The capability of the integrin VLA-3 to function as a receptor for collagen (Coll), laminin (Lm), and fibronectin (Fn) was addressed using both whole cell adhesion assays and ligand affinity columns. Analysis of VLA-3-mediated cell adhesion was facilitated by the use of a small cell lung carcinoma line (NCI-H69), which expresses VLA-3 but few other integrins. While VLA-3 interaction with Fn was often low or undetectable in cells having both VLA-3 and VLA-5, NCI-H69 cells readily attached to Fn in a VLA-3-dependent manner. Both Arg-Gly-Asp (RGD) peptide inhibition studies, and Fn fragment affinity columns suggested that VLA-3, like VLA-5, may bind to the RGD site in human Fn. However, unlike Fn, both Coll and Lm supported VLA-3-mediated adhesion that was not inhibited by RGD peptide, and was totally unaffected by the presence of VLA-5. In addition, VLA-3-mediated binding to Fn was low in the presence of Ca++, but was increased 6.6-fold with Mg++, and 30-fold in the presence of Mn++. In contrast, binding to Coll was increased only 1.2-fold with Mg++, and 1.7-fold in Mn++, as compared to the level seen with Ca++. Together, these experiments indicate that VLA- 3 can bind Coll, Lm, and Fn, and also show that (a) VLA-3 can recognize both RGD-dependent and RGD-independent ligands, and (b) different VLA-3 ligands have distinctly dissimilar divalent cation sensitivities.
The integrin heterodimer VLA-2, previously known as a collagen receptor, is now shown also to be a laminin receptor. Adhesion of the human melanoma cell line LOX to laminin was inhibited by anti-VLA a2 antibodies. Because VLA-2-mediated LOX cell attachment to laminin was not inhibited by digestion with collagenase, collagen contamination of laminin was not a factor. In addition, VLA-2 from LOX cells bound to immobilized laminin, and binding was disrupted by EDTA but not by Arg-Gly-Asp (RGD) peptides. VLA-3 also bound to lammnin-Sepharose, although less avidly than VLA-2.Thus, at least four separate members of the integrin .,8 subfamily serve as laminin receptors-i.e., VLA-2 and VLA-3 (this study) together with VLA-1 and VLA-6 (other reports). Whereas LOX and other cell lines used VLA-2 as both a laminin and collagen receptor, fibroblast VLA-2 mediated collagen but not laminin binding. Likewise, VLA-2 from platelets did not interact with laminin. Despite this functional discordancy, VLA-2 from laminin-binding and nonbinding sources was indistinguishable by all immunochemical and biochemical criteria examined. Thus, functional differences in VLA-2 may be due to cell type-specific modulation.Laminin, a major constituent of basement membranes, promotes adhesion, growth, migration, and differentiation of cells (1). The laminin glycoprotein (Mr 900,000) consists of three subunits arranged in the shape of a cross with one long and three short arms (1). Distinct functions of laminin, such as promotion of neurite outgrowth, interaction with basement membrane components, and attachment and spreading of cells, each reside within separate structural domains (1). In particular, cell adhesion appears to be mediated by at least three different regions in laminin (2-5), and a peptide derived from one of the cell-binding sites has been shown to inhibit experimental metastasis (6).Thus far, specific cell-adhesion receptors for laminin include a 68-kDa protein (7-9), as well as other surface molecules that belong to the integrin family. Integrins are a group of af3 heterodimers involved in cell-cell and cell-extracellular matrix interactions, some of which occur via recognition of an Arg-Gly-Asp (RGD) tripeptide sequence present in their ligands (10,11). Members of the integrin family implicated as laminin receptors include a rat cell complex that resembles VLA-3 (12), human VLA-6 from platelets (13), and a rat cell complex that may be the homolog of human VLA-1 (14).VLA-2, another member of the integrin ,1 subfamily, has been shown to act as a cell-surface receptor for collagen in platelets (15-18), fibroblasts (19, 20), and melanoma cells (21). However, in the platelet system VLA-2 was clearly unable to mediate cell binding to laminin (13,18). In the present report, a role for VLA-2 as a laminin receptor in human LOX melanoma cells, as well as other cell lines, is unequivocally demonstrated, despite finding that neither platelet nor fibroblast VLA-2 appears to interact with laminin. METHODS Antibodies and Cells. The mono...
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