There are two classes of folate binding proteins (FBP)--soluble and membrane-associated. Since their discovery, it has been hypothesized that these proteins play some role in the uptake, metabolism or storage of folate although, thus far, no precise function has been defined. The long term goal is to establish the relationship between these two classes of FBP and to investigate their roles in folate metabolism. The cultured human KB cell possesses a high level of membrane-associated FBP and also releases a soluble FBP in the culture medium and is therefore an excellent model in which to study these questions. We have described several forms of FBP associated with the KB cell and this proposal will further investigate their relationship. Pulse labeling of KB cells with (35S) methionine and immunoprecipitation of the labeled FBP will be used to measure the turnover rate of the membrane-associated FBP, the rate of release of FBP into the medium, and the biosynthesis and the subcellular localization of the various forms of FBP. The topography of the FBP in situ on the cell membrane will also be investigated using pulse labeling of cells followed by controlled proteolysis. To investigate the role that the FBP may play in folate uptake, intact KB cells will be covalently labeled with activated folate in order to block the folate binding site of the membrane-associated FBP and determine the effect this block has on the uptake of folate into intact cells in either folate replete or deficient medium. Monoclonal antibodies to the folate binding site of the FBP will be produced using standard hybridoma techniques and these antibodies will also be used to specifically block the folate binding site of the FBP in order to evaluate the effect of this blockage on the uptake of folate into the cell and on the cell's survival. The KB cell is able to survive in folate deficient medium for a prolonged period of time. In contrast, K562 cells, which contain very little FBP, are unable to grow in folate deficient medium for more than 14 days. These observations suggest that FBP may play an important role in the uptake of folate. To investigate this hypothesis, K562 cells will be transfected with the DNA extracted from KB cells. After transfection, the K562 cells will be cultured in folate deficient medium and cells that survive will be examined for the presence of FBP by cytoimmunofluorescence. Successful transfection of the K562 cells, resulting in increased expression of FBP and improved survival in folate deficient medium, would be strong evidence that the FBP plays a vital role in folate uptake.
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