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Distinct domains of MuSK mediate its abilities to induce and to associate with postsynaptic specializations.

Zhou H, Glass DJ, Yancopoulos GD, Sanes JR - J. Cell Biol. (1999)

Bottom Line: Using this system, we found that sequences in or near the first of four extracellular immunoglobulin-like domains in MuSK are required for agrin responsiveness, whereas sequences in or near the fourth immunoglobulin-like domain are required for interaction with rapsyn.Together, our results indicate that the ectodomain of MuSK mediates both agrin- dependent activation of a complex signal transduction pathway and agrin-independent association of the kinase with other postsynaptic components.These interactions allow MuSK not only to induce a multimolecular AChR-containing complex, but also to localize that complex to a primary scaffold in the postsynaptic membrane.

View Article: PubMed Central - PubMed

Affiliation: Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Agrin released from motor nerve terminals activates a muscle-specific receptor tyrosine kinase (MuSK) in muscle cells to trigger formation of the skeletal neuromuscular junction. A key step in synaptogenesis is the aggregation of acetylcholine receptors (AChRs) in the postsynaptic membrane, a process that requires the AChR-associated protein, rapsyn. Here, we mapped domains on MuSK necessary for its interactions with agrin and rapsyn. Myotubes from MuSK(-/)- mutant mice form no AChR clusters in response to agrin, but agrin-responsiveness is restored by the introduction of rat MuSK or a Torpedo orthologue. Thus, MuSK(-/)- myotubes provide an assay system for the structure-function analysis of MuSK. Using this system, we found that sequences in or near the first of four extracellular immunoglobulin-like domains in MuSK are required for agrin responsiveness, whereas sequences in or near the fourth immunoglobulin-like domain are required for interaction with rapsyn. Analysis of the cytoplasmic domain revealed that a recognition site for the phosphotyrosine binding domain-containing proteins is essential for MuSK activity, whereas consensus binding sites for the PSD-95/Dlg/ZO-1-like domain-containing proteins and phosphatidylinositol-3-kinase are dispensable. Together, our results indicate that the ectodomain of MuSK mediates both agrin- dependent activation of a complex signal transduction pathway and agrin-independent association of the kinase with other postsynaptic components. These interactions allow MuSK not only to induce a multimolecular AChR-containing complex, but also to localize that complex to a primary scaffold in the postsynaptic membrane.

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Tyrosine phosphorylation of MuSK cytoplasmic mutants. MuSK−/− myotubes were transfected with indicated constructs (Fig. 6 a), treated with agrin for 10 min (+), or left untreated (−), and then subjected to immunoprecipitation with anti-MuSK. Immunoblots were probed with antiphosphotyrosine (a), and then stripped and reprobed with anti-MuSK (b). Constructs 16 and 17, which do not mediate agrin-dependent tyrosine phosphorylation, are not detectably phosphorylated. Constructs 18 and 19 mediate lower levels of agrin-induced AChR clustering than wild-type MuSK; agrin-dependent tyrosine phosphorylation is greatly reduced for construct 18 but not construct 19.
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Figure 7: Tyrosine phosphorylation of MuSK cytoplasmic mutants. MuSK−/− myotubes were transfected with indicated constructs (Fig. 6 a), treated with agrin for 10 min (+), or left untreated (−), and then subjected to immunoprecipitation with anti-MuSK. Immunoblots were probed with antiphosphotyrosine (a), and then stripped and reprobed with anti-MuSK (b). Constructs 16 and 17, which do not mediate agrin-dependent tyrosine phosphorylation, are not detectably phosphorylated. Constructs 18 and 19 mediate lower levels of agrin-induced AChR clustering than wild-type MuSK; agrin-dependent tyrosine phosphorylation is greatly reduced for construct 18 but not construct 19.

Mentions: As a first step, we tested a construct in which a critical lysine residue in the consensus ATP binding site was replaced with alanine (Fig. 6 a, construct 16). Such mutations in other receptor tyrosine kinases abolish tyrosine kinase activity (Chou et al. 1987; Honegger et al. 1987), and we showed previously that this MuSK mutant inhibits AChR clustering when expressed in wild-type myotubes (Glass et al. 1997), suggesting that it had reduced activity. Therefore, if MuSK is acting as a receptor tyrosine kinase, construct 16 should be unable to mediate AChR clustering. Alternatively, if MuSK were only a substrate for other kinases, this mutation might not abolish activity. Here, we transfected construct 16 into MuSK−/− cells, and then assayed for tyrosine phosphorylation of the mutant by immunoblotting and AChR clustering by staining with rBTX. Construct 16 was devoid of detectable phosphotyrosine (Fig. 7, lanes 1 and 2) and was completely unable to induce formation of AChR clusters, either spontaneously or in response to agrin. Inactivity did not reflect poor expression or misrouting because construct 16 was expressed at levels similar to those of wild-type MuSK and reached the cell-surface without impediment (data not shown). These results indicate that the kinase activity of MuSK is essential for its biological activity.


Distinct domains of MuSK mediate its abilities to induce and to associate with postsynaptic specializations.

Zhou H, Glass DJ, Yancopoulos GD, Sanes JR - J. Cell Biol. (1999)

Tyrosine phosphorylation of MuSK cytoplasmic mutants. MuSK−/− myotubes were transfected with indicated constructs (Fig. 6 a), treated with agrin for 10 min (+), or left untreated (−), and then subjected to immunoprecipitation with anti-MuSK. Immunoblots were probed with antiphosphotyrosine (a), and then stripped and reprobed with anti-MuSK (b). Constructs 16 and 17, which do not mediate agrin-dependent tyrosine phosphorylation, are not detectably phosphorylated. Constructs 18 and 19 mediate lower levels of agrin-induced AChR clustering than wild-type MuSK; agrin-dependent tyrosine phosphorylation is greatly reduced for construct 18 but not construct 19.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169478&req=5

Figure 7: Tyrosine phosphorylation of MuSK cytoplasmic mutants. MuSK−/− myotubes were transfected with indicated constructs (Fig. 6 a), treated with agrin for 10 min (+), or left untreated (−), and then subjected to immunoprecipitation with anti-MuSK. Immunoblots were probed with antiphosphotyrosine (a), and then stripped and reprobed with anti-MuSK (b). Constructs 16 and 17, which do not mediate agrin-dependent tyrosine phosphorylation, are not detectably phosphorylated. Constructs 18 and 19 mediate lower levels of agrin-induced AChR clustering than wild-type MuSK; agrin-dependent tyrosine phosphorylation is greatly reduced for construct 18 but not construct 19.
Mentions: As a first step, we tested a construct in which a critical lysine residue in the consensus ATP binding site was replaced with alanine (Fig. 6 a, construct 16). Such mutations in other receptor tyrosine kinases abolish tyrosine kinase activity (Chou et al. 1987; Honegger et al. 1987), and we showed previously that this MuSK mutant inhibits AChR clustering when expressed in wild-type myotubes (Glass et al. 1997), suggesting that it had reduced activity. Therefore, if MuSK is acting as a receptor tyrosine kinase, construct 16 should be unable to mediate AChR clustering. Alternatively, if MuSK were only a substrate for other kinases, this mutation might not abolish activity. Here, we transfected construct 16 into MuSK−/− cells, and then assayed for tyrosine phosphorylation of the mutant by immunoblotting and AChR clustering by staining with rBTX. Construct 16 was devoid of detectable phosphotyrosine (Fig. 7, lanes 1 and 2) and was completely unable to induce formation of AChR clusters, either spontaneously or in response to agrin. Inactivity did not reflect poor expression or misrouting because construct 16 was expressed at levels similar to those of wild-type MuSK and reached the cell-surface without impediment (data not shown). These results indicate that the kinase activity of MuSK is essential for its biological activity.

Bottom Line: Using this system, we found that sequences in or near the first of four extracellular immunoglobulin-like domains in MuSK are required for agrin responsiveness, whereas sequences in or near the fourth immunoglobulin-like domain are required for interaction with rapsyn.Together, our results indicate that the ectodomain of MuSK mediates both agrin- dependent activation of a complex signal transduction pathway and agrin-independent association of the kinase with other postsynaptic components.These interactions allow MuSK not only to induce a multimolecular AChR-containing complex, but also to localize that complex to a primary scaffold in the postsynaptic membrane.

View Article: PubMed Central - PubMed

Affiliation: Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Agrin released from motor nerve terminals activates a muscle-specific receptor tyrosine kinase (MuSK) in muscle cells to trigger formation of the skeletal neuromuscular junction. A key step in synaptogenesis is the aggregation of acetylcholine receptors (AChRs) in the postsynaptic membrane, a process that requires the AChR-associated protein, rapsyn. Here, we mapped domains on MuSK necessary for its interactions with agrin and rapsyn. Myotubes from MuSK(-/)- mutant mice form no AChR clusters in response to agrin, but agrin-responsiveness is restored by the introduction of rat MuSK or a Torpedo orthologue. Thus, MuSK(-/)- myotubes provide an assay system for the structure-function analysis of MuSK. Using this system, we found that sequences in or near the first of four extracellular immunoglobulin-like domains in MuSK are required for agrin responsiveness, whereas sequences in or near the fourth immunoglobulin-like domain are required for interaction with rapsyn. Analysis of the cytoplasmic domain revealed that a recognition site for the phosphotyrosine binding domain-containing proteins is essential for MuSK activity, whereas consensus binding sites for the PSD-95/Dlg/ZO-1-like domain-containing proteins and phosphatidylinositol-3-kinase are dispensable. Together, our results indicate that the ectodomain of MuSK mediates both agrin- dependent activation of a complex signal transduction pathway and agrin-independent association of the kinase with other postsynaptic components. These interactions allow MuSK not only to induce a multimolecular AChR-containing complex, but also to localize that complex to a primary scaffold in the postsynaptic membrane.

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