This Program Project Grant began 25 years ago when we defined the LDL receptor pathway for the control of cholesterol metabolism and showed that defects in this pathway produce Familial Hypercholesterolemia and its attendant atherosclerosis. After 25 years, our goals remain the same: to understand the genetic and metabolic defects that produce hypercholesterolemia and accelerated atherosclerosis and to use this knowledge to prevent and treat the disease. During the last grant period ( Years 20-24), we have made considerable progress with the following notable achievements: 1) discovery of the SREBP pathway as the central regulatory mechanism for cholesterol homeostasis; 2) appreciation of the role of the SREBP pathway in overproduction of fatty acids in diabetic states; 3) molecular delineation of the pathways for bile acid biosynthesis, and elucidation of the molecular defects responsible for two forms of neonatal intrahepatic cholestasis in humans; 4) discovery that two members of the LDL receptor gene family are required for the development of normal brain architecture; and 5) elucidation of the role of cholesterol-rich caveolae and other cholesterol-rich structures in concentrating signaling receptors at the cell surface. We now apply for a 5-year renewal of our Program Project Grant (Years 26-30) that will allow us to further study these phenomena through an integrated and multidisciplinary approach. We propose to focus on 25 key molecules involved in three biological processes: 1) cholesterol and fatty acid metabolism (SREBP-1a, SREBP-1c, SREBP-2, SCAP, Site-1 protease, Site-2 protease, LDL receptor, HMG CoA reductase, fatty acid synthase, acetyl CoA synthase, IRS-1, IRS-2); 2) oxysterol and bile acid metabolism (cholesterol 7alpha-hydroxylase, oxysterol 7alpha-hydroxylase-1 and -2, cholesterol 24-hydroxylase, cholesterol 25-hydroxylase, sterol 27-hydroxylase); and 3) caveolae membrane system and cell signaling (caveolin-1, LRP, VLDL receptor, ApoER2, Reelin, RAP, HDL receptor). A series of model systems will be used to study the mechanisms by which these proteins operate at the molecular level (i.e., the gene, the mRNA, and the protein), at the level of the intact cell, at the level of the whole animal, and at the level of the human patient. In conducting these studies, we will use a wide variety of techniques, including biochemistry, immunology, molecular biology, genetics, cell biology, electron microscopy, transgenic and knockout mice, animal physiology, clinical genetics, and human genomics.