Endocytic Trafficking Motifs in Syndecan & LDL receptor

Syndecan 中的内吞转运基序

基本信息

批准号：
7234006
负责人：
Kevin Jon Williams
金额：
$ 29.58万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
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/7234006
关键词：
Actins Acyl Coenzyme A Adenoviruses Apolipoprotein E Apolipoproteins B Binding Biochemical Biological Assay C-terminal Catabolism Cell Fractionation Cell membrane Cell surface Cells Chimera organism Chinese Hamster Ovary Cell Cholesterol Class Clathrin Cluster Analysis Confocal Microscopy Consensus Cytoplasmic Tail Cytoskeleton Data Detergents Endocytosis Enzymes Epitopes Exhibits Family Family member Fc Receptor Gene Family Heparan Sulfate Proteoglycan Hepatocyte Human Immune Immunoglobulin G In Vitro Knockout Mice LDL-Receptor Related Proteins Ligands Link Lipoproteins Literature Localized Low Density Lipoprotein Receptor Low-Density Lipoproteins Lysosomes Maps Mediating Mediation Membrane Modeling Molecular Movement Mutation Nutrient PTPRC gene Participant Pathway interactions Plasma Play Process Protein Tyrosine Kinase Proteins Publishing Receptor Gene Regulation Reporting Research Personnel Role Signal Transduction Site Staging Structure Surface System Techniques Testing Transferase Transmembrane Domain VLDL receptor Variant Viral Work base cell type coated pit design interest low density lipoprotein inhibitor macrophage member novel programs receptor receptor binding stem syndecan trafficking uptake

项目摘要

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酰肝素蛋白聚糖 (HSPG) - 是血浆脂蛋白疏水性营养物质运输的关键参与者。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是由多配体 HSPG 直接介导的。使用嵌合体 FcR-Synd，该嵌合体由与 syndecan-1 的跨膜 (TM) 和胞质区域连接的 IgG Fc 受体胞外域组成，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、不溶于去污剂的膜筏中，然后实际摄取到细胞中需要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类时同样好地定位于筏。序列比较揭示了多配体聚糖和 LDLr TM 结构域的内部（C 端）部分之间存在意外的 15 残基一致性，而从筏中排除的蛋白质则不共享该一致性。重要的是，这一共识可能解释了这两种分子处理多价配体（例如富含 apoE 的大残留脂蛋白）的方式的不寻常特征。因此，该提议的中心假设是多配体的特定基序，包括与 LDL 受体共享的筏定位片段，指导营养配体的亚细胞运输，产生特定的功能后果。有两个目标。目标 1：详细定义 LDLr 基因家族和多聚糖中的新型运输基序。在目标 1a，我们将使用 CHO 细胞和 McArdle 肝细胞来绘制 LDLr、LDLr 基因家族其他成员和 Syndecan 的 TM 结构域内筏定位的决定因素。在目标 1b 中，我们将绘制多配体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，巨噬细胞因其通过 LDLr 的变异内吞途径而特别令人感兴趣。目标 2：LDLr 跨膜结构域中新型筏定位基序的功能作用。在目标 2a 中，我们将确定来自目标 1 的 TM 筏定位基序在 LDLr 结合多价脂蛋白时巨噬细胞中发生的 ACAT 显着刺激中的作用。在目标 2b 中，将在肝细胞中研究这些 TM 基序在 LDLr 介导的通过再摄取调节 apoB 分泌中的作用。这些拟议的研究将阐明 LDLr 和多配体中这些新型内吞决定簇的基本机制和功能后果，包括营养输送过程中筏定位的作用。