Abstract. The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH: terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent.Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NHe and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, otl/31 and oe2/$1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors o¢1/~1 and o~2B1.
Laminin, a basement membrane glycoprotein promotes both cell attachment and neurite outgrowth. Separate domains on laminin elicit these responses, suggesting that distinct receptors occur on the surface of cells. NG108-15 neuroblastoma-glioma cells rapidly extend long processes in the presence of laminin. We report here that '251-labeled laminin specifically binds to these cells and to three membrane proteins of 67, 110, and 180 kDa. These proteins were isolated by affinity chromatography on laminin-Sepharose. The 67-kDa protein reacted with antibody to the previously characterized receptor for cell attachment to laminin. Antibodies to the 110-kDa and 180-kDa bands demonstrated that the 110-kDa protein was found in a variety of epithilial cell lines and in brain, whereas the 180-kDa protein was neural specific. Antibodies prepared against the 110-kDa and 180-kDa proteins inhibited neurite outgrowth induced by the neuritepromoting domain of laminin, whereas antibodies to the 67-kDa laminin receptor had no effect on neurite outgrowth. We conclude that neuronal cells have multiple cell-surface laminin receptors and that the 110-kDa and 180-kDa proteins are involved in neurite formation.Laminin, a major glycoprotein of basement membranes, induces a variety of cellular responses, including cell attachment (1-3) and neurite outgrowth (4)(5)(6)(7)(8)(9). Laminin is composed of three chains, including the A chain (400 kDa), B1 chain (210 kDa), and B2 chain (200 kDa), which are arranged in a cross-shaped structure. A large fragment of laminin with cell-attachment activity has been produced by proteolysis and localized to the central portion of the cross where the three chains intersect (10). Subsequently, a synthetic pentapeptide, Tyr-Ile-Gly-Ser-Arg, was found to exhibit cell attachment activity for a variety of epithelial cells (11). However, this is not the only biologically active domain in laminin, since a separate site near the globule at the end of the long arm of the cross has neurite-promoting activity and cell attachment activity for certain cells (12,13). Since these separate domains elicit distinct activities, it is likely that different receptors mediate these activities.Previous studies have shown that there is a specific laminin receptor on cells (67 kDa) that recognizes the synthetic peptide Tyr-Ile-Gly-Ser-Arg from a sequence in the B1 chain (11,(14)(15)(16). Evidence that this receptor mediates cell attachment has been shown with antibody to the receptor, the Tyr-Ile-Gly-Ser-Arg peptide, and proteolytic fragments of laminin, all of which inhibit the attachment of a variety of cells to laminin (11). However, Tyr-Ile-Gly-SerArg peptide and synthetic fragments from the intersection of the chains lack neurite-promoting activity. The cell-surface ligand responsible for laminin-mediated neurite outgrowth has not been identified, but several candidates exist, including integrin, sulfatides, and gangliosides (17-20).Here we have used laminin affinity chromatography to identify receptors on neuronal cell...
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