A variety of functional and evolutionally diverse proteins are anchored to the cell surface by a C-terminal glycolipid, termed glycosyl- phosphatidylinositol (GPI). Despite the diversity of the protein ectodomains, all GPI anchors contain a conserved core structure: EtN-P- Man3-GicN-Inositol-P. Using the model renal epithelial cell line MDCK, we have demonstrated that GPI-protein anchoring behaves as a apical targeting signal in polarized epithelial cells. This finding represents the first function assigned to the GPI anchor and also the first well- defined post-Golgi targeting signal in epithelial cells.
The aim of this proposal is to characterize the molecular machinery responsible for the recognition of GPI as an apical sorting signal. Using innovative, complementary molecular and cellular approaches, we will identify and clone GPI-adaptor/sorter molecules and other components of the sorting process related to GPI-recognition.
Our specific aims are: 1) As the anchor structure of epithelial anchored protein expressed in parental MDCK cells and in a mutant MDCK cell line which missorts GPI-anchored proteins. 2) Using a thyroid epithelial cell line (FRT) which missorts GPI-anchored proteins, we will employ an expression cloning strategy to identify the gene product that complements the missorting phenotype. 3) Using a purified subcellular fraction we believe contains the GPI-sorting machinery (GPI-rich membrane microdomains), we will generate monoclonal antibodies to dissect its molecular components. 4) We will prepare radio-labeled photo-reactive GPI molecules to identify candidate GPI sorting/recognition molecules by photo-affinity labeling. It is expected that these studies will contribute fundamental knowledge to the understanding of epithelial cell polarity and differentiation.
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