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Neurite fasciculation mediated by complexes of axonin-1 and Ng cell adhesion molecule.

Kunz S, Spirig M, Ginsburg C, Buchstaller A, Berger P, Lanz R, Rader C, Vogt L, Kunz B, Sonderegger P - J. Cell Biol. (1998)

Bottom Line: In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding.These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM.The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.

ABSTRACT
Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

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Binding of NgCAM-microspheres to wild-type NgCAM, the domain deletion mutants, and the NgCAM variants  deficient in proteolytic processing. Binding of NgCAM-conjugated TRITC Covaspheres to COS cells expressing wild-type NgCAM and the domain deletion mutants (A) and the mutants NgCAM PM1-3 and NgCAMΔP871-V888 (B). NgCAM expressing  cells were identified as described in Fig. 2. For each mutant 100  green fluorescing cells were analyzed. Green fluorescing cells  that bound four or more red fluorescing Covaspheres were  scored as positive cells. Cells that did not express NgCAM were  used as internal negative controls and were found to bind on the  average 0.1 NgCAM-conjugated Covaspheres per cell. Each column corresponds to three independent experiments (± SD).
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Figure 6: Binding of NgCAM-microspheres to wild-type NgCAM, the domain deletion mutants, and the NgCAM variants deficient in proteolytic processing. Binding of NgCAM-conjugated TRITC Covaspheres to COS cells expressing wild-type NgCAM and the domain deletion mutants (A) and the mutants NgCAM PM1-3 and NgCAMΔP871-V888 (B). NgCAM expressing cells were identified as described in Fig. 2. For each mutant 100 green fluorescing cells were analyzed. Green fluorescing cells that bound four or more red fluorescing Covaspheres were scored as positive cells. Cells that did not express NgCAM were used as internal negative controls and were found to bind on the average 0.1 NgCAM-conjugated Covaspheres per cell. Each column corresponds to three independent experiments (± SD).

Mentions: For the identification of the NgCAM domains involved in homophilic binding we investigated the binding of NgCAM-conjugated microspheres to both series of domain deletion mutants of NgCAM (Fig. 6). The data derived from both sets of NgCAM deletion mutants show clearly that deletions of the four NH2-terminal Ig domains of NgCAM resulted in a strong reduction of the homophilic binding. In addition, a significant reduction of the homophilic binding was observed with ΔFn2. In contrast, no reduction of binding was found with the mutant lacking the domain tandem Fn1+Fn2, ΔFn12. This result resembles the situation regarding the binding of axonin-1 described above. The reduced homophilic binding of ΔFn2 could reflect an impaired folding of the NgCAM mutant due to the deletion of Fn2 rather than an involvement of Fn2 in the homophilic interaction.


Neurite fasciculation mediated by complexes of axonin-1 and Ng cell adhesion molecule.

Kunz S, Spirig M, Ginsburg C, Buchstaller A, Berger P, Lanz R, Rader C, Vogt L, Kunz B, Sonderegger P - J. Cell Biol. (1998)

Binding of NgCAM-microspheres to wild-type NgCAM, the domain deletion mutants, and the NgCAM variants  deficient in proteolytic processing. Binding of NgCAM-conjugated TRITC Covaspheres to COS cells expressing wild-type NgCAM and the domain deletion mutants (A) and the mutants NgCAM PM1-3 and NgCAMΔP871-V888 (B). NgCAM expressing  cells were identified as described in Fig. 2. For each mutant 100  green fluorescing cells were analyzed. Green fluorescing cells  that bound four or more red fluorescing Covaspheres were  scored as positive cells. Cells that did not express NgCAM were  used as internal negative controls and were found to bind on the  average 0.1 NgCAM-conjugated Covaspheres per cell. Each column corresponds to three independent experiments (± SD).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132982&req=5

Figure 6: Binding of NgCAM-microspheres to wild-type NgCAM, the domain deletion mutants, and the NgCAM variants deficient in proteolytic processing. Binding of NgCAM-conjugated TRITC Covaspheres to COS cells expressing wild-type NgCAM and the domain deletion mutants (A) and the mutants NgCAM PM1-3 and NgCAMΔP871-V888 (B). NgCAM expressing cells were identified as described in Fig. 2. For each mutant 100 green fluorescing cells were analyzed. Green fluorescing cells that bound four or more red fluorescing Covaspheres were scored as positive cells. Cells that did not express NgCAM were used as internal negative controls and were found to bind on the average 0.1 NgCAM-conjugated Covaspheres per cell. Each column corresponds to three independent experiments (± SD).
Mentions: For the identification of the NgCAM domains involved in homophilic binding we investigated the binding of NgCAM-conjugated microspheres to both series of domain deletion mutants of NgCAM (Fig. 6). The data derived from both sets of NgCAM deletion mutants show clearly that deletions of the four NH2-terminal Ig domains of NgCAM resulted in a strong reduction of the homophilic binding. In addition, a significant reduction of the homophilic binding was observed with ΔFn2. In contrast, no reduction of binding was found with the mutant lacking the domain tandem Fn1+Fn2, ΔFn12. This result resembles the situation regarding the binding of axonin-1 described above. The reduced homophilic binding of ΔFn2 could reflect an impaired folding of the NgCAM mutant due to the deletion of Fn2 rather than an involvement of Fn2 in the homophilic interaction.

Bottom Line: In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding.These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM.The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.

ABSTRACT
Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

Show MeSH
Related in: MedlinePlus