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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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Binding sites predicted to recognize p85 and PDZ domain-containing proteins are inessential for MuSK to cluster AChRs or to associate with AChR clusters. (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). +, spontaneous clustering; −, no clustering; ↑↑, wild-type level of agrin sensitivity; and ↑, reduced clustering relative to wild-type. In constructs 20–23, large segments of the cytoplasmic domain were deleted. In constructs 24 and 25, putative binding sites for p85 and PDZ domains were mutated. Constructs 20–23 were completely inactive, whereas constructs 24 and 25 showed activity equivalent to that of wild-type MuSK. (b and c) Cells that had been transfected with construct 25, incubated with or without agrin, and then stained with rBTX. (d–g) Cells that had been transfected with construct 17 (d and e) or 25 (f and g), treated with agrin, and then doubly stained with rBTX (d and f) and anti-MuSK antibodies (e and g). The MuSK mutant with a three residue carboxy-terminal truncation (construct 25) is unable to bind PDZ domains in PICK (data not shown) but can induce and associate with AChR clusters. MuSK and AChRs are both diffusely distributed in myotubes transfected with construct 17. (h) Quantitation of the extent to which MuSK constructs 24 and 25 rescued the ability of MuSK−/− myotubes to form AChR clusters in the absence or presence of agrin. Cultures were doubly stained with rBTX and anti-MuSK, and only MuSK-positive (i.e., successfully transfected) myotubes were scored. Bar, 20 μm.
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Figure 8: Binding sites predicted to recognize p85 and PDZ domain-containing proteins are inessential for MuSK to cluster AChRs or to associate with AChR clusters. (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). +, spontaneous clustering; −, no clustering; ↑↑, wild-type level of agrin sensitivity; and ↑, reduced clustering relative to wild-type. In constructs 20–23, large segments of the cytoplasmic domain were deleted. In constructs 24 and 25, putative binding sites for p85 and PDZ domains were mutated. Constructs 20–23 were completely inactive, whereas constructs 24 and 25 showed activity equivalent to that of wild-type MuSK. (b and c) Cells that had been transfected with construct 25, incubated with or without agrin, and then stained with rBTX. (d–g) Cells that had been transfected with construct 17 (d and e) or 25 (f and g), treated with agrin, and then doubly stained with rBTX (d and f) and anti-MuSK antibodies (e and g). The MuSK mutant with a three residue carboxy-terminal truncation (construct 25) is unable to bind PDZ domains in PICK (data not shown) but can induce and associate with AChR clusters. MuSK and AChRs are both diffusely distributed in myotubes transfected with construct 17. (h) Quantitation of the extent to which MuSK constructs 24 and 25 rescued the ability of MuSK−/− myotubes to form AChR clusters in the absence or presence of agrin. Cultures were doubly stained with rBTX and anti-MuSK, and only MuSK-positive (i.e., successfully transfected) myotubes were scored. Bar, 20 μm.

Mentions: Next, we tested the function of the cytoplasmic sequence NPMY, which is perfectly conserved among rat, mouse, human, Xenopus, and Torpedo MuSKs (Jennings et al. 1993; Valenzuala et al., 1995; Ganju et al. 1995; Fu et al. 1999), and which corresponds to a consensus binding site (NPXY) for signaling proteins that contain a PTB domain (for review see van der Geer and Pawson 1995; Borg and Margolis 1998). We changed the critical tyrosine residue to phenylalanine (construct 17), a mutation known to abolish binding of most PTB domain proteins. This mutation had no detectable effect on the expression level or the ability of MuSK to reach the cell surface (Fig. 8 d). However, myotubes transfected with construct 17 formed neither spontaneous nor agrin-induced AChR clusters (Fig. 6d, Fig. e, and Fig. h). Moreover, construct 17 was not detectably phosphorylated either spontaneously or after addition of agrin (Fig. 7, lanes 3 and 4). These results suggest that PTB domain–containing proteins are required for activation of MuSK's kinase 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)

Binding sites predicted to recognize p85 and PDZ domain-containing proteins are inessential for MuSK to cluster AChRs or to associate with AChR clusters. (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). +, spontaneous clustering; −, no clustering; ↑↑, wild-type level of agrin sensitivity; and ↑, reduced clustering relative to wild-type. In constructs 20–23, large segments of the cytoplasmic domain were deleted. In constructs 24 and 25, putative binding sites for p85 and PDZ domains were mutated. Constructs 20–23 were completely inactive, whereas constructs 24 and 25 showed activity equivalent to that of wild-type MuSK. (b and c) Cells that had been transfected with construct 25, incubated with or without agrin, and then stained with rBTX. (d–g) Cells that had been transfected with construct 17 (d and e) or 25 (f and g), treated with agrin, and then doubly stained with rBTX (d and f) and anti-MuSK antibodies (e and g). The MuSK mutant with a three residue carboxy-terminal truncation (construct 25) is unable to bind PDZ domains in PICK (data not shown) but can induce and associate with AChR clusters. MuSK and AChRs are both diffusely distributed in myotubes transfected with construct 17. (h) Quantitation of the extent to which MuSK constructs 24 and 25 rescued the ability of MuSK−/− myotubes to form AChR clusters in the absence or presence of agrin. Cultures were doubly stained with rBTX and anti-MuSK, and only MuSK-positive (i.e., successfully transfected) myotubes were scored. Bar, 20 μm.
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Figure 8: Binding sites predicted to recognize p85 and PDZ domain-containing proteins are inessential for MuSK to cluster AChRs or to associate with AChR clusters. (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). +, spontaneous clustering; −, no clustering; ↑↑, wild-type level of agrin sensitivity; and ↑, reduced clustering relative to wild-type. In constructs 20–23, large segments of the cytoplasmic domain were deleted. In constructs 24 and 25, putative binding sites for p85 and PDZ domains were mutated. Constructs 20–23 were completely inactive, whereas constructs 24 and 25 showed activity equivalent to that of wild-type MuSK. (b and c) Cells that had been transfected with construct 25, incubated with or without agrin, and then stained with rBTX. (d–g) Cells that had been transfected with construct 17 (d and e) or 25 (f and g), treated with agrin, and then doubly stained with rBTX (d and f) and anti-MuSK antibodies (e and g). The MuSK mutant with a three residue carboxy-terminal truncation (construct 25) is unable to bind PDZ domains in PICK (data not shown) but can induce and associate with AChR clusters. MuSK and AChRs are both diffusely distributed in myotubes transfected with construct 17. (h) Quantitation of the extent to which MuSK constructs 24 and 25 rescued the ability of MuSK−/− myotubes to form AChR clusters in the absence or presence of agrin. Cultures were doubly stained with rBTX and anti-MuSK, and only MuSK-positive (i.e., successfully transfected) myotubes were scored. Bar, 20 μm.
Mentions: Next, we tested the function of the cytoplasmic sequence NPMY, which is perfectly conserved among rat, mouse, human, Xenopus, and Torpedo MuSKs (Jennings et al. 1993; Valenzuala et al., 1995; Ganju et al. 1995; Fu et al. 1999), and which corresponds to a consensus binding site (NPXY) for signaling proteins that contain a PTB domain (for review see van der Geer and Pawson 1995; Borg and Margolis 1998). We changed the critical tyrosine residue to phenylalanine (construct 17), a mutation known to abolish binding of most PTB domain proteins. This mutation had no detectable effect on the expression level or the ability of MuSK to reach the cell surface (Fig. 8 d). However, myotubes transfected with construct 17 formed neither spontaneous nor agrin-induced AChR clusters (Fig. 6d, Fig. e, and Fig. h). Moreover, construct 17 was not detectably phosphorylated either spontaneously or after addition of agrin (Fig. 7, lanes 3 and 4). These results suggest that PTB domain–containing proteins are required for activation of MuSK's kinase 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