The specific aims of the proposed research are to identify retinal pigment epithelial (RPE) cell surface receptors which participate in the recognition, attachment and ingestion of rod outer segment (ROS).
These aims are directed towards understanding the biochemical and molecular events which participate in the phagocytosis of ROS by the RPE, and towards elucidating the molecular cause of the failure of this proocess in the RCS rat model of retinal degeneration. Because of the greatly reduced ability of the RCS rat to phagocytize ROS, this animal provides an excellent model for probing a fundamental cellular interaction, the maintenance of which is vital to the persistence of vision. By understanding the cause of a retinal degeneration in the rat, we may open avenues for understanding one of the possible causes of retinal degeneration in man. Utilizing RPE cells grown in tissue culture from normal and dystrophic rats, the interaction of ROS with receptors on the RPE cell surface will be studied. Nonionic detergents will be used to extract ligand-receptor-cytoskeleton complexes, which will be analyzed by SDS-PAGE, isoelectric focusing and sucrose density gradient centrifugation. Transmembrane signalling will be studied during the binding and ingestion of ROS. Classes of protein kinases present in normal and dystrophic RPE cells will be identified, as will the endogenous substrates phosphorylated by the various kinases. Phosphorylation of these substrates will be analyzed in response to extracellular signals, particularly the phagocytosis of ROS. Classification of glycoproteins in the RPE cell plasma membrane will be studied after labeling with 3H-sugars. The effect of inhibitors of glycosylation, or of oligosaccharide processing, on ROS binding and ingestion will be examined in normal and dystrophic RPE cells. The reinsertion of fully processed glycopeptides into the plasma membrane will be correlated with the binding and ingestion of ROS. Additionally, a library of monoclonal antibodies (MAbs) to RPE plasma membrane proteins will be developed. The specific antigens which these Mabs recognize will be characterized with respect to molecular weight, presence of oligosaccharide chains, association with the cytoskeleton, and where possible, function.
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