Phosphatidylinositol transfer protein (PITP) catalyzes an in vitro transfer of phosphatidylinositol and phosphatidylcholine between membranes and plays a significant role in several cellular processes including signal transduction pathways that proceed through G protein-coupled and growth factor receptors and formation of secretory vesicles and their fusion with the plasma membrane. A common threat in these processes is the participation of PITP in synthesis of critical lipids such as phosphatidylinositol 4,5- bisphosphate and phosphatidylinositol 3,4,5-triphosphate, possibly by substrate presentation to phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase. Multiple, highly conserved isoforms of PITP have been identified in mammals and other species, including PITPalpha and PITPbeta, both of which are cytosolic proteins, the N-terminal domain (rdgB-PITP) of the retinal degeneration )rdgB) gene product, a transmembrane protein localized to the retina and central nervous system, and the functionally related (though not structurally homologous( sec14 gene product in yeasts. Cytosolic holo PITPs contain one molecule of non- covalently bound phospholipid that becomes a substrates for intermembrane transfer. Hence, within the protein architecture there must be domains for phospholipid monomer binding and for interactions with other macrormolecular organizations, such as membranes or signal transduction complexes.
The specific aims of the experiments we propose are: 1) express, purify, and characterize mouse rdB-PITP with respect to lipid transfer properties and membrane interactions; 2) determine the three-dimensional crystal structures of PITPs and correlate their structures with biological activities; 3) describe the effects of PITPs on the catalytic activities of purified phospholipid kinases; and 4) define the complexes, affinities, and amino acids involved in the interactions between membrane surfaces and PTPs. These experiments will provide critical new information on the structure, function, and catalytic mechanism of an increasingly important class of cellular proteins. Moreover, they may yield valuable insight into the underlying causes of some forms of retinal degeneration and other visual abnormalities.
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