Limits...
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.

Show MeSH

Related in: MedlinePlus

Meltrin β does not affect the expression of EphA4 on the cell surface nor the initial association between ephrin-A5 and EphA4, but decreases accessibility of antibodies that recognize the EphA4 C-terminus.(A) EphA4-HA transformant cells were transfected with Meltrin β expression plasmids (meltrin β) or with control vectors, and biotinylated proteins on the cell surface were precipitated with streptavidin beads. The proteins were separated by SDS-PAGE, and EphA4 was detected with an anti-HA antibody (HA-7). Caveolin 2 was used as an internal control. (B) NIH3T3 cells were transfected with EphA4-HA expression plasmids and the various plasmids listed at the top of the lanes and stimulated with ephrin-A5-Fc fusion proteins as indicated. The cell lysates were divided into a Triton-X100–solubilized fraction (soluble fraction) and Triton-X100–resistant raft microdomains (insoluble fraction). After SDS-PAGE separation, EphA4 was detected with HA-7. EphA4 proteins in the soluble fractions were also immunoprecipitated with HA-7 (the bottom panel) and detected with HA-7 after SDS-PAGE. The amount of EphA4 precipitated was reduced dramatically when the Meltrin β WT or EQ mutant was coexpressed with EphA4, regardless of stimulation with ephrin-A5. (C) (Upper Panel) NIH3T3 cells were transfected with EphA4-HA expression plasmids and the various plasmids listed at the top of the lanes and stimulated with ephrin-A5-Fc fusion proteins as indicated. EphA4 proteins in the soluble fractions (whole cell lysate) were immunoblotted with anti-HA antibody (HA-7) or immunoprecipitated with an anti-EphA4 antibody and detected with HA-7 after SDS-PAGE. Immunoprecipitation of EphA4 with anti-EphA4 was inefficient when the meltrin β WT or EQ mutant were coexpressed, regardless of the stimulation with ephrin-A5-Fc. (Lower Panel) An anti-EphA4 antibody from Zymed recognizes the SAM domain in the EphA4-C terminus. NIH3T3 cells were transfected with plasmids that express EphA4-HA or SAM domain–deleted EphA4-HA, and expressed proteins were detected either with HA-7 or with the anti-EphA4 antibody after SDS-PAGE of the cell lysates. (D) EphA4 transformants transfected with meltrin β or a control vector were seeded onto plastic dishes coated with a human Fc fragment or ephrin-A5-Fc. The binding activity of EphA4-expressing cells (detected with DAPI) on ephrin-A5-coated plates was not changed by coexpression of Meltrin β.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2552171&req=5

pone-0003322-g005: Meltrin β does not affect the expression of EphA4 on the cell surface nor the initial association between ephrin-A5 and EphA4, but decreases accessibility of antibodies that recognize the EphA4 C-terminus.(A) EphA4-HA transformant cells were transfected with Meltrin β expression plasmids (meltrin β) or with control vectors, and biotinylated proteins on the cell surface were precipitated with streptavidin beads. The proteins were separated by SDS-PAGE, and EphA4 was detected with an anti-HA antibody (HA-7). Caveolin 2 was used as an internal control. (B) NIH3T3 cells were transfected with EphA4-HA expression plasmids and the various plasmids listed at the top of the lanes and stimulated with ephrin-A5-Fc fusion proteins as indicated. The cell lysates were divided into a Triton-X100–solubilized fraction (soluble fraction) and Triton-X100–resistant raft microdomains (insoluble fraction). After SDS-PAGE separation, EphA4 was detected with HA-7. EphA4 proteins in the soluble fractions were also immunoprecipitated with HA-7 (the bottom panel) and detected with HA-7 after SDS-PAGE. The amount of EphA4 precipitated was reduced dramatically when the Meltrin β WT or EQ mutant was coexpressed with EphA4, regardless of stimulation with ephrin-A5. (C) (Upper Panel) NIH3T3 cells were transfected with EphA4-HA expression plasmids and the various plasmids listed at the top of the lanes and stimulated with ephrin-A5-Fc fusion proteins as indicated. EphA4 proteins in the soluble fractions (whole cell lysate) were immunoblotted with anti-HA antibody (HA-7) or immunoprecipitated with an anti-EphA4 antibody and detected with HA-7 after SDS-PAGE. Immunoprecipitation of EphA4 with anti-EphA4 was inefficient when the meltrin β WT or EQ mutant were coexpressed, regardless of the stimulation with ephrin-A5-Fc. (Lower Panel) An anti-EphA4 antibody from Zymed recognizes the SAM domain in the EphA4-C terminus. NIH3T3 cells were transfected with plasmids that express EphA4-HA or SAM domain–deleted EphA4-HA, and expressed proteins were detected either with HA-7 or with the anti-EphA4 antibody after SDS-PAGE of the cell lysates. (D) EphA4 transformants transfected with meltrin β or a control vector were seeded onto plastic dishes coated with a human Fc fragment or ephrin-A5-Fc. The binding activity of EphA4-expressing cells (detected with DAPI) on ephrin-A5-coated plates was not changed by coexpression of Meltrin β.

Mentions: Meltrin β reaches lipid rafts to cleave the neuregulin-β1 extracellular region [19] , and is expressed mainly in the Golgi apparatus and the intracellular vesicles around it [20]. The molecular interaction between Meltrin β and EphA4 in neural cells led us to investigate intracellular localization of Meltrin β in the existence of EphA4. When Meltrin β was expressed together with EphA4, some of the Meltrin β proteins were translocated to the cell surface (Figure S2). Then, we examined whether Meltrin β affects the membrane localization of EphA4 on the cellular membrane. We compared the amount of EphA4 expressed on the cell surface; this was estimated by cell surface biotinylation, in NIH3T3 cells transformed with EphA4-HA, with or without the exogenous expression of Meltrin β (Fig. 5A). Meltrin β expression did not affect the expression of EphA4 on the cell surface (Fig. 5A, arrowhead). Next, we investigated the distribution of EphA4 in lipid raft and non-raft fractions (Fig. 5B). EphA4-HA was expressed together with wild-type Meltrin β (Meltrin β WT); Meltrin β EQ, which contains a point mutation in the active site of its metalloprotease domain; or the empty plasmid as a control, and total cell lysates were prepared from each transfectant with Triton-X100 lysis buffer. We divided the cellular lysates into two fractions, a Triton-X100–soluble fraction and a Triton-X100–resistant insoluble fraction, and analyzed them with anti-HA antibodies. The distribution pattern of EphA4 in the soluble and insoluble fractions was not altered by the expression of Meltrin β WT or the EQ mutant, regardless of stimulation with ephrin-A5-Fc, a chimeric protein of a soluble form of ephrin A5 and a human Fc fragment. However, the amount of EphA4 protein in the soluble fraction that was precipitated with an anti-HA antibody (clone HA-7) was reduced significantly when Meltrin β WT or the EQ mutant was expressed in the cells (Fig. 5B, bottom). Similar results were obtained with an anti-EphA4 antibody that recognizes the SAM domain in the cytoplasmic region of EphA4 (Fig. 5C). These results suggest that the C-terminus, especially SAM domain, of EphA4 becomes less accessible to these antibodies when Eph4A interacts with Meltrin β, independent of ephrin-A5 stimulation, and that the interaction of these two proteins does not require the protease activity of Meltrin β.


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 does not affect the expression of EphA4 on the cell surface nor the initial association between ephrin-A5 and EphA4, but decreases accessibility of antibodies that recognize the EphA4 C-terminus.(A) EphA4-HA transformant cells were transfected with Meltrin β expression plasmids (meltrin β) or with control vectors, and biotinylated proteins on the cell surface were precipitated with streptavidin beads. The proteins were separated by SDS-PAGE, and EphA4 was detected with an anti-HA antibody (HA-7). Caveolin 2 was used as an internal control. (B) NIH3T3 cells were transfected with EphA4-HA expression plasmids and the various plasmids listed at the top of the lanes and stimulated with ephrin-A5-Fc fusion proteins as indicated. The cell lysates were divided into a Triton-X100–solubilized fraction (soluble fraction) and Triton-X100–resistant raft microdomains (insoluble fraction). After SDS-PAGE separation, EphA4 was detected with HA-7. EphA4 proteins in the soluble fractions were also immunoprecipitated with HA-7 (the bottom panel) and detected with HA-7 after SDS-PAGE. The amount of EphA4 precipitated was reduced dramatically when the Meltrin β WT or EQ mutant was coexpressed with EphA4, regardless of stimulation with ephrin-A5. (C) (Upper Panel) NIH3T3 cells were transfected with EphA4-HA expression plasmids and the various plasmids listed at the top of the lanes and stimulated with ephrin-A5-Fc fusion proteins as indicated. EphA4 proteins in the soluble fractions (whole cell lysate) were immunoblotted with anti-HA antibody (HA-7) or immunoprecipitated with an anti-EphA4 antibody and detected with HA-7 after SDS-PAGE. Immunoprecipitation of EphA4 with anti-EphA4 was inefficient when the meltrin β WT or EQ mutant were coexpressed, regardless of the stimulation with ephrin-A5-Fc. (Lower Panel) An anti-EphA4 antibody from Zymed recognizes the SAM domain in the EphA4-C terminus. NIH3T3 cells were transfected with plasmids that express EphA4-HA or SAM domain–deleted EphA4-HA, and expressed proteins were detected either with HA-7 or with the anti-EphA4 antibody after SDS-PAGE of the cell lysates. (D) EphA4 transformants transfected with meltrin β or a control vector were seeded onto plastic dishes coated with a human Fc fragment or ephrin-A5-Fc. The binding activity of EphA4-expressing cells (detected with DAPI) on ephrin-A5-coated plates was not changed by coexpression of Meltrin β.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2552171&req=5

pone-0003322-g005: Meltrin β does not affect the expression of EphA4 on the cell surface nor the initial association between ephrin-A5 and EphA4, but decreases accessibility of antibodies that recognize the EphA4 C-terminus.(A) EphA4-HA transformant cells were transfected with Meltrin β expression plasmids (meltrin β) or with control vectors, and biotinylated proteins on the cell surface were precipitated with streptavidin beads. The proteins were separated by SDS-PAGE, and EphA4 was detected with an anti-HA antibody (HA-7). Caveolin 2 was used as an internal control. (B) NIH3T3 cells were transfected with EphA4-HA expression plasmids and the various plasmids listed at the top of the lanes and stimulated with ephrin-A5-Fc fusion proteins as indicated. The cell lysates were divided into a Triton-X100–solubilized fraction (soluble fraction) and Triton-X100–resistant raft microdomains (insoluble fraction). After SDS-PAGE separation, EphA4 was detected with HA-7. EphA4 proteins in the soluble fractions were also immunoprecipitated with HA-7 (the bottom panel) and detected with HA-7 after SDS-PAGE. The amount of EphA4 precipitated was reduced dramatically when the Meltrin β WT or EQ mutant was coexpressed with EphA4, regardless of stimulation with ephrin-A5. (C) (Upper Panel) NIH3T3 cells were transfected with EphA4-HA expression plasmids and the various plasmids listed at the top of the lanes and stimulated with ephrin-A5-Fc fusion proteins as indicated. EphA4 proteins in the soluble fractions (whole cell lysate) were immunoblotted with anti-HA antibody (HA-7) or immunoprecipitated with an anti-EphA4 antibody and detected with HA-7 after SDS-PAGE. Immunoprecipitation of EphA4 with anti-EphA4 was inefficient when the meltrin β WT or EQ mutant were coexpressed, regardless of the stimulation with ephrin-A5-Fc. (Lower Panel) An anti-EphA4 antibody from Zymed recognizes the SAM domain in the EphA4-C terminus. NIH3T3 cells were transfected with plasmids that express EphA4-HA or SAM domain–deleted EphA4-HA, and expressed proteins were detected either with HA-7 or with the anti-EphA4 antibody after SDS-PAGE of the cell lysates. (D) EphA4 transformants transfected with meltrin β or a control vector were seeded onto plastic dishes coated with a human Fc fragment or ephrin-A5-Fc. The binding activity of EphA4-expressing cells (detected with DAPI) on ephrin-A5-coated plates was not changed by coexpression of Meltrin β.
Mentions: Meltrin β reaches lipid rafts to cleave the neuregulin-β1 extracellular region [19] , and is expressed mainly in the Golgi apparatus and the intracellular vesicles around it [20]. The molecular interaction between Meltrin β and EphA4 in neural cells led us to investigate intracellular localization of Meltrin β in the existence of EphA4. When Meltrin β was expressed together with EphA4, some of the Meltrin β proteins were translocated to the cell surface (Figure S2). Then, we examined whether Meltrin β affects the membrane localization of EphA4 on the cellular membrane. We compared the amount of EphA4 expressed on the cell surface; this was estimated by cell surface biotinylation, in NIH3T3 cells transformed with EphA4-HA, with or without the exogenous expression of Meltrin β (Fig. 5A). Meltrin β expression did not affect the expression of EphA4 on the cell surface (Fig. 5A, arrowhead). Next, we investigated the distribution of EphA4 in lipid raft and non-raft fractions (Fig. 5B). EphA4-HA was expressed together with wild-type Meltrin β (Meltrin β WT); Meltrin β EQ, which contains a point mutation in the active site of its metalloprotease domain; or the empty plasmid as a control, and total cell lysates were prepared from each transfectant with Triton-X100 lysis buffer. We divided the cellular lysates into two fractions, a Triton-X100–soluble fraction and a Triton-X100–resistant insoluble fraction, and analyzed them with anti-HA antibodies. The distribution pattern of EphA4 in the soluble and insoluble fractions was not altered by the expression of Meltrin β WT or the EQ mutant, regardless of stimulation with ephrin-A5-Fc, a chimeric protein of a soluble form of ephrin A5 and a human Fc fragment. However, the amount of EphA4 protein in the soluble fraction that was precipitated with an anti-HA antibody (clone HA-7) was reduced significantly when Meltrin β WT or the EQ mutant was expressed in the cells (Fig. 5B, bottom). Similar results were obtained with an anti-EphA4 antibody that recognizes the SAM domain in the cytoplasmic region of EphA4 (Fig. 5C). These results suggest that the C-terminus, especially SAM domain, of EphA4 becomes less accessible to these antibodies when Eph4A interacts with Meltrin β, independent of ephrin-A5 stimulation, and that the interaction of these two proteins does not require the protease activity of Meltrin β.

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