Limits...
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.

Show MeSH

Related in: MedlinePlus

The NH2-terminal, laminin-binding domain of agrin  causes AChR clusters to be small. (a) Fluorescence micrographs of  cultured chick myotubes labeled with rhodamine–α-bungarotoxin.  Myotubes were grown for 12 h in the presence of conditioned  medium of transiently transfected COS cells containing either  100 pM cAgrin7A4B8 or cΔN15AgrinA4B8. AChR clusters induced  by cAgrin7A4B8 are considerably smaller than those induced by  cΔN15AgrinA4B8. In the presence of cN257Fc, the size of  cAgrin7A4B8-induced AChR clusters increases. (b) Quantification  of the effect on the size of the AChR aggregates. The presence of  500 nM cN257Fc (+ cN257Fc) increases the size of the AChR  clusters induced by cAgrin7A4B8 twofold and makes them indistinguishable from AChR clusters induced by cΔN15AgrinA4B8. In  contrast, addition of 500 nM cN257Fc does not alter the size of  cΔN15AgrinA4B8-induced AChR aggregates. The result (mean ±  SEM) of one representative experiment is shown where the size  of AChR clusters in 20 myotube segments was determined. Bar,  50 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139873&req=5

Figure 9: The NH2-terminal, laminin-binding domain of agrin causes AChR clusters to be small. (a) Fluorescence micrographs of cultured chick myotubes labeled with rhodamine–α-bungarotoxin. Myotubes were grown for 12 h in the presence of conditioned medium of transiently transfected COS cells containing either 100 pM cAgrin7A4B8 or cΔN15AgrinA4B8. AChR clusters induced by cAgrin7A4B8 are considerably smaller than those induced by cΔN15AgrinA4B8. In the presence of cN257Fc, the size of cAgrin7A4B8-induced AChR clusters increases. (b) Quantification of the effect on the size of the AChR aggregates. The presence of 500 nM cN257Fc (+ cN257Fc) increases the size of the AChR clusters induced by cAgrin7A4B8 twofold and makes them indistinguishable from AChR clusters induced by cΔN15AgrinA4B8. In contrast, addition of 500 nM cN257Fc does not alter the size of cΔN15AgrinA4B8-induced AChR aggregates. The result (mean ± SEM) of one representative experiment is shown where the size of AChR clusters in 20 myotube segments was determined. Bar, 50 μm.

Mentions: AChR aggregates induced by the active full-length splice variant cAgrin7A4B8 are more than twofold smaller than those induced by the fragment cΔN15AgrinA4B8 lacking the first 130 NH2-terminal amino acids (Denzer et al., 1995). Aggregation of AChRs induced by agrin is mainly based on the lateral migration of diffusely distributed molecules (Godfrey et al., 1984). We have speculated that the binding of cAgrin7A4B8 to ECM influences this process by immobilizing agrin in the ECM. If this were so, inhibition of the binding of cAgrin7A4B8 to muscle laminins should result in AChR clusters with the same size as AChR clusters induced by cΔN15AgrinA4B8. Myotubes incubated with cAgrin7A4B8 had considerably smaller AChR aggregates than those incubated with cΔN15AgrinA4B8 (Fig. 9 a). When available laminin-binding sites for full-length agrin were blocked by 500 nM cN257Fc, cAgrin7A4B8-induced AChR clusters became indistinguishable from those induced by cΔN15AgrinA4B8 (Fig. 9 a). In contrast to this, 500 nM of cN257Fc did not alter the shape of AChR aggregates induced by cΔN15AgrinA4B8. Quantification of this effect with a computerized image analysis system confirmed that the presence of cN257Fc increased the average size of AChR aggregates induced by cAgrin7A4B8 approximately twofold, while the average size of cΔN15AgrinA4B8induced AChR clusters was not affected (Fig. 9 b). These experiments provide evidence that the smaller size of the AChR clusters induced by full-length agrin is mainly based on its binding to laminin isoforms expressed by the myotubes.


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

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

The NH2-terminal, laminin-binding domain of agrin  causes AChR clusters to be small. (a) Fluorescence micrographs of  cultured chick myotubes labeled with rhodamine–α-bungarotoxin.  Myotubes were grown for 12 h in the presence of conditioned  medium of transiently transfected COS cells containing either  100 pM cAgrin7A4B8 or cΔN15AgrinA4B8. AChR clusters induced  by cAgrin7A4B8 are considerably smaller than those induced by  cΔN15AgrinA4B8. In the presence of cN257Fc, the size of  cAgrin7A4B8-induced AChR clusters increases. (b) Quantification  of the effect on the size of the AChR aggregates. The presence of  500 nM cN257Fc (+ cN257Fc) increases the size of the AChR  clusters induced by cAgrin7A4B8 twofold and makes them indistinguishable from AChR clusters induced by cΔN15AgrinA4B8. In  contrast, addition of 500 nM cN257Fc does not alter the size of  cΔN15AgrinA4B8-induced AChR aggregates. The result (mean ±  SEM) of one representative experiment is shown where the size  of AChR clusters in 20 myotube segments was determined. Bar,  50 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139873&req=5

Figure 9: The NH2-terminal, laminin-binding domain of agrin causes AChR clusters to be small. (a) Fluorescence micrographs of cultured chick myotubes labeled with rhodamine–α-bungarotoxin. Myotubes were grown for 12 h in the presence of conditioned medium of transiently transfected COS cells containing either 100 pM cAgrin7A4B8 or cΔN15AgrinA4B8. AChR clusters induced by cAgrin7A4B8 are considerably smaller than those induced by cΔN15AgrinA4B8. In the presence of cN257Fc, the size of cAgrin7A4B8-induced AChR clusters increases. (b) Quantification of the effect on the size of the AChR aggregates. The presence of 500 nM cN257Fc (+ cN257Fc) increases the size of the AChR clusters induced by cAgrin7A4B8 twofold and makes them indistinguishable from AChR clusters induced by cΔN15AgrinA4B8. In contrast, addition of 500 nM cN257Fc does not alter the size of cΔN15AgrinA4B8-induced AChR aggregates. The result (mean ± SEM) of one representative experiment is shown where the size of AChR clusters in 20 myotube segments was determined. Bar, 50 μm.
Mentions: AChR aggregates induced by the active full-length splice variant cAgrin7A4B8 are more than twofold smaller than those induced by the fragment cΔN15AgrinA4B8 lacking the first 130 NH2-terminal amino acids (Denzer et al., 1995). Aggregation of AChRs induced by agrin is mainly based on the lateral migration of diffusely distributed molecules (Godfrey et al., 1984). We have speculated that the binding of cAgrin7A4B8 to ECM influences this process by immobilizing agrin in the ECM. If this were so, inhibition of the binding of cAgrin7A4B8 to muscle laminins should result in AChR clusters with the same size as AChR clusters induced by cΔN15AgrinA4B8. Myotubes incubated with cAgrin7A4B8 had considerably smaller AChR aggregates than those incubated with cΔN15AgrinA4B8 (Fig. 9 a). When available laminin-binding sites for full-length agrin were blocked by 500 nM cN257Fc, cAgrin7A4B8-induced AChR clusters became indistinguishable from those induced by cΔN15AgrinA4B8 (Fig. 9 a). In contrast to this, 500 nM of cN257Fc did not alter the shape of AChR aggregates induced by cΔN15AgrinA4B8. Quantification of this effect with a computerized image analysis system confirmed that the presence of cN257Fc increased the average size of AChR aggregates induced by cAgrin7A4B8 approximately twofold, while the average size of cΔN15AgrinA4B8induced AChR clusters was not affected (Fig. 9 b). These experiments provide evidence that the smaller size of the AChR clusters induced by full-length agrin is mainly based on its binding to laminin isoforms expressed by the myotubes.

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