Interaction of agrin with laminin requires a coiled-coil conformation of the agrin-binding site within the laminin gamma 1 chain

Abstract

Coiled-coil domains are found in a wide variety of proteins, where they typically specify subunit oligomerization. Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in the development of the nerve-muscle synapse, binds to the three-stranded coiled-coil domain of laminin-1. The interaction with laminin mediates the integration of agrin into basement membranes. Here we characterize the binding site within the laminin-1 coiled coil in detail. Binding assays with individual laminin-1 full-length chains and fragments revealed that agrin specifically interacts with the γ1 subunit of laminin-1, whereas no binding to α1 and β1 chains was detected. By using recombinant γ1 chain fragments, we mapped the binding site to a sequence of 20 residues. Furthermore, we demonstrate that a coiled-coil conformation of this binding site is required for its interaction with agrin. The finding that recombinant γ1 fragments bound at least 10-fold less than native laminin-1 indicates that the structure of the three-stranded coiled-coil domain of laminin is required for high-affinity agrin binding. Interestingly, no binding to a chimeric γ2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.

Cross-linking reveals laminin coiled-coil architecture

“…The binding site of the heparan sulfate proteoglycan agrin was mapped to residues γ1329 to γ1348 in the laminin coiled coil (11). A coiled-coil framework was necessary for binding activity (11), but further progress in understanding the physical basis for this interaction has been hampered by the unknown register of the three subunits (41).…”

“…A coiled-coil framework was necessary for binding activity (11), but further progress in understanding the physical basis for this interaction has been hampered by the unknown register of the three subunits (41). The laminin coiled-coil simulations, restrained by one BS3 and two zero-length links in this region, place the agrin-binding stretch of the γ subunit opposite residues 1863-1880 of the α subunit and 1517-1533 of the β subunit, with a precision of about one heptad.…”

“…Due to the centrality of laminin in cell-ECM interactions, efforts have been made to analyze the functional regions of the trimer, to determine which α, β, and γ paralogs associate into physiological heterotrimers and to understand how trimers self-assemble into higher-order networks. Laminin fragments have been generated to assess their binding properties (11,12) and as targets for structure determination by X-ray crystallography (13)(14)(15)(16)(17)(18)(19)(20).…”

Basement Membranes: Cell Scaffoldings and Signaling Platforms

“…A high-affinity binding interaction was discovered among the upper coiled-coil segment of the g1 subunit with the amino-terminal NtA domain of agrin (Denzer et al 1998;Kammerer et al 1999). The LG domains of agrin were also found to bind strongly to a-dystroglycan and to sulfatides (Gesemann et al 1998).…”

Disruption of Glomerular Basement Membrane Charge through Podocyte-Specific Mutation of Agrin Does Not Alter Glomerular Permselectivity

“…26 BM-associated agrin binds to the laminin ␥1 chain via a globular domain (NtA) at its N terminus and to dystroglycan and integrin receptors through its C terminus. [27][28][29] By virtue of these interactions, agrin is thought to be an integral part of the molecular complex linking podocytes to the GBM. 30 Agrin-deficient mice are grossly normal but die at birth with severe neuromuscular defects.…”