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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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Soluble TN-R and TN-C differentially compete for  ligand binding to neurofascin. Microspheres coated with NrCAM  (A and B), axonin-1 (C), or F11 (D and E; indicated on the top of  the panel) are incubated with COS7 cells expressing neurofascin  variants NF17 or NF22 (see bottom row). Competitor indicates  TN-R or TN-C in solution at a concentraion of 150 μg/ml. The interpretation of the results is illustrated schematically in the lower  half of the figure. Circles indicate fluorescent beads conjugated  with NrCAM, axonin-1, or F11. NF22 is shown as a filled and  NF17 as a hatched ellipse. The indication that F11 binds to TN-R  while NrCAM and axonin-1 do not is based on previous studies  (Nörenberg et al., 1995). Bars on the columns indicate SEM.
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Figure 5: Soluble TN-R and TN-C differentially compete for ligand binding to neurofascin. Microspheres coated with NrCAM (A and B), axonin-1 (C), or F11 (D and E; indicated on the top of the panel) are incubated with COS7 cells expressing neurofascin variants NF17 or NF22 (see bottom row). Competitor indicates TN-R or TN-C in solution at a concentraion of 150 μg/ml. The interpretation of the results is illustrated schematically in the lower half of the figure. Circles indicate fluorescent beads conjugated with NrCAM, axonin-1, or F11. NF22 is shown as a filled and NF17 as a hatched ellipse. The indication that F11 binds to TN-R while NrCAM and axonin-1 do not is based on previous studies (Nörenberg et al., 1995). Bars on the columns indicate SEM.

Mentions: One explanation for the finding that NrCAM is replaced as receptor for cell adhesion and neurite extension on the neurofascin-Fc/TN-R complex might be that TN-R sterically blocks the NrCAM binding region within neurofascin, but still allows binding of axonin-1 and F11 expressed by tectal cells to neurofascin or to the neurofascin/TN-R complex. To mediate the attachment of tectal cells, axonin-1 may directly interact with neurofascin and not with TN-R of the complex since no direct binding between axonin-1 and TN-R has been detected so far in different binding assays (Nörenberg et al., 1995 and data not shown). F11 may either bind to TN-R as shown in a previous communication (Brümmendorf et al., 1993; Rathjen et al., 1991; Nörenberg et al., 1995) or to neurofascin as shown above. To address these questions we made use of the observation that some neurofascin isoforms bind TN-R, and others do not. COS7 cells were transfected with the TN-R–binding isoform NF22 and the nonbinding isoform NF17 (see Fig. 2 B), followed by an incubation with F11-, axonin-1-, or NrCAM-coated microspheres in the presence or absence of soluble TN-R or of soluble TN-C as a control (Fig. 5).


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)

Soluble TN-R and TN-C differentially compete for  ligand binding to neurofascin. Microspheres coated with NrCAM  (A and B), axonin-1 (C), or F11 (D and E; indicated on the top of  the panel) are incubated with COS7 cells expressing neurofascin  variants NF17 or NF22 (see bottom row). Competitor indicates  TN-R or TN-C in solution at a concentraion of 150 μg/ml. The interpretation of the results is illustrated schematically in the lower  half of the figure. Circles indicate fluorescent beads conjugated  with NrCAM, axonin-1, or F11. NF22 is shown as a filled and  NF17 as a hatched ellipse. The indication that F11 binds to TN-R  while NrCAM and axonin-1 do not is based on previous studies  (Nörenberg et al., 1995). Bars on the columns indicate SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132869&req=5

Figure 5: Soluble TN-R and TN-C differentially compete for ligand binding to neurofascin. Microspheres coated with NrCAM (A and B), axonin-1 (C), or F11 (D and E; indicated on the top of the panel) are incubated with COS7 cells expressing neurofascin variants NF17 or NF22 (see bottom row). Competitor indicates TN-R or TN-C in solution at a concentraion of 150 μg/ml. The interpretation of the results is illustrated schematically in the lower half of the figure. Circles indicate fluorescent beads conjugated with NrCAM, axonin-1, or F11. NF22 is shown as a filled and NF17 as a hatched ellipse. The indication that F11 binds to TN-R while NrCAM and axonin-1 do not is based on previous studies (Nörenberg et al., 1995). Bars on the columns indicate SEM.
Mentions: One explanation for the finding that NrCAM is replaced as receptor for cell adhesion and neurite extension on the neurofascin-Fc/TN-R complex might be that TN-R sterically blocks the NrCAM binding region within neurofascin, but still allows binding of axonin-1 and F11 expressed by tectal cells to neurofascin or to the neurofascin/TN-R complex. To mediate the attachment of tectal cells, axonin-1 may directly interact with neurofascin and not with TN-R of the complex since no direct binding between axonin-1 and TN-R has been detected so far in different binding assays (Nörenberg et al., 1995 and data not shown). F11 may either bind to TN-R as shown in a previous communication (Brümmendorf et al., 1993; Rathjen et al., 1991; Nörenberg et al., 1995) or to neurofascin as shown above. To address these questions we made use of the observation that some neurofascin isoforms bind TN-R, and others do not. COS7 cells were transfected with the TN-R–binding isoform NF22 and the nonbinding isoform NF17 (see Fig. 2 B), followed by an incubation with F11-, axonin-1-, or NrCAM-coated microspheres in the presence or absence of soluble TN-R or of soluble TN-C as a control (Fig. 5).

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