The N-CAMs (or neural cell adhesion molecules) are a group of structurally similar but not identical neural cell surface peptides. They have been proposed to mediate a number of developmental processes by increasing cell-cell adhesion. The hypothesis of this proposal is that N-CAM is one member of a small family of neural cell surface proteins which all mediate adhesion and which are differentially distributed among neural cells. In previous work we have demonstrated that clonal rat neural cell types express immunologically distinct N-CAM forms. Further, two categories of rat cDNAs have been isolated. Rat cDNA clone 18 is strongly related or identical to rat N-CAM as it is homologous throughout its length to the small mouse N-CAM related cDNA pM1.3 and also to a chicken N-CAM related cDNA. Rat cDNA clone 13.2 has a short region of perfect homology to pM1.3 immediately adjacent to a divergent coding sequence and thus appears to be more distantly related to N-CAM. Clones 18 and 13.2 appear to represent independent rat genes with homologs in other mammalian species. RNA hybridizing to these cDNAs is present in neural but not non-neural cells. We propose to determine the complete sequence of these rat cDNAs in order to more firmly establish their relationship to N-CAM and to analyze the predicted properties of the polypeptides they encode. Segments of the cDNAs will be used to produce polypeptides in bacterial expression vectors. Antibodies produced to these polypeptides will be used to further determine their relationship to N-CAM and their distribution among neural cell types. At the same time previous protocols will be adapted to isolate full length cDNA clones for rat N-CAM and the related clone 13.2 encoded protein. These cDNA clones will be transfected into mouse L cells to determine if the N-CAM amino acid sequence alone is sufficient to mediate cell-cell adhesion. Neural cells which phenotypically lack N-CAM will be isolated by immunoselection. The N-CAM related cDNAs will be transfected into these cells as well in order to determine if neural specific carbohydrate or glycosaminoglycan groups are essential for, or aid in, N-CAM mediated adhesion. In an independent approach to the role of carbohydrate groups in N-CAM mediated adhesion, N-CAM lacking individual glycosylation sites or the heparin binding domain will be produced by in vitro mutagenesis of the cDNA. These will be transfected into L cells and their adhesion tested.
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