Agrin Interactions at Neuromuscular Junction

The development of the neuromuscular junction (NMJ) is triggered by agrin, a signaling factor that is deposited by the nerve terminal at the site of contact with the muscle cell. Motor neuron-derived agrin induces many aspects of synaptic differentiation and is required for the postsynaptic localization of several synapse-specific proteins in the basal lamina, plasma membrane, and cytoplasm. Most notably, agrin induces a rapid aggregation of acetylcholine receptors (AChR), initially through a redistribution of preexisting receptors in the muscle membrane. This agrin-induced clustering of the neurotransmitter receptor allows synaptic transmission from early stages of NMJ formation.At the NMJ, neuronal agrin bound to the extracellular matrix protein laminin induces phosphorylation of muscle-specific tyrosine kinase (MuSK). MuSK is a component of a multisubunit receptor complex to which agrin connects through a putative myotube-associated specificity component (MASC). Phosphorylated MuSK initiates a signaling cascade that requires the AChR-associated protein RAPSYN 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 AChRs. The protein RAPSYN-associated transmembrane linker (RATL) links the RAPSYN-based scaffold to MuSK. At the MuSK-Dvl scaffold, PAK is phosphorylated through agrin-MuSK-Rac/CDC42 and drives aggregation of AChRs. Integrins engaged by agrin directly or indirectly (through binding to laminin) participate in cytoskeletal re-organization through FAK and Src. It is also thought that agrin stimulates ErbB receptors indirectly by concentrating neuregulins at synaptic sites. 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 and JNK. This ultimately leads to activation of c-Jun and phosphorylation of the Ets-related transcription factor GABP, which 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. Therefore, activation of common transcription factors increases expression of a multitude of proteins of the postsynaptic apparatus. Cortactin functions as a checkpoint for several pathways involved in the cytoskeleton rearrangement. α-Dystroglycan (α-DG) 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 PKL-PAK-PIX scaffold. Muscle agrin plays a role in stabilizing AChRs by binding to laminin and α-DG and subsequently through β-DG to proteins such as Dystrophin and Utrophin, which interact with the cortical F-actin cytoskeleton. AChR clustering in myotubes is also induced by Laminin-1 and Laminin-2/4.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 the elderly population.