the target sequence, i.e., a 9-bp sequence in the rat c-myc intron 1. MIBP1 is most likely the rat counterpart of human MHC binding protein-2 (MBP-2/HIV-EP2), based on the 86% similarity in nucleotide sequence and 93% similarity in amno acid sequence. Northern blotting revealed a high level of MIBP1 mRNA in the brain.     

ADAM12-mediated focal adhesion formation is differently regulated by beta1 and beta3 integrins

ADAM12, adisintegrin and metalloprotease, has been demonstrated to be upregulated in human malignant tumors and to accelerate the malignant phenotype in a mouse model for breast cancer. ADAM12 is a substrate for beta1 integrins and may affect tumor and stromal cell behavior through its binding to beta1 integrins. Here, we report that cells deficient in beta1 integrin or overexpressing beta3 integrin can bind to recombinant full-length human ADAM12 via beta3 integrin. Furthermore, cell binding to ADAM12 via beta3 integrin results in the formation of focal adhesions, which are not formed upon beta1 integrin-mediated cell attachment. We also show that RhoA is involved in beta3 integrin-mediated focal adhesion formation.     

Identification of the first prokaryotic collagen sequence motif that mediates binding to human collagen receptors, integrins alpha2beta1 and alpha11beta1

Many pathogenic bacteria interact with human integrins to enter host cells and to augment host colonization. Group A Streptococcus (GAS) employs molecular mimicry by direct interactions between the cell surface streptococcal collagen-like protein-1 (Scl1) and the human collagen receptor, integrin alpha2beta1. The collagen-like (CL) region of the Scl1 protein mediates integrin-binding, although, the integrin binding motif was not defined. Here, we used molecular cloning and site-directed mutagenesis to identify the GLPGER sequence as the alpha2beta1 and the alpha11beta1 binding motif. Electron microscopy experiments mapped binding sites of the recombinant alpha2-integrin-inserted domain to the GLPGER motif of the recombinant Scl (rScl) protein. rScl proteins and a synthetic peptide harboring the GLPGER motif mediated the attachment of C2C12-alpha2+myoblasts expressing the alpha2beta1 integrin as the sole collagen receptor. The C2C12-alpha11+myoblasts expressing the alpha11beta1 integrin also attached to GLPGER-harboring rScl proteins. Furthermore, the C2C12-alpha11+cells attached to rScl1 more efficiently than C2C12-alpha2+cells, suggesting that the alpha11beta1 integrin may have a higher binding affinity for the GLPGER sequence. Human endothelial cells and dermal fibroblasts adhered to rScl proteins, indicating that multiple cell types may recognize and bind the Scl proteins via their collagen receptors. This work is a stepping stone toward defining the utilization of collagen receptors by microbial collagen-like proteins that are expressed by pathogenic bacteria.     

Proteolytic shedding of ST6Gal-I by BACE1 regulates the glycosylation and function of alpha4beta1 integrins

Differentiation of monocytes into macrophages is accompanied by increased cell adhesiveness, due in part to the activation of alpha4beta1 integrins. Here we report that the sustained alpha4beta1 activation associated with macrophage differentiation results from expression of beta1 integrin subunits that lack alpha2-6-linked sialic acids, a carbohydrate modification added by the ST6Gal-I sialyltransferase. During differentiation of U937 monocytic cells and primary human CD14(+) monocytes, ST6Gal-I is down-regulated, leading to beta1 hyposialylation and enhanced alpha4beta1-dependent VCAM-1 binding. Importantly, ST6Gal-I down-regulation results from cleavage by the BACE1 secretase, which we show is dramatically up-regulated during macrophage differentiation. BACE1 up-regulation, ST6Gal-I shedding, beta1 hyposialylation, and alpha4beta1-dependent VCAM-1 binding are all temporally correlated and share the same signaling mechanism (protein kinase C/Ras/ERK). Preventing ST6Gal-I down-regulation (and therefore integrin hyposialylation), through BACE1 inhibition or ST6Gal-I constitutive overexpression, eliminates VCAM-1 binding. Similarly, preventing integrin hyposialylation inhibits a differentiation-induced increase in the expression of an activation-dependent conformational epitope on the beta1 subunit. Collectively, these results describe a novel mechanism for alpha4beta1 regulation and further suggest an unanticipated role for BACE1 in macrophage function.     

A streptococcal collagen-like protein interacts with the alpha2beta1 integrin and induces intracellular signaling

The streptococcal collagen-like proteins Scl1 and Scl2 are prokaryotic members of a large protein family with domains containing the repeating amino acid sequence (Gly-Xaa-Yaa)(n) that form a collagen-like triple-helical structure. Here, we test the hypothesis that Scl variant might interact with mammalian collagen-binding integrins. We show that the recombinant Scl protein p176 promotes adhesion and spreading of human lung fibroblast cells through an alpha2beta1 integrin-mediated interaction as shown in cell adhesion inhibition assays using anti-alpha2beta1 and anti-beta1 integrins monoclonal antibodies. Accordingly, C2C12 cells stably expressing alpha2beta1 integrin as the only collagen-binding integrin show productive cell adhesion activities on p176 that can be blocked by an anti-alpha2beta1 integrin antibody. In addition, p176 promotes tyrosine phosphorylation of p125(FAK) of C2C12 cells expressing alpha2beta1 integrin, whereas parental cells do not. Furthermore, C2C12 adhesion of human lung fibroblast cells to p176 induces phosphorylation of p125FAK, p130CAS, and p68Paxillin proteins. In a domain swapping experiment, we show that integrin binds to the collagenous domain of the Scl protein. Moreover, the recombinant inserted domain of the alpha2 integrin interacts with p176 with a relatively high affinity (K(D) = 17 nm). Attempts to identify the integrin sites in p176 suggest that more than one site may be involved. These studies, for the first time, suggest that the collagen-like proteins of prokaryotes retained not only structural but also functional characteristics of their eukaryotic counterparts.     

A tumor-associated beta 1 integrin mutation that abrogates epithelial differentiation control

SCC4 human keratinocytes are derived from a squamous cell carcinoma of the tongue and undergo very little spontaneous differentiation. Introduction of a wild-type beta 1 integrin subunit into SCC4 cells stimulates differentiation, suggesting either that the cells have a defect in the integrin signaling pathways that control differentiation or that the beta1 subunit itself is defective. Here we describe a heterozygous mutation in the SCC4 beta 1 subunit. The mutation, T188I, maps to the I-like domain. It results in constitutive activation of ligand binding, irrespective of the partner alpha subunit, in solid phase assays with recombinant protein and in living cells. The mutation promotes cell spreading, but not proliferation, motility, or invasiveness. It results in sustained activation of Erk MAPK independent of cell spreading. When introduced into SCC4 keratinocytes, the wild-type beta1 integrin stimulates differentiation, whereas the mutant is inactive. Activation of beta 1 integrins in normal keratinocytes also suppresses differentiation. These results establish, for the first time, mutation as a mechanism by which integrins can contribute to neoplasia, because the degree of differentiation in epithelial cancers is inversely correlated with prognosis. They also provide new insights into how integrins regulate keratinocyte differentiation.     

Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion

ADAM12 belongs to the transmembrane metalloprotease ADAM ("a disintegrin and metalloprotease") family. ADAM12 has been implicated in muscle cell differentiation and fusion, but its precise function remains unknown. Here, we show that ADAM12 is dramatically up-regulated in regenerated, newly formed fibers in vivo. In C2C12 cells, ADAM12 is expressed at low levels in undifferentiated myoblasts and is transiently up-regulated at the onset of differentiation when myoblasts fuse into multinucleated myotubes, whereas other ADAMs, such as ADAMs 9, 10, 15, 17, and 19, are expressed at all stages of differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for alpha-actinin-2 was mapped to a short sequence in the membrane-proximal region of ADAM12 cytoplasmic tail; a second binding site was identified in the membrane-distal region. Co-immunoprecipitation experiments confirm the in vivo association of ADAM12 cytoplasmic domain with alpha-actinin-2. Overexpression of the entire cytosolic ADAM12 tail acted in a dominant negative fashion by inhibiting fusion of C2C12 cells, whereas expression of a cytosolic ADAM12 lacking the major alpha-actinin-2 binding site had no effect on cell fusion. Our results suggest that interaction of ADAM12 with alpha-actinin-2 is important for ADAM12 function.     

Cooperation of the metalloprotease, disintegrin, and cysteine-rich domains of ADAM12 during inhibition of myogenic differentiation

The extracellular domain of the mature form of ADAM12 consists of the metalloprotease, disintegrin, cysteine-rich, and epidermal growth factor (EGF)-like domains. The disintegrin, cysteine-rich, and EGF-like fragments have been shown previously to support cell adhesion via activated integrins or proteoglycans. In this study, we report that the entire extracellular domain of mouse ADAM12 produced in Drosophila S2 cells supported efficient adhesion and spreading of C2C12 myoblasts even in the absence of exogenous integrin activators. This adhesion was not mediated by beta1 integrins or proteoglycans, was myoblast-specific, and required the presence of both the metalloprotease and disintegrin/cysteine-rich domains of ADAM12. Analysis of the recombinant proteins by far-UV circular dichroism suggested that the secondary structures of the autonomously expressed metalloprotease domain and the disintegrin/cysteine-rich/EGF-like domains differ from the structures present in the intact extracellular domain. Furthermore, the intact extracellular domain (but not the metalloprotease domain or the disintegrin/cysteine-rich/EGF-like fragment alone) decreased the expression of the cell cycle inhibitor p21 and myogenin, two markers of differentiation, and inhibited C2C12 myoblast fusion. Thus, the novel protein-protein interaction reported here involving the extracellular domain of ADAM12 may have important biological consequences during myoblast differentiation.     

Hyposialylation of integrins stimulates the activity of myeloid fibronectin receptors

Despite numerous reports suggesting that beta(1) integrin receptors undergo differential glycosylation, the potential role of N-linked carbohydrates in modulating integrin function has been largely ignored. In the present study, we find that beta(1) integrins are differentially glycosylated during phorbol ester (PMA)-stimulated differentiation of myeloid cells along the monocyte/macrophage lineage. PMA treatment of two myeloid cell lines, U937 and THP-1, induces a down-regulation in expression of the ST6Gal I sialyltransferase. Correspondingly, the beta(1) integrin subunit becomes hyposialylated, suggesting that the beta(1) integrin is a substrate for this enzyme. The expression of hyposialylated beta(1) integrin isoforms is temporally correlated with enhanced binding of myeloid cells to fibronectin, and, importantly, fibronectin binding is inhibited when the Golgi disrupter, brefeldin A, is used to block the expression of the hyposialylated form. Consistent with the observation that cells with hyposialylated integrins are more adhesive to fibronectin, we demonstrate that the enzymatic removal of sialic acid residues from purified alpha(5)beta(1) integrins stimulates fibronectin binding by these integrins. These data support the hypothesis that unsialylated beta(1) integrins are more adhesive to fibronectin, although desialylation of alpha(5) subunits could also contribute to increased fibronectin binding. Collectively our results suggest a novel mechanism for regulation of the beta(1) integrin family of cell adhesion receptors.     

Discoidin domain receptor 1 is activated independently of beta(1) integrin

Various types of collagen have been identified as potential ligands for the two mammalian discoidin domain receptor (DDR) tyrosine kinases, DDR1 and DDR2. It is presently unclear whether collagen-induced DDR receptor activation, which occurs with very slow kinetics, involves additional proteins with kinase activity or membrane-anchored proteins serving as coreceptors. In particular, the role of the collagen-binding integrins alpha(1)beta(1) or alpha(2)beta(1) in the DDR activation process is undefined. Here, we provide three lines of evidence suggesting that DDR1 signaling is distinct from integrin activation. First we demonstrate that the enzymatic activity of DDR1 is essential for receptor tyrosine phosphorylation. Collagen-induced DDR receptor autophosphorylation can be blocked either by a dominant negative mutant or by a preparation of recombinant extracellular domain. Second, we show DDR1 signals independent of the epidermal growth factor (EGF) receptor. In cells that endogenously express both DDR1 and the EGF receptor, stimulation with EGF does not induce DDR activation. Third, we detected full DDR1 activation after collagen stimulation in cells that have been treated with blocking antibodies for alpha(2)beta(1) integrin or in cells with a targeted deletion of the beta(1) integrin gene. Finally, we show that overexpression of dominant negative DDR1 in the myoblast cell line C2C12 blocks cellular differentiation and the formation of myofibers.     

Integrins alpha2beta1 and alpha4beta1 can mediate SA11 rotavirus attachment and entry into cells

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins alpha2beta1 and alpha4beta1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing alpha2beta1 or alpha4beta1 integrins via transfection is increased over virus binding to and infection of cells transfected with alpha3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of alpha2beta1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing alpha2beta1 was elevated over binding to control cells and was specifically blocked by the anti-alpha2 monoclonal antibody AK7. Virus growth in alpha4-transfected K562 cells which had also been induced to express alpha2beta1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, alpha2beta1 and alpha4beta1, are capable of acting as cellular receptors for SA11 rotavirus.     

Interaction of integrins alpha 3 beta 1 and alpha 2 beta 1: potential role in keratinocyte intercellular adhesion

The colocalization of integrins alpha 2 beta 1 and alpha 3 beta 1 at intercellular contact sites of keratinocytes in culture and in epidermis suggests that these integrins may mediate intercellular adhesion (ICA). P1B5, an anti-alpha 3 beta 1 mAb previously reported to inhibit keratinocyte adhesion to epiligrin, was also found to induce ICA. Evidence that P1B5-induced ICA was mediated by alpha 2 beta 1 and alpha 3 beta 1 was obtained using both ICA assays and assays with purified, mAb-immobilized integrins. Selective binding of alpha 2 beta 1-coated beads to epidermal cells or plate-bound alpha 3 beta 1 was observed. This binding was inhibited by mAbs to integrin alpha 3, alpha 2, or beta 1 subunits and could be stimulated by P1B5. We also demonstrate a selective and inhibitable interaction between affinity- purified integrins alpha 2 beta 1 and alpha 3 beta 1. Finally, we show that expression of alpha 2 beta 1 by CHO fibroblasts results in the acquisition of collagen and alpha 3 beta 1 binding. Binding to both of these ligands is inhibited by P1H5, an anti-alpha 2 beta 1 specific mAb. Results of these in vitro experiments suggest that integrins alpha 2 beta 1 and alpha 3 beta 1 can interact and may do so to mediate ICA in vivo. Thus, alpha 3 beta 1 mediates keratinocyte adhesion to epiligrin and plays a second role in ICA via alpha 2 beta 1.