The specific aims of this proposal are to identify pigment epithelial (PE) cell surface receptors which participate in the recognition and attachment of rod outer segments (ROS) and receptors which are responsible for initiating and controlling the ingestion phase of phagocytosis. These goals are directed towards understanding the biochemical and molecular events which participate in the phagocytosis of rod outer segments by the pigment epithelium, and towards elucidating the molecular cause of the failure of this process in the RCS rate model of retinal degeneration. Because of the reduced capability of the RCS rat PE to phagocytize ROS, this animal provides a convenient model for probing a fundamental cellular interaction, the maintenance of which is vital to the persistence of vision. By unravelling the cause of a retinal degeneration in the rat, we may better understand one of the possible causes of retinal degeneration in man. These specific goals are possible because of the discovery that the mutation in the RCS rat PE affects the ingestion phase of phagocytosis. Additionally, I have developed an antiserum to rat PE cells which inhibits the phagocytosis of ROS by these cells. Thus it is now possible to design experiments to identify the cell surface receptors which participate in the several separate steps of phagocytosis, and to identify those receptors which are absent or defective in the RCS rat PE. Monoclonal antibodies (MCA) will be produced against PE cell plasma membranes, and the specific antibodies which inhibit the phagocytosis of ROS will be selected. PE cell surface antigens will be separated by 2D-SDS PAGE and transblotting. After reaction with antiserum or MCA and 125I-protein A, specific antigens will be identified by autoradiography. Cell surface receptors for ROS binding will be radiolabeled and cross-linked to ROS surface proteins. The receptors will then be identified as outlined above. The role of actin in ROS disk shedding will be explored using actin antibodies immunoferritin labeling, and electron microscopy.
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