Signaling By The Ecm In Synaptogenesis Agrin

Studies on synaptogenesis in regenerating nerve and muscle preparations have illustrated that the cue or cues directing synapse formation are to be found in the ECM, or "basal lamina ghosts"1,2. Many putative signaling molecules have been tested, but agrin quickly emerged as the strongest candidate. Agrin was identified and named based upon its ability to aggregate AChRs into clusters on cultured myotubes, forming sites of postsynaptic differentiation that closely resembled the sites seen on developing muscle fibers in vivo3. Although this AChR clustering activity was detectable in protein homogenates from many sources, it was ultimately purified by a combination of biochemical and antibody approaches using ECM prepared from the T. californica electric organ. The cDNA sequence was determined shortly thereafter4,5. In 1990, Dr. U. J. McMahan put forward the "Agrin hypothesis," stating that agrin is the nerve-derived organizer of postsynaptic differentiation at the NMJ6.

The agrin hypothesis has been beset by various challenges in the last 15 years, but has largely proven correct. Initial alarm was caused by the finding that agrin was made by muscle as well as motor neurons, thus making it unclear how it could be the nerve-derived organizer of the NMJ. This quandary was quickly resolved with the discovery of alternatively spliced forms of agrin (Figure 1.2).

Figure 1.2. The Agrin Protein. Agrin is a heparan sulfate proteoglycan of almost 2,000 amino acids. At its N-terminus, agrin has nine follistatin-like repeats that also have homology with Kazal-type protease inhibitor domains. Also in the N-terminal half are the sites of glycosaminoglycan (GAG) addition. This post-translational modification is usually heparan sulfate, but may sometimes include chondroitin sulfate. The SEA domain is proposed to mediate interactions with other extracellular O-linked glycoproteins. In the C-terminal half of the protein are four EGF-like repeats and three laminin-type globular G-domains (G). Agrin is alternatively spliced in its C-terminal half. The X-splice site is an alternative splice acceptor site in exon 20 of the agrin gene of unknown significance. The Y-splice site includes an exon of just 12 base pairs, encoding four amino acids. Inclusion of these amino acids confers heparin-binding activity to agrin. The Z-splice site involves two exons of 24 and 33 bp, resulting in the inclusion of eight, 11, or the combined 19 amino acids. The inclusion of these amino acids is specific for neuronally expressed isoforms of this protein, and these amino acids are necessary for MuSK activation and the AChR clustering activity of agrin. The N-terminus of agrin has two variants resulting from alternative transcriptional start sites and thus different translational start sites. The LN form of agrin is encoded by two distinct exons before joining the common sequence, resulting in an isoform of the protein that has a signal peptide for secretion from the cell and a domain that interacts with the y1 chain of laminins for assembly into the ECM. This is the predominant nonneuronal form of agrin, and it is also expressed by motor neurons, where it becomes anchored into the ECM of the NMJ. The other isoform, SN agrin, is encoded by a single unique exon before its transcript rejoins the common sequence at the same point as LN agrin. SN agrin is the primary form of agrin found in the brain. The protein is a type-2 transmembrane protein that remains associated with the cell surface. Its role in the brain and on the surface of neurons is under investigation.

Isoforms containing an insertion at a C-terminal alternative splice site (called the Z+ splice forms) were found to be active at inducing AChR clusters in cultured muscle fibers, while isoforms that did not include these amino acids (Z- isoforms) were inactive7. Furthermore, the active isoforms were made only in the nervous system, while muscle and other non-neuronal tissues made the inactive isoforms8,9. Thus, while agrin is made by both nerves and muscles, only nerves made isoforms of the protein that were active in AChR clustering, refining but not disproving the agrin hypothesis.

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