TRAFFICKING, SHREDDING, AND FUNCTIONS OF APOER2

ApoE受体介导apoE在CNS中的许多功能;包括脂蛋白的内吞作用， 激酶信号传导途径的活化，以及突触信号传导和神经胶质活化的调节。一 apoE受体生物学的一个重要方面是其通过受调节的切割机制从细胞脱落。 在这个项目中，我们将集中在这些apoE受体之一，ApoER 2。在目标1中，我们将测试蛋白水解 酶产生可溶性受体和细胞质片段，分析亚当斯和BACE。我们将 确定这些裂解的机制，测试它们是否被选择性剪接的O- 连接ApoER 2的糖基化结构域，并被内源性金属蛋白酶抑制剂TIMP-3抑制。 我们推测负责APR切割的蛋白酶也作用于apoE受体。在目标2中， 将集中在这些裂解事件的调节。我们将测试细胞质衔接蛋白（FE 65， Dab-1，SNX-17），影响ApoER 2的运输，以及配体结合（特别是apoE亚型）如何影响 受体代谢我们将研究脱落在ApoER 2内吞作用中的作用 和信号传导，包括神经毒性的机制。在目标3中，我们将测试分泌的apoE受体是如何 清除，检查几种可能的介质的内吞作用。通过这三个目标，我们将确定 可溶性ApoER 2的产生、功能和清除，以及脱落过程如何影响功能 全长ApoER 2在目标4中，我们将使用primary 神经元和转基因小鼠。我们将测试配体（apoE-脂蛋白）如何影响ApoER 2 体内蛋白水解，以及可溶性和全长ApoER 2如何影响AH斑块形成。我们假设 由于APP和apoE受体共享配体、衔接蛋白和蛋白水解途径， 受体如ApoER 2对体内APP运输和蛋白水解具有重要作用。我们将在 与其他项目合作，以实现这些目标：项目1，了解如何 人apoE同种型的生理学相关形式影响ApoER 2功能，包括神经毒性; 项目2：受体剪接变体分析;项目3：ApoER 2贩运和加工分析; 和项目5，用于确定ApoER 2对神经元信号传导和APP加工的影响， vivo.总之，这些研究将检查apoE亚型通过ApoER 2在CNS中的功能作用。

ApoE receptors mediate many of the functions of apoE in the CNS; including endocytosis of lipoproteins, activation of kinase signaling pathways, and modulation of synaptic signaling and glial activation. One important aspect of apoE receptor biology is its shedding from the cell via regulated cleavage mechanisms. In this project, we will focus on one of these apoE receptors, ApoER2. In Aim 1, we will test how proteolytic enzymes generate soluble receptors and cytoplasmic fragments, analyzing ADAMs and BACE. We will determine the mechanism of these cleavages, testing whether they are altered by the alternatively spliced O- linked glycosylation domain of ApoER2 and inhibited by the endogenous metalloproteinase inhibitor, TIMP-3. We hypothesize that the proteases responsible for APR cleavage also act on apoE receptors. In Aim 2, we will focus on the regulation of these cleavage events. We will test how cytoplasmic adaptor proteins (FE65, Dab-1, SNX-17), influence trafficking of ApoER2, and how ligand binding (specifically apoE isoforms) affect receptor metabolism. We will examine the role shedding plays in the functions of ApoER2 in endocytosis and signaling, inlcuding mechanisms of neurotoxicity. In Aim 3, we will test how secreted apoE receptors are cleared, examining several possible mediators of endocytosis. Through these three aims, we will define the production, function, and clearance of soluble ApoER2, and how the shedding process affects the functions of full-length ApoER2. In Aim 4, we will examine the connections between ApoER2 and APP, using primary neurons and genetically modified mice. We will test how ligands (apoE-lipoproteins) affect ApoER2 proteolysis in vivo, and how soluble and full length ApoER2 affect AH plaque formation. We hypothesizethat because APP and apoE receptors share ligands, adaptor proteins, and proteolytic pathways, alteration of receptors such as ApoER2 have important effects on APP trafficking and proteolysis in vivo. We will work in concert with the other projects to progress each of these aims: Project 1for understanding how physiologically relevant forms of human apoE isoforms affect ApoER2 function including neurotoxicity; Project 2 for analysis of receptor splice variants; Project 3 for analysis of ApoER2 trafficking and processing; and Project 5 for determining the effects of ApoER2 on neuronal signaling and APP processing in vivo. Overall, these studies will examine the functional effects of apoE isoforms via ApoER2 in the CNS.