Cell adhesion molecules (CAMs) belonging to the immunoglobulin (Ig) superfamily play critical roles in development and in adult tissues. Despite extensive studies, the mechanisms by which they mediate homophilic binding (a CAM on one cell binds the same CAM on another) and how binding is linked to the ability of CAMs to generate intracellular signals are unknown. Our studies are designed to make this connection by examining the mechanisms involved in cell-cell binding and proximal signaling events by the neural CAM (N-CAM), the first Ig CAM to be characterized.
Our first aim i s to characterize the interactions between the extracellular five Ig domains and two fibronectin-like repeats of N-CAM. Recombinant proteins and soluble fragments of native N-CAM will be used to identify specific domain-domain interactions using physico-chemical methods, NMR spectroscopy, and X-ray crystallography. Chemical cross-linking of these proteins in solution and native N-CAM in lipid vesicles and on cells will also be used. To evaluate the models derived from these studies, site-directed mutagenesis will be used to create N-CAM variants which will be tested for their ability to mediate homophilic adhesion of transfected cells.
Our second aim will examine the mechanism by which N-CAM binding activates the signal cascade involving the nuclear factor kB (NF-KB) that leads to changes in gene expression. Fluorescence resonance energy transfer (FRET) will be used to look for interactions among the extracellular and cytoplasmic domains of N-CAM molecules on the same cell. In addition, cellular transfection of a dominant-negative cytoplasmic construct will be used in conjunction with site-directed mutagenesis to define critical regions in the N-CAM cytoplasmic domain involved in NF-kB signaling. Affinity chromatography and the yeast two-hybrid system will be used to identify molecules in the cytoplasm that interact with the N-CAM cytoplasmic region and may initiate the signal cascade. The ability of N-CAM to inhibit astrocyte proliferation, to activate NF-kB in neurons and to stimulate neurite outgrowth will be examined in terms of the mechanism defined for its activation of NF-KB in astrocytes. These studies should provide new insight into the mechanisms by which CAMs influence cells at a variety of levels and provide the basis for diagnostic and therapeutic agents for the identification and treatment of CAM-related diseases.
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