Membrane boundaries define distinct structural and functional compartments between the eukaryotic cell and its environment, and within the cell itself. Movement of material and information between these compartments is mediated by vesicular transport and protein coats, of which the clathrin coated membrane is a paradigm, are invariably associated with these processes. Recently, phosphoinositides have also been recognized to play important roles in the regulation of membrane trafficking. Important questions have been the mechanism by which inositides are locally accumulated or generated at sites of action, and how they interact with effector proteins. During the last award period, we found that clathrin binds specifically to a novel Class II PI 3-kinase, PI3K-C2a, and that this binding enhances the enzymatic activity of the protein. This leads to the hypothesis that clathrin recruitment and activation of PI3K-C2alpha induces local generation of inositides. We also showed that 3-phosphoinositides bind specifically to a discrete site on the AP-2cx subunit, and that the integrity of this site is required both for proper recruitment of AP-2 to coated pits and for receptor-mediated endocytosis. A major goal will be to determine the membrane trafficking pathways in which PI3K-C2a participates and how it interacts with clathrin to accomplish these functions. This will be attained by determining the ultrastructural localization of PI3K-C2a in cells, examining the possibility that it is preferentially associated with specific stages of CP formation, and dissecting the role in intact cell membrane trafficking pathways of the lipid kinase and clathrin binding activities of PI3K-C2a. Study of determinants mediating the clathrin-PI3K-C2alpha interaction will be carried out to provide dominant-negative reagents for these studies, and to provide material for crystallization and structural analysis. Finally, the inositide binding properties of the AP-2alpha binding domain will be examined and structural studies initiated. In addressing the mode of regulation of coat function and vesicular transport in intact cells, this project focus upon a central issue in cell biology with relevance for normal biological processes in all eukaryotic cells and derangement in many diseases.
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