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MuSK induces in vivo acetylcholine receptor clusters in a ligand-independent manner.

Sander A, Hesser BA, Witzemann V - J. Cell Biol. (2001)

Bottom Line: Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters.Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters.This process does not require additional determinants located in the ectodomain.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Zellphysiologie, Max-Planck-Institut für Medizinische Forschung, D-69120 Heidelberg, Germany.

ABSTRACT
Muscle-specific receptor tyrosine kinase (MuSK) is required for the formation of the neuromuscular junction. Using direct gene transfer into single fibers, MuSK was expressed extrasynaptically in innervated rat muscle in vivo to identify its contribution to synapse formation. Spontaneous MuSK kinase activity leads, in the absence of its putative ligand neural agrin, to the appearance of epsilon-subunit-specific transcripts, the formation of acetylcholine receptor clusters, and acetylcholinesterase aggregates. Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters. The contribution of endogenous MuSK was excluded by using genetically altered mice, where the kinase domain of the MuSK gene was flanked by loxP sequences and could be deleted upon expression of Cre recombinase. This allowed the conditional inactivation of endogenous MuSK in single muscle fibers and prevented the induction of ectopic AChR clusters. Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters. This process does not require additional determinants located in the ectodomain.

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MuSK and MuSK mutant constructs used for direct gene transfer. MuSK and MuSK mutant constructs are shown schematically. SP, signal peptide; TM, transmembrane domain. K608 of the wild-type kinase (o) is mutated to K608A (x) in the kinase-inactive MuSK mutants. The cDNAs cloned into pRK5 expression vectors were injected ectopically in single muscle fibers to express the transgenic proteins. Their ability to induce AChR clusters is indicated on the right.
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fig2: MuSK and MuSK mutant constructs used for direct gene transfer. MuSK and MuSK mutant constructs are shown schematically. SP, signal peptide; TM, transmembrane domain. K608 of the wild-type kinase (o) is mutated to K608A (x) in the kinase-inactive MuSK mutants. The cDNAs cloned into pRK5 expression vectors were injected ectopically in single muscle fibers to express the transgenic proteins. Their ability to induce AChR clusters is indicated on the right.

Mentions: Expression of the kinase-inactive mutant MuSKK608A (Glass et al., 1997) in single muscle fibers confirmed that no AChR clusters were induced (Fig. 2). To visualize and demonstrate directly the expression of recombinant MuSK in the injected muscle fibers, we fused GFP with MuSK (MuSK–GFP) and with the kinase-inactive mutant MuSKK608A (MuSKK608A–GFP), as schematically summarized in Fig. 2. Fig. 3, A and D, demonstrate that both constructs were expressed with similar efficiency. MuSK–GFP gave rise to AChR clusters which were clearly identified with rhodamine-labeled α-bungarotoxin (r-bgt) (Fig. 3 B). The overlay of the green and red fluorescence confocal images (Fig. 3 C) shows that MuSK–GFP was not strictly colocalized with the AChR clusters. Statistical analysis revealed that in all transgene-expressing fibers, as marked by GFP, AChR clusters were formed (Fig. 3 F). No AChR clusters, however, were induced by the kinase-inactive mutant MuSKK608A–GFP (Fig. 3, D, E, and G and Fig. 2), demonstrating that kinase activity is required for the induction of AChR clusters. Rapsyn coinjected with the MuSK mutant was unable to form AChR clusters.


MuSK induces in vivo acetylcholine receptor clusters in a ligand-independent manner.

Sander A, Hesser BA, Witzemann V - J. Cell Biol. (2001)

MuSK and MuSK mutant constructs used for direct gene transfer. MuSK and MuSK mutant constructs are shown schematically. SP, signal peptide; TM, transmembrane domain. K608 of the wild-type kinase (o) is mutated to K608A (x) in the kinase-inactive MuSK mutants. The cDNAs cloned into pRK5 expression vectors were injected ectopically in single muscle fibers to express the transgenic proteins. Their ability to induce AChR clusters is indicated on the right.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199313&req=5

fig2: MuSK and MuSK mutant constructs used for direct gene transfer. MuSK and MuSK mutant constructs are shown schematically. SP, signal peptide; TM, transmembrane domain. K608 of the wild-type kinase (o) is mutated to K608A (x) in the kinase-inactive MuSK mutants. The cDNAs cloned into pRK5 expression vectors were injected ectopically in single muscle fibers to express the transgenic proteins. Their ability to induce AChR clusters is indicated on the right.
Mentions: Expression of the kinase-inactive mutant MuSKK608A (Glass et al., 1997) in single muscle fibers confirmed that no AChR clusters were induced (Fig. 2). To visualize and demonstrate directly the expression of recombinant MuSK in the injected muscle fibers, we fused GFP with MuSK (MuSK–GFP) and with the kinase-inactive mutant MuSKK608A (MuSKK608A–GFP), as schematically summarized in Fig. 2. Fig. 3, A and D, demonstrate that both constructs were expressed with similar efficiency. MuSK–GFP gave rise to AChR clusters which were clearly identified with rhodamine-labeled α-bungarotoxin (r-bgt) (Fig. 3 B). The overlay of the green and red fluorescence confocal images (Fig. 3 C) shows that MuSK–GFP was not strictly colocalized with the AChR clusters. Statistical analysis revealed that in all transgene-expressing fibers, as marked by GFP, AChR clusters were formed (Fig. 3 F). No AChR clusters, however, were induced by the kinase-inactive mutant MuSKK608A–GFP (Fig. 3, D, E, and G and Fig. 2), demonstrating that kinase activity is required for the induction of AChR clusters. Rapsyn coinjected with the MuSK mutant was unable to form AChR clusters.

Bottom Line: Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters.Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters.This process does not require additional determinants located in the ectodomain.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Zellphysiologie, Max-Planck-Institut für Medizinische Forschung, D-69120 Heidelberg, Germany.

ABSTRACT
Muscle-specific receptor tyrosine kinase (MuSK) is required for the formation of the neuromuscular junction. Using direct gene transfer into single fibers, MuSK was expressed extrasynaptically in innervated rat muscle in vivo to identify its contribution to synapse formation. Spontaneous MuSK kinase activity leads, in the absence of its putative ligand neural agrin, to the appearance of epsilon-subunit-specific transcripts, the formation of acetylcholine receptor clusters, and acetylcholinesterase aggregates. Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters. The contribution of endogenous MuSK was excluded by using genetically altered mice, where the kinase domain of the MuSK gene was flanked by loxP sequences and could be deleted upon expression of Cre recombinase. This allowed the conditional inactivation of endogenous MuSK in single muscle fibers and prevented the induction of ectopic AChR clusters. Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters. This process does not require additional determinants located in the ectodomain.

Show MeSH
Related in: MedlinePlus