We have studied the reconstitution of basement membrane molecules from extracts prepared from the basement membrane of the EHS tumor. Under physiological conditions and in the presence of added type IV collagen and heparan sulfate proteoglycan, gellike structures form whose ultrastructure appears as interconnected thin sheets resembling the lamina dense zone of basement membrane. The major components of the reconstituted structures include laminin, type IV collagen, heparan sulfate proteoglycan, entactin, and nidogen. These components polymerize in constant proportions on reconstitution, suggesting that they interact in defined proportions. Molecular sieve studies on the soluble extract demonstrate that laminin, entactin, and nidogen are associated in large but dissociable complexes which may be a necessary intermediate in the deposition of basement membrane. The reconstituted matrix was biologically active and stimulated the growth and differentiation of certain cells.

Laminin is a multifunctional protein with diverse biological activities. Like fibronectin, it can influence cell adhesion, growth, morphology, differentiation, migration, and agglutination as well as the assembly of the extracellular matrix. Laminin primarily affects cells of epithelial origin, and the response varies depending on the cell. Because most differentiated cells are difficult to maintain in culture, laminin may be an important supplement in studies on cell differentiation in vitro.

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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