The goal of this proposal is to identify portions of the molecule. In the CNS, NILE is a neuron-specific adhesion molecule that appears to be important for the ability of developing axons to fasciculate into fiber tracts. In the PNS, NILE is also found on Schwann cells and may be important in interactions that lead to myelination. In humans, defects in NILE are thought to be associated with the onset of X-linked hydrocephalus. NILE is a transmembrane protein belonging to the immunoglobulin superfamily of neural cell adhesion molecules. Its ectodomain contains six C2 immunoglobulin-like loops and five fibronectin type III repeats, while the cytoplasmic domain contains consensus sites for phosphorylation and motifs for possible interaction with cytoskeletal elements. The planned research will attempt to identify sequences in these various domains that are involved in binding to specific extracellular and intracellular ligands. The experimental approach will involve transfection of NILE negative cells with normal NILE cDNA and with NILE cDNA constructs containing specific deletions. Transfected cells expressing mutant forms of NILE will be compared to wild-type NILE transfectants in several functional assays. (1) The loss of ability to promote neurite outgrowth from cerebellar neurons will be used to identify NILE deletion mutants lacking domains that are critical for interaction with developing axons (neurites). (2) Ectodomain deletion mutants found to be defective in promoting neurite outgrowth will be further analyzed to determine if the missing domains participate in homophilic NILE-NILE interactions or in heterophilic interactions with other ligands. (3) For domains that participate in heterophilic interactions, attempts will be made to identify the heterophilic ligands. (4) Cytoplasmic functions of NILE will be investigated using cytoplasmic domain deletion mutants. Emphasis will be place on identification of sequences that interact with the cytoskeleton to stabilize the pattern of NILE expression on the cell surface and sequences that are involved in signal transduction from the ectodomain to suspected second messenger systems in the cytoplasm.
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