This proposal represents an effort to identify proteins or factors, which may be linked with the HDL receptor (scavenger receptor subtype BI, SR-BI) to facilitate the uptake of lipoprotein-donated cholesteryl esters through the unique selective cholesterol uptake pathway. This is a pathway in which circulating (i.e., exogenous) lipoproteins are able to contribute bulk cholesterol to many types of cells for synthetic purposes. Recent studies indicate that cystolic extracts from selective pathway-competent rodent steriodogenic tissues (such as luteinized ovary) enhance selective uptake in purified SR-BI-containing proteoliposomes by 8-10 fold. The proposed studies will capitalize on this finding and attempt to identify and purify the active agents in luteal tissue using a sensitive cell-free membrane reconstitution system. Potentially important known factors associated with molecular transport machinery (SNARES, sphingomyelin, caveolin etc.), will also be tested by the membrane reconstitution system using selective cholesteryl ester transport as an assay, or may be tested in cell systems containing sufficient quantities of SR-BI, but overexpressing (or lacking) certain other proteins. The idea that caveolin may be a negative regulator for SR-BI function in the selected cholesteryl ester uptake process will be thoroughly investigated, as will the idea that specialized domains of microvillar channel membranes (membrane rafts) in stereridogenic tissues represent distinct regions of the microvillar compartment specialized for efficient uptake of neutral lipids. All tissue/cell models described are available in this laboratory. Currently used technologies permit us to faithfully track proteins both biochemically and morphologically, and the integrated system we propose to study should yield detailed information on potential protein links between the SR-BI receptor and the selective uptake of lipoprotein-donated cholesterol.
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