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M Ferns

Publications and source records attributed to M Ferns.

13 recordsLinked to original sources
PubMed2005

Agrin becomes concentrated at neuroeffector junctions in developing rodent urinary bladder.

The formation of somatic neuromuscular junctions in skeletal muscle is regulated by an extracellular matrix protein called agrin. Here, we have examined the expression and localization of agrin during development of the rodent urinary bladder, as a first step to examining its possible role at autonomic neuroeffector junctions in smooth muscle. We have found that agrin is expressed on the surface of developing smooth muscle cells and in the basement membrane underlying the urothelium. More importantly, agrin is progressively concentrated at parasympathetic varicosities during postnatal development and is present at virtually all junctions in mature muscle. Reverse transcription/polymerase chain reaction analysis has shown that pelvic ganglion neurons that innervate the bladder express LN/z8 agrin, whereas bladder smooth muscle expresses LN/z- agrin. Together, these results demonstrate that nerve and/or muscle agrin becomes localized at cholinergic parasympathetic varicosities in smooth muscle, where it could play a role in the maturation of the neuroeffector junction.

Agrin↗
PubMed2001

Expression and localization of agrin during sympathetic synapse formation in vitro.

Agrin is a motoneuron-derived signaling factor that plays a key organizing role in the initial stages of neuromuscular synapse formation. Agrin is expressed in other regions of the developing central and peripheral nervous systems, however, raising the possibility that it also directs the formation of some interneuronal synapses. To address this question, we have examined the expression and localization of agrin during formation of cholinergic, interneuronal synapses in the sympathetic system. In the superior cervical ganglia (SCG) in vivo, we found that agrin is highly expressed, and that it is present at, but is not limited to, synapses. In SCG neuronal cultures that were treated with ciliary neurotrophic factor to induce a uniform cholinergic phenotype, we found that agrin immunostaining colocalized precisely with cholinergic terminals and aggregates of neuronal acetylcholine receptor on the neuronal cell bodies and dendrites. Moreover, we found that alpha-dystroglycan, which is a potential receptor for agrin, is also concentrated at these cholinergic synaptic contacts. Finally, the SCG neurons expressed the C-terminal isoform of agrin that is neural-specific and highly active in synaptogenesis, and also the N-terminal splice isoform that occurs as a type II transmembrane protein. These findings show that agrin is specifically localized at sympathetic synapses in vitro, and are consistent with it playing a role in interneuronal synapse formation.

Agrin↗
PubMed2001

Determination of the upper size limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo.

The asialoglycoprotein receptor (ASGPr) on hepatocytes plays a role in the clearance of desialylated proteins from the serum. Although its sugar preference (N-acetylgalactosamine (GalNAc) >> galactose) and the effects of ligand valency (tetraantennary > triantennary >> diantennary >> monoantennary) and sugar spacing (20 A 10 A 4 A) are well documented, the effect of particle size on recognition and uptake of ligands by the receptor is poorly defined. In the present study, we assessed the maximum ligand size that still allows effective processing by the ASGPr of mouse hepatocytes in vivo and in vitro. Here too, we synthesized a novel glycolipid, which possesses a highly hydrophobic steroid moiety for stable incorporation into liposomes, and a triantennary GalNAc(3)-terminated cluster glycoside with a high nanomolar affinity (2 nm) for the ASGPr. Incorporation of the glycolipid into small (30 nm) [(3)H]cholesteryl oleate-labeled long circulating liposomes (1-50%, w/w) caused a concentration-dependent increase in particle clearance that was liver-specific (reaching 85 +/- 7% of the injected dose at 30 min after injection) and mediated by the ASGPr on hepatocytes, as shown by competition studies with asialoorosomucoid in vivo. By using glycolipid-laden liposomes of various sizes between 30 and 90 nm, it was demonstrated that particles with a diameter of >70 nm could no longer be recognized and processed by the ASGPr in vivo. This threshold size for effective uptake was not related to the physical barrier raised by the fenestrated sinusoidal endothelium, which shields hepatocytes from the circulation, because similar results were obtained by studying the uptake of liposomes on isolated mouse hepatocytes in vitro. From these data we conclude that in addition to the species, valency, and orientation of sugar residues, size is also an important determinant for effective recognition and processing of substrates by the ASGPr. Therefore, these data have important implications for the design of ASGPr-specific carriers that are aimed at hepatocyte-directed delivery of drugs and genes.

Animals↗
PubMed2001

Agrin-induced phosphorylation of the acetylcholine receptor regulates cytoskeletal anchoring and clustering.

At the developing neuromuscular junction, a motoneuron-derived factor called agrin signals through the muscle-specific kinase receptor to induce postsynaptic aggregation of the acetylcholine receptor (AChR). The agrin signaling pathway involves tyrosine phosphorylation of the AChR beta subunit, and we have tested its role in receptor localization by expressing tagged, tyrosine-minus forms of the beta subunit in mouse Sol8 myotubes. We find that agrin-induced phosphorylation of the beta subunit occurs only on cell surface AChR, and that AChR-containing tyrosine-minus beta subunit is targeted normally to the plasma membrane. Surface AChR that is tyrosine phosphorylated is less detergent extractable than nonphosphorylated AChR, indicating that it is preferentially linked to the cytoskeleton. Consistent with this, we find that agrin treatment reduces the detergent extractability of AChR that contains tagged wild-type beta subunit but not tyrosine-minus beta subunit. In addition, agrin-induced clustering of AChR containing tyrosine-minus beta subunit is reduced in comparison to wild-type receptor. Thus, we find that agrin-induced phosphorylation of AChR beta subunit regulates cytoskeletal anchoring and contributes to the clustering of the AChR, and this is likely to play an important role in the postsynaptic localization of the receptor at the developing synapse.

Agrin↗
PubMed1999

Agrin-independent activation of the agrin signal transduction pathway.

The neural factor agrin induces the aggregation of acetylcholine receptors (AChRs) and other synaptic molecules on cultured myotubes. This aggregating activity can be mimicked by experimental manipulations that include treatment with neuraminidase or elevated calcium. We report evidence that neuraminidase and calcium act through the agrin signal transduction pathway. The effects of neuraminidase and calcium on AChR clustering are additive with that of agrin at low concentrations and cosaturating at high concentrations. In addition, like agrin, both neuraminidase and calcium cause rapid tyrosine phosphorylation of the muscle-specific kinase (MuSK) and the AChR-beta subunit. Our results argue that all three agents act directly on components of the same signal transduction complex. We suggest that sialic acids on components of the complex inhibit interactions necessary for signal transduction and that disinhibition can result in activation. In such a model, agrin could activate signal transduction by disinhibition or by circumventing the inhibition.

Agrin↗
PubMed1999

A mechanism for acetylcholine receptor clustering distinct from agrin signaling.

Acetylcholine receptors (AChRs) and other postsynaptic molecules cluster spontaneously on cultured C2 myotubes. The frequency of clustering is enhanced by neural agrin, neuraminidase, or calcium through a signaling pathway which includes tyrosine phosphorylation of a muscle-specific kinase (MuSK) and the AChR beta-subunit. Vicia villosa agglutinin (VVA) lectin, previously shown to potentiate agrin-induced clustering on C2 myotubes, is shown here to also potentiate neuraminidase- and calcium-induced clustering of AChRs, while having no effect on the level of tyrosine phosphorylation of MuSK or the AChR beta-subunit. We propose that VVA lectin increases the frequency of AChR clustering through a mechanism that is distinct from agrin signaling, and that may involve alpha-dystroglycan.

Agrin↗
PubMed1998

alpha-Dystroglycan functions in acetylcholine receptor aggregation but is not a coreceptor for agrin-MuSK signaling.

alpha-dystroglycan (alpha-DG) is an agrin-binding protein that has been implicated in acetylcholine receptor (AChR) clustering, but it is unclear whether it acts as a coreceptor involved in initial agrin signaling or as a component involved in later events. To investigate its role, we have generated antisense derivatives of the C2 mouse muscle cell line, which have reduced alpha-DG expression. When compared with wild-type cells, the alpha-DG-deficient myotubes have a dramatic reduction in the number of spontaneous and agrin-induced AChR clusters. Several findings suggest that this decrease in AChR clustering is likely not because of a defect in agrin signaling through the MuSK receptor tyrosine kinase. Compared with wild-type cells, the alpha-DG-deficient cell lines showed only a transient reduction in the level of agrin-induced MuSK tyrosine phosphorylation and no reduction in AChR beta-subunit tyrosine phosphorylation. Additionally, agrin-induced phosphorylation of MuSK in wild-type myotubes was not decreased using agrin fragments that lack the domain primarily responsible for binding to alpha-DG. Finally, neural agrin-induced phosphorylation of MuSK was unaffected by treatments such as excess muscle agrin or anti-alpha-DG antibodies, both of which block agrin-alpha-DG binding. Together, these results suggest that alpha-DG is not required for agrin-MuSK signaling but rather that it may play a role elsewhere in the clustering pathway, such as in the downstream consolidation or maintenance of AChR clusters.

Agrin↗
PubMed1996

Neural agrin activates a high-affinity receptor in C2 muscle cells that is unresponsive to muscle agrin.

During synaptogenesis, agrin, released by motor nerves, causes the clustering of acetylcholine receptors (AChRs) in the skeletal muscle membrane. Although muscle alpha-dystroglycan has been postulated to be the receptor for the activity of agrin, previous experiments have revealed a discrepancy between the biological activity of soluble fragments of two isoforms of agrin produced by nerves and muscles, respectively, and their ability to bind alpha-dystroglycan. We have determined the specificity of the signaling receptor by investigating whether muscle agrin can block the activity of neural agrin on intact C2 myotubes. We find that a large excess of muscle agrin failed to inhibit either the number of AChR clusters or the phosphorylation of the AChR induced by picomolar concentrations of neural agrin. These results indicate that neural, but not muscle, agrin interacts with the signaling receptor. Muscle agrin did block the binding of neural agrin to isolated alpha-dystroglycan, however, suggesting either that alpha-dystroglycan is not the signaling receptor or that its properties in the membrane are altered. Direct assay of the binding of muscle or neural agrin to intact myotubes revealed only low-affinity binding. We conclude that the signaling receptor for agrin is a high-affinity receptor that is highly specific for the neural form.

Agrin↗
PubMed1996

Agrin-induced acetylcholine receptor clustering in mammalian muscle requires tyrosine phosphorylation.

Agrin is thought to be the nerve-derived factor that initiates acetylcholine receptor (AChR) clustering at the developing neuromuscularjunction. We have investigated the signaling pathway in mouse C2 myotubes and report that agrin induces a rapid but transient tyrosine phosphorylation of the AChR beta subunit. As the beta-subunit tyrosine phosphorylation occurs before the formation of AChR clusters, it may serve as a precursor step in the clustering mechanism. Consistent with this, we observed that tyrosine phosphorylation of the beta subunit correlated precisely with the presence or absence of clustering under several experimental conditions. Moreover, two tyrosine kinase inhibitors, herbimycin and staurosporine, that blocked beta-subunit phosphorylation also blocked agrin-induced clustering. Surprisingly, the inhibitors also dispersed preformed AChR clusters, suggesting that the tyrosine phosphorylation of other proteins may be required for the maintenance of receptor clusters. These findings indicate that in mammalian muscle, agrin-induced AChR clustering occurs through a mechanism that requires tyrosine phosphorylation and may involve tyrosine phosphorylation of the AChR itself.

Agrin↗
PubMed1993

Developmental regulation of highly active alternatively spliced forms of agrin.

Agrin is an extracellular matrix protein involved in clustering acetylcholine receptors during development of the neuromuscular junction. We have previously shown that alternative splicing at three sites generates multiple forms of rat agrin and that a novel 8 amino acid insert is the most important in determining biological activity. In the present study we have examined the expression of agrin during development with particular emphasis on determining the tissue distribution of the splicing variants at each site. Our principal observation is that the variants containing the sequence most responsible for biological activity are expressed exclusively in neural tissue and that their expression is highly regulated during development. We also show that muscle expresses less active forms and that agrin immunoreactivity during synaptogenesis is initially not limited to synaptic sites, but becomes progressively restricted to the synapse as development proceeds.

Aging↗
PubMed1992

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

Agrin is a component of the synaptic basal lamina that induces the clustering of acetylcholine receptors (AChRs) on muscle fibers. A region near the carboxyl terminus of the protein exists in four forms that are generated by alternative RNA splicing. All four alternatively spliced forms of agrin are active in inducing AChR clusters on rat primary and C2-derived muscle fibers. In contrast, only two forms of the protein, each containing an 8 amino acid insert, are capable of inducing clusters on myotubes of S27 cells, a C2 variant that has defective proteoglycans. These two forms are also most active in inducing clusters on chick myotubes. This pattern of differential activity suggests that RNA splicing of agrin transcripts and interactions with proteoglycans or other components of basal lamina have important roles in regulating the localization of neurotransmitter receptors at synaptic sites.

Agrin↗