RNA splicing regulates agrin-mediated acetylcholine receptor clustering activity on cultured myotubes

Ferns, Michael J.; Hoch, Werner; Campanelli, James T.; Rupp, Fabio; Hall, Zach W.; Scheller, Richard H.

doi:10.1016/0896-6273(92)90129-2

Cited by 201 publications

(189 citation statements)

References 35 publications

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“…PCR products were radiolabeled by inclusion of ϳ2 ϫ 10 5 cpm of 32 P-labeled forward primer in the reaction mixture, separated by electrophoresis on 8% polyacrylamide gels, and the relative abundance determined by analysis of the gel with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). To investigate the pattern of alternative splicing at either the Y or Z sites, samples were subjected to a single round of amplification (35 cycles) using oligonucleotide primers F111/ B112 or F24/B2 flanking the Y and Z sites, respectively (18,19). To examine alternative splicing in the Z region of agrin transcripts containing the 4-amino acid insert at the Y site, two rounds of amplification were performed using nested primers.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, although agrin is encoded by a single gene, alternative splicing of agrin pre-mRNA gives rise to multiple protein isoforms that differ in their tissue distribution and biological activity. In particular, two sites of alternative splicing in the carboxyl-terminal half of the protein, referred to in the rat gene as Y and Z (15,18) and in chicken as A and B (7), respectively, have been identified. Optional exclusion of sequences encoding 4 amino acids at the Y site and 8 and 11 amino acids at the Z site result in mRNAs encoding agrin proteins with unique functional properties and tissue-specific patterns of expression.…”

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confidence: 99%

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Selective Regulation of Agrin mRNA Induction and Alternative Splicing in PC12 Cells by Ras-dependent Actions of Nerve Growth Factor

Smith¹,

Fanger²,

O'Connor³

et al. 1997

Journal of Biological Chemistry

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The extracellular matrix protein agrin plays an important role in the formation and maintenance of the neuromuscular junction. However, regulation of agrin gene expression and pre-mRNA splicing, important in determining the biological actions of agrin, is not well understood. To begin to identify mechanisms controlling agrin expression, quantitative polymerase chain reaction techniques were used to analyze the effect of growth factors on the expression of agrin mRNA isoforms in rat pheochromocytoma (PC12) cells. Agrin transcripts in untreated cells lacked inserts in the Y and Z sites (agrin y0z0 ), encoding agrin isoforms with low acetylcholine receptor aggregating activity and a primarily non-neuronal tissue distribution. Transcripts encoding isoforms with high aggregating activity and neuronal tissue distribution (agrin y4z8 , agrin y4z11 , and agrin y4z19 ) were not detected. Treatment of PC12 cells with nerve growth factor (NGF) caused a significant increase in total agrin mRNA. In contrast, exposure to epidermal growth factor had no effect. Analysis of alternative splicing of agrin mRNA revealed that NGF elicited a specific increase in agrin y4 and agrin z8 mRNAs that did not occur in the presence of epidermal growth factor, insulin, dexamethasone, or retinoic acid. Analysis of PC12 sublines stably overexpressing a dominant inhibitory form of p21 Ras indicated that NGF induced changes in levels of agrin mRNA and alternative splicing required Ras activity. The results show that NGF can influence important aspects of neuronal differentiation by regulating alternative splicing. Furthermore, these data provide insight into the mechanisms governing agrin gene expression and suggest that neurotrophic factors may play a role in regulating agrin expression in vivo.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Selective Regulation of Agrin mRNA Induction and Alternative Splicing in PC12 Cells by Ras-dependent Actions of Nerve Growth Factor

Smith¹,

Fanger²,

O'Connor³

et al. 1997

Journal of Biological Chemistry

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“…On primary rat myotubes and on the mouse C2C12 cell line, all rat agrin isoforms are active in aggregating AChRs (Ferns et al, 1992. On the other hand, on chick primary myotubes and on variants of the C2C12 cell line that are defective in glycosaminoglycan synthesis, only isoforms containing amino acids at sites A and B are active (Ferns et al, 1992.…”

Section: D-h)mentioning

confidence: 99%

“…When tested in a qualitative way, this activity was dependent on the presence of amino acids at both sites A and B . Similarly, AChRaggregating activity of rat agrin differs between isoforrns, although the extent of the changes in activity depended on the experimental conditions, such as the origin of muscle cells and the size of rat agrin (Ferns et al, 1992.…”

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confidence: 99%

Acetylcholine receptor-aggregating activity of agrin isoforms and mapping of the active site.

Gesemann

Denzer

Rüegg

1995

The Journal of Cell Biology

220

228

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Abstract. Agrin is a basal lamina protein that induces aggregation of acetylcholine receptors (AChRs) and other molecules at the developing neuromuscular junction. Alternative splicing of chick agrin mRNA at two sites, A and B, 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, we have determined the ECs0 (effective concentration to induce half-maximal AChR clustering) of each agrin isoform and of truncation mutants. On chick myotubes, ECs0 of the COOH-terminal, 95-kD fragment of agrinA4a8 was ,'~35 pM, of agrinA4aZ9 ~110 pM and of agrinA4a, --5 nM. While some AChR clusters were observed with 64 nM of agrinA4aO, no activity was detected for agrinAoa0. Recombinant fulllength chick agrin and a 100-kD fragment of ray agrin showed similar ECso values. A 45-kD, COOH-terminal fragment of agrinA4u retained high activity (ECso ----130 pM) and a 21-kD fragment was still active, but required higher concentrations (ECs0 ---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 agrinA4nS and agrinA4mg, expressed in motor neurons, are most active, while no activity is detected in agrinAoBo, the dominant isoform synthesized by muscle cells. Furthermore, our results show that a fragment comprising site Bs and the most COOHterminal G-like domain is sufficient for this activity, and that agrin domains required for binding to heparin and those for AChR aggregation are distinct from each other.T HE formation of chemical synapses in the nervous system requires exchange of local signals between preand postsynaptic cells. At the neuromuscular junction (NMJ) ~, muscle cells aggregate several proteins, such as acetylcholine receptors (AChRs), acetylcholinesterase, and heparan sulfate proteoglycans at the site of contact between nerve and muscle cell. This process is, at least in part, initiated by the release of molecules from the motor axon. Several lines of evidence suggest that agrin, a component of synaptic basal lamina, is such a molecule. On cultured myotubes, agrin induces aggregation of several molecules that are concentrated at the NMJ including AChRs (Nitkin et al., 1987;Wallace, 1989). Motor neurons synthesize agrin, transport it to the nerve terminal from where it is released to induce AChR aggregation (Magill

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“…However, when presented in a cell-attached form, rat agrin lacking inserts at sites A and B was shown to have some AChR-aggregating activity (C ampanelli et al, 1991;Ferns et al, 1992Ferns et al, , 1993. To address whether the agrin A0B0 isoform, when expressed in a physiological context, would have such aggregating activity, we also injected cDNA constructs encoding cAgrin 7A0B0 into extrasynaptic fiber regions.…”

Section: Induction Of Postsynaptic Specializations In Vivo Is Splice-mentioning

confidence: 99%

Neural Agrin Induces Ectopic Postsynaptic Specializations in Innervated Muscle Fibers

Meier¹,

et al. 1997

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Neural agrin, in the absence of a nerve terminal, can induce the activity-resistant expression of acetylcholine receptor (AChR) subunit genes and the clustering of synapse-specific adult-type AChR channels in nonsynaptic regions of adult skeletal muscle fibers. Here we show that, when expression plasmids for neural agrin are injected into the extrasynaptic region of innervated muscle fibers, the following components of the postsynaptic apparatus are aggregated and colocalized with ectopic agrininduced AChR clusters: laminin-␤2, MuSK, phosphotyrosinecontaining proteins, ␤-dystroglycan, utrophin, and rapsyn. These components have been implicated to play a role in the differentiation of neuromuscular junctions. Furthermore, ErbB2 and ErbB3, which are thought to be involved in the regulation of neurally induced AChR subunit gene expression, were colocalized with agrin-induced AChR aggregates at ectopic nerve-free sites. The postsynaptic muscle membrane also contained a high concentration of voltage-gated Na ϩ channels as well as deep, basal lamina-containing invaginations comparable to the secondary synaptic folds of normal endplates. The ability to induce AChR aggregation in vivo was not observed in experiments with a muscle-specific agrin isoform. Thus, a motor neuron-specific agrin isoform is sufficient to induce a full ectopic postsynaptic apparatus in muscle fibers kept electrically active at their original endplate sites.

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RNA splicing regulates agrin-mediated acetylcholine receptor clustering activity on cultured myotubes

Cited by 201 publications

References 35 publications

Selective Regulation of Agrin mRNA Induction and Alternative Splicing in PC12 Cells by Ras-dependent Actions of Nerve Growth Factor

Selective Regulation of Agrin mRNA Induction and Alternative Splicing in PC12 Cells by Ras-dependent Actions of Nerve Growth Factor

Acetylcholine receptor-aggregating activity of agrin isoforms and mapping of the active site.

Neural Agrin Induces Ectopic Postsynaptic Specializations in Innervated Muscle Fibers

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