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Dissection of complex molecular interactions of neurofascin with axonin-1, F11, and tenascin-R, which promote attachment and neurite formation of tectal cells.

Volkmer H, Zacharias U, Nörenberg U, Rathjen FG - J. Cell Biol. (1998)

Bottom Line: In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11.Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding.In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.

View Article: PubMed Central - PubMed

Affiliation: Max-Delbrück-Centrum für Molekulare Medizin, D-13122 Berlin, Germany.

ABSTRACT
Neurofascin is a member of the L1 subgroup of the Ig superfamily that promotes axon outgrowth by interactions with neuronal NgCAM-related cell adhesion molecule (NrCAM). We used a combination of cellular binding assays and neurite outgrowth experiments to investigate mechanisms that might modulate the interactions of neurofascin. In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11. Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding. While interactions of neurofascin with F11 are only slightly modulated, binding to axonin-1 and TN-R is strongly regulated by alternatively spliced stretches located in the NH2-terminal half, and by the proline-alanine-threonine-rich segment. In vitro neurite outgrowth and cell attachment assays on a neurofascin-Fc substrate reveal a shift of cellular receptor usage from NrCAM to axonin-1, F11, and at least one additional protein in the presence of TN-R, presumably due to competition of the neurofascin- NrCAM interaction. Thereby, F11 binds to TN-R of the neurofascin/TN-R complex, but not to neurofascin, whereas axonin-1 is not able to bind directly to the neurofascin/TN-R complex as shown by competition binding assays. In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.

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(A–I) Binding of TN-R, axonin-1, and F11 by neurofascin isoform NF22 expressed on the surface of transfected COS7  cells. Double fluorescence images obtained using a confocal microscope are shown. The right half of each micrograph of A–I reveals neurofascin expression (NF expr.) by indirect immunofluorescence in the FITC channel, while the left half depicts the  binding of F11- (A, D, and G), of axonin-1–(B, E, and H) or of  TN-R– (C, F, and I) conjugated microspheres in the same microscopic field as detected in the Texas Red channel. Each microscopic field also contains unstained cells that do not express neurofascin, and concomitantly do not bind beads. Beads were  incubated in the absence (A–C) or presence of Fab fragments of  polyclonal antibodies to neurofascin (D–F), to F11 (G), to axonin-1 (H), or to TN-R (I), which is indicated at the left of each  panel. (A)Bar, 100 μm. (K–M) F11-coated beads bind to the Ig-like domains of neurofascin. COS7 cells transfected with plasmid  NF17 (K) expressing both Ig- and FNIII-like domains of neurofascin, deletion mutant NF21 lacking the Ig-like domains (L), or  deletion mutant NF13 lacking the FNIII-like repeats (M) were incubated with F11-coated beads. (K) Bar, 100 μm. NF17 and the  deletion constructs NF21 and NF13 are shown schematically at  the right of each panel. (N) Neurofascin and F11 or neurofascin  and axonin-1 coprecipitate in immunocomplexes. Retinae from  embryonic day 11/12 were solubilized in detergent containing  buffer, and were subjected to immunoprecipitation using an mAb  to neurofascin. Immunocomplexes were separated by SDS-PAGE and transferred to nitrocellulose, followed by staining  with antibodies to neurofascin (lane 1), F11 (lane 2), axonin-1  (lane 3), NCAM (lane 4), or NgCAM (lane 5). Binding of antibodies was visualized by alkaline-conjugated secondary antibody.  The arrow indicates the heavy chain of the mAb used to immunoprecipitate neurofascin.
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Figure 1: (A–I) Binding of TN-R, axonin-1, and F11 by neurofascin isoform NF22 expressed on the surface of transfected COS7 cells. Double fluorescence images obtained using a confocal microscope are shown. The right half of each micrograph of A–I reveals neurofascin expression (NF expr.) by indirect immunofluorescence in the FITC channel, while the left half depicts the binding of F11- (A, D, and G), of axonin-1–(B, E, and H) or of TN-R– (C, F, and I) conjugated microspheres in the same microscopic field as detected in the Texas Red channel. Each microscopic field also contains unstained cells that do not express neurofascin, and concomitantly do not bind beads. Beads were incubated in the absence (A–C) or presence of Fab fragments of polyclonal antibodies to neurofascin (D–F), to F11 (G), to axonin-1 (H), or to TN-R (I), which is indicated at the left of each panel. (A)Bar, 100 μm. (K–M) F11-coated beads bind to the Ig-like domains of neurofascin. COS7 cells transfected with plasmid NF17 (K) expressing both Ig- and FNIII-like domains of neurofascin, deletion mutant NF21 lacking the Ig-like domains (L), or deletion mutant NF13 lacking the FNIII-like repeats (M) were incubated with F11-coated beads. (K) Bar, 100 μm. NF17 and the deletion constructs NF21 and NF13 are shown schematically at the right of each panel. (N) Neurofascin and F11 or neurofascin and axonin-1 coprecipitate in immunocomplexes. Retinae from embryonic day 11/12 were solubilized in detergent containing buffer, and were subjected to immunoprecipitation using an mAb to neurofascin. Immunocomplexes were separated by SDS-PAGE and transferred to nitrocellulose, followed by staining with antibodies to neurofascin (lane 1), F11 (lane 2), axonin-1 (lane 3), NCAM (lane 4), or NgCAM (lane 5). Binding of antibodies was visualized by alkaline-conjugated secondary antibody. The arrow indicates the heavy chain of the mAb used to immunoprecipitate neurofascin.

Mentions: An interesting feature of several axonal members of the L1- and F11-subgroups of the IgSF is their complex binding pattern with other surface-associated proteins or ECM glycoproteins (Brümmendorf and Rathjen, 1996). As neurofascin-mediated neurite extension and fasciculation might be modulated by distinct molecular interactions, we are interested in defining novel binding partners of this protein. As a first step we therefore analyzed whether neurofascin also binds to the axon-associated IgSF members NCAM, NgCAM, F11, axonin-1, or the ECM glycoproteins TN-C or TN-R. To this end, binding of protein-coated fluorescent microspheres to COS7 cells that express neurofascin on their surface was examined. Since neurofascin is generated in several isoforms by alternative splicing (Hassel et al., 1997) in the initial screen for novel binding partners, a neurofascin isoform (NF22, see Fig. 2 D) was used for transfection that shows the strongest binding activity to NrCAM beads (Volkmer et al., 1996). While incubation of microspheres coated with TN-C, NgCAM or NCAM did not lead to detectable binding to neurofascin-expressing COS7 cells (data not shown), beads conjugated with F11 (Fig. 1, A, D, and G), axonin-1 (Fig. 1, B, E, and H), or TN-R (Fig. 1, C, F, and I) were found to bind to neurofascin-expressing COS7 cells. Binding to untransfected cells within the same culture or to mock-transfected cells was not observed (data not shown). To provide additional data on the specificity, microspheres were incubated with transfected COS7 cells in the presence of Fab fragments of polyclonal antibodies to the proteins under investigation. While anti-neurofascin antibodies blocked binding of F11-, axonin-1–, or TN-R–coated microspheres to neurofascin-expressing COS7 cells (Fig. 1, D–F), Fab fragments to F11, axonin-1, or TN-R specifically blocked the interaction between neurofascin and the respective protein (Fig. 1, G–I). Incubating F11-coated beads with COS7 cells expressing neurofascin deletion mutants reveals that F11 specifically interacts with the Ig-like domains (Fig. 1 M), but not with the FNIII-like repeats of neurofascin (Fig. 1 L). TN-R or axonin-1–coated beads did not bind to either deletion mutant, suggesting that binding of these proteins to neurofascin is more complex, and that both proteins most likely require an intact neurofascin polypeptide to bind (data not shown).


Dissection of complex molecular interactions of neurofascin with axonin-1, F11, and tenascin-R, which promote attachment and neurite formation of tectal cells.

Volkmer H, Zacharias U, Nörenberg U, Rathjen FG - J. Cell Biol. (1998)

(A–I) Binding of TN-R, axonin-1, and F11 by neurofascin isoform NF22 expressed on the surface of transfected COS7  cells. Double fluorescence images obtained using a confocal microscope are shown. The right half of each micrograph of A–I reveals neurofascin expression (NF expr.) by indirect immunofluorescence in the FITC channel, while the left half depicts the  binding of F11- (A, D, and G), of axonin-1–(B, E, and H) or of  TN-R– (C, F, and I) conjugated microspheres in the same microscopic field as detected in the Texas Red channel. Each microscopic field also contains unstained cells that do not express neurofascin, and concomitantly do not bind beads. Beads were  incubated in the absence (A–C) or presence of Fab fragments of  polyclonal antibodies to neurofascin (D–F), to F11 (G), to axonin-1 (H), or to TN-R (I), which is indicated at the left of each  panel. (A)Bar, 100 μm. (K–M) F11-coated beads bind to the Ig-like domains of neurofascin. COS7 cells transfected with plasmid  NF17 (K) expressing both Ig- and FNIII-like domains of neurofascin, deletion mutant NF21 lacking the Ig-like domains (L), or  deletion mutant NF13 lacking the FNIII-like repeats (M) were incubated with F11-coated beads. (K) Bar, 100 μm. NF17 and the  deletion constructs NF21 and NF13 are shown schematically at  the right of each panel. (N) Neurofascin and F11 or neurofascin  and axonin-1 coprecipitate in immunocomplexes. Retinae from  embryonic day 11/12 were solubilized in detergent containing  buffer, and were subjected to immunoprecipitation using an mAb  to neurofascin. Immunocomplexes were separated by SDS-PAGE and transferred to nitrocellulose, followed by staining  with antibodies to neurofascin (lane 1), F11 (lane 2), axonin-1  (lane 3), NCAM (lane 4), or NgCAM (lane 5). Binding of antibodies was visualized by alkaline-conjugated secondary antibody.  The arrow indicates the heavy chain of the mAb used to immunoprecipitate neurofascin.
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Figure 1: (A–I) Binding of TN-R, axonin-1, and F11 by neurofascin isoform NF22 expressed on the surface of transfected COS7 cells. Double fluorescence images obtained using a confocal microscope are shown. The right half of each micrograph of A–I reveals neurofascin expression (NF expr.) by indirect immunofluorescence in the FITC channel, while the left half depicts the binding of F11- (A, D, and G), of axonin-1–(B, E, and H) or of TN-R– (C, F, and I) conjugated microspheres in the same microscopic field as detected in the Texas Red channel. Each microscopic field also contains unstained cells that do not express neurofascin, and concomitantly do not bind beads. Beads were incubated in the absence (A–C) or presence of Fab fragments of polyclonal antibodies to neurofascin (D–F), to F11 (G), to axonin-1 (H), or to TN-R (I), which is indicated at the left of each panel. (A)Bar, 100 μm. (K–M) F11-coated beads bind to the Ig-like domains of neurofascin. COS7 cells transfected with plasmid NF17 (K) expressing both Ig- and FNIII-like domains of neurofascin, deletion mutant NF21 lacking the Ig-like domains (L), or deletion mutant NF13 lacking the FNIII-like repeats (M) were incubated with F11-coated beads. (K) Bar, 100 μm. NF17 and the deletion constructs NF21 and NF13 are shown schematically at the right of each panel. (N) Neurofascin and F11 or neurofascin and axonin-1 coprecipitate in immunocomplexes. Retinae from embryonic day 11/12 were solubilized in detergent containing buffer, and were subjected to immunoprecipitation using an mAb to neurofascin. Immunocomplexes were separated by SDS-PAGE and transferred to nitrocellulose, followed by staining with antibodies to neurofascin (lane 1), F11 (lane 2), axonin-1 (lane 3), NCAM (lane 4), or NgCAM (lane 5). Binding of antibodies was visualized by alkaline-conjugated secondary antibody. The arrow indicates the heavy chain of the mAb used to immunoprecipitate neurofascin.
Mentions: An interesting feature of several axonal members of the L1- and F11-subgroups of the IgSF is their complex binding pattern with other surface-associated proteins or ECM glycoproteins (Brümmendorf and Rathjen, 1996). As neurofascin-mediated neurite extension and fasciculation might be modulated by distinct molecular interactions, we are interested in defining novel binding partners of this protein. As a first step we therefore analyzed whether neurofascin also binds to the axon-associated IgSF members NCAM, NgCAM, F11, axonin-1, or the ECM glycoproteins TN-C or TN-R. To this end, binding of protein-coated fluorescent microspheres to COS7 cells that express neurofascin on their surface was examined. Since neurofascin is generated in several isoforms by alternative splicing (Hassel et al., 1997) in the initial screen for novel binding partners, a neurofascin isoform (NF22, see Fig. 2 D) was used for transfection that shows the strongest binding activity to NrCAM beads (Volkmer et al., 1996). While incubation of microspheres coated with TN-C, NgCAM or NCAM did not lead to detectable binding to neurofascin-expressing COS7 cells (data not shown), beads conjugated with F11 (Fig. 1, A, D, and G), axonin-1 (Fig. 1, B, E, and H), or TN-R (Fig. 1, C, F, and I) were found to bind to neurofascin-expressing COS7 cells. Binding to untransfected cells within the same culture or to mock-transfected cells was not observed (data not shown). To provide additional data on the specificity, microspheres were incubated with transfected COS7 cells in the presence of Fab fragments of polyclonal antibodies to the proteins under investigation. While anti-neurofascin antibodies blocked binding of F11-, axonin-1–, or TN-R–coated microspheres to neurofascin-expressing COS7 cells (Fig. 1, D–F), Fab fragments to F11, axonin-1, or TN-R specifically blocked the interaction between neurofascin and the respective protein (Fig. 1, G–I). Incubating F11-coated beads with COS7 cells expressing neurofascin deletion mutants reveals that F11 specifically interacts with the Ig-like domains (Fig. 1 M), but not with the FNIII-like repeats of neurofascin (Fig. 1 L). TN-R or axonin-1–coated beads did not bind to either deletion mutant, suggesting that binding of these proteins to neurofascin is more complex, and that both proteins most likely require an intact neurofascin polypeptide to bind (data not shown).

Bottom Line: In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11.Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding.In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.

View Article: PubMed Central - PubMed

Affiliation: Max-Delbrück-Centrum für Molekulare Medizin, D-13122 Berlin, Germany.

ABSTRACT
Neurofascin is a member of the L1 subgroup of the Ig superfamily that promotes axon outgrowth by interactions with neuronal NgCAM-related cell adhesion molecule (NrCAM). We used a combination of cellular binding assays and neurite outgrowth experiments to investigate mechanisms that might modulate the interactions of neurofascin. In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11. Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding. While interactions of neurofascin with F11 are only slightly modulated, binding to axonin-1 and TN-R is strongly regulated by alternatively spliced stretches located in the NH2-terminal half, and by the proline-alanine-threonine-rich segment. In vitro neurite outgrowth and cell attachment assays on a neurofascin-Fc substrate reveal a shift of cellular receptor usage from NrCAM to axonin-1, F11, and at least one additional protein in the presence of TN-R, presumably due to competition of the neurofascin- NrCAM interaction. Thereby, F11 binds to TN-R of the neurofascin/TN-R complex, but not to neurofascin, whereas axonin-1 is not able to bind directly to the neurofascin/TN-R complex as shown by competition binding assays. In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.

Show MeSH
Related in: MedlinePlus