Functional analysis of posttranslational cleavage products of the neuron-glia cell adhesion molecule, Ng-CAM

Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM₂₀₀) with posttranslational cleavage products of 135 kD (F₁₃₅, which contains most of the extracellular region) and 80 kD (F₈₀, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM₂₀₀, F₁₃₅, and F₈₀ in murine L cells, and F₁₃₅ and F₈₀ as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F₁₃₅ was also found in the media of cells transfected with Ng-CAM₂₀₀ and F₁₃₅. In addition to binding homophilically, cells transfected with Ng-CAM₂₀₀ and F₁₃₅ bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F₁₃₅ fragment of the molecule. The results also indicated that proteolytic cleavage of Ng-CAM₂₀₀ is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F₁₃₅ on L cells (and presumably on neurons). In contrast to the cell binding results, the F₈₀ but not the F₁₃₅ fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the Fn_III repeats of F₈₀. The observations that a protein corresponding to F₁₃₅ contains the cell aggregation sites whereas one corresponding to the F₈₀ has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.