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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

The enzyme ␤1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting GlcNAc residue to glycoproteins, resulting in a modulation in biological function. Our previous studies showed that the transfection of the GnT-III gene into B16 melanoma cells results in a suppression of invasive ability and lung colonization. The suppression has been postulated to be due to an increased level of E-cadherin expression on the cell surface, which in turn leads to the up-regulation of cell-cell adhesion. In this study, we report on the effects of overexpression of GnT-III on cell-matrix adhesion. The overexpression of GnT-III, but not that of an enzymatic inactive GnT-III (D323A), inhibits cell spreading and migration on fibronectin, a specific ligand for integrin ␣ 5 ␤ 1 , and the focal adhesion kinase phosphorylation. E 4 -PHA lectin blot analyses showed that the levels of bisecting GlcNAc structures on the integrin ␣ 5 subunit as well as ␣ 2 and ␣ 3 subunits immunoprecipitated from GnT-III transfectants were substantially increased. In addition, the affinity of the binding of integrin ␣ 5 ␤ 1 to fibronectin was significantly reduced by the introduction of the bisecting GlcNAc, to the ␣ 5 subunit. These findings suggest that the modification of N-glycan of integrin by GnT-III inhibits its ligand binding ability, subsequently leading to the down-regulation of integrin-mediated signaling.Nearly all secreted and cell surface proteins are glycosylated. The attached sugar chains have many biological functions, for example, cell-cell communication, signal transduction, protein folding, and stability (1). Oligosaccharides are synthesized through the action of glycosyltransferase, which catalyzes the formation of glycosidic bonds. ␤1,4-N-Acetylglucosaminyltransferase (GnT-III) 1 catalyzes the transfer of GlcNAc residues in a ␤1,4-linkage to the ␤1,4-mannose residue in the core region of N-glycans, producing a bisecting GlcNAc (Fig. 1). We previously purified GnT-III from rat kidney and cloned the cDNA from both the rat and human (2, 3). In a quest for the biological significance of GnT-III, we found that GnT-III suppresses the dimerization of TrkA, the receptor for nerve growth factor, and EGF receptor phosphorylation (4, 5). It is interesting to note that the metastatic capabilities of B16 mouse melanoma cell are down-regulated by the introduction of the GnT-III gene (6). This anti-metastatic effect has been partly ascribed to the effect of GnT-III on an increase in E-cadherinmediated homotypic adhesion and a suppression of the phosphorylation of the E-cadherin-␤-catenin complex on the cell-cell adhesion (7,8).Cell-extracellular matrix (ECM) interactions play essential roles during the acquisition of migration and invasive behavior of the cells. The integrin family consists of ␣ and ␤ heterodimeric transmembrane receptors for ECM and connects many biological functions, such as development, control of cell proliferation, protection against apoptosis, and malignant transformation (9). The fibronectin (FN) ...

The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as ␣-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their ␣ chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (␣5␤1␥1) for ␣ 3 ␤ 1 and ␣ 6 ␤ 1 integrins and ␣-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4 -5 modules did not compromise the binding of laminin-10 to ␣ 3 ␤ 1 and ␣ 6 ␤ 1 integrins but completely abrogated its binding to ␣-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to ␣-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the ␣-dystroglycan binding by the LG4 module.

Integrins alpha3beta1 and alpha6beta1 are two major laminin receptors expressed on the surface of mammalian cells. Interactions of cells with laminins through these integrins play important roles in cell adhesion, differentiation, motility, and matrix assembly. To determine the binding specificity and affinity of these integrins toward various types of laminins at the level of direct protein-protein interactions, we purified integrins alpha3beta1 and alpha6beta1 from human placenta, and examined their binding to a panel of laminin isoforms, each containing distinct alpha chains (i.e., laminin-1, laminin-2/4, laminin-5, laminin-8, and laminin-10/11). Integrin alpha3beta1 showed clear specificity for laminin-5 and laminin-10/11, with no significant binding to laminin-1, laminin-2/4, and laminin-8. In contrast, integrin alpha6beta1 showed a broad spectrum of specificity, with apparent binding affinity in the following order: laminin-10/11 > laminin-5 > laminin-1 > laminin-2/4 congruent with laminin-8. Integrin titration assays demonstrated that laminin-10/11 was the most preferred ligand among the five distinct laminin isoforms for both alpha3beta1 and alpha6beta1 integrins. Given that laminin-10/11 is the major basement membrane component of many adult tissues, the interaction of laminin-10/11 with these integrins should play a central role in the adhesive interactions of epithelial cells with underlying basement membranes.

CD151 (PETA-3 ⁄ SFA-1) is a member of the tetraspanin family of proteins, possessing four membranespanning domains [1]. CD151 was initially identified in platelets [2] and T-cell leukemic cells [3] and has been found to be expressed in a wide variety of cells, including epithelial, endothelial, muscle, Schwann and dendritic cells. Studies using antibodies and small interfering RNA (siRNA) against CD151 have revealed that treatments with these antibodies and siRNA attenuate cell adhesion to and migration on substrates, and disturb epithelial cell-cell adhesion and polarization in cultured cells [4][5][6][7][8][9][10], indicating that CD151 is involved in the regulation of cell-cell and cell-substratum adhesions. Several studies performed in vivo highlight the physiological importance of CD151: in humans, a nonsense mutation in CD151 causes diseases, including end-stage hereditary nephropathy, pretibial epidermolysis bullosa and sensorineural deafness [11]. CD151-null mice show defects in platelet aggregation, keratinocyte migration, T-cell proliferation and pathological angiogenesis, although these mice are viable and fertile [12][13][14][15]. Recently, Sachs et al. [16] reported that CD151 knockout mice show strain-dependent severe renal defects caused by abnormalities of the glomerular basement membrane, loss of podocyte foot processes, glomerulosclerosis and cystic tubular dilation.CD151 interacts with various membrane proteins, including the laminin-binding integrins a3b1, a6b1, a6b4 and a7b1, and other tetraspanin family members, The tetraspanin CD151 forms a stable complex with integrin a3b1, a widely expressed laminin receptor, and is implicated in the regulation of integrin a3b1-mediated cellular responses, including cell attachment, spreading and migration. However, the molecular mechanism by which CD151 regulates integrin a3b1 functions remains unclear. To address this issue, we knocked down CD151 expression in A549 human lung adenocarcinoma cells by RNA interference. When plated on laminin-511 (laminin-10), the CD151-knocked-down cells showed aberrant membrane protrusions and exhibited reductions in the tyrosine phosphorylation of focal adhesion kinase, Src, p130Cas and paxillin. The formation of membrane protrusions was attenuated when the cells were either plated on surfaces coated with higher concentrations of laminin-511 or treated with the integrin b1-activating mAb TS2 ⁄ 16; however, neither treatment could rescue the reduced tyrosine phosphorylation. These results indicate that CD151 knockdown weakens the integrin a3b1-mediated adhesion to laminin-511 and thereby provokes an aberrant morphology, but this reduced adhesive activity is not involved in the decline of signaling events in CD151-knocked-down cells. Thus, our results suggest that CD151 regulates integrin a3b1 functions in two independent aspects: potentiation of integrin a3b1-mediated cell adhesion and promotion of integrin a3b1-stimulated signaling events involving tyrosine phosphorylation.Abbreviations EGFP, enhanced green fluorescent...

A novel fibronectin (FN) isoform lacking the segment from IIICS (type III connecting segment) through the I-10 module is expressed predominantly in normal cartilaginous tissues. We expressed and purified recombinant cartilage-type FN using a mammalian expression system and characterized its molecular and biological properties. Although FNs have been shown to be secreted as disulfide-bonded dimers, cartilage-type FN was secreted mainly as a monomer. It was less potent than plasma-type FN in promoting cell adhesion and binding to integrin alpha5beta1, although it was more active than plasma-type FN in binding to chondroitin sulfate E. When added exogenously, cartilage-type FN was poorly assembled into the fibrillar FN matrix, mostly because of its monomeric structure. Given that cartilage is characterized by its non-fibrillar matrix with abundant chondroitin sulfate-containing proteoglycans, it is likely that cartilage-type FN has evolved to adapt itself to the non-fibrillar structure of the cartilage matrix through acquisition of a novel mechanism of alternative pre-mRNA splicing.

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