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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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Agrin and laminin-1  induce AChR aggregation  over different time courses.  C2 myotubes were treated  with 150 pM soluble neural  agrin (solid bars) or with 120  nM laminin-1 (striped bars)  for 4, 12, or 18 h. Whereas  agrin quickly induces receptor aggregation within 4 h  and has a peak effect by 12 h,  laminin-1 acts with a slower  time course, and half-maximal clustering does not occur  until ∼12 h. Data averaged  from three (Laminin-1) or  four (Agrin) experiments are  shown as means ± SEM (n = 3 or 4). Asterisks denote significant  difference between untreated and agrin-treated (**P < 0.001) or  laminin-treated (*P < 0.01) cultures according to analysis of  variance (ANOVA) and a Student-Newman-Keuls test.
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Figure 5: Agrin and laminin-1 induce AChR aggregation over different time courses. C2 myotubes were treated with 150 pM soluble neural agrin (solid bars) or with 120 nM laminin-1 (striped bars) for 4, 12, or 18 h. Whereas agrin quickly induces receptor aggregation within 4 h and has a peak effect by 12 h, laminin-1 acts with a slower time course, and half-maximal clustering does not occur until ∼12 h. Data averaged from three (Laminin-1) or four (Agrin) experiments are shown as means ± SEM (n = 3 or 4). Asterisks denote significant difference between untreated and agrin-treated (**P < 0.001) or laminin-treated (*P < 0.01) cultures according to analysis of variance (ANOVA) and a Student-Newman-Keuls test.

Mentions: Agrin acts quickly to aggregate surface AChRs; after the addition of agrin to C2 myotubes, AChR clusters are first apparent after 3 h, and their number reaches a peak within 6 to 8 h (Ferns et al., 1996). To determine if laminin-1 acts with the same time course, we treated C2 myotubes with either neural agrin (150 pM) or laminin-1 (120 nM) for 4, 12, or 18 h. We then stained the myotubes with rhodamine– α-bungarotoxin to visualize the receptor clusters and counted the number of AChR clusters in random fields (Fig. 5). After 4 h of treatment, agrin-treated cultures exhibited a large increase in the number of AChR clusters, whereas laminin-1–treated myotubes displayed no increase over the background level. After 12 h, however, C2 cultures treated with laminin-1 exhibited roughly a twofold increase in AChR cluster number, and after 18 h of treatment, the number of laminin-1–induced clusters approached the number of clusters induced by 150 pM of neural agrin. Thus, the induction of AChR clusters by laminin-1 occurs significantly more slowly than by neural agrin.


Laminin-induced acetylcholine receptor clustering: an alternative pathway.

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

Agrin and laminin-1  induce AChR aggregation  over different time courses.  C2 myotubes were treated  with 150 pM soluble neural  agrin (solid bars) or with 120  nM laminin-1 (striped bars)  for 4, 12, or 18 h. Whereas  agrin quickly induces receptor aggregation within 4 h  and has a peak effect by 12 h,  laminin-1 acts with a slower  time course, and half-maximal clustering does not occur  until ∼12 h. Data averaged  from three (Laminin-1) or  four (Agrin) experiments are  shown as means ± SEM (n = 3 or 4). Asterisks denote significant  difference between untreated and agrin-treated (**P < 0.001) or  laminin-treated (*P < 0.01) cultures according to analysis of  variance (ANOVA) and a Student-Newman-Keuls test.
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Figure 5: Agrin and laminin-1 induce AChR aggregation over different time courses. C2 myotubes were treated with 150 pM soluble neural agrin (solid bars) or with 120 nM laminin-1 (striped bars) for 4, 12, or 18 h. Whereas agrin quickly induces receptor aggregation within 4 h and has a peak effect by 12 h, laminin-1 acts with a slower time course, and half-maximal clustering does not occur until ∼12 h. Data averaged from three (Laminin-1) or four (Agrin) experiments are shown as means ± SEM (n = 3 or 4). Asterisks denote significant difference between untreated and agrin-treated (**P < 0.001) or laminin-treated (*P < 0.01) cultures according to analysis of variance (ANOVA) and a Student-Newman-Keuls test.
Mentions: Agrin acts quickly to aggregate surface AChRs; after the addition of agrin to C2 myotubes, AChR clusters are first apparent after 3 h, and their number reaches a peak within 6 to 8 h (Ferns et al., 1996). To determine if laminin-1 acts with the same time course, we treated C2 myotubes with either neural agrin (150 pM) or laminin-1 (120 nM) for 4, 12, or 18 h. We then stained the myotubes with rhodamine– α-bungarotoxin to visualize the receptor clusters and counted the number of AChR clusters in random fields (Fig. 5). After 4 h of treatment, agrin-treated cultures exhibited a large increase in the number of AChR clusters, whereas laminin-1–treated myotubes displayed no increase over the background level. After 12 h, however, C2 cultures treated with laminin-1 exhibited roughly a twofold increase in AChR cluster number, and after 18 h of treatment, the number of laminin-1–induced clusters approached the number of clusters induced by 150 pM of neural agrin. Thus, the induction of AChR clusters by laminin-1 occurs significantly more slowly than by neural agrin.

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