Our objectives are to identify, by a combination of direct force measurements and the genetic manipulation of protein structure, the fundamental physical mechanisms of homophilic adhesion between neural cell adhesion molecules (NCAM) and the regulation of NCAM adhesion by post-translational modification. NCAM is a large, multidomain protein that mediates cell-cell interactions through homophilic binding. It is one of the most abundant and widespread cell adhesion proteins of the immunoglobulin superfamily, and the molecule's adhesive function has been shown to influence a number of important developmental processes. The protein is unusual in that it exhibits two structural forms: one of which exhibits adhesive activity and the other which displays anti-adhesive function. Although extensive research addressed the biological role of these functions, neither of the underlying mechanisms has been defined in molecular detail. The anti-adhesive property is linked to the protein glycosylation, which produces polysialylic acid (PSA). This modification affects not only NCAM adhesion but also cell interactions mediated by other adhesion molecules. These proposed studies will quantify the distance dependence of the forces between membranes displaying the different forms of NCAM, in order to determine the molecular mechanism by which polysialic acid alters both interactions between NCAM as well as the interactions of two other adhesion proteins N-cadherin and Li. The measurements will directly test the hypothesis that PSA sterically impedes the close apposition of cells and thereby exerts a general regulatory influence on cell interactions. With regard to the adhesive function, additional force/distance measurements with both NCAM and NCAM fragments lacking specific domains will further test several current hypotheses regarding the specific domain interactions that mediate homophilic binding. These proposed force-distance measurements will generate unique, molecular level information concerning the mechanisms of NCAM adhesion that cannot be determined from studies with whole cells, solution binding measurements, or crystal structures.
