Contributions of the LG modules and furin processing to laminin-2 functions

The alpha2-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 alpha2 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 (alpha(2)beta(1)gamma(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 alpha-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 alpha(6)beta(1) integrin and alpha-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 alpha-dystroglycan and alpha(6)beta(1) integrin binding.