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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 does not induce tyrosine phosphorylation of  the AChR β subunit. C2 myotubes were treated with neural agrin  (1.5 pM to 15 nM) or with laminin-1 (4 to 120 nM) for 45 min at  37°C. AChRs were isolated on α-bungarotoxin Sepharose beads,  and nitrocellulose blots containing AChRs were probed with  anti-phosphotyrosine antibodies. Laminin-1 did not induce β subunit phosphorylation at any concentration. The same concentrations of neural agrin that induced both AChR clustering and  MuSK phosphorylation also induced β subunit phosphorylation.  Untreated and muscle agrin-treated cultures did not exhibit β  subunit phosphorylation. The nitrocellulose blot was stripped  and reprobed with an antibody against the β subunit (mAb 124)  to ensure that similar amounts of β subunit were present in each  sample.
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Figure 9: Laminin-1 does not induce tyrosine phosphorylation of the AChR β subunit. C2 myotubes were treated with neural agrin (1.5 pM to 15 nM) or with laminin-1 (4 to 120 nM) for 45 min at 37°C. AChRs were isolated on α-bungarotoxin Sepharose beads, and nitrocellulose blots containing AChRs were probed with anti-phosphotyrosine antibodies. Laminin-1 did not induce β subunit phosphorylation at any concentration. The same concentrations of neural agrin that induced both AChR clustering and MuSK phosphorylation also induced β subunit phosphorylation. Untreated and muscle agrin-treated cultures did not exhibit β subunit phosphorylation. The nitrocellulose blot was stripped and reprobed with an antibody against the β subunit (mAb 124) to ensure that similar amounts of β subunit were present in each sample.

Mentions: Because tyrosine phosphorylation of the β subunit of the AChR is an early response to agrin that precedes, and may be required for, agrin-induced AChR clustering, we were interested to determine whether laminin-1 also caused tyrosine phosphorylation of the AChR. C2 myotubes were incubated with either 4, 12, 40, or 120 nM of laminin-1, 1.5 pM to 15 nM of soluble neural agrin (Ag4,8), or 15 nM of soluble muscle agrin (Ag0,0) as described above, and detergent extracts were prepared. The extracts were then incubated with α-bungarotoxin conjugated to Sepharose beads to isolate AChRs. Bound AChR was removed from the toxin beads with SDS and the subunits separated by SDS-PAGE and transferred to nitrocellulose blots. The blots were assayed for tyrosine phosphorylation of the AChR β subunit as described above and then reprobed with mAb 124, an antibody against the β subunit. Immunoblots with mAb 124 showed that equal amounts of AChR had been isolated on the α-bungarotoxin beads. Whereas treatment with neural agrin induced β subunit phosphorylation, neither laminin-1 treatment, at any concentration tested, nor muscle agrin treatment caused tyrosine phosphorylation of the β subunit (Fig. 9). Thus, tyrosine phosphorylation of the β subunit is not required for AChR clustering.


Laminin-induced acetylcholine receptor clustering: an alternative pathway.

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

Laminin-1 does not induce tyrosine phosphorylation of  the AChR β subunit. C2 myotubes were treated with neural agrin  (1.5 pM to 15 nM) or with laminin-1 (4 to 120 nM) for 45 min at  37°C. AChRs were isolated on α-bungarotoxin Sepharose beads,  and nitrocellulose blots containing AChRs were probed with  anti-phosphotyrosine antibodies. Laminin-1 did not induce β subunit phosphorylation at any concentration. The same concentrations of neural agrin that induced both AChR clustering and  MuSK phosphorylation also induced β subunit phosphorylation.  Untreated and muscle agrin-treated cultures did not exhibit β  subunit phosphorylation. The nitrocellulose blot was stripped  and reprobed with an antibody against the β subunit (mAb 124)  to ensure that similar amounts of β subunit were present in each  sample.
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Related In: Results  -  Collection

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Figure 9: Laminin-1 does not induce tyrosine phosphorylation of the AChR β subunit. C2 myotubes were treated with neural agrin (1.5 pM to 15 nM) or with laminin-1 (4 to 120 nM) for 45 min at 37°C. AChRs were isolated on α-bungarotoxin Sepharose beads, and nitrocellulose blots containing AChRs were probed with anti-phosphotyrosine antibodies. Laminin-1 did not induce β subunit phosphorylation at any concentration. The same concentrations of neural agrin that induced both AChR clustering and MuSK phosphorylation also induced β subunit phosphorylation. Untreated and muscle agrin-treated cultures did not exhibit β subunit phosphorylation. The nitrocellulose blot was stripped and reprobed with an antibody against the β subunit (mAb 124) to ensure that similar amounts of β subunit were present in each sample.
Mentions: Because tyrosine phosphorylation of the β subunit of the AChR is an early response to agrin that precedes, and may be required for, agrin-induced AChR clustering, we were interested to determine whether laminin-1 also caused tyrosine phosphorylation of the AChR. C2 myotubes were incubated with either 4, 12, 40, or 120 nM of laminin-1, 1.5 pM to 15 nM of soluble neural agrin (Ag4,8), or 15 nM of soluble muscle agrin (Ag0,0) as described above, and detergent extracts were prepared. The extracts were then incubated with α-bungarotoxin conjugated to Sepharose beads to isolate AChRs. Bound AChR was removed from the toxin beads with SDS and the subunits separated by SDS-PAGE and transferred to nitrocellulose blots. The blots were assayed for tyrosine phosphorylation of the AChR β subunit as described above and then reprobed with mAb 124, an antibody against the β subunit. Immunoblots with mAb 124 showed that equal amounts of AChR had been isolated on the α-bungarotoxin beads. Whereas treatment with neural agrin induced β subunit phosphorylation, neither laminin-1 treatment, at any concentration tested, nor muscle agrin treatment caused tyrosine phosphorylation of the β subunit (Fig. 9). Thus, tyrosine phosphorylation of the β subunit is not required for AChR 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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