For the past 14 years this grant has been directed at characterizing a variety of cell adhesion molecules and their genes. The proposed studies refocus on the neural cell adhesion molecule, N-CAM, which was the first cell adhesion molecule to be characterized in detail. The protein contains five immunoglobulin-like domains (Ig I-V) and two fibronectin type III repeats (FnIII 1-2) and is expressed in a variety of alternatively spliced forms. It has been studied extensively in a variety of species and biological systems and is the exemplar of a large and continually growing family of cell adhesion molecules. Despite the extensive characterization of N-CAM and its gene, a number of significant features of the molecule remain to be understood. These include the detailed structures of its domains, their role in N-CAM-mediated binding and in neurite outgrowth, and the ability of N-CAM to interact with other molecules, particularly in the cytoplasm. The overall goal of these studies is to use recombinant proteins corresponding to each of the Ig domains and the FnIII repeats and a combination of chemical, NMR, and molecular biological techniques to analyze these features of N-CAM in detail.
Our specific aims are: 1) to determine in collaborative NMR studies the three-dimensional structures of recombinant proteins corresponding to the individual Ig domains as a basis for understanding N-CAM structure and binding; 2) to use binding assays, chemical cross-linking and neurite outgrowth assays to identify activities of each domain and the interactions of the specific domains with each other; and 3) to use the yeast two-hybrid system and chemical techniques to identify molecules with which N-CAM interacts in the cell cytoplasm. Given its diverse influences on neural development and its potential role in neural regeneration, describing the mechanism of N-CAM binding would significantly improve our understanding of how N-CAM influences these processes. Identifying cytoplasmic components that interact with N-CAM would help identify the signals generated by N-CAM-binding that affect cellular functions and gene expression. Together, the results should allow the design of specific reagents to perturb N-CAM binding and signaling; such reagents would be valuable for research purposes, and may also aid in the design of new diagnostic or therapeutic agents.
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