Endocytic Trafficking Motifs in Syndecan & LDL receptor

Syndecan 中的内吞转运基序

• 批准号: 7056775
• 负责人: Kevin Jon Williams
• 金额: $ 30.47万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7056775
• 关键词: CHO cells; apolipoprotein B; cell population study; endocytosis; genetic mapping; genetically modified animals; intracellular transport; laboratory mouse; liver cells; low density lipoprotein receptor; macrophage; protein structure function; receptor binding; syndecan

Two classes of molecules - the LDL receptor (LDLr) family and cell-surface heparan sulfate proteoglycans (HSPGs) - are key participants in the transport of hydrophobic nutrients by plasma lipoproteins. We have discovered a novel pathway, in which endocytosis of lipoproteins and other ligands is mediated directly by syndecan HSPGs. Using a chimera, FcR-Synd, that consists of an IgG Fc receptor ectodomain linked to the transmembrane (TM) and cytoplasmic regions of syndecan-1, we found that efficient endocytosis is triggered by clustering of syndecan or the chimera. Clustering causes rapid movement into cholesterol-rich, detergent-insoluble, membrane rafts, and then the actual uptake into the cell requires recruitment of tyrosine kinases and the actin cytoskeleton. Surprisingly, we found that constructs containing either the LDLr TM or the syndecan-1 TM domain localized equally well to rafts upon clustering. Sequence comparisons revealed an unexpected 15- residue consensus between the inner (C-terminal) portions of the syndecan and LDLr TM domains, which was not shared by a protein excluded from rafts. Importantly, this consensus may explain unusual features of the way these two molecules have been shown to process multivalent ligands, such as large apoE-rich remnant lipoproteins. Thus, the central hypothesis of this proposal is that specific motifs of syndecan, including the raft-localizing segment shared with the LDL receptor, direct the sub-cellular trafficking of nutrient-bearing ligands, with specific functional consequences. There are two Aims. Aim 1: Detailed definition of novel trafficking motifs in the LDLr gene family and in syndecan. In Aim 1a, we will use CHO cells andMcArdle hepatocytes to map determinants of raft localization within the TM domain of the LDLr, other members of the LDLr gene family, and syndecan. In Aim 1b, we will map trafficking determinants in the syndecan cytoplasmic tail. In Aim Ic, we will test these determinants in another key cell type, the macrophage, which is of particular interest becauseofits variant endocytic pathway through the LDLr. Aim 2: Functional roles for the novel raft-localizing motif in the LDLr transmembrane domain. In Aim 2a, we will determine the role of TM raft-localizing motifs from Aim 1 in the marked stimulation of ACAT that occurs in macrophages when the LDLr binds multivalent lipoproteins. In Aim 2b, the role of these TM motifs in LDLr-mediated regulation of apoB secretion via re-uptake will be investigated in hepatocytes. These proposed studies will clarify basic mechanisms and functional consequences of these novel endocytic determinants within the LDLr and syndecans, including the role of raft localization during nutrient delivery.

两类分子-低密度脂蛋白受体(LDLR)家族和细胞表面硫酸乙酰肝素蛋白多糖(HSPGs)-是血浆脂蛋白运输疏水性营养物质的关键参与者。我们发现了一种新的途径，其中脂蛋白和其他配体的内吞作用直接由Syndecan HSPGs介导。使用嵌合体FCR-Synd，它由连接到syndecan-1的跨膜区(TM)和细胞质区域的Ig G Fc受体外区组成，我们发现有效的内吞作用是由Syndecan或嵌合体的聚集触发的。聚集导致快速移动到富含胆固醇的、不溶于洗涤剂的膜筏中，然后实际摄取到细胞中需要重新招募酪氨酸激酶和肌动蛋白细胞骨架。令人惊讶的是，我们发现包含LDLR TM或syndecan-1 TM结构域的构建体在聚集时同样能很好地定位于RAFT。序列比较显示，Syndecan和LDLR TM结构域的内部(C-末端)部分之间存在意想不到的15个残基的一致性，这不是从筏子中排除的蛋白质所共有的。重要的是，这一共识可能解释了这两个分子处理多价配体的方式的不寻常特征，例如大的富含apoE的残余脂蛋白。因此，这一建议的中心假设是，Syndecan的特定基序，包括与LDL受体共享的RAFT定位片段，指导营养配体的亚细胞运输，具有特定的功能后果。有两个目标。 目的1：详细定义LDLR基因家族和Syndecan中新的交易基序。在……里面 目的1a，我们将使用CHO细胞和McArdle肝细胞在LDLR、LDLR基因家族的其他成员和Syndecan的TM区域内定位RAFT定位的决定因素。在目标1b中，我们将定位Syndecan细胞质尾部的运输决定因素。在AIM IC中，我们将在另一种关键细胞类型-巨噬细胞中测试这些决定因素，巨噬细胞是特别感兴趣的，因为它通过LDLR的不同内吞途径。 目的2：新的RAFT定位基序在LDLR跨膜结构域中的功能作用。 在目标2a中，我们将确定目标1中的TM RAFT定位基序在LDLR与多价脂蛋白结合时巨噬细胞中ACAT的显著刺激中的作用。在AIM 2b中，将研究这些TM基序在LDLR介导的通过重新摄取在肝细胞中调节apoB分泌的作用。 这些拟议的研究将阐明这些新的内吞决定因素在LDLR和Syndecans中的基本机制和功能后果，包括RAFT在营养输送过程中的定位作用。