The LDL receptor family contains several members, including the large cell surface receptors, gp330 and LRP, and the newly discovered very low density lipoprotein (VLDL) receptor. A 39 kDa receptor associated protein (RAP) binds reversibly to LRP, gp330, and the VLDL receptor with high affinity. Its association with these receptors antagonizes the binding of all known ligands. At this time, the biological function of the VLDL receptor is not completely understood. While sharing considerable sequence homology with the LDL receptor, the VLDL receptor differs from the LDL receptor in its tissue distribution, and its ligand binding specificity. Our recent studies indicate that like LRP and gp330, the VLDL receptor is a multiligand receptor, and mediates the cellular catabolism of apoE containing lipoproteins as well as uPA complexed to its inhibitor, PAI-1. The central hypothesis of this application is that regulation of apoE- lipoprotein levels and cell surface urokinase levels are important physiological pathways, and that alterations in the activity or levels of this receptor may contribute to the pathology of certain diseases such as atherosclerosis. The specific hypotheses to be tested are: 1) That failure of the VLDL receptor to remove inhibited uPA (i.e. uPA:PAI-1 complexes) from the cell surface greatly diminishes the capacity of the cell to activate plasminogen, thereby producing a thrombotic state, 2) that the VLDL receptor is responsible for the catabolism of Lp(a) and may be expressed in endothelial cells and in macrophages and smooth muscle cells in atherosclerotic lesions, and 3) that RAP plays an important role in modulating VLDL receptor function. These hypothesis will be tested in three specific aims. The first specific aim proposes to investigate the role of the VLDL receptor in regulating cell surface uPA activity. These studies will be facilitated by use of an adenoviral vector system to express functional VLDL receptor in cells. The second specific aim will investigate the role of the VLDL receptor in the catabolism of Lp(a), and determine if the VLDL receptor is expressed in human atherosclerotic lesions, and in lesion present in the apoE-deficient mouse. In vitro binding studies and cellular uptake assays in cells over-expressing the VLDL receptor will be utilized to address these questions. The third specific aim will explore the hypothesis that RAP functions as a chaperone or attendant protein in the biosynthesis or intracellular transport of the VLDL receptor. To accomplish these goals antisense strategies will be utilized to prepare RAP-deficient cell lines. The intracellular processing of the VLDL receptor in these cells will be contrasted with cells that over-express RAP. Together, these studies should give insight into the role of the VLDL receptor and RAP.
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