MOLECULAR CLONING OF CDNA ENCODING OXIDIZED LDL RECEPTOR

THE LONG TERM OBJECTIVE OF THIS RESEARCH IS TO DEVELOP DRUGS TO PREVENT PROGRESSION/FORMATION OF ATHEROSCLEROTIC LESIONS IN HUMANS. LIPID DEPOSITION IN THE ARTERIAL WALL HAS BEEN SHOWN IN ANIMAL MODELS TO INVOLVE THE INITIAL ACCUMULATION OF LIPID BY MACROPHAGES. EVIDENCE TO DATE SUGGESTS THAT THIS LIPID IS TAKEN UP IN THE FORM OF OXIDIZED LOW-DENSITY LIPOPROTEIN (LDL), BY TWO MACROPHAGE RECEPTORS, THE ACETYL-LDL RECEPTOR, AND A DISTINCT OXIDIZED-LDL RECEPTOR. THE INTENT IS TO IDENTIFY COMPOUNDS WHICH WILL BLOCK LIPID UPTAKE BY THE OXIDIZED-LDL RECEPTOR. DURING PHASE I, WE WILL CLONE THE CDNA ENCODING THE OXIDIZED-LDL RECEPTOR FROM MACROPHAGES. A MOUSE MACROPHAGE CDNA LIBRARY WILL BE EXPRESSED TRANSIENTLY IN MAMMALIAN CELLS, AND THE CLONE FOR THE RECEPTOR WILL BE ISOLATED BY SCREENING FOR THE CELL SURFACE EXPRESSION OF RECEPTORS WHICH BIND OXIDIZED-LDL. IN ORDER NOT TO CLONE THE ACETYL-LDL RECEPTOR, BINDING STUDIES WILL BE CARRIED OUT UNDER CONDITIONS IN WHICH THE ACETYL-LDL RECEPTOR IS BLOCKED. DURING PHASE II, THE OXIDIZED-LDL RECEPTOR WILL BE STABLY EXPRESSED IN A CELL LINE, AND THIS LINE WILL BE USED TO SCREEN LIBRARIES OF COMPOUNDS FOR BLOCKING ACTIVITY.