Endocytic Trafficking Motifs in Syndecan & LDL receptor

两类分子——低密度脂蛋白受体（LDLr）家族和细胞表面硫酸肝素蛋白聚糖（HSPGs）——是血浆脂蛋白运输疏水营养物质的关键参与者。我们发现了一种新的途径，在这种途径中，脂蛋白和其他配体的内吞作用是由syndecan HSPGs直接介导的。利用嵌合体FcR-Synd，其中IgG Fc受体外结构域连接到syndecan-1的跨膜（TM）和细胞质区域，我们发现syndecan或嵌合体的聚集可以触发有效的内吞作用。聚集导致快速移动到富含胆固醇、不溶清洁剂的膜筏中，然后实际进入细胞需要酪氨酸激酶和肌动蛋白细胞骨架的招募。令人惊讶的是，我们发现含有LDLr TM或syndecan-1 TM结构域的结构体在聚类时同样很好地定位于筏。序列比较揭示了syndecan和LDLr TM结构域的内部（c端）部分之间出人意料的15个残基一致性，这是被排除在筏外的蛋白质所不共享的。重要的是，这一共识可能解释了这两种分子处理多价配体的方式的不寻常特征，例如大型富含载脂蛋白的残余脂蛋白。因此，该建议的中心假设是syndecan的特定基序，包括与LDL受体共享的筏定位片段，指导携带营养配体的亚细胞运输，具有特定的功能后果。有两个目的。

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.