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

Coiled-coil domains are found in a wide variety of proteins, where they typ ically specify subunit oligomerization, Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in t he development of the nerve-muscle synapse, binds to the three-stranded coi led-coil domain of laminin-1, The interaction with laminin mediates the int egration of agrin into basement membranes. Here we characterize the binding site within the laminin-1 coiled coil in detail. Binding assays with indiv idual laminin-1 full-length chains and fragments revealed that agrin specif ically interacts with the gamma 1 subunit of laminin-1, whereas no binding to alpha 1 and beta 1 chains was detected. By using recombinant gamma 1 cha in fragments, we mapped the binding site to a sequence of 20 residues. Furt hermore, we demonstrate that a coiled-coil conformation of this binding sit e is required for its interaction with agrin, The finding that recombinant gamma 1 fragments bound at least 10-fold less than native laminin-1 indicat es that the structure of the three-stranded coiled-coil domain of laminin i s required for high-affinity agrin binding. Interestingly, no binding to a chimeric gamma 2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.