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.