Contributions of the LG Modules and Furin Processing to Laminin-2 Functions

Abstract

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 ...

Endorepellin, a Novel Inhibitor of Angiogenesis Derived from the C Terminus of Perlecan

“…Moreover, proteolytic remodeling of the extracellular matrix can expose cryptic sites within collagen type IV that are required for angiogenesis in vivo (67), Thus, it is likely that perlecan might undergo a similar proteolytic processing in vivo, thereby liberating endorepellin through an endogenous processing mechanism common to most LG domains of laminin (43,68,69). The modular nature of perlecan protein core is particularly well suited for selective proteolysis (17,66) and subsequent release of peptides with biological activity.…”

Basement Membranes: Cell Scaffoldings and Signaling Platforms

“…Laminins differ based on their complement of domains, ability to polymerize, proteolytic processing, receptor-binding repertoire, and receptor affinities. Relative strong (heavy solid and dashed lines) and weak (thin dashed lines) interactions are indicated with heavy and thin lines with approximate dissociation constants are indicated where known (small numbers in nM values) (Denzer et al 1998;Gesemann et al 1998;Hopf et al 1999;Talts et al 1999;Talts et al 2000;Hopf et al 2001;Nielsen and Yamada 2001;Ries et al 2001;Garbe et al 2002;Smirnov et al 2002;Nishiuchi et al 2006;Harrison et al 2007). …”

Laminin-Induced Activation of Rac1 and JNKp46 Is Initiated by Src Family Kinases and Mimics the Effects of Skeletal Muscle Contraction

“…It binds to both dystroglycan and integrins in five globular domains (i.e., LG domains) of laminin's α-subunit. Laminin α2-chain LG modules 4-5 bind to the acidic polysaccharide chains of αDG (4); the integrin binding site in the LG1-5 region has not been mapped in detail (5). The binding site for αDG also localizes to the LG4-5 modules of laminin α5, however the binding site for α3β1 and α6β1 integrins localizes to LG1-3 (6).…”