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Meltrin beta/ADAM19 interacting with EphA4 in developing neural cells participates in formation of the neuromuscular junction.

Yumoto N, Wakatsuki S, Kurisaki T, Hara Y, Osumi N, Frisén J, Sehara-Fujisawa A - PLoS ONE (2008)

Bottom Line: Meltrin beta plays a regulatory role in formation of the NMJ.The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph.We propose that Meltrin beta stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Shogo-in, Kyoto, Japan.

ABSTRACT

Background: Development of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously.

Principal findings: We report here that Meltrin beta, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor alpha mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin beta-deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin beta-deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin beta and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin beta and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin beta and EphA4 strongly blocked vesicular internalization of ephrin-A5-EphA4 complexes without requiring the protease activity of Meltrin beta, suggesting a regulatory role of Meltrin beta in ephrin-A5-Eph signaling.

Conclusion: Meltrin beta plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin beta stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

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Meltrin β interacts with EphA4 in the peripheral nervous system.(A–D) Sequential sections of the spinal cord at E12.5 were double-stained with antibodies against EphA4 (A) and islet 1/2 (A, inbox) or against Meltrin β (B) and Islet 1/2 (C). EphA4 and Meltrin β proteins were expressed in Islet 1/2–positive motor neurons (indicated by arrows). Dotted lines show dorsal root ganglia (DRG). Bar: 50 µm. (E) Immunoprecipitation was performed with HEK293T cells transfected with HA-tagged EphA4 and myc-tagged Meltrin β. EphA4 coimmunoprecipitated with Meltrin β (anti-myc, left panel), and full-length Meltrin β coimmunoprecipitated with EphA4 (anti-HA, right panel). (F) Immunoprecipitation was performed with NIH3T3 cells expressing. EphA4 and meltrin β domain-deletion mutants tagged with HA. The ectodomain of Meltrin β, but not its cytoplasmic domain, is required for interactions with EphA4 (open arrow). (G) Coimmunoprecipitation was performed with lysates prepared from E13.5 embryonic spinal cords and DRG using the anti-EphA4 antibody used in (F). Endogenous Meltrin β (open arrow) was coimmunoprecipitated with EphA4 (closed arrow) in vivo. The bands for IgG and non-specific bands (asterisks) in the lane for control mouse IgG were higher than those in the lane for EphA4 because the concentration of control IgG used in this experiment was higher in this experiment, further excluding that the band for EphA4 is one of non-specific bands.
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pone-0003322-g004: Meltrin β interacts with EphA4 in the peripheral nervous system.(A–D) Sequential sections of the spinal cord at E12.5 were double-stained with antibodies against EphA4 (A) and islet 1/2 (A, inbox) or against Meltrin β (B) and Islet 1/2 (C). EphA4 and Meltrin β proteins were expressed in Islet 1/2–positive motor neurons (indicated by arrows). Dotted lines show dorsal root ganglia (DRG). Bar: 50 µm. (E) Immunoprecipitation was performed with HEK293T cells transfected with HA-tagged EphA4 and myc-tagged Meltrin β. EphA4 coimmunoprecipitated with Meltrin β (anti-myc, left panel), and full-length Meltrin β coimmunoprecipitated with EphA4 (anti-HA, right panel). (F) Immunoprecipitation was performed with NIH3T3 cells expressing. EphA4 and meltrin β domain-deletion mutants tagged with HA. The ectodomain of Meltrin β, but not its cytoplasmic domain, is required for interactions with EphA4 (open arrow). (G) Coimmunoprecipitation was performed with lysates prepared from E13.5 embryonic spinal cords and DRG using the anti-EphA4 antibody used in (F). Endogenous Meltrin β (open arrow) was coimmunoprecipitated with EphA4 (closed arrow) in vivo. The bands for IgG and non-specific bands (asterisks) in the lane for control mouse IgG were higher than those in the lane for EphA4 because the concentration of control IgG used in this experiment was higher in this experiment, further excluding that the band for EphA4 is one of non-specific bands.

Mentions: Ephrin-A5 is a glycosylphosphatidylinositol-anchored protein expressed in muscles and is a ligand for a group of membrane-bound receptor tyrosine kinases, the Ephs. Motor neurons express EphA4 receptors [27]–[30], and EphA4 is localized at the NMJ [31]. Islet 1/2–positive motor neurons of E12.5 spinal cords expressed Meltrin β as well as EphA4 (Fig. 4A–D). We asked whether Meltrin β interacts with EphA4 in motor neurons to regulate ephrin–Eph signaling. As shown in Fig. 4E–G, immunoprecipitation analyses were performed. When myc-tagged Meltrin β and HA-tagged EphA4 were co-expressed in HEK293T cells, these two proteins interacted with each other (Fig. 4E). When HA-tagged Meltrin β lacking its ectodomain was co-expressed with EphA4, the mutant Meltrin β did not precipitate EphA4; in contrast, EphA4 did co-precipitate with Meltrin β lacking the cytoplasmic domain (Fig. 4F). This suggested that Meltrin β, through its ectodomain, interacts with EphA4. We tested whether Meltrin β interacts with EphA4 in developing neural cells. We dissected spinal cords and dorsal root ganglia (DRG) from embryos at E13.5 and lysed them with Triton-X100 lysis buffer. Proteins were immunoprecipitated with the anti-EphA4 antibody and immunoblotted with the anti-Meltrin β antibody. Meltrin β co-immunoprecipitated with EphA4 (Fig. 4G, open arrow), suggesting that Meltrin β interacts with EphA4 in neural cells during development.


Meltrin beta/ADAM19 interacting with EphA4 in developing neural cells participates in formation of the neuromuscular junction.

Yumoto N, Wakatsuki S, Kurisaki T, Hara Y, Osumi N, Frisén J, Sehara-Fujisawa A - PLoS ONE (2008)

Meltrin β interacts with EphA4 in the peripheral nervous system.(A–D) Sequential sections of the spinal cord at E12.5 were double-stained with antibodies against EphA4 (A) and islet 1/2 (A, inbox) or against Meltrin β (B) and Islet 1/2 (C). EphA4 and Meltrin β proteins were expressed in Islet 1/2–positive motor neurons (indicated by arrows). Dotted lines show dorsal root ganglia (DRG). Bar: 50 µm. (E) Immunoprecipitation was performed with HEK293T cells transfected with HA-tagged EphA4 and myc-tagged Meltrin β. EphA4 coimmunoprecipitated with Meltrin β (anti-myc, left panel), and full-length Meltrin β coimmunoprecipitated with EphA4 (anti-HA, right panel). (F) Immunoprecipitation was performed with NIH3T3 cells expressing. EphA4 and meltrin β domain-deletion mutants tagged with HA. The ectodomain of Meltrin β, but not its cytoplasmic domain, is required for interactions with EphA4 (open arrow). (G) Coimmunoprecipitation was performed with lysates prepared from E13.5 embryonic spinal cords and DRG using the anti-EphA4 antibody used in (F). Endogenous Meltrin β (open arrow) was coimmunoprecipitated with EphA4 (closed arrow) in vivo. The bands for IgG and non-specific bands (asterisks) in the lane for control mouse IgG were higher than those in the lane for EphA4 because the concentration of control IgG used in this experiment was higher in this experiment, further excluding that the band for EphA4 is one of non-specific bands.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2552171&req=5

pone-0003322-g004: Meltrin β interacts with EphA4 in the peripheral nervous system.(A–D) Sequential sections of the spinal cord at E12.5 were double-stained with antibodies against EphA4 (A) and islet 1/2 (A, inbox) or against Meltrin β (B) and Islet 1/2 (C). EphA4 and Meltrin β proteins were expressed in Islet 1/2–positive motor neurons (indicated by arrows). Dotted lines show dorsal root ganglia (DRG). Bar: 50 µm. (E) Immunoprecipitation was performed with HEK293T cells transfected with HA-tagged EphA4 and myc-tagged Meltrin β. EphA4 coimmunoprecipitated with Meltrin β (anti-myc, left panel), and full-length Meltrin β coimmunoprecipitated with EphA4 (anti-HA, right panel). (F) Immunoprecipitation was performed with NIH3T3 cells expressing. EphA4 and meltrin β domain-deletion mutants tagged with HA. The ectodomain of Meltrin β, but not its cytoplasmic domain, is required for interactions with EphA4 (open arrow). (G) Coimmunoprecipitation was performed with lysates prepared from E13.5 embryonic spinal cords and DRG using the anti-EphA4 antibody used in (F). Endogenous Meltrin β (open arrow) was coimmunoprecipitated with EphA4 (closed arrow) in vivo. The bands for IgG and non-specific bands (asterisks) in the lane for control mouse IgG were higher than those in the lane for EphA4 because the concentration of control IgG used in this experiment was higher in this experiment, further excluding that the band for EphA4 is one of non-specific bands.
Mentions: Ephrin-A5 is a glycosylphosphatidylinositol-anchored protein expressed in muscles and is a ligand for a group of membrane-bound receptor tyrosine kinases, the Ephs. Motor neurons express EphA4 receptors [27]–[30], and EphA4 is localized at the NMJ [31]. Islet 1/2–positive motor neurons of E12.5 spinal cords expressed Meltrin β as well as EphA4 (Fig. 4A–D). We asked whether Meltrin β interacts with EphA4 in motor neurons to regulate ephrin–Eph signaling. As shown in Fig. 4E–G, immunoprecipitation analyses were performed. When myc-tagged Meltrin β and HA-tagged EphA4 were co-expressed in HEK293T cells, these two proteins interacted with each other (Fig. 4E). When HA-tagged Meltrin β lacking its ectodomain was co-expressed with EphA4, the mutant Meltrin β did not precipitate EphA4; in contrast, EphA4 did co-precipitate with Meltrin β lacking the cytoplasmic domain (Fig. 4F). This suggested that Meltrin β, through its ectodomain, interacts with EphA4. We tested whether Meltrin β interacts with EphA4 in developing neural cells. We dissected spinal cords and dorsal root ganglia (DRG) from embryos at E13.5 and lysed them with Triton-X100 lysis buffer. Proteins were immunoprecipitated with the anti-EphA4 antibody and immunoblotted with the anti-Meltrin β antibody. Meltrin β co-immunoprecipitated with EphA4 (Fig. 4G, open arrow), suggesting that Meltrin β interacts with EphA4 in neural cells during development.

Bottom Line: Meltrin beta plays a regulatory role in formation of the NMJ.The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph.We propose that Meltrin beta stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Shogo-in, Kyoto, Japan.

ABSTRACT

Background: Development of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously.

Principal findings: We report here that Meltrin beta, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor alpha mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin beta-deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin beta-deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin beta and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin beta and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin beta and EphA4 strongly blocked vesicular internalization of ephrin-A5-EphA4 complexes without requiring the protease activity of Meltrin beta, suggesting a regulatory role of Meltrin beta in ephrin-A5-Eph signaling.

Conclusion: Meltrin beta plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin beta stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

Show MeSH
Related in: MedlinePlus