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regenerating islet derived-3
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approved gene symbol AGRN) Agrin is a major large, highly negatively charged multidomain heparan sulfate proteoglycan (> 500 kDa) of the extracellular matrix (ECM) with a calculated relative molecular mass of more than 200 kDa for the protein core (Tsen et al, 1995). The protein contains a number of structural domains, including regions of homology to laminin, Kazal protease inhibitors, and EGF repeats.
Agrin is synthesized and secreted by motoneurons at the axon terminal after anterograde axonal transport and binds to receptors present on muscle cells. In addition to motor neurons, agrin is expressed by many other neuronal populations throughout the nervous system (Tsen et al, 1995; Stone and Nikolics, 1995).
Agrin was identified originally as an electric organ-aggregating factor in extracts from basal lamina-containing fractions of the synapse-rich electric organ of Torpedo californica (Nitkin et al, 1987). Agrin causes the formation of patches on cultured chicken myotubes at which acetylcholine receptors, acetylcholinesterase, and butyrylcholinesterase are concentrated. These specialized structures resemble the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction and Agrin is thought to play a key role in synaptic differentiation (Ruegg et al, 1992). Agrin also induces acetylcholine receptor phosphorylation and this process is inhibited by Staurosporine (Wallace et al, 1994; Bowe and Fallon, 1995; Patthy and Nikolics, 1993). The induction of acetylcholine receptors subunit gene expression is governed mainly by neuregulins. It has been suggested that agrin may mediate acetylcholine receptor clustering by interacting with muscle-bound heparin binding growth factors such as HB-GAM.
That neurotrophic factors may play a role in regulating agrin expression in vivo is suggested by experiments with rat pheochromocytoma PC12 cells. Significant increases in total agrin mRNA are observed upon treatment with NGF. NGF induces the production of transcripts encoding isoforms with high aggregating activity and neuronal tissue distribution that are not expressed in untreated cells (Smith et al, 1997). EGF, insulin, dexamethasone, or retinoic acid do not have these effects. Wells et al (1999) have shown that BDNF and NT-4 inhibit Agrin induced AChR clustering on cultured myotubes.
The human gene has been mapped to chromosome 1pter-p32 (Rupp et al, 1992). Alternative splicing of agrin mRNA is regulated during development and in a cell-specific manner and particular splice variants can differ in function and distribution (Ruegg et al, 1992; McMahan et al, 1992).
Agrin-0 (no inserts of amino acids), Agrin-8 (insert of 8 amino acids), Agrin-11 (insert of 11 amino acids), and Agrin-19 (insert of 8 amino acids plus insert of 11 amino acids) encode splice variants of the agrin proteins that are active in acetylcholine receptor aggregating assays. The insert-containing variants are neuron-specific and encode agrin proteins with greater synapse-organizing activity than the Agrin-0 variant, which is a nonneuronally expressed isoform of agrin. Rat cerebellar granule cells express and secrete an isoform of agrin into the conditioned medium that lacks the acetylcholine receptor aggregating activity. Some splice variants synthesized by Schwann cells lack one (Agrin-related protein-1) or two (Agrin-related protein-2) regions in agrin that are required for its activity and are inactive in standard acetylcholine receptor aggregation assays.
Burgess et al (2000) have shown that two proteins with different aminotermini obtained from the murine Agrin gene (SN-Agrin, short isoform; LN-Agrin, long isoform) differ in subcellular localization, tissue distribution, and function. LN-Agrin is expressed in both neural and nonneural tissues and in motorneurons of the spinal cord. SN-Agrin is expressed selectively in the nervous system but is widely distributed in many neuronal cell types. LN-Agrin assembles into basal laminae and SN-Agrin remains cell-associated. In mice lacking LN-Agrin transcripts the Agrin protein is absent from all basal laminae and this leads to a drastic impairment of neuromuscular junctions.
Agrin has been shown to act via a receptor complex that includes the receptor tyrosine kinase MuSK as well as a myotube-specific accessory component (Glass et al, 1996; Herbst and Burden, 2000). Signaling throught the agrin receptor involves the participation of a cytoplasmic 43 kDa protein, rapsyn, a synaptic peripheral membrane protein which is necessary for the clustering of acetylcholine receptors and all other postsynaptic membrane components studied to date.
Some isoforms of agrin also bind to alpha-dystroglycan, which functions as a dual receptor for agrin and laminin-2 in the membranes of Schwann cells (Gee et al, 1994; O'Tool et al, 1996; Yamada et al, 1996). The interaction of agrin with alpha-dystroglycan can be modulated by cell surface glycosaminoglycans in an isoform-dependent manner. Some, but not all, isoforms possess the ability to bind heparin, which then inhibits binding of agrin to alpha-dystroglycan.
Cotman et al (1999) have shown that Agrin binds FGF-2, thrombospondin, merosin, laminin, and tenascin, suggesting a function of these interactions in the extracellular matrix and possibly a role for Agrin in axon pathway development.
Khan et al (2001) have demonstrated that Agrin is expressed also in lymphocytes and is important for the formation of the immunological synapse, i.e., the contact site between T-cells and antigen-presenting cells. Agrin is important for setting the threshold of T-cell signaling and its mechanism of action involves the reorganization of membrane lipid microdomains.
TRANSGENIC ANIMALS, KNOCK-OUT, AND ANTISENSE STUDIES
Transgenic mice with a targeted disruption of the gene encoding the receptor tyrosine kinase MuSK, which is localized selectively to the postsynaptic muscle surface, do not form neuromuscular synapses. Mice lacking agrin exhibit similar defects in their neuromuscular junctions. Intramuscular nerve branching and presynaptic differentiation are abnormal in the mutant. These experiments confirm the essential role of agrin and its receptor in signaling between developing nerve and muscle.
Introduction of an agrin antisense RNA into the neuroblastoma-glioma hybrid cell line, NG108-15 abolishes the ability of the hybrid cells to induce the aggregation of acetylcholine receptors when co-cultured with myotubes demonstrating that the aggregation of receptors on cultured myotubes is mediated by neuron-derived agrin.
Suppression of Agrin expression in cultured hippocampal neurons by means of antisense RNA treatment results in the impairment of dendritic development, the formation of fewer synapses, and a selective inhibition of the clustering of GABA receptors.
Li et al (1999) have studied synapse formation between cultured cortical neurons isolated from Agrin deficient mouse embryos. They show that functional glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses form between mutant neurons. Therefore, the mechanisms of interneuronal synaptogenesis may be distinct from those regulating synapse formation at the neuromuscular junction.
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ENTRY LAST MODIFIED: March 2002
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