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Agrin binds to the nerve-muscle basal lamina via laminin.

Denzer AJ, Brandenberger R, Gesemann M, Chiquet M, Ruegg MA - J. Cell Biol. (1997)

Bottom Line: After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood.In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment.These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Biozentrum, University of Basel, Switzerland.

ABSTRACT
Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel. The first 130 amino acids from the NH2 terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH2-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

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Binding sites for agrin on cultured chick myotubes colocalize with AChRs and laminin. Cultured chick myotubes were induced to form AChR clusters with 200 nM c21B8. Simultaneously,  20 nM cN257Fc were included. AChR aggregates were stained by  rhodamine–α-bungarotoxin, and cN257Fc bound to the myotubes  was visualized with biotinylated goat anti–mouse IgG followed by  fluorescein-conjugated streptavidin. cN257Fc is concentrated in  AChR clusters and is distributed along the edges of the myotubes  (arrowhead). No staining is seen in the absence of this fragment  (−cN257Fc). Consistent with the idea that cN257Fc binds to laminin, the distribution of myotube-bound cN257Fc resembles the  staining pattern obtained with anti-β/γ-specific antiserum 648 (β/ γ). The β2 chain of laminin, also called s-laminin, is concentrated  in AChR clusters. In light of the colocalization of cN257Fc and  AChR clusters, laminin-4 (α2, β2, γ1) is a binding partner of  agrin in these clusters. Bar, 40 μm.
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Figure 8: Binding sites for agrin on cultured chick myotubes colocalize with AChRs and laminin. Cultured chick myotubes were induced to form AChR clusters with 200 nM c21B8. Simultaneously, 20 nM cN257Fc were included. AChR aggregates were stained by rhodamine–α-bungarotoxin, and cN257Fc bound to the myotubes was visualized with biotinylated goat anti–mouse IgG followed by fluorescein-conjugated streptavidin. cN257Fc is concentrated in AChR clusters and is distributed along the edges of the myotubes (arrowhead). No staining is seen in the absence of this fragment (−cN257Fc). Consistent with the idea that cN257Fc binds to laminin, the distribution of myotube-bound cN257Fc resembles the staining pattern obtained with anti-β/γ-specific antiserum 648 (β/ γ). The β2 chain of laminin, also called s-laminin, is concentrated in AChR clusters. In light of the colocalization of cN257Fc and AChR clusters, laminin-4 (α2, β2, γ1) is a binding partner of agrin in these clusters. Bar, 40 μm.

Mentions: Many proteins that are highly concentrated at the NMJ in vivo are also found in AChR clusters in vitro (for review see Bowe and Fallon, 1995). These include proteins of the ECM, such as HSPG (Wallace, 1989) and laminin (Nitkin and Rothschild, 1990). Consequently, the laminin-binding fragment, cN257Fc, should colocalize with AChR clusters. To test this, AChR aggregation was induced on cultured chick myotubes with 200 nM c21B8, the minimal COOHterminal agrin fragment required for AChR aggregation (Gesemann et al., 1995), and 20 nM of cN257Fc was included. After 16 h, AChR clusters were visualized with rhodamine–α-bungarotoxin and myotube-bound cN257Fc was stained with biotinylated goat anti–mouse IgG followed by fluorescein-conjugated streptavidin. As shown in Fig. 8 (first row), cN257Fc localized to the agrin-induced AChR clusters. cN257Fc also displayed a more widespread distribution, often along the edge of the myotubes (Fig. 8, arrowhead). In myotubes, where AChR aggregation had been induced with c21B8, but no cN257Fc was included, no specific staining was seen (Fig. 8, second row). These results illustrate that binding sites for the NH2-terminal fragment of agrin are concentrated in AChR clusters but are also found on the remaining surface of the myotube.


Agrin binds to the nerve-muscle basal lamina via laminin.

Denzer AJ, Brandenberger R, Gesemann M, Chiquet M, Ruegg MA - J. Cell Biol. (1997)

Binding sites for agrin on cultured chick myotubes colocalize with AChRs and laminin. Cultured chick myotubes were induced to form AChR clusters with 200 nM c21B8. Simultaneously,  20 nM cN257Fc were included. AChR aggregates were stained by  rhodamine–α-bungarotoxin, and cN257Fc bound to the myotubes  was visualized with biotinylated goat anti–mouse IgG followed by  fluorescein-conjugated streptavidin. cN257Fc is concentrated in  AChR clusters and is distributed along the edges of the myotubes  (arrowhead). No staining is seen in the absence of this fragment  (−cN257Fc). Consistent with the idea that cN257Fc binds to laminin, the distribution of myotube-bound cN257Fc resembles the  staining pattern obtained with anti-β/γ-specific antiserum 648 (β/ γ). The β2 chain of laminin, also called s-laminin, is concentrated  in AChR clusters. In light of the colocalization of cN257Fc and  AChR clusters, laminin-4 (α2, β2, γ1) is a binding partner of  agrin in these clusters. Bar, 40 μm.
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Figure 8: Binding sites for agrin on cultured chick myotubes colocalize with AChRs and laminin. Cultured chick myotubes were induced to form AChR clusters with 200 nM c21B8. Simultaneously, 20 nM cN257Fc were included. AChR aggregates were stained by rhodamine–α-bungarotoxin, and cN257Fc bound to the myotubes was visualized with biotinylated goat anti–mouse IgG followed by fluorescein-conjugated streptavidin. cN257Fc is concentrated in AChR clusters and is distributed along the edges of the myotubes (arrowhead). No staining is seen in the absence of this fragment (−cN257Fc). Consistent with the idea that cN257Fc binds to laminin, the distribution of myotube-bound cN257Fc resembles the staining pattern obtained with anti-β/γ-specific antiserum 648 (β/ γ). The β2 chain of laminin, also called s-laminin, is concentrated in AChR clusters. In light of the colocalization of cN257Fc and AChR clusters, laminin-4 (α2, β2, γ1) is a binding partner of agrin in these clusters. Bar, 40 μm.
Mentions: Many proteins that are highly concentrated at the NMJ in vivo are also found in AChR clusters in vitro (for review see Bowe and Fallon, 1995). These include proteins of the ECM, such as HSPG (Wallace, 1989) and laminin (Nitkin and Rothschild, 1990). Consequently, the laminin-binding fragment, cN257Fc, should colocalize with AChR clusters. To test this, AChR aggregation was induced on cultured chick myotubes with 200 nM c21B8, the minimal COOHterminal agrin fragment required for AChR aggregation (Gesemann et al., 1995), and 20 nM of cN257Fc was included. After 16 h, AChR clusters were visualized with rhodamine–α-bungarotoxin and myotube-bound cN257Fc was stained with biotinylated goat anti–mouse IgG followed by fluorescein-conjugated streptavidin. As shown in Fig. 8 (first row), cN257Fc localized to the agrin-induced AChR clusters. cN257Fc also displayed a more widespread distribution, often along the edge of the myotubes (Fig. 8, arrowhead). In myotubes, where AChR aggregation had been induced with c21B8, but no cN257Fc was included, no specific staining was seen (Fig. 8, second row). These results illustrate that binding sites for the NH2-terminal fragment of agrin are concentrated in AChR clusters but are also found on the remaining surface of the myotube.

Bottom Line: After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood.In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment.These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Biozentrum, University of Basel, Switzerland.

ABSTRACT
Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel. The first 130 amino acids from the NH2 terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH2-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

Show MeSH
Related in: MedlinePlus