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 bet ween neurons homophilically and between neurons and glia heterophilica lly; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM(200)) with posttr anslational cleavage products of 135 kD (F-135, which contains most of the extracellular region) and 80 kD (F-80, which includes the transme mbrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM(200), F-135, and F-80 in murine L cells, and F-135 and F-80 as GST fusion proteins in the pGEX vector in bacteria, Appropriately transfected L cells expressed each of these proteins on their surfaces; F-135 was also found in the media of cells transfected with Ng-CAM(200) and F-135. In addition to bindi ng homophilically, cells transfected with Ng-CAM(200) and F-135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial liga nd. Detailed studies using the transfected cells and the fusion protei ns indicated that both the hemophilic and the heterophilic binding act ivities of Ng-CAM are localized in the F-135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng-CAM(200) i s not required either for its expression on the cell surface or for ce ll adhesion and that there is an ''anchor'' for F-135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F -80 but not the F-135 fusion protein enhanced the outgrowth of neurite s from dorsal root ganglion cells; this activity was associated with t he Fn(III) repeats of F-80. The observations that a protein correspond ing to F-135 contains the cell aggregation sites whereas one correspon ding to the F-80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating thes e Ng-CAM activities during embryonic development and neural regenerati on.