CDB15:0000311 CNTN2 — NRCAM

ABSTRACT: Axonin-1 is an axon-associated cell adhesion molecule with dualistic expression, one form being glycophosphatidylinositol-anchored to the axonal membrane, the other secreted from axons in a soluble form. When presented as a substratum for neuronal cultures it strongly promotes neurite outgrowth from chicken embryonic dorsal root ganglia neurons. In this study, the axon-associated cell adhesion molecule G4, which is identical with Ng-CAM and 8D9, and homologous or closely related to L1 of the mouse and NILE of the rat, was investigated with respect to a receptor function for axonin-1. Using fluorescent microspheres with covalently coupled axonin-1 or L1(G4) at their surface we showed that these proteins bind to each other. Within the sensitivity of this microsphere assay, no interaction of axonin-1 with itself could be detected. Axonin-1-coated microspheres also bound to the neurites of cultured dorsal root ganglia neurons. This interaction was exclusively mediated by L1(G4), as indicated by complete binding suppression by monovalent anti-L1(G4) antibodies. The interaction between neuritic L1(G4) and immobilized axonin-1 was found to mediate the promotion of neurite growth on axonin-1, as evidenced by the virtually complete arrest of neurite outgrowth in the presence of anti-L1(G4) antibodies. Convincing evidence has recently been presented that neurite growth on L1(8D9) is mediated by the homophilic binding of neuritic L1(G4) (1989. Neuron. 2: 1597-1603). Thus, both L1(G4)- and axonin-1-expressing axons may serve as "substrate pathways" for the guidance of following axons expressing L1(G4) into their target area. Conceivably, differences in the concentration of axonin-1 and L1(G4), and/or modulatory influences on their specific binding parameters in leading pathways and following axons could represent elements in the control of axonal pathway selection.

ABSTRACT: The neuronal cell adhesion molecule axonin-1 is composed of six immunoglobulin and four fibronectin type III domains. Axonin-1 promotes neurite outgrowth, when presented as a substratum for neurons in vitro, via a neuronal receptor that has been identified as the neuron-glia cell adhesion molecule, NgCAM, based on the blocking effect of polyclonal antibodies directed to NgCAM. Here we report the identification of axonin-1 domains involved in NgCAM binding. NgCAM-conjugated microspheres were tested for binding to COS cells expressing domain deletion mutants of axonin-1. In addition, monoclonal antibodies directed to axonin-1 were assessed for their ability to block the axonin-1-NgCAM interaction, and their epitopes were mapped using the domain deletion mutants. The results suggest that the four amino-terminal immunoglobulin domains of axonin-1 form a domain conglomerate which is necessary and sufficient for NgCAM binding. Surprisingly, NgCAM binding to membrane-bound axonin-1 was increased strongly by deletion of the fifth or sixth immunoglobulin domains of axonin-1. Based on these results and on negative staining electron microscopy, we propose a horseshoe-shaped domain arrangement of axonin-1 that obscures the NgCAM binding site. Neurite outgrowth studies with truncated forms of axonin-1 show that axonin-1 is a neurite outgrowth-promoting substratum in the absence of the NgCAM binding site.