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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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Laminin-1 induces AChR clustering and increases agrin-induced AChR clustering in an additive manner. C2 myotubes were  untreated (A), treated with 150 pM soluble neural agrin (B), 40 nM laminin-1 (C), or treated with both 150 pM neural agrin and 40 nM  laminin-1 (D). Cultures were then stained with rhodamine-conjugated α-bungarotoxin. Neural agrin and laminin-1 induce the formation  of AChR clusters, while treatment with both further increases the number of AChR clusters. Bar, 25 μm.
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Figure 1: Laminin-1 induces AChR clustering and increases agrin-induced AChR clustering in an additive manner. C2 myotubes were untreated (A), treated with 150 pM soluble neural agrin (B), 40 nM laminin-1 (C), or treated with both 150 pM neural agrin and 40 nM laminin-1 (D). Cultures were then stained with rhodamine-conjugated α-bungarotoxin. Neural agrin and laminin-1 induce the formation of AChR clusters, while treatment with both further increases the number of AChR clusters. Bar, 25 μm.

Mentions: Previous experiments have shown that laminin-1 can induce AChR clustering on primary rat myotubes or on G8-1 clonal rat muscle cells (Vogel et al., 1983). We compared laminin-1 and agrin-induced AChR clustering on C2 myotubes using laminin-1 isolated from the basement membrane of the mouse EHS tumor, and a soluble COOH-terminal truncated agrin fragment (Ferns et al., 1993). When cultures of C2 mouse myotubes were treated with either 40 nM (33 μg/ml) of laminin-1 or 150 pM soluble neural agrin (C-Ag4,8), the number of AChR clusters observed after labeling with rhodamine-conjugated α-bungarotoxin was dramatically increased compared to the small number of spontaneous AChR clusters seen in untreated C2 myotubes (Fig. 1, A–C). These results were further analyzed by counting the number of AChR clusters induced by various concentrations of laminin-1. The effects of laminin-1 were detectable at concentrations as low as 12 nM and reached maximal levels at concentrations of ∼120 nM (Fig. 2 A). When saturating concentrations were used, laminin-1 was nearly as effective in inducing the formation of AChR clusters as agrin (compare Figs. 2, A and B). Clusters produced by laminin-1, however, appeared more intensely stained than those produced by agrin, suggesting that the density of the AChRs in the laminin-1–induced clusters may be higher. When the fluorescence intensity of rhodamine-labeled AChR clusters was quantitated using an image analysis program, we found that the AChR clusters induced by 40 nM laminin-1 were over two times more brightly stained than those induced by 15 pM agrin (Fig. 3 A). In addition, the length of the laminin-1–induced AChR clusters appeared to be greater than those induced by higher concentrations (1.5 nM) of agrin (Fig. 3 B).


Laminin-induced acetylcholine receptor clustering: an alternative pathway.

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

Laminin-1 induces AChR clustering and increases agrin-induced AChR clustering in an additive manner. C2 myotubes were  untreated (A), treated with 150 pM soluble neural agrin (B), 40 nM laminin-1 (C), or treated with both 150 pM neural agrin and 40 nM  laminin-1 (D). Cultures were then stained with rhodamine-conjugated α-bungarotoxin. Neural agrin and laminin-1 induce the formation  of AChR clusters, while treatment with both further increases the number of AChR clusters. Bar, 25 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139811&req=5

Figure 1: Laminin-1 induces AChR clustering and increases agrin-induced AChR clustering in an additive manner. C2 myotubes were untreated (A), treated with 150 pM soluble neural agrin (B), 40 nM laminin-1 (C), or treated with both 150 pM neural agrin and 40 nM laminin-1 (D). Cultures were then stained with rhodamine-conjugated α-bungarotoxin. Neural agrin and laminin-1 induce the formation of AChR clusters, while treatment with both further increases the number of AChR clusters. Bar, 25 μm.
Mentions: Previous experiments have shown that laminin-1 can induce AChR clustering on primary rat myotubes or on G8-1 clonal rat muscle cells (Vogel et al., 1983). We compared laminin-1 and agrin-induced AChR clustering on C2 myotubes using laminin-1 isolated from the basement membrane of the mouse EHS tumor, and a soluble COOH-terminal truncated agrin fragment (Ferns et al., 1993). When cultures of C2 mouse myotubes were treated with either 40 nM (33 μg/ml) of laminin-1 or 150 pM soluble neural agrin (C-Ag4,8), the number of AChR clusters observed after labeling with rhodamine-conjugated α-bungarotoxin was dramatically increased compared to the small number of spontaneous AChR clusters seen in untreated C2 myotubes (Fig. 1, A–C). These results were further analyzed by counting the number of AChR clusters induced by various concentrations of laminin-1. The effects of laminin-1 were detectable at concentrations as low as 12 nM and reached maximal levels at concentrations of ∼120 nM (Fig. 2 A). When saturating concentrations were used, laminin-1 was nearly as effective in inducing the formation of AChR clusters as agrin (compare Figs. 2, A and B). Clusters produced by laminin-1, however, appeared more intensely stained than those produced by agrin, suggesting that the density of the AChRs in the laminin-1–induced clusters may be higher. When the fluorescence intensity of rhodamine-labeled AChR clusters was quantitated using an image analysis program, we found that the AChR clusters induced by 40 nM laminin-1 were over two times more brightly stained than those induced by 15 pM agrin (Fig. 3 A). In addition, the length of the laminin-1–induced AChR clusters appeared to be greater than those induced by higher concentrations (1.5 nM) of agrin (Fig. 3 B).

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.

Show MeSH