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
Carboxy-terminal regions of the MuSK ectodomain are required for association with AChR clusters in myotubes. (a) MuSK constructs, numbered as in Fig. 2, which were tested for their ability to associate with AChR clusters in MuSK−/− muscle cells. To the right of each construct is indicated whether it was concentrated at spontaneous or agrin-induced clusters. NA, not applicable, because agrin did not induce AChR clusters in these cells. (b–i) MuSK and AChR localization in cells transfected with expression vectors encoding MuSK constructs that colocalize (b and c, construct 1; d and e, construct 2; f and g, construct 5) or do not colocalize (h and i, construct 6) with AChRs. Cells were treated with agrin for 18 h, and then doubly stained with anti-mouse MuSK antibodies (b, d, f, and h) and rBTX (c, e, g, and i). Arrows indicate corresponding points in the two images of each field. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169478&req=5

Figure 5: Carboxy-terminal regions of the MuSK ectodomain are required for association with AChR clusters in myotubes. (a) MuSK constructs, numbered as in Fig. 2, which were tested for their ability to associate with AChR clusters in MuSK−/− muscle cells. To the right of each construct is indicated whether it was concentrated at spontaneous or agrin-induced clusters. NA, not applicable, because agrin did not induce AChR clusters in these cells. (b–i) MuSK and AChR localization in cells transfected with expression vectors encoding MuSK constructs that colocalize (b and c, construct 1; d and e, construct 2; f and g, construct 5) or do not colocalize (h and i, construct 6) with AChRs. Cells were treated with agrin for 18 h, and then doubly stained with anti-mouse MuSK antibodies (b, d, f, and h) and rBTX (c, e, g, and i). Arrows indicate corresponding points in the two images of each field. Bar, 20 μm.

Mentions: Together, the results in Fig. 2 and Fig. 4 show that sequences in the MuSK ectodomain required for colocalization with rapsyn are dispensable for activation by agrin. What role might this juxtamembranous region play in muscle cells? One obvious possibility is that it is required for the association of MuSK with AChR rich aggregates. To test this hypothesis, we transfected MuSK−/− cells with MuSK mutants, and then stained nonpermeabilized cells with anti-MuSK and rBTX to determine whether the MuSK constructs reached the cell surface and whether they colocalized with AChRs. As shown in Fig. 5, constructs that contained the fourth immunoglobulin-like domain of the MuSK ectodomain (constructs 1, 2, 4, and 5) were concentrated at AChR clusters. In contrast, the two constructs that lacked sequences within the fourth immunoglobulin-like domain (constructs 6 and 7) were present on the myotube surface, but not concentrated at significantly higher levels in AChR rich than in AChR poor areas. Thus, the domain required for MuSK to associate with rapsyn in QT-6 cells is required for association with rapsyn–AChR clusters in myotubes.


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)

Carboxy-terminal regions of the MuSK ectodomain are required for association with AChR clusters in myotubes. (a) MuSK constructs, numbered as in Fig. 2, which were tested for their ability to associate with AChR clusters in MuSK−/− muscle cells. To the right of each construct is indicated whether it was concentrated at spontaneous or agrin-induced clusters. NA, not applicable, because agrin did not induce AChR clusters in these cells. (b–i) MuSK and AChR localization in cells transfected with expression vectors encoding MuSK constructs that colocalize (b and c, construct 1; d and e, construct 2; f and g, construct 5) or do not colocalize (h and i, construct 6) with AChRs. Cells were treated with agrin for 18 h, and then doubly stained with anti-mouse MuSK antibodies (b, d, f, and h) and rBTX (c, e, g, and i). Arrows indicate corresponding points in the two images of each field. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Carboxy-terminal regions of the MuSK ectodomain are required for association with AChR clusters in myotubes. (a) MuSK constructs, numbered as in Fig. 2, which were tested for their ability to associate with AChR clusters in MuSK−/− muscle cells. To the right of each construct is indicated whether it was concentrated at spontaneous or agrin-induced clusters. NA, not applicable, because agrin did not induce AChR clusters in these cells. (b–i) MuSK and AChR localization in cells transfected with expression vectors encoding MuSK constructs that colocalize (b and c, construct 1; d and e, construct 2; f and g, construct 5) or do not colocalize (h and i, construct 6) with AChRs. Cells were treated with agrin for 18 h, and then doubly stained with anti-mouse MuSK antibodies (b, d, f, and h) and rBTX (c, e, g, and i). Arrows indicate corresponding points in the two images of each field. Bar, 20 μm.
Mentions: Together, the results in Fig. 2 and Fig. 4 show that sequences in the MuSK ectodomain required for colocalization with rapsyn are dispensable for activation by agrin. What role might this juxtamembranous region play in muscle cells? One obvious possibility is that it is required for the association of MuSK with AChR rich aggregates. To test this hypothesis, we transfected MuSK−/− cells with MuSK mutants, and then stained nonpermeabilized cells with anti-MuSK and rBTX to determine whether the MuSK constructs reached the cell surface and whether they colocalized with AChRs. As shown in Fig. 5, constructs that contained the fourth immunoglobulin-like domain of the MuSK ectodomain (constructs 1, 2, 4, and 5) were concentrated at AChR clusters. In contrast, the two constructs that lacked sequences within the fourth immunoglobulin-like domain (constructs 6 and 7) were present on the myotube surface, but not concentrated at significantly higher levels in AChR rich than in AChR poor areas. Thus, the domain required for MuSK to associate with rapsyn in QT-6 cells is required for association with rapsyn–AChR clusters in myotubes.

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