RnoEX0007999 @ rn6

Alternative splicing of chick agrin mRNA at two sites, A (GgaEX0002487) and B (GgaEX1007051 and GgaEX1007055), gives rise to eight possible isoforms of which five are expressed in vivo. Motor neurons express high levels of isoforms with inserts at sites A and B, muscle cells synthesize isoforms that lack amino acids at the B-site. To obtain further insights into the mechanism of agrin-induced AChR aggregation, the authors determined the EC50 (effective concentration to induce half-maximal AChR clustering) of each agrin isoform and of truncation mutants. On chick myotubes, EC50 of the COOH-terminal, 95-kD fragment of agrinA4B8 was approximately 35 pM, of agrinA4B19 approximately 110 pM and of agrinA4B11 approximately 5 nM. While some AChR clusters were observed with 64 nM of agrinA4B0, no activity was detected for agrinA0B0. Recombinant full-length chick agrin and a 100-kD fragment of ray agrin showed similar EC50 values. A 45-kD, COOH-terminal fragment of agrinA4B8 retained high activity (EC50 approximately equal to 130 pM) and a 21-kD fragment was still active, but required higher concentrations (EC50 approximately equal to 13 nM). Unlike the 45-kD fragment, the 21-kD fragment neither bound to heparin nor did heparin inhibit its capability to induce AChR aggregation. These data show quantitatively that agrinA4B8 and agrinA4B19, expressed in motor neurons, are most active, while no activity is detected in agrinA0B0, the dominant isoform synthesized by muscle cells. A4 = HsaEX0003154/GgaEX0002487, B8 = HsaEX0003157, B11 = HsaEX0003152/GgaEX1007055, B19= HsaEX0003152/GgaEX1007055+HsaEX0003157.

In the experiments reported here the authors investigate the regions of agrin necessary for nAChR clustering activity using two different methods. First, the authors expressed truncated soluble forms of the agrin protein in mammalian cells and assessed their clustering activity. Second, the authors generated a panel of monoclonal antibodies (mAbs) against agrin and mapped their epitopes. Several mAbs block agrin-induced aggregation of nAChRs. One of the mAbs, Agr86, binds exclusively to the CNS-specific splicing variants and thus identifies an epitope common only to these more active isoforms. Mapping of the Agr86 epitope suggests that alternative splicing results in a distributed conformational change in the agrin protein. Taken together these data suggest that four domains in the C-terminal 55 kDa of agrin contribute to its nAChR clustering activity.

Agrin is a heparan sulfate proteoglycan that induces aggregation of acetylcholine receptors (AChRs) at the neuromuscular synapse. This aggregating activity is modulated by alternative splicing. Here, the authors compared binding of agrin isoforms to heparin, alpha-dystroglycan, and cultured myotubes. The authors find that the alternatively spliced 4 amino acid insert (KSRK) is required for heparin binding (encoded by HsaEX0003154). The binding affinity of agrin isoforms to alpha-dystroglycan correlates neither with binding to heparin nor with their AChR-aggregating activities. Moreover, the minimal fragment sufficient to induce AChR aggregation does not bind to alpha-dystroglycan. Nevertheless, this fragment still binds to cultured muscle cells. (HsaEX003157 is also examined, it negatively affects binding to putative agrin receptor).

Agrin is a component of the extracellular matrix that regulates aspects of neuromuscular junction differentiation. Identification of agrin-binding proteins has lead to the suggestion that alpha-dystroglycan is a muscle cell surface proteoglycan that mediates agrin activity. To further test this hypothesis, the authors have compared the ability of differentially active agrin isoforms to interact with a model component of proteoglycans, heparin, as well as with the putative proteoglycan alpha-dystroglycan. The authors demonstrate that an alternately spliced exon (encoding the sequence lysine, serine, arginine, lysine: Y site) (HsaEX0003154) is necessary for agrin-heparin interactions. The authors also show that alternate splicing at another site (Z site) (HsaEX0003157) dramatically affects interaction of alpha-dystroglycan with agrin. A bigger Z-site insert (inclusion of both HsaEX003157 and HsaEX0003152) shows that it is only the HsaEX0003157 insert that affects the alpha-dystroglycan interaction. None of the exons in the Z-site insert appears to affect the heparain binding.

Here, the binding of four recombinant agrin isoforms to heparin, to alpha-dystroglycan (a component of an agrin receptor), and to myoblasts was tested. The presence of a four-amino acid insert at the y site (encoded by cassette exon HsaEX0003154) is necessary and sufficient to confer heparin binding ability to agrin. Moreover, the binding of agrin to alpha-dystroglycan is inhibited by heparin when this insert is present. Agrin binding to the cell surface showed analogous properties: heparin inhibits the binding of only those agrin isoforms containing this four-amino acid insert. The z site insert is also examined (encoded by HsaEX0003157), this insert doesn't impact the examined interactions.

Overexpression of A4B11 and A4B8, but not A0B0 or A4B0, induces MuSK phosphorylation activity. The conserved tripeptide asparagineglutamate-isoleucine in the eight-amino acid long insert at the B-site (HsaEX0003157) is necessary and sufficient for full MuSK phosphorylation activity. However, even if all eight amino acids were replaced by alanines, this agrin mutant still has significantly higher MuSK phosphorylation activity than the splice version lacking any insert. The study also shows that binding to alpha-dystroglycan requires at least two LG domains and that amino acid inserts at the A and the B splice sites negatively affect binding. A4 = HsaEX0003154, B8 = HsaEX0003157, B11 = HsaEX0003152.

The most active forms are expressed in neural cells, and muscle cells express less active forms (i.e. skipping form)

Neural agrin, an extracellular matrix protein secreted by motor neurons, plays a key role in clustering of nicotinic acetylcholine receptors (AChR) on postsynaptic membranes of the neuromuscular junction. The insertion of this MIC (8aa) at the z site is particularly important for AChR clustering, whereas insertion of the 11 aa encoded by HsaEX0003152 has a much smaller effect.

Multiple alternatively spliced forms of agrin that differ in binding characteristics and bioactivity are synthesized by nerve and muscle cells. The authors used surgical chimeras, isoform-specific mutant mice, and nerve-muscle cocultures to determine the origins and nature of the agrin required for synaptogenesis. The authors show that agrin containing Z exons (Z+) is a critical nerve-derived inducer of postsynaptic differentiation, whereas neural isoforms containing a heparin binding site (Y+) and all muscle-derived isoforms are dispensable for major steps in synaptogenesis. These results also suggest that the requirement of agrin for presynaptic differentiation is mediated indirectly by its ability to promote postsynaptic production or localization of appropriate retrograde signals.

Eight-amino acid insert (z8 insert) in the third of three consecutive laminin-like globular (G3) domains near the C terminus of neural agrin. The insertion is necessary for Ca2+ binding, which is necessary for AChR clustering.