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Laminin-induced acetylcholine receptor clustering: an alternative pathway.

Sugiyama JE, Glass DJ, Yancopoulos GD, Hall ZW - J. Cell Biol. (1997)

Bottom Line: Most importantly, laminin- 1-induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin.Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK-/- mice that do not respond to agrin.In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The induction of acetylcholine receptor (AChR) clustering by neurally released agrin is a critical, early step in the formation of the neuromuscular junction. Laminin, a component of the muscle fiber basal lamina, also induces AChR clustering. We find that induction of AChR clustering in C2 myotubes is specific for laminin-1; neither laminin-2 (merosin) nor laminin-11 (a synapse-specific isoform) are active. Moreover, laminin-1 induces AChR clustering by a pathway that is independent of that used by neural agrin. The effects of laminin-1 and agrin are strictly additive and occur with different time courses. Most importantly, laminin- 1-induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin. Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK-/- mice that do not respond to agrin. In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes. Laminin-1 thus acts by a mechanism that is independent of that used by agrin and may provide a supplemental pathway for AChR clustering during synaptogenesis.

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AChR clustering activity is specific for the laminin-1 isoform. C2 myotubes were untreated (A) or treated with 60 nM of laminin-2 (B), laminin-11 (C), or laminin-1 (D). Cultures were stained with rhodamine-conjugated α-bungarotoxin and examined under  400×. Unlike laminin-1, laminin-2 and -11 were both unable to induce AChR aggregation over background levels. Bar, 25 μm.
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Figure 4: AChR clustering activity is specific for the laminin-1 isoform. C2 myotubes were untreated (A) or treated with 60 nM of laminin-2 (B), laminin-11 (C), or laminin-1 (D). Cultures were stained with rhodamine-conjugated α-bungarotoxin and examined under 400×. Unlike laminin-1, laminin-2 and -11 were both unable to induce AChR aggregation over background levels. Bar, 25 μm.

Mentions: To determine whether the laminin effect on AChR clustering is specfic for particular isoforms, we tested whether other laminins expressed in muscle could cause AChRs to aggregate. Accordingly, C2 myotubes were treated with either laminin-1, -2 (merosin), or -11 (a synapse-specific isoform containing the β2 chain). We found that only the laminin-1 isoform could induce AChR clustering (Fig. 4). Neither laminin-2 nor -11 was active, even at concentrations of 120 nM (the concentration of laminin-1 that produced a maximal clustering response). Both laminin-2 and -11 used in these experiments were shown to be active in other biological assays (Kleinman, H., and A. Chiu, personal communication). The failure of laminin-11 to induce clustering is surprising since it is localized at the adult neuromuscular junction (Patton, B.L., and J.R. Sanes, personal communication). Induction of AChR clustering on myotubes is thus not a general property of laminins, but appears to be specific for laminin-1 (α1β1γ1). As α1 is the only chain of laminin-1 that is not shared with the other two isoforms, it appears to confer specificity for clustering.


Laminin-induced acetylcholine receptor clustering: an alternative pathway.

Sugiyama JE, Glass DJ, Yancopoulos GD, Hall ZW - J. Cell Biol. (1997)

AChR clustering activity is specific for the laminin-1 isoform. C2 myotubes were untreated (A) or treated with 60 nM of laminin-2 (B), laminin-11 (C), or laminin-1 (D). Cultures were stained with rhodamine-conjugated α-bungarotoxin and examined under  400×. Unlike laminin-1, laminin-2 and -11 were both unable to induce AChR aggregation over background levels. Bar, 25 μm.
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Related In: Results  -  Collection

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Figure 4: AChR clustering activity is specific for the laminin-1 isoform. C2 myotubes were untreated (A) or treated with 60 nM of laminin-2 (B), laminin-11 (C), or laminin-1 (D). Cultures were stained with rhodamine-conjugated α-bungarotoxin and examined under 400×. Unlike laminin-1, laminin-2 and -11 were both unable to induce AChR aggregation over background levels. Bar, 25 μm.
Mentions: To determine whether the laminin effect on AChR clustering is specfic for particular isoforms, we tested whether other laminins expressed in muscle could cause AChRs to aggregate. Accordingly, C2 myotubes were treated with either laminin-1, -2 (merosin), or -11 (a synapse-specific isoform containing the β2 chain). We found that only the laminin-1 isoform could induce AChR clustering (Fig. 4). Neither laminin-2 nor -11 was active, even at concentrations of 120 nM (the concentration of laminin-1 that produced a maximal clustering response). Both laminin-2 and -11 used in these experiments were shown to be active in other biological assays (Kleinman, H., and A. Chiu, personal communication). The failure of laminin-11 to induce clustering is surprising since it is localized at the adult neuromuscular junction (Patton, B.L., and J.R. Sanes, personal communication). Induction of AChR clustering on myotubes is thus not a general property of laminins, but appears to be specific for laminin-1 (α1β1γ1). As α1 is the only chain of laminin-1 that is not shared with the other two isoforms, it appears to confer specificity for clustering.

Bottom Line: Most importantly, laminin- 1-induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin.Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK-/- mice that do not respond to agrin.In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The induction of acetylcholine receptor (AChR) clustering by neurally released agrin is a critical, early step in the formation of the neuromuscular junction. Laminin, a component of the muscle fiber basal lamina, also induces AChR clustering. We find that induction of AChR clustering in C2 myotubes is specific for laminin-1; neither laminin-2 (merosin) nor laminin-11 (a synapse-specific isoform) are active. Moreover, laminin-1 induces AChR clustering by a pathway that is independent of that used by neural agrin. The effects of laminin-1 and agrin are strictly additive and occur with different time courses. Most importantly, laminin- 1-induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin. Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK-/- mice that do not respond to agrin. In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes. Laminin-1 thus acts by a mechanism that is independent of that used by agrin and may provide a supplemental pathway for AChR clustering during synaptogenesis.

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