The vertebrate neuromuscular junction (NMJ) remains the best-studied model for understanding the mechanisms involved in synaptogenesis, due to its relatively large size, its simplicity of patterning, and its unparalleled experimental accessibility. During neuromuscular development, each skeletal myofiber secretes and deposits around its extracellular surface an assemblage of extracellular matrix (ECM) proteins that ultimately form a basal lamina. The differentiation of the neuromuscular junction is a multistep process requiring coordinated interactions between nerve terminals and muscle. Although innervation is not needed for muscle production, the formation of nerve-muscle contacts, intramuscular nerve branching, and neuronal survival require reciprocal signals from nerve and muscle to regulate the formation of synapses (Ref.1 and 2). The neuromuscular junction (NMJ), a synapse formed between a motoneuron and a muscle fiber, has contributed greatly to understanding of the general principles of synaptogenesis as well as of neuromuscular disorders. Agrin, a basal lamina proteoglycan, is a critical factor secreted by motoneurons to direct NMJ formations by three laminin globular (LG) domains at the C terminus (Ref.3). Intriguingly, muscles also secrete Agrin; however, the muscle Agrin isoform lacks a specific alternative splicing sequence at the B/z site within the LG3 domain and is incapable of inducing AChR clustering. Neural Agrin activates muscle-specific kinase (MuSK), a type I receptor tyrosine kinase (RTK), which is a key organizer of NMJ formation. Unlike most conventional RTKs that are activated by a bound ligand, MuSK does not bind directly to neural Agrin, suggesting the existence of a coreceptor and a unique mechanism of activation of MuSK (Ref.4).
MuSK is a component of a multisubunit receptor complex to which Agrin connects through a putative MASC (Myotube-Associated Specificity Component). Phosphorylated MuSK initiates a signaling cascade that requires the AChR (Acetylcholine Receptor)-associated protein RAPSYN (Receptor-Associated Protein of the Synapse) to cause clustering of AChRs followed by their aggregation and attachment to the cytoskeleton. Via this pathway, Agrin also induces clustering of many other postsynaptic proteins, leading to coextensive aggregates of these proteins with AChR (Ref.3). The RATL (RAPSYN-Associated Transmembrane Linker) is another putative protein that links the RAPSYN-based scaffold to MuSK. At the MuSK–Dvl (Dishevelled) scaffold, the PAK (p21-Activated Kinase) is phosphorylated through Agrin–MuSK-activated Rac or CDC42 and drives aggregation of AChRs (Ref.10). Integrins engaged by Agrin directly or indirectly (through binding to laminin) participate in cytoskeletal re-organization through FAK (Focal adhesion kinase) and Src. Agrin probably stimulates ErbB receptors indirectly by concentrating neuregulins (ARIA) at synaptic sites. This effect is mediated by heparan sulfate binding sites in the ARIA molecule to which Agrin as a proteoglycan can bind (Ref.8). Activated MuSK, ErbB receptors and integrins in turn may synergise and result in triggering of signaling pathways that involve Rho-GTPases such as Ras, Rac and CDC42 and their effector molecules ERK1/2 (Extracellular Signal-Regulated Kinase) and JNK(c-Jun Kinase). This ultimately leads to activation of c-Jun and phosphorylation of the Ets-related transcription factor GABP (Guanidine and Adenosine-Binding Protein) that induces transcription of synapse-specific genes through binding to the N-box. The N-box contains a consensus motif for binding of Ets family members and is present not only in the genes encoding the subunits of the AChR, but also utrophine and other synapse-specific proteins. Activation of common transcription factors therefore increases expression of a multitude of proteins of the postsynaptic apparatus. Cortactin functions as a checkpoint for several pathways involved in the cytoskeleton rearrangement. DG (Dystroglycan) stabilizes the mature synapse by connecting the basal lamina to the cortical F-Actin cytoskeleton. Integrins also contribute to the stability of the NMJ by regulating the turnover of focal contacts through Src and PAK. PAK is recruited to integrin through a Paxillin–PKL (Paxillin Kinase Linker)–PIX (PAK-Interacting Exchange Factor) scaffold. Muscle Agrin plays a role in stabilizing AChR by binding to laminin and Alpha-DG and subsequently through Beta-DG to proteins such as dystrophin and utrophin, which interact with the cortical F-Actin cytoskeleton (Ref.9). AChR clustering in myotubes is also induced by Laminin-1 and Laminin-2/Laminin-4.
Agrin plays a key role in the formation and maintenance of the vertebrate NMJ and is the key neural factor that controls muscle postsynaptic differentiation, including the induction of synapse-specific transcription via neuregulins. Agrin has recently also been implicated in the formation of the immunological synapse, the organization of the cytoskeleton and the amelioration of function in diseased muscle. Recent studies suggest the possibility that Agrin plays a role in the etiology of Alzheimer’s disease, the most frequent cause of dementia in older persons (Ref.7).