Limits...
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.

Show MeSH

Related in: MedlinePlus

Roles of binding sites for ATP and for PTB domain proteins in the MuSK cytoplasmic domain. (a) Mutant constructs. To the right of each construct is indicated whether or not it rescued the ability of MuSK−/− myotubes to form AChR clusters in the absence of agrin (−Ag) or to form additional clusters in the presence of agrin (+Ag). Plus sign, spontaneous clustering; −, no clustering; ↑↑, wild-type level of agrin sensitivity; and ↑, reduced clustering relative to wild-type. In constructs 16 and 17, a conserved ATP binding site and a binding site for PTB domain proteins, respectively, were mutated. In constructs 18 and 19, the specificity of the PTB site was altered, as detailed in Results. (b–g) Distribution of AChRs on MuSK−/− myotubes that had been transfected with mutant constructs, treated, and stained as in Fig. 1. (b and c) Construct 1; (d and e) construct 17; and (f and g) construct 19. (h) Quantitation of the extent to which MuSK constructs 17, 18, and 19 rescued the ability of MuSK−/− myotubes to form AChR clusters in the absence or presence of agrin. Constructs 16 and 17 were inactive, whereas constructs 18 and 19 showed reduced activity relative to wild-type (compare with Fig. 2 h and 8 h). Cultures were doubly stained with rBTX and anti-MuSK, and only MuSK-positive (i.e., successfully transfected) myotubes were scored. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169478&req=5

Figure 6: Roles of binding sites for ATP and for PTB domain proteins in the MuSK cytoplasmic domain. (a) Mutant constructs. To the right of each construct is indicated whether or not it rescued the ability of MuSK−/− myotubes to form AChR clusters in the absence of agrin (−Ag) or to form additional clusters in the presence of agrin (+Ag). Plus sign, spontaneous clustering; −, no clustering; ↑↑, wild-type level of agrin sensitivity; and ↑, reduced clustering relative to wild-type. In constructs 16 and 17, a conserved ATP binding site and a binding site for PTB domain proteins, respectively, were mutated. In constructs 18 and 19, the specificity of the PTB site was altered, as detailed in Results. (b–g) Distribution of AChRs on MuSK−/− myotubes that had been transfected with mutant constructs, treated, and stained as in Fig. 1. (b and c) Construct 1; (d and e) construct 17; and (f and g) construct 19. (h) Quantitation of the extent to which MuSK constructs 17, 18, and 19 rescued the ability of MuSK−/− myotubes to form AChR clusters in the absence or presence of agrin. Constructs 16 and 17 were inactive, whereas constructs 18 and 19 showed reduced activity relative to wild-type (compare with Fig. 2 h and 8 h). Cultures were doubly stained with rBTX and anti-MuSK, and only MuSK-positive (i.e., successfully transfected) myotubes were scored. Bar, 20 μm.

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)

Roles of binding sites for ATP and for PTB domain proteins in the MuSK cytoplasmic domain. (a) Mutant constructs. To the right of each construct is indicated whether or not it rescued the ability of MuSK−/− myotubes to form AChR clusters in the absence of agrin (−Ag) or to form additional clusters in the presence of agrin (+Ag). Plus sign, spontaneous clustering; −, no clustering; ↑↑, wild-type level of agrin sensitivity; and ↑, reduced clustering relative to wild-type. In constructs 16 and 17, a conserved ATP binding site and a binding site for PTB domain proteins, respectively, were mutated. In constructs 18 and 19, the specificity of the PTB site was altered, as detailed in Results. (b–g) Distribution of AChRs on MuSK−/− myotubes that had been transfected with mutant constructs, treated, and stained as in Fig. 1. (b and c) Construct 1; (d and e) construct 17; and (f and g) construct 19. (h) Quantitation of the extent to which MuSK constructs 17, 18, and 19 rescued the ability of MuSK−/− myotubes to form AChR clusters in the absence or presence of agrin. Constructs 16 and 17 were inactive, whereas constructs 18 and 19 showed reduced activity relative to wild-type (compare with Fig. 2 h and 8 h). Cultures were doubly stained with rBTX and anti-MuSK, and only MuSK-positive (i.e., successfully transfected) myotubes were scored. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169478&req=5

Figure 6: Roles of binding sites for ATP and for PTB domain proteins in the MuSK cytoplasmic domain. (a) Mutant constructs. To the right of each construct is indicated whether or not it rescued the ability of MuSK−/− myotubes to form AChR clusters in the absence of agrin (−Ag) or to form additional clusters in the presence of agrin (+Ag). Plus sign, spontaneous clustering; −, no clustering; ↑↑, wild-type level of agrin sensitivity; and ↑, reduced clustering relative to wild-type. In constructs 16 and 17, a conserved ATP binding site and a binding site for PTB domain proteins, respectively, were mutated. In constructs 18 and 19, the specificity of the PTB site was altered, as detailed in Results. (b–g) Distribution of AChRs on MuSK−/− myotubes that had been transfected with mutant constructs, treated, and stained as in Fig. 1. (b and c) Construct 1; (d and e) construct 17; and (f and g) construct 19. (h) Quantitation of the extent to which MuSK constructs 17, 18, and 19 rescued the ability of MuSK−/− myotubes to form AChR clusters in the absence or presence of agrin. Constructs 16 and 17 were inactive, whereas constructs 18 and 19 showed reduced activity relative to wild-type (compare with Fig. 2 h and 8 h). Cultures were doubly stained with rBTX and anti-MuSK, and only MuSK-positive (i.e., successfully transfected) myotubes were scored. Bar, 20 μm.
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.

Show MeSH
Related in: MedlinePlus