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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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(A) Laminin-1–induced AChR clusters are more  intensely stained than agrin-induced AChRs. C2 myotubes  were treated with 15 pM neural agrin, 40 nM laminin-1, or  with the two together, and the fluorescence intensity was  measured as described in Materials and Methods. The laminin-1–induced clusters are more intensely stained than the  agrin-induced AChRs, while treatment with both agrin and  laminin-1 produced brightly stained clusters more similar to  those induced by laminin-1. Data are averaged from two different experiments (10 fields/experiment) and shown as  means ± SD (n = 2). (B) AChR clusters induced by high  concentrations of agrin are shorter than those induced by  low concentrations of agrin or by laminin-1. C2 myotubes  were treated with 15 pM or 1.5 nM neural agrin, 12 or 120  nM laminin-1, or with both 1.5 nM neural agrin and 120 nM  laminin-1. Clusters induced by high (1.5 nM) concentrations  of agrin are smaller than those induced by low (15 pM) concentrations. In contrast, both concentrations of laminin- 1–induced large AChR clusters to form. Treatment with high concentrations of both agrin and laminin-1 produced large clusters, similar in size to the laminin-induced clusters. Data are averaged from three different experiments (10 fields/experiment) and shown as means  ± SEM (n = 3). Asterisks denote significant difference (*P < 0.05 or **P < 0.01) between high concentrations of agrin and other treatments according to analysis of variance (ANOVA) and a Student-Newman-Keuls test.
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Figure 3: (A) Laminin-1–induced AChR clusters are more intensely stained than agrin-induced AChRs. C2 myotubes were treated with 15 pM neural agrin, 40 nM laminin-1, or with the two together, and the fluorescence intensity was measured as described in Materials and Methods. The laminin-1–induced clusters are more intensely stained than the agrin-induced AChRs, while treatment with both agrin and laminin-1 produced brightly stained clusters more similar to those induced by laminin-1. Data are averaged from two different experiments (10 fields/experiment) and shown as means ± SD (n = 2). (B) AChR clusters induced by high concentrations of agrin are shorter than those induced by low concentrations of agrin or by laminin-1. C2 myotubes were treated with 15 pM or 1.5 nM neural agrin, 12 or 120 nM laminin-1, or with both 1.5 nM neural agrin and 120 nM laminin-1. Clusters induced by high (1.5 nM) concentrations of agrin are smaller than those induced by low (15 pM) concentrations. In contrast, both concentrations of laminin- 1–induced large AChR clusters to form. Treatment with high concentrations of both agrin and laminin-1 produced large clusters, similar in size to the laminin-induced clusters. Data are averaged from three different experiments (10 fields/experiment) and shown as means ± SEM (n = 3). Asterisks denote significant difference (*P < 0.05 or **P < 0.01) between high concentrations of agrin and other treatments according to analysis of variance (ANOVA) and a Student-Newman-Keuls test.

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)

(A) Laminin-1–induced AChR clusters are more  intensely stained than agrin-induced AChRs. C2 myotubes  were treated with 15 pM neural agrin, 40 nM laminin-1, or  with the two together, and the fluorescence intensity was  measured as described in Materials and Methods. The laminin-1–induced clusters are more intensely stained than the  agrin-induced AChRs, while treatment with both agrin and  laminin-1 produced brightly stained clusters more similar to  those induced by laminin-1. Data are averaged from two different experiments (10 fields/experiment) and shown as  means ± SD (n = 2). (B) AChR clusters induced by high  concentrations of agrin are shorter than those induced by  low concentrations of agrin or by laminin-1. C2 myotubes  were treated with 15 pM or 1.5 nM neural agrin, 12 or 120  nM laminin-1, or with both 1.5 nM neural agrin and 120 nM  laminin-1. Clusters induced by high (1.5 nM) concentrations  of agrin are smaller than those induced by low (15 pM) concentrations. In contrast, both concentrations of laminin- 1–induced large AChR clusters to form. Treatment with high concentrations of both agrin and laminin-1 produced large clusters, similar in size to the laminin-induced clusters. Data are averaged from three different experiments (10 fields/experiment) and shown as means  ± SEM (n = 3). Asterisks denote significant difference (*P < 0.05 or **P < 0.01) between high concentrations of agrin and other treatments according to analysis of variance (ANOVA) and a Student-Newman-Keuls test.
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Figure 3: (A) Laminin-1–induced AChR clusters are more intensely stained than agrin-induced AChRs. C2 myotubes were treated with 15 pM neural agrin, 40 nM laminin-1, or with the two together, and the fluorescence intensity was measured as described in Materials and Methods. The laminin-1–induced clusters are more intensely stained than the agrin-induced AChRs, while treatment with both agrin and laminin-1 produced brightly stained clusters more similar to those induced by laminin-1. Data are averaged from two different experiments (10 fields/experiment) and shown as means ± SD (n = 2). (B) AChR clusters induced by high concentrations of agrin are shorter than those induced by low concentrations of agrin or by laminin-1. C2 myotubes were treated with 15 pM or 1.5 nM neural agrin, 12 or 120 nM laminin-1, or with both 1.5 nM neural agrin and 120 nM laminin-1. Clusters induced by high (1.5 nM) concentrations of agrin are smaller than those induced by low (15 pM) concentrations. In contrast, both concentrations of laminin- 1–induced large AChR clusters to form. Treatment with high concentrations of both agrin and laminin-1 produced large clusters, similar in size to the laminin-induced clusters. Data are averaged from three different experiments (10 fields/experiment) and shown as means ± SEM (n = 3). Asterisks denote significant difference (*P < 0.05 or **P < 0.01) between high concentrations of agrin and other treatments according to analysis of variance (ANOVA) and a Student-Newman-Keuls test.
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
Related in: MedlinePlus