FUNCTIONAL STUDIES ON THE VERY LOW DENSITY LIPOPROTEIN RECEPTOR

低密度脂蛋白受体家族包含几个成员，包括大细胞 表面受体，gp330和lrp，以及新发现的极低 密度脂蛋白(VLDL)受体。一种39 kDa受体相关蛋白 (RAP)与LRP、gp330和极低密度脂蛋白受体可逆性结合 亲和力。它与这些受体的结合可拮抗 所有已知的配基。此时，极低密度脂蛋白的生物学功能 受体还没有完全被理解。在共享相当大的序列的同时 与低密度脂蛋白受体同源，极低密度脂蛋白受体不同于低密度脂蛋白 受体在其组织中的分布，以及其与配体结合的特异性。 我们最近的研究表明，与lrp和gp330一样，极低密度脂蛋白受体 一种多配体受体，介导载脂蛋白E的细胞分解代谢 含有脂蛋白和uPA与其抑制物PAI-1络合。 这一应用的中心假设是apoE- 脂蛋白水平和细胞表面尿激酶水平是重要的 生理途径，以及活动或水平的变化 这种受体可能与某些疾病的病理有关，例如 动脉硬化。需要检验的具体假设是：1)失败 清除被抑制的uPA(即uPA：PAI-1复合体) 极大地降低了细胞的能力 激活纤溶酶原，从而产生血栓状态，2) 极低密度脂蛋白受体负责Lp(A)的分解代谢，并可能是 表达于内皮细胞、巨噬细胞和平滑肌细胞 在动脉粥样硬化病变中，以及3)RAP在 调节极低密度脂蛋白受体功能。这些假设将在#年进行检验。 三个具体目标。第一个具体目标是建议调查 极低密度脂蛋白受体在调节细胞表面uPA活性中的作用。这些 将通过使用腺病毒载体系统促进研究 在细胞中表达功能性极低密度脂蛋白受体。第二个具体目标是 研究极低密度脂蛋白受体在脂蛋白(A)分解代谢中的作用 确定极低密度脂蛋白受体是否在人类动脉粥样硬化中表达 病变，以及在apoE缺陷小鼠中存在的病变。离体 过表达该基因的细胞的结合研究和细胞摄取分析 我们将利用极低密度脂蛋白受体来解决这些问题。第三 《特定目的》将探索说唱歌手作为监护人的假设 或在生物合成或细胞内运输中伴随的蛋白质 极低密度脂蛋白受体。为了实现这些目标，反义策略将是 用于制备RAP缺陷细胞系。细胞内加工 这些细胞中极低密度脂蛋白受体的含量将与 过度表达的说唱。总而言之，这些研究应该会让我们深入了解 极低密度脂蛋白受体和RAP的作用。

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.