Endocytic Trafficking Motifs in Syndecan & LDL receptor

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）-是血浆脂蛋白转运疏水营养素的关键参与者。我们已经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多配体蛋白聚糖HSPGs介导。使用嵌合体FcR-Synd，其由连接到多配体蛋白聚糖-1的跨膜（TM）和胞质区域的IgG Fc受体胞外域组成，我们发现有效的内吞作用由多配体蛋白聚糖或嵌合体的聚集触发。聚集导致快速移动到富含胆固醇、不溶于洗涤剂的膜筏中，然后实际吸收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，无论是LDLr TM或syndecan-1 TM结构域的构建体定位同样很好的筏聚类。序列比较发现了一个意想不到的15个残基之间的共识syndecan和LDLr TM结构域的内部（C-末端）部分，这是不共享的蛋白质排除筏。重要的是，这一共识可能解释了这两种分子处理多价配体（如富含apoE的大残基脂蛋白）的方式的不寻常特征。因此，该提议的中心假设是，syndecan的特定基序，包括与LDL受体共享的筏定位片段，指导营养物承载配体的亚细胞运输，具有特定的功能后果。有两个目标。 目的1：详细定义LDLr基因家族和多配体蛋白聚糖中的新型转运基序。在 目的1a，我们将使用CHO细胞和McArdle肝细胞来定位LDLr、LDLr基因家族的其他成员和多配体聚糖的TM结构域内的筏定位决定因素。在目标1b中，我们将绘制多配体蛋白质尾区的运输决定簇。在目标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，巨噬细胞由于其通过LDL r的内吞途径的变体而特别感兴趣。 目的2：LDLr跨膜结构域中新的筏定位基序的功能作用。 在目标2a中，我们将确定目标1中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显著刺激中的作用。在目的2b中，将在肝细胞中研究这些TM基序在LDL r介导的apoB分泌调节（通过再摄取）中的作用。 这些拟议的研究将阐明这些新的内吞决定因素内的LDLr和syndecans的基本机制和功能后果，包括筏定位在营养输送过程中的作用。

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.