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

Amino-terminal regions in the MuSK ectodomain are required to mediate agrin-dependent AChR clustering. (a) Mutant constructs. The top line shows motifs in the MuSK ectodomain, including four immunoglobulin-like domains (Ig I–IV) and a region containing six phylogenetically conserved cysteine residues (C6). Subsequent lines show structures of wild-type and mutant constructs tested by expression in MuSK−/− myotubes. 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). All mutants permitted spontaneous clustering (+), but mutants lacking amino-terminal regions (Ig-I and -II) were unable to respond to agrin (=, spontaneous level of clustering; ↑, reduced relative to wild-type; ↑↑, wild-type level of clustering). (b–g) Examples of AChR clusters on MuSK−/−myotubes that had been transfected with mutant constructs, treated, and stained as in Fig. 1. (b and c) Construct 2. (d and e) Construct 3. (f and g) Construct 10. (h) Quantitation of the extent to which MuSK constructs 1, 2, and 3 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 as in Fig. 1 (i and j), and only MuSK-positive (i.e., successfully transfected) myotubes were scored. Bar is 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169478&req=5

Figure 2: Amino-terminal regions in the MuSK ectodomain are required to mediate agrin-dependent AChR clustering. (a) Mutant constructs. The top line shows motifs in the MuSK ectodomain, including four immunoglobulin-like domains (Ig I–IV) and a region containing six phylogenetically conserved cysteine residues (C6). Subsequent lines show structures of wild-type and mutant constructs tested by expression in MuSK−/− myotubes. 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). All mutants permitted spontaneous clustering (+), but mutants lacking amino-terminal regions (Ig-I and -II) were unable to respond to agrin (=, spontaneous level of clustering; ↑, reduced relative to wild-type; ↑↑, wild-type level of clustering). (b–g) Examples of AChR clusters on MuSK−/−myotubes that had been transfected with mutant constructs, treated, and stained as in Fig. 1. (b and c) Construct 2. (d and e) Construct 3. (f and g) Construct 10. (h) Quantitation of the extent to which MuSK constructs 1, 2, and 3 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 as in Fig. 1 (i and j), and only MuSK-positive (i.e., successfully transfected) myotubes were scored. Bar is 20 μm.

Mentions: The extracellular segment of MuSK contains five distinct domains: four immunoglobulin-like domains and a cysteine rich region called a C6 box. All five domains are conserved in sequence and arrangement in rat, mouse, human, Xenopus, chicken, and Torpedo MuSK (Jennings et al. 1993; Ganju et al. 1995; Valenzuela et al. 1995; Fu et al. 1999; D.J. Glass, and G.D. Yancopoulos, unpublished). To determine which portions of the MuSK ectodomain are required for its activation, we generated the mutants diagrammed in Fig. 2 a. In a first series (constructs 2–6), sequences were deleted that encoded either the first, second, or third immunoglobulin-like domain, the C6 box, or the fourth immunoglobulin-like domain. In a second series (constructs 7–13), nested deletions extended variable distances toward the amino terminus from a common site just upstream of the predicted transmembrane domain. Each mutant, as well as wild-type MuSK (construct 1), was transfected into MuSK−/− myoblasts, which were fused, treated, and stained as described above.


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)

Amino-terminal regions in the MuSK ectodomain are required to mediate agrin-dependent AChR clustering. (a) Mutant constructs. The top line shows motifs in the MuSK ectodomain, including four immunoglobulin-like domains (Ig I–IV) and a region containing six phylogenetically conserved cysteine residues (C6). Subsequent lines show structures of wild-type and mutant constructs tested by expression in MuSK−/− myotubes. 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). All mutants permitted spontaneous clustering (+), but mutants lacking amino-terminal regions (Ig-I and -II) were unable to respond to agrin (=, spontaneous level of clustering; ↑, reduced relative to wild-type; ↑↑, wild-type level of clustering). (b–g) Examples of AChR clusters on MuSK−/−myotubes that had been transfected with mutant constructs, treated, and stained as in Fig. 1. (b and c) Construct 2. (d and e) Construct 3. (f and g) Construct 10. (h) Quantitation of the extent to which MuSK constructs 1, 2, and 3 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 as in Fig. 1 (i and j), and only MuSK-positive (i.e., successfully transfected) myotubes were scored. Bar is 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Amino-terminal regions in the MuSK ectodomain are required to mediate agrin-dependent AChR clustering. (a) Mutant constructs. The top line shows motifs in the MuSK ectodomain, including four immunoglobulin-like domains (Ig I–IV) and a region containing six phylogenetically conserved cysteine residues (C6). Subsequent lines show structures of wild-type and mutant constructs tested by expression in MuSK−/− myotubes. 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). All mutants permitted spontaneous clustering (+), but mutants lacking amino-terminal regions (Ig-I and -II) were unable to respond to agrin (=, spontaneous level of clustering; ↑, reduced relative to wild-type; ↑↑, wild-type level of clustering). (b–g) Examples of AChR clusters on MuSK−/−myotubes that had been transfected with mutant constructs, treated, and stained as in Fig. 1. (b and c) Construct 2. (d and e) Construct 3. (f and g) Construct 10. (h) Quantitation of the extent to which MuSK constructs 1, 2, and 3 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 as in Fig. 1 (i and j), and only MuSK-positive (i.e., successfully transfected) myotubes were scored. Bar is 20 μm.
Mentions: The extracellular segment of MuSK contains five distinct domains: four immunoglobulin-like domains and a cysteine rich region called a C6 box. All five domains are conserved in sequence and arrangement in rat, mouse, human, Xenopus, chicken, and Torpedo MuSK (Jennings et al. 1993; Ganju et al. 1995; Valenzuela et al. 1995; Fu et al. 1999; D.J. Glass, and G.D. Yancopoulos, unpublished). To determine which portions of the MuSK ectodomain are required for its activation, we generated the mutants diagrammed in Fig. 2 a. In a first series (constructs 2–6), sequences were deleted that encoded either the first, second, or third immunoglobulin-like domain, the C6 box, or the fourth immunoglobulin-like domain. In a second series (constructs 7–13), nested deletions extended variable distances toward the amino terminus from a common site just upstream of the predicted transmembrane domain. Each mutant, as well as wild-type MuSK (construct 1), was transfected into MuSK−/− myoblasts, which were fused, treated, and stained as described above.

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