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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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Neurofascin–TN-R interaction in the presence of domain-specific antibodies to TN-R. (A) Micrographs of neurofascin NF22–expressing COS7 cells incubated with TN-R–coated  beads in the presence of Fab fragment of polyclonal antibodies  (concentration 200 μg/ml) specific for individual TN-R domains  is shown. Only Fab fragments to FNIII-like repeats 2-3 and 4-A  block TN-R bead binding. Neurofascin expression is shown in the  left columns, while TN-R binding is revealed in the right columns. Fab fragments of polyclonal antibodies to invidual domains to TN-R applied are indicated at the left and right of the  panel. (B) Quantification of cell attachment of tectal cells to the  combined neurofascin-Fc/TN-R substrate in the presence of Fab  fragments of polyclonal antibodies to individual domains of TN-R  or in the presence of mAb 23–13 or mAb 23–14 directed to  TN-R. Bars on the columns indicate SEM. (C) Scheme of the  TN-R polypeptide. Alternative spliced segments are indicated in  black. FNIII-like repeats are indicated as rectangles, and are  numbered. EGF-like domains are depicted as small circles, and  the fibrinogen-like segment as large circle. (D) Comparison of  the inhibition of TN-R binding to neurofascin, and of the inhibition of cell attachment of tectal cells to the neurofascin/TN-R  complex.
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Figure 6: Neurofascin–TN-R interaction in the presence of domain-specific antibodies to TN-R. (A) Micrographs of neurofascin NF22–expressing COS7 cells incubated with TN-R–coated beads in the presence of Fab fragment of polyclonal antibodies (concentration 200 μg/ml) specific for individual TN-R domains is shown. Only Fab fragments to FNIII-like repeats 2-3 and 4-A block TN-R bead binding. Neurofascin expression is shown in the left columns, while TN-R binding is revealed in the right columns. Fab fragments of polyclonal antibodies to invidual domains to TN-R applied are indicated at the left and right of the panel. (B) Quantification of cell attachment of tectal cells to the combined neurofascin-Fc/TN-R substrate in the presence of Fab fragments of polyclonal antibodies to individual domains of TN-R or in the presence of mAb 23–13 or mAb 23–14 directed to TN-R. Bars on the columns indicate SEM. (C) Scheme of the TN-R polypeptide. Alternative spliced segments are indicated in black. FNIII-like repeats are indicated as rectangles, and are numbered. EGF-like domains are depicted as small circles, and the fibrinogen-like segment as large circle. (D) Comparison of the inhibition of TN-R binding to neurofascin, and of the inhibition of cell attachment of tectal cells to the neurofascin/TN-R complex.

Mentions: In contrast to axonin-1 or F11, application of soluble TN-R to the immobilized neurofascin-Fc protein resulted in considerable stimulation of neural long-term cell attachment (Fig. 3, A and B), whereas analysis of the length distribution of neurites performed in the presence of soluble TN-R remained unchanged (Fig. 4 E; see below). Increased neural attachment was also observed after preincubating the neurofascin-Fc substrate with TN-R, followed by removal of unbound TN-R by washing before adding tectal cells, whereas immobilized TN-R alone does not allow long-term cell attachment (Fig. 3 A) and neurite extension of tectal cells of embryonic day 6 (Fig. 4 E; Rathjen et al., 1991; Nörenberg et al., 1995). Attachment of tectal cells to the neurofascin-Fc/TN-R complex could be blocked by polyclonal antibodies to TN-R to levels using neurofascin-Fc alone, while antibodies to neurofascin inhibited attachment completely (Fig. 3 A). TN-R did not enhance neural attachment on the Fc portion without neurofascin sequences, collagen, or F11 substrates (Fig. 3 A). Application of soluble TN-C, which does not bind to a neurofascin substrate, did not result in an enhancement of cell attachment (data not shown), emphasizing the specificity of the interaction between neurofascin and TN-R (see Fig. 6 B). These findings indicate that the increase of attached cells was due to the presence of TN-R and to an interaction between soluble TN-R with immobilized neurofascin-Fc.


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)

Neurofascin–TN-R interaction in the presence of domain-specific antibodies to TN-R. (A) Micrographs of neurofascin NF22–expressing COS7 cells incubated with TN-R–coated  beads in the presence of Fab fragment of polyclonal antibodies  (concentration 200 μg/ml) specific for individual TN-R domains  is shown. Only Fab fragments to FNIII-like repeats 2-3 and 4-A  block TN-R bead binding. Neurofascin expression is shown in the  left columns, while TN-R binding is revealed in the right columns. Fab fragments of polyclonal antibodies to invidual domains to TN-R applied are indicated at the left and right of the  panel. (B) Quantification of cell attachment of tectal cells to the  combined neurofascin-Fc/TN-R substrate in the presence of Fab  fragments of polyclonal antibodies to individual domains of TN-R  or in the presence of mAb 23–13 or mAb 23–14 directed to  TN-R. Bars on the columns indicate SEM. (C) Scheme of the  TN-R polypeptide. Alternative spliced segments are indicated in  black. FNIII-like repeats are indicated as rectangles, and are  numbered. EGF-like domains are depicted as small circles, and  the fibrinogen-like segment as large circle. (D) Comparison of  the inhibition of TN-R binding to neurofascin, and of the inhibition of cell attachment of tectal cells to the neurofascin/TN-R  complex.
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Figure 6: Neurofascin–TN-R interaction in the presence of domain-specific antibodies to TN-R. (A) Micrographs of neurofascin NF22–expressing COS7 cells incubated with TN-R–coated beads in the presence of Fab fragment of polyclonal antibodies (concentration 200 μg/ml) specific for individual TN-R domains is shown. Only Fab fragments to FNIII-like repeats 2-3 and 4-A block TN-R bead binding. Neurofascin expression is shown in the left columns, while TN-R binding is revealed in the right columns. Fab fragments of polyclonal antibodies to invidual domains to TN-R applied are indicated at the left and right of the panel. (B) Quantification of cell attachment of tectal cells to the combined neurofascin-Fc/TN-R substrate in the presence of Fab fragments of polyclonal antibodies to individual domains of TN-R or in the presence of mAb 23–13 or mAb 23–14 directed to TN-R. Bars on the columns indicate SEM. (C) Scheme of the TN-R polypeptide. Alternative spliced segments are indicated in black. FNIII-like repeats are indicated as rectangles, and are numbered. EGF-like domains are depicted as small circles, and the fibrinogen-like segment as large circle. (D) Comparison of the inhibition of TN-R binding to neurofascin, and of the inhibition of cell attachment of tectal cells to the neurofascin/TN-R complex.
Mentions: In contrast to axonin-1 or F11, application of soluble TN-R to the immobilized neurofascin-Fc protein resulted in considerable stimulation of neural long-term cell attachment (Fig. 3, A and B), whereas analysis of the length distribution of neurites performed in the presence of soluble TN-R remained unchanged (Fig. 4 E; see below). Increased neural attachment was also observed after preincubating the neurofascin-Fc substrate with TN-R, followed by removal of unbound TN-R by washing before adding tectal cells, whereas immobilized TN-R alone does not allow long-term cell attachment (Fig. 3 A) and neurite extension of tectal cells of embryonic day 6 (Fig. 4 E; Rathjen et al., 1991; Nörenberg et al., 1995). Attachment of tectal cells to the neurofascin-Fc/TN-R complex could be blocked by polyclonal antibodies to TN-R to levels using neurofascin-Fc alone, while antibodies to neurofascin inhibited attachment completely (Fig. 3 A). TN-R did not enhance neural attachment on the Fc portion without neurofascin sequences, collagen, or F11 substrates (Fig. 3 A). Application of soluble TN-C, which does not bind to a neurofascin substrate, did not result in an enhancement of cell attachment (data not shown), emphasizing the specificity of the interaction between neurofascin and TN-R (see Fig. 6 B). These findings indicate that the increase of attached cells was due to the presence of TN-R and to an interaction between soluble TN-R with immobilized neurofascin-Fc.

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