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 - LDL受体（LDLR）家族和细胞表面硫酸乙二醇蛋白聚糖（HSPG） - 血浆脂蛋白在疏水性养分中运输的关键参与者。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 HSPG介导。使用嵌合体FCR-同步，由与Syndecan-1的跨膜（TM）（TM）和细胞质区相关的IgG FC受体外域组成，我们发现有效的内吞作用是由Syndecan或Syndecan或Chimera的聚类触发的。聚集会导致快速运动进入富含胆固醇，洗涤剂不溶的，膜木筏，然后实际摄取对细胞的实际摄取需要募集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类时固定在木筏上。序列比较揭示了Syndecan和LDLR TM结构域之间（C末端）部分之间意外的15-残基共识，这并未被筏中排除的蛋白质共享。重要的是，这种共识可能解释了这两个分子被证明可以处理多价配体的方式，例如富含Apoe的富含Apoe的脂蛋白。因此，该提案的中心假设是，辛迪克的特定基序，包括与LDL受体共有的RAFT-LOCALIZE片段，指导养分含有养分配体的亚细胞运输，具有特定的功能后果。有两个目标。 目标1：在LDLR基因家族和Syndecan中的新型贩运图案的详细定义。在 AIM 1A，我们将使用CHO细胞和肝细胞来映射LDLR TM域内筏定位的决定因素，LDLR基因家族的其他成员和Syndecan。在AIM 1B中，我们将绘制syndecan细胞质尾部中的运输决定因素。在AIM IC中，我们将在另一种关键的细胞类型（巨噬细胞）中测试这些决定因素，这特别是因为通过LDLR的变异性内吞途径。 AIM 2：在LDLR跨膜结构域中新型筏定位基序的功能作用。 在AIM 2A中，我们将确定AIM 1的TM筏 - 位置基序在明显的ACAT刺激中，当LDLR结合多价脂蛋白时发生的ACAT刺激。在AIM 2B中，将研究这些TM基序在LDLR介导的通过再摄取的APOB分泌调节中的作用。 这些提出的研究将阐明LDLR和Syndecan中这些新型内吞决定因素的基本机制和功能后果，包括在营养递送过程中筏定位的作用。