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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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Carboxy-terminal regions of the MuSK ectodomain are required for association with rapsyn in heterologous cells. (a) MuSK constructs tested for their ability to cocluster with rapsyn in QT-6 cells. In all constructs, the ectodomain was derived from MuSK and the transmembrane and cytoplasmic domains were derived from TrkC. Abbreviations are as in Fig. 2, and areas of the MuSK ectodomain included in constructs 1T, 2T, 5T, and 6T are identical to those in 1, 2, 5, and 6, respectively, in Fig. 2. To the right of each construct is indicated whether it coclustered with rapsyn in QT-6 cells. (b–i) Localization of MuSK and rapsyn in cells transfected with expression vectors encoding MuSK construct 1T alone, (b and c) rapsyn plus MuSK constructs that cocluster well (d and e, 1T; f and g, 2T), or rapsyn plus a MuSK construct that does not cocluster (h and i, 6T). Cells were doubly stained with antibodies specific for MuSK (b, d, f, and h) and rapsyn (c, e, g, and i). Bar, 10 μm.
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Figure 4: Carboxy-terminal regions of the MuSK ectodomain are required for association with rapsyn in heterologous cells. (a) MuSK constructs tested for their ability to cocluster with rapsyn in QT-6 cells. In all constructs, the ectodomain was derived from MuSK and the transmembrane and cytoplasmic domains were derived from TrkC. Abbreviations are as in Fig. 2, and areas of the MuSK ectodomain included in constructs 1T, 2T, 5T, and 6T are identical to those in 1, 2, 5, and 6, respectively, in Fig. 2. To the right of each construct is indicated whether it coclustered with rapsyn in QT-6 cells. (b–i) Localization of MuSK and rapsyn in cells transfected with expression vectors encoding MuSK construct 1T alone, (b and c) rapsyn plus MuSK constructs that cocluster well (d and e, 1T; f and g, 2T), or rapsyn plus a MuSK construct that does not cocluster (h and i, 6T). Cells were doubly stained with antibodies specific for MuSK (b, d, f, and h) and rapsyn (c, e, g, and i). Bar, 10 μm.

Mentions: We asked whether the portions of MuSK required for association with rapsyn were distinguishable from those required for activation by agrin. For this purpose, we modified construct 2, which lacks the first immunoglobulin-like domain and is agrin unresponsive. In construct 2T (Fig. 4 a), the ectodomain from construct 2 was fused to the transmembrane and cytoplasmic domains of trkC, to exclude any contribution of cytoplasmic MuSK sequences to its localization. We used trkC as a negative control and the chimera between wild-type MuSK and trkC (construct 1T) as a positive control. These constructs were transfected into QT-6 cells either alone or with an expression vector encoding rapsyn. 2 d later, cells were fixed, permeabilized, and doubly stained with antibodies specific for rapsyn and MuSK or trkC. Rapsyn formed small aggregates when transfected by itself, whereas construct 1T, construct 2T, and trkC were all diffusely distributed when introduced alone (Fig. 4b and Fig. c, and data not shown). Cotransfection of rapsyn and trkC had no effect on the distribution of either component, whereas cotransfection of rapsyn with constructs 1T or 2T led to nearly perfect colocalization of the two components in most cells (Fig. 4, d–g, and data not shown). No differences were detected between constructs 1T and 2T in this assay. Thus, sequences required for activation by agrin are not required for colocalization with rapsyn.


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 rapsyn in heterologous cells. (a) MuSK constructs tested for their ability to cocluster with rapsyn in QT-6 cells. In all constructs, the ectodomain was derived from MuSK and the transmembrane and cytoplasmic domains were derived from TrkC. Abbreviations are as in Fig. 2, and areas of the MuSK ectodomain included in constructs 1T, 2T, 5T, and 6T are identical to those in 1, 2, 5, and 6, respectively, in Fig. 2. To the right of each construct is indicated whether it coclustered with rapsyn in QT-6 cells. (b–i) Localization of MuSK and rapsyn in cells transfected with expression vectors encoding MuSK construct 1T alone, (b and c) rapsyn plus MuSK constructs that cocluster well (d and e, 1T; f and g, 2T), or rapsyn plus a MuSK construct that does not cocluster (h and i, 6T). Cells were doubly stained with antibodies specific for MuSK (b, d, f, and h) and rapsyn (c, e, g, and i). Bar, 10 μm.
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Figure 4: Carboxy-terminal regions of the MuSK ectodomain are required for association with rapsyn in heterologous cells. (a) MuSK constructs tested for their ability to cocluster with rapsyn in QT-6 cells. In all constructs, the ectodomain was derived from MuSK and the transmembrane and cytoplasmic domains were derived from TrkC. Abbreviations are as in Fig. 2, and areas of the MuSK ectodomain included in constructs 1T, 2T, 5T, and 6T are identical to those in 1, 2, 5, and 6, respectively, in Fig. 2. To the right of each construct is indicated whether it coclustered with rapsyn in QT-6 cells. (b–i) Localization of MuSK and rapsyn in cells transfected with expression vectors encoding MuSK construct 1T alone, (b and c) rapsyn plus MuSK constructs that cocluster well (d and e, 1T; f and g, 2T), or rapsyn plus a MuSK construct that does not cocluster (h and i, 6T). Cells were doubly stained with antibodies specific for MuSK (b, d, f, and h) and rapsyn (c, e, g, and i). Bar, 10 μm.
Mentions: We asked whether the portions of MuSK required for association with rapsyn were distinguishable from those required for activation by agrin. For this purpose, we modified construct 2, which lacks the first immunoglobulin-like domain and is agrin unresponsive. In construct 2T (Fig. 4 a), the ectodomain from construct 2 was fused to the transmembrane and cytoplasmic domains of trkC, to exclude any contribution of cytoplasmic MuSK sequences to its localization. We used trkC as a negative control and the chimera between wild-type MuSK and trkC (construct 1T) as a positive control. These constructs were transfected into QT-6 cells either alone or with an expression vector encoding rapsyn. 2 d later, cells were fixed, permeabilized, and doubly stained with antibodies specific for rapsyn and MuSK or trkC. Rapsyn formed small aggregates when transfected by itself, whereas construct 1T, construct 2T, and trkC were all diffusely distributed when introduced alone (Fig. 4b and Fig. c, and data not shown). Cotransfection of rapsyn and trkC had no effect on the distribution of either component, whereas cotransfection of rapsyn with constructs 1T or 2T led to nearly perfect colocalization of the two components in most cells (Fig. 4, d–g, and data not shown). No differences were detected between constructs 1T and 2T in this assay. Thus, sequences required for activation by agrin are not required for colocalization with rapsyn.

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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