The objective of the proposed research is to undertake a detailed analysis of a long-recognized, but remarkably underinvestigated, class of proteins: the mammalian phosphatidylinositol/phosphatidylcholine transfer proteins (PITPs). These ubiquitous proteins catalyze the transport of either phosphatidylinositol or phosphatidylcholine, as monomers, between membrane bilayers in vitro. Only in the past seven years have any informative clues bee forthcoming as to the physiological function of these proteins, however. The proposed studies represent a comprehensive and multidisciplinary effort designed to identify the function(s) executed by PITPs in mammalian cells, and to determine the relationship between phospholipid binding/transfer and mammalian PITP function. Individual aims will include the use of genetically altered embryonic stem (ES) cells to dissect the role of PITP in reinoic acid-induced differentiation to the neuronal lineage, and the characterization of PITP knockout mice to determine the general physiology that results from PITP insufficiency. These studies will be coupled to the use of novel mutant PITPs (and natural PITP variants) in complementation experiments designed to address which phospholipid binding/transfer property is relevant to any particular physiological function of PITP. In a more directed approach for defining PITP function, the investigators will characterize fully developed mice induced for central nervous system-specific PITP deficiencies. Finally, they will biochemically characterize distinct PITP isoforms with a view toward linking specific biochemical properties with function. The Bankaitis laborator is in a unique position to establish this line of investigation as it has developed facile genetic and biochemical systems for the comprehensive analyse proposed. The available evidence suggests that PITPs play central and previously unrecognized roles in phospholipid-mediated signal transduction processes that interface with such diverse cellular processes as protein secretion, phototransduction, and receptor-mediated signalling. As at least two cases of inherited PITP insufficiency in higher eukaryotes result in neurodegeneration, the proposed studies will provide new and fundamental information that will bear directly on the molecular mechanisms by which PITPs protect the mammalian nervous system from neurodegenerative disease.
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