The ␣2-laminin subunit contributes to basement membrane functions in muscle, nerve, and other tissues, and mutations in its gene are causes of congenital muscular dystrophy. The ␣2 G-domain modules, mutated in several of these disorders, are thought to mediate different cellular interactions. To analyze these contributions, we expressed recombinant laminin-2 (␣ 2  1 ␥ 1 ) with LG4 -5, LG1-3, and LG1-5 modular deletions. Wild-type and LG4 -5 deleted-laminins were isolated from medium intact and cleaved within LG3 by a furin-like convertase. Myoblasts adhered predominantly through LG1-3 while ␣-dystroglycan bound to both LG1-3 and LG4 -5. Recombinant laminin stimulated acetylcholine receptor (AChR) clustering; however, clustering was induced only by the proteolytic processed form, even in the absence of LG4 -5. Furthermore, clustering required ␣ 6  1 integrin and ␣-dystroglycan binding activities available on LG1-3, acting in concert with laminin polymerization. The ability of the modified laminins to mediate basement membrane assembly was also evaluated in embryoid bodies where it was found that both LG1-3 and LG4 -5, but not processing, were required. In conclusion, there is a division of labor among LG-modules in which (i) LG4 -5 is required for basement membrane assembly but not for AChR clustering, and (ii) laminin-induced AChR clustering requires furin cleavage of LG3 as well as ␣-dystroglycan and ␣ 6  1 integrin binding.The laminin ␣2-chain, a subunit of laminin-2 (␣ 2  1 ␥ 1 ) and laminin-4 (␣ 2  2 ␥ 1 ), is expressed in the basement membranes of skeletal muscle, peripheral nerve, brain, and placenta (1-3). ␣2-Laminins, similar in molecular morphology and functional attributes to ␣1-laminin, are thought to play important roles in basement membrane assembly and the maintenance of the neuromuscular axis (reviewed in Ref. 4). Mutations in the laminin ␣2-subunit are a cause of a human congenital muscular dystrophy typically characterized by early onset, severity, and involvement of peripheral nerve and brain (5, 6). Some of these dystrophies have mutations within the part of the gene coding for LG modules (7). The dystrophic phenotype in mouse models of the disease is one of defective basement membranes in muscle and nerve, muscle necrosis with poor regeneration, patchy peripheral nerve dysmyelination, and decreased complexity of infoldings and post-junctional membrane lengths of the NMJ motor endplate (6, 8 -12).Studies on cultured myotubes have revealed that ␣1-and ␣2-laminin polymerization and G-domain contributions drive laminin assembly on the cell surface and direct a redistribution of interacting cytoskeletal components (13). The ability of laminin to induce such cytoskeletal reorganizations is thought to reflect an important receptor-dependent property of laminin during early steps in assembly of a basement membrane. It has also been shown that laminins can induce the clustering of the acetylcholine receptor (AChR), 1 a property shared with agrin (14). However, while agrin is secreted by terminal ...