Limits...
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.

Show MeSH

Related in: MedlinePlus

Binding of F11-, axonin-1–, or TN-R–conjugated microspheres to different isoforms of neurofascin. After transfection of neurofascin plasmids into COS7 cells and incubation with  fluorescent beads coated with F11 (A), TN-R (B), or axonin-1  (C), the binding of beads on individual cells was quantified and  normalized with respect to the expression level of neurofascin as  described in Materials and Methods. Bars indicate SEM. (D)  Overview of neurofascin isoforms encoded by individual plasmids to transfect COS7 cells. Alternatively expressed segments  are hatched. Ig-like domains are indicated by loops, while FNIII-like repeats are shown as rectangles. All identified neurofascin  sequences (Hassel et al., 1997) are summarized at the left as NF  and isoforms tested in the assay are indicated by NF and a number. The left schematic version containing all spliced segments  has not been detected in brain tissue (Hassel et al., 1997).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132869&req=5

Figure 2: Binding of F11-, axonin-1–, or TN-R–conjugated microspheres to different isoforms of neurofascin. After transfection of neurofascin plasmids into COS7 cells and incubation with fluorescent beads coated with F11 (A), TN-R (B), or axonin-1 (C), the binding of beads on individual cells was quantified and normalized with respect to the expression level of neurofascin as described in Materials and Methods. Bars indicate SEM. (D) Overview of neurofascin isoforms encoded by individual plasmids to transfect COS7 cells. Alternatively expressed segments are hatched. Ig-like domains are indicated by loops, while FNIII-like repeats are shown as rectangles. All identified neurofascin sequences (Hassel et al., 1997) are summarized at the left as NF and isoforms tested in the assay are indicated by NF and a number. The left schematic version containing all spliced segments has not been detected in brain tissue (Hassel et al., 1997).

Mentions: Within the L1 subgroup, neurofascin is unique as it contains a proline-alanine-threonine-rich segment (termed PAT domain) between the fourth and fifth FNIII-like repeats that may be the target of O-glycosylation (Volkmer et al., 1992). cDNA cloning and genomic analysis of neurofascin indicated that at least 50 different isoforms are generated by alternate usage of nine alternatively spliced exons spread over the complete gene (Hassel et al., 1997). There are three alternatively spliced sequences comprising complete domains, the third as well as the fifth FNIII-like repeat and the PAT domain, and four short stretches of 4–15 amino acid residues in length (see Fig. 2 D). They are located at the NH2 terminus between the second and third Ig-like domain at the junction between the Ig- and the FNIII-like domains and in the cytoplasmic segment. Isoforms that contain either the third or the fifth FNIII-like repeat are selectively localized either on unmyelinated axons or on the nodes of Ranvier in the peripheral nervous system, respectively (Davis et al., 1996).


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)

Binding of F11-, axonin-1–, or TN-R–conjugated microspheres to different isoforms of neurofascin. After transfection of neurofascin plasmids into COS7 cells and incubation with  fluorescent beads coated with F11 (A), TN-R (B), or axonin-1  (C), the binding of beads on individual cells was quantified and  normalized with respect to the expression level of neurofascin as  described in Materials and Methods. Bars indicate SEM. (D)  Overview of neurofascin isoforms encoded by individual plasmids to transfect COS7 cells. Alternatively expressed segments  are hatched. Ig-like domains are indicated by loops, while FNIII-like repeats are shown as rectangles. All identified neurofascin  sequences (Hassel et al., 1997) are summarized at the left as NF  and isoforms tested in the assay are indicated by NF and a number. The left schematic version containing all spliced segments  has not been detected in brain tissue (Hassel et al., 1997).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132869&req=5

Figure 2: Binding of F11-, axonin-1–, or TN-R–conjugated microspheres to different isoforms of neurofascin. After transfection of neurofascin plasmids into COS7 cells and incubation with fluorescent beads coated with F11 (A), TN-R (B), or axonin-1 (C), the binding of beads on individual cells was quantified and normalized with respect to the expression level of neurofascin as described in Materials and Methods. Bars indicate SEM. (D) Overview of neurofascin isoforms encoded by individual plasmids to transfect COS7 cells. Alternatively expressed segments are hatched. Ig-like domains are indicated by loops, while FNIII-like repeats are shown as rectangles. All identified neurofascin sequences (Hassel et al., 1997) are summarized at the left as NF and isoforms tested in the assay are indicated by NF and a number. The left schematic version containing all spliced segments has not been detected in brain tissue (Hassel et al., 1997).
Mentions: Within the L1 subgroup, neurofascin is unique as it contains a proline-alanine-threonine-rich segment (termed PAT domain) between the fourth and fifth FNIII-like repeats that may be the target of O-glycosylation (Volkmer et al., 1992). cDNA cloning and genomic analysis of neurofascin indicated that at least 50 different isoforms are generated by alternate usage of nine alternatively spliced exons spread over the complete gene (Hassel et al., 1997). There are three alternatively spliced sequences comprising complete domains, the third as well as the fifth FNIII-like repeat and the PAT domain, and four short stretches of 4–15 amino acid residues in length (see Fig. 2 D). They are located at the NH2 terminus between the second and third Ig-like domain at the junction between the Ig- and the FNIII-like domains and in the cytoplasmic segment. Isoforms that contain either the third or the fifth FNIII-like repeat are selectively localized either on unmyelinated axons or on the nodes of Ranvier in the peripheral nervous system, respectively (Davis et al., 1996).

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