The objective of this proposal is to better understand the molecular mechanisms involved in the transferrin-mediated uptake of the essential mineral iron in the form of Fe(III). Transferrin, a serum glycoprotein that binds Fe(III), is the major carrier by which nutrient iron is delivered to cells. The delivery process involves receptor mediated endocytosis whereby transferrin binds to cell surface receptors and is endocytosed, release bound Fe(III), and is recycled out of the cell to bind more Fe(III). To investigate this process we are isolating and characterizing mutants in cultured cells that are deficient in the transferrin-mediated iron uptake mechanism. Research into this process is related to two aspects of health care. The first is to provide basic information about endocytosis. The endocytic process is a fundamental activity of cells that is required in the uptake of a variety of nutrients (in addition to iron) and in responding to extracellular signals that control a diverse spectrum of activities ranging from biosynthetic pathways to aspects of the immune response. Moreover, many infectious agents, such as certain viruses and bacteria, use the endocytic process to help exert their cytopathic effects. A better understanding of endocytosis may provide new information that can be applied clinically to the recognition and treatment of disease conditions related to the endocytic process. A second area in which this project impacts health care concerns iron toxicitiy. There are a variety of clinical conditions, such as hereditary hemochromatosis and transfusion-induced iron overload, that are related to iron toxicity. By studying transferrin-mediated iron uptake by mutants that might aberrantly accumulate iron, we may learn something about how the toxic effects of iron overload are expressed at the cellular level. To isolate mutants, we expose cultured cells to a cytotoxic form of transferrin prepared by covalently coupling diptheria toxin to transferrin. Cells that survive exposure to this reagent are likely to be defective in the transferrin uptake pathway and we have indeed found mutants with this phenotype. Work in the future will focus on analyzing these mutants.
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