The applicants note that the composition and organization of phospholipids across the bilayer membrane of the human erythrocyte are critical to the initiation and regulation of many cellular processes. While most membrane-related enzymatic and transport functions are associated with normal membrane lipid asymmetry, the redistribution of phosphatidylserine (PS) from its preferential location in the cell's inner leaflet to the outer leaflet results in a sequence of events that appears to be regulated by its display. For example, PS at the cell surface is critical to hemostasis, and may play a role in cell aging, membrane fusion, and the recognition and elimination of senescent and apoptotic cells. Although considerable progress has been made toward understanding aminophospholipid movement in red blood cells, the mechanism of lipid movement and its regulation are not understood, nor have the protein(s) involved in its control or recognition been identified. This proposal focuses on the organization, dynamics and spatial distribution of PS in the erythrocyte membrane and the mechanism by which reticuloendothelial cells recognize aged, PS-expressing cells. Particular emphasis is placed on identifying and characterizing the proteins that regulate the distribution and control the movement of PS in erythrocytes and PS binding proteins present in macrophage membranes. The proposed studies are based on results that have led to the concept that a 32 kDa membrane polypeptide, related to the Rh family of proteins, is involved in the generation and regulation of membrane lipid asymmetry. The main objectives of the application are to isolate, identify, and characterize PS binding proteins and the proteins responsible for transbilayer lipid movement. This will be done by a combination of techniques including isolation of transport protein from artificially- generated erythrocyte vesicles. Macrophage/monocyte PS binding proteins will be isolated by PS affinity reagents. The isolated proteins will be mapped and sequenced. Antibodies against PS binding proteins and the transporter as well as oligonucleotides generated from information of the sequenced protein will be used to screen appropriate cDNA expression libraries. Positive clones will be sequenced to identify the entire coding region of the transporter and PS binding proteins. The results of these studies will contribute toward understanding the regulatory processes that maintain specific transbilayer lipid distributions in human erythrocytes and mediate the recognition and ultimate elimination of aged, PS-expressing cells.
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