Agrin Binds to the Nerve–Muscle Basal Lamina via Laminin

Abstract

Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel™. The first 130 amino acids from the NH2 terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH2-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel™ and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

Neural Agrin Induces Ectopic Postsynaptic Specializations in Innervated Muscle Fibers

“…In culture, recombinant cAgrin 7A4B8 is capable of inducing AChR aggregates, and it binds to the BL component laminin (Denzer et al, 1997). On its secretion from the injected fibers, such agrin presumably became attached to extracellular matrix.…”

“…8 A). Because agrin attaches to laminin (Denzer et al, 1997) and laminin-␤2 is closely colocalized with AChR clusters, agrin may become bound preferentially at such sites. Our experiments further demonstrate that agrin alone can induce the differentiation of an ectopic postsynapticlike structure locally, containing molecular components of normal postsynaptic specializations.…”

“…PCR reactions were conducted on partial cDNA clones (provided by Dr. A. D. Reith, L udwig Institute for C ancer Research, L ondon, UK) encoding the extracellular domain of Nsk2 (Ganju et al, 1995). PCR products were ligated into pcDNAI (Invitrogen) containing a tag consisting of the constant region of mouse immunoglobulin (Fc), which was generated by restriction digestion of pcN25 7 Fc (Denzer et al, 1997) with EcoRI and BamHI. The resulting construct, pNsk2Fc, was digested with EcoRI and XbaI restriction endonucleases and ligated into EcoRI /XbaI cut pcI N EO expression vector (Promega, Madison, W I), giving rise to pI N Nsk2Fc.…”

“…We have shown previously that AChE activity is tightly colocalized with agrin-induced ectopic AChR clusters (Jones et al, 1997). Full-length cAgrin 7A4B8 binds to laminin-␤2 (Denzer et al, 1997) and to ␣-dystroglycan (Bowe et al, 1994;C ampanelli et al, 1994;Gee et al, 1994;Sugiyama et al, 1994;Gesemann et al, 1996). We therefore asked whether cAgrin 7A4B8 would induce accumulation of laminin-␤2 or dystroglycans at ectopic AChR aggregates.…”

The Process-inducing Activity of Transmembrane Agrin Requires Follistatin-like Domains

“…The secreted form of agrin binds to the laminin ␥1-chain via its NtA domain resulting in stable association of this agrin isoform with basal laminae (19). In contrast, in TM-agrin the NtA domain is replaced by a noncleaved signal anchor that converts agrin into a type II transmembrane protein and localizes the agrin protein in N cyto /C exo orientation in the plasma membrane (17,18).…”