FUNCTIONAL STUDIES ON THE VERY LOW DENSITY LIPOPROTEIN RECEPTOR

LDL受体家族包含几个成员，包括大细胞 表面受体，gp 330和LRP，以及新发现的非常低的 密度脂蛋白（VLDL）受体。一种39 kDa受体相关蛋白 (RAP)可逆结合LRP，gp 330和VLDL受体， 亲和力 它与这些受体的结合拮抗 所有已知的配体。此时，VLDL的生物学功能 受体尚未完全了解。虽然有相当多的序列 由于VLDL受体与LDL受体具有同源性，因此VLDL受体不同于LDL受体。 受体在其组织中的分布及其配体结合特异性。 我们最近的研究表明，像LRP和gp 330一样，VLDL受体也是一种 一种多配体受体，介导apoE的细胞催化作用 含有脂蛋白以及与其抑制剂派-1复合的uPA。 本申请的中心假设是apoE的调节- 脂蛋白水平和细胞表面尿激酶水平是重要的 生理途径，以及活动或水平的改变， 这种受体可能导致某些疾病的病理学 动脉粥样硬化需要检验的具体假设是：1）失败 VLDL受体，以去除受抑制的uPA（即uPA：派-1复合物） 从细胞表面大大降低了细胞的能力， 激活纤溶酶原，从而产生血栓形成状态，2） VLDL受体负责Lp（a）的催化作用， 在内皮细胞、巨噬细胞和平滑肌细胞中表达 在动脉粥样硬化病变中，以及3）RAP在 调节VLDL受体功能。这些假设将在 三个具体目标。第一个具体目标是调查 VLDL受体在调节细胞表面uPA活性中的作用。这些 通过使用腺病毒载体系统， 在细胞中表达功能性VLDL受体。第二个具体目标将 研究VLDL受体在Lp（a）催化剂中的作用， 确定VLDL受体是否在人类动脉粥样硬化中表达， 病变，以及存在于apoE缺陷小鼠中的病变。 体外 结合研究和细胞摄取试验， VLDL受体将用于解决这些问题。第三 一个具体的目标将探讨假设RAP功能作为一个伴侣 或伴随蛋白质的生物合成或细胞内运输的 VLDL受体。为了实现这些目标， 用于制备RAP缺陷型细胞系。细胞内加工 这些细胞中VLDL受体的表达将与 过表达RAP。总之，这些研究应该让我们深入了解 VLDL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.