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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 ectodomain mutants. MuSK−/− myotubes were transfected with the indicated constructs (Fig. 2 a), treated with agrin for 10 min (+) or left untreated (−), and then subjected to immunoprecipitation with anti-MuSK. Immunoblots were probed with antiphosphotyrosine (a), stripped, and reprobed with anti-MuSK (b). (lanes 1 and 2) Untransfected myoblasts; (lanes 3 and 4) myoblasts transfected with wild-type MuSK (construct 1); (lanes 5 and 6) myoblasts transfected with construct 11; and (lanes 7 and 8) myoblasts transfected with construct 13. Wild-type MuSK and construct 11, both of which mediate agrin-induced AChR clustering, also show agrin-dependent tyrosine phosphorylation. Mutant 13, which does not mediate agrin-dependent tyrosine phosphorylation, shows a high level of agrin-independent phosphorylation but only slight agrin-dependent phosphorylation.
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Figure 3: Tyrosine phosphorylation of MuSK ectodomain mutants. MuSK−/− myotubes were transfected with the indicated constructs (Fig. 2 a), treated with agrin for 10 min (+) or left untreated (−), and then subjected to immunoprecipitation with anti-MuSK. Immunoblots were probed with antiphosphotyrosine (a), stripped, and reprobed with anti-MuSK (b). (lanes 1 and 2) Untransfected myoblasts; (lanes 3 and 4) myoblasts transfected with wild-type MuSK (construct 1); (lanes 5 and 6) myoblasts transfected with construct 11; and (lanes 7 and 8) myoblasts transfected with construct 13. Wild-type MuSK and construct 11, both of which mediate agrin-induced AChR clustering, also show agrin-dependent tyrosine phosphorylation. Mutant 13, which does not mediate agrin-dependent tyrosine phosphorylation, shows a high level of agrin-independent phosphorylation but only slight agrin-dependent phosphorylation.

Mentions: As shown in Fig. 3, MuSK was undetectable in untransfected MuSK−/− cells, but readily detectable in transfected cells. Wild-type MuSK was phosphorylated at a low level in the absence of added agrin, and its phosphorylation was increased >10-fold 10 min after the addition of agrin to the cells (lanes 3 and 4). Tyrosine phosphorylation of construct 11 (which lacks immunoglobulin-like domains 3 and 4 and the C6 box yet remains responsive to agrin) was also greatly stimulated by agrin (lanes 5 and 6). In contrast, construct 13, which lacks most of the ectodomain and is agrin unresponsive, was highly phosphorylated in the absence of agrin and showed only slightly increased phosphorylation after treatment with agrin (lanes 7 and 8). This result raises the possibility that the ectodomain of MuSK keeps the cytoplasmic domain inactive in the absence of agrin; ligand binding would alter the conformation of the ectodomain to relieve the inhibition.


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 ectodomain mutants. MuSK−/− myotubes were transfected with the indicated constructs (Fig. 2 a), treated with agrin for 10 min (+) or left untreated (−), and then subjected to immunoprecipitation with anti-MuSK. Immunoblots were probed with antiphosphotyrosine (a), stripped, and reprobed with anti-MuSK (b). (lanes 1 and 2) Untransfected myoblasts; (lanes 3 and 4) myoblasts transfected with wild-type MuSK (construct 1); (lanes 5 and 6) myoblasts transfected with construct 11; and (lanes 7 and 8) myoblasts transfected with construct 13. Wild-type MuSK and construct 11, both of which mediate agrin-induced AChR clustering, also show agrin-dependent tyrosine phosphorylation. Mutant 13, which does not mediate agrin-dependent tyrosine phosphorylation, shows a high level of agrin-independent phosphorylation but only slight agrin-dependent phosphorylation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169478&req=5

Figure 3: Tyrosine phosphorylation of MuSK ectodomain mutants. MuSK−/− myotubes were transfected with the indicated constructs (Fig. 2 a), treated with agrin for 10 min (+) or left untreated (−), and then subjected to immunoprecipitation with anti-MuSK. Immunoblots were probed with antiphosphotyrosine (a), stripped, and reprobed with anti-MuSK (b). (lanes 1 and 2) Untransfected myoblasts; (lanes 3 and 4) myoblasts transfected with wild-type MuSK (construct 1); (lanes 5 and 6) myoblasts transfected with construct 11; and (lanes 7 and 8) myoblasts transfected with construct 13. Wild-type MuSK and construct 11, both of which mediate agrin-induced AChR clustering, also show agrin-dependent tyrosine phosphorylation. Mutant 13, which does not mediate agrin-dependent tyrosine phosphorylation, shows a high level of agrin-independent phosphorylation but only slight agrin-dependent phosphorylation.
Mentions: As shown in Fig. 3, MuSK was undetectable in untransfected MuSK−/− cells, but readily detectable in transfected cells. Wild-type MuSK was phosphorylated at a low level in the absence of added agrin, and its phosphorylation was increased >10-fold 10 min after the addition of agrin to the cells (lanes 3 and 4). Tyrosine phosphorylation of construct 11 (which lacks immunoglobulin-like domains 3 and 4 and the C6 box yet remains responsive to agrin) was also greatly stimulated by agrin (lanes 5 and 6). In contrast, construct 13, which lacks most of the ectodomain and is agrin unresponsive, was highly phosphorylated in the absence of agrin and showed only slightly increased phosphorylation after treatment with agrin (lanes 7 and 8). This result raises the possibility that the ectodomain of MuSK keeps the cytoplasmic domain inactive in the absence of agrin; ligand binding would alter the conformation of the ectodomain to relieve the inhibition.

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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