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A panel of human tumor cell lines was screened for selective expression of laminin ␣5 chain, a newly identified laminin subunit comprising laminin-10 (␣5␤1␥1) and -11 (␣5␤2␥1). The lung adenocarcinoma cell line A549 was found to express the ␣5 chain at relatively high levels but no detectable amounts of other ␣ chains. The laminin variants containing ␣5 chain were purified from the conditioned medium of A549 cells by immunoaffinity chromatography using the anti-laminin monoclonal antibody 4C7 which was shown recently to recognize the laminin ␣5 chain (Tiger, C.-F., Champliaud, M.-F., Pedrosa-Domellof, F., Thornell, L.-E., Ekblom, P., and Gullberg, D. (1997) J. Biol. Chem. 272, 28590 -28595). The purified laminin variants consisted of three chains with molecular masses of 350, 220, and 210 kDa. The 350-kDa chain was specifically recognized by another anti-␣5 chain monoclonal antibody capable of recognizing denatured ␣5 chain on immunoblots, whereas the 210-kDa chain was recognized by an anti-␥1 chain antibody. The purified ␣5 chain-containing laminin variants (hereafter referred to as laminin-10/11) were highly active in mediating adhesion of A549 cells to the substratum with potency as high as that of laminin-5 and significantly higher than those of laminin-1, laminin-2/4, or fibronectin. Adhesion to substrata coated with laminin-10/11 was specifically inhibited by anti-integrin antibodies directed against the integrin ␣3 or ␤1 subunit but not by those against ␣2 or ␣6 subunit, indicating that laminin-10/11 is specifically recognized by integrin ␣3␤1. Given the wide distribution of laminin-10/11 in the basement membrane of various tissue types and dominant expression of integrin ␣3␤1 in most epithelial cells, specific interaction of laminin-10/11 with integrin ␣3␤1 may play an important role in in vivo regulation of proliferation and differentiation of epithelial cells through the basement membrane.

The ␣ 5 chain-containing laminin isoforms, laminins-10 and -11 (laminin-10/11), are the major components of the basement membrane, having potent cell-adhesive activity. We examined the cell-adhesive and integrin-mediated signaling activities of laminin-10/11 in comparison to fibronectin, the best characterized extracellular adhesive ligand. We found that laminin-10/11 are more active than fibronectin in promoting cell migration and preferentially activate Rac, not Rho, via the p130CasCrkII-DOCK180 pathway. Cells adhering to fibronectin develop stress fibers and focal contacts, whereas cells adhering to laminin-10/11 do not, consistent with the high cell migration-promoting activity of laminin-10/11. Pull-down assays of GTP-loaded Rac and Rho demonstrated the preferential activation of Rac on laminin-10/ 11, in contrast to the activation of Rho on fibronectin. Activation of Rac by laminin-10/11 was associated with the phosphorylation of p130Cas and an increased formation of a p130Cas -CrkII-DOCK180 complex. Cell migration on laminin-10/11 was suppressed by the expression of either a dominant-negative Rac or CrkII mutants defective in p130Cas or DOCK180 binding. This is the first report demonstrating a distinct activation of Rho family GTPases resulting from adhesion to different extracellular ligands.

Anti-p200 pemphigoid has been characterized by autoantibodies to an unidentified 200-kDa protein (p200) of the dermal؊epidermal junction. The objective of this study was to identify p200. We performed 2D gel electrophoresis of dermal extracts and immunoblotting with patients' sera, followed by MS analysis of a unique protein band. The protein band corresponded to laminin ␥1. Anti-laminin ␥1 mAb reacted with the anti-p200 immunoprecipitates by immunoblotting. Sera from 32 patients with anti-p200 pemphigoid showed 90% reactivity to the recombinant products of laminin ␥1. None of the healthy control sera reacted with laminin ␥1. By immunoblotting, reactivity of a patient's serum with p200 was competitively inhibited by adding anti-laminin ␥1 C-terminus mAb. Purified anti-p200 IgG also inhibited the reactivity of this mAb to dermal laminin ␥1. Most laminin ␥1-positive sera showed reactivity with recombinant laminin ␥1 C-terminal E8 fragment. Reactivity of patients' sera and purified IgG to dermal laminin ␥1 was higher than reactivity to blood vessel laminin ␥1 under reducing conditions. These results suggest that laminin ␥1 is the autoantigen for patients with anti-p200 pemphigoid. The autoantibodies may specifically recognize dermal laminin ␥1 with unique posttranslational modifications. The epitope is localized to the 246 C-terminal amino acids within the coiled-coil domain. The 9 C-terminal residues are known to be critically involved in laminin recognition by integrins.autoimmune disease ͉ basement membrane ͉ bullous pemphigoid ͉ proteomics

CD151, one of the tetraspanins, forms a stable complex with integrin ␣3␤1, the major laminin receptor on the cell surface. We found that 8C3, an anti-CD151 mAb obtained by screening for reactivity with integrin ␣3␤1-CD151 complexes, was capable of dissociating CD151 from integrin ␣3␤1, thereby allowing us to deplete CD151 from purified integrin ␣3␤1-CD151 complexes. The CD151-free integrin ␣3␤1 thus obtained showed a significant reduction in its ability to bind to laminin-10͞11, a high-affinity ligand for integrin ␣3␤1, with a concomitant reduction in its reactivity with mAb AG89, which recognizes activated ␤1-containing integrins. These results raised the possibility that the association of integrin ␣3␤1 with CD151 potentiates the ligand-binding activity of the integrin through sustaining its activated conformation. In support of this possibility, the ligand-binding activity was restored when CD151-free integrin ␣3␤1 was reassociated with purified CD151. 8C3-induced dissociation of CD151 from integrin ␣3␤1 was also demonstrated on the surface of living cells by fluorescent resonance energy transfer imaging, accompanied by a concomitant reduction in the cell adhesion to laminin-10͞11-coated substrates. CD151 knock-down by RNA interference also resulted in a reduction in the adhesive activity of the cells. Taken together, these results indicate that CD151 association modulates the ligandbinding activity of integrin ␣3␤1 through stabilizing its activated conformation not only with purified proteins but also in a physiological context. cell adhesion ͉ laminin

CD151, a member of the tetraspanin family proteins, tightly associates with integrin α3β1 and localizes at basolateral surfaces of epithelial cells. We found that overexpression of CD151 in A431 cells accelerated intercellular adhesion, whereas treatment of cells with anti-CD151 mAb perturbed the integrity of cortical actin filaments and cell polarity. E-Cadherin puncta formation, indicative of filopodia-based adhesion zipper formation, as well as E-cadherin anchorage to detergent-insoluble cytoskeletal matrix, was enhanced in CD151-overexpressing cells. Levels of GTP-bound Cdc42 and Rac were also elevated in CD151-overexpressing cells, suggesting the role of CD151 in E-cadherin–mediated cell–cell adhesion as a modulator of actin cytoskeletal reorganization. Consistent with this possibility, engagement of CD151 by the substrate-adsorbed anti-CD151 mAb induced prominent Cdc42-dependent filopodial extension, which along with E-cadherin puncta formation, was strongly inhibited by calphostin C, a protein kinase C (PKC) inhibitor. Together, these results indicate that CD151 is involved in epithelial cell–cell adhesion as a modulator of PKC- and Cdc42-dependent actin cytoskeletal reorganization.

Extracellular matrix (ECM), which provides critical scaffolds for all adhesive cells, regulates proliferation, differentiation, and apoptosis. Different cell types employ customized ECMs, which are thought to play important roles in the generation of so-called niches that contribute to cell-specific functions. The molecular entities of these customized ECMs, however, have not been elucidated. Here, we describe a strategy for transcriptome-wide identification of ECM proteins based on computational screening of >60,000 full-length mouse cDNAs for secreted proteins, followed by in vitro functional assays. These assays screened the candidate proteins for ECM-assembling activities, interactions with other ECM molecules, modifications with glycosaminoglycans, and cell-adhesive activities, and were then complemented with immunohistochemical analysis. We identified 16 ECM proteins, of which seven were localized in basement membrane (BM) zones. The identification of these previously unknown BM proteins allowed us to construct a body map of BM proteins, which represents the comprehensive immunohistochemistry-based expression profiles of the tissue-specific customization of BMs. basement membrane ͉ body map ͉ niche ͉ cell adhesion ͉ glycosaminoglycan