Cytoskeletal Regulation Of Vesicle Transport In Liver

肝脏囊泡运输的细胞骨架调节

• 批准号: 7800477
• 负责人: MARK A. MC NIVEN
• 金额: $ 29.44万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7800477
• 关键词: Actin-Binding Protein; Actins; Adaptor Signaling Protein; Affect; Arts; Binding; Biochemical; Capsid Proteins; Cell Survival; Cell membrane; Clathrin; Clathrin Adaptors; Complex; Cytoskeleton; Data; Dissection; Dynamin; Dynamin 2; Endocytosis; Epidermal Growth Factor Receptor; Golgi Apparatus; Growth Factor Receptors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hepatocyte; Homeostasis; Hydrolase; Hydrolysis; Image; Iron; Lead; Liver; Liver diseases; Lysosomes; Mediating; Membrane; Membrane Lipids; Methods; Molecular; Morphology; Nutrient; Organelles; Play; Polymers; Process; Proteins; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Recycling; Regulation; Research Personnel; Role; Sorting - Cell Movement; Structure; Tertiary Protein Structure; Testing; Transferrin; Transferrin Receptor; Transport Vesicles; Ubiquitin; Vesicle; Work; base; bile formation; disorder prevention; human EMS1 protein; information gathering; innovation; insight; liver function; nexin; novel; protein complex; protein transport; receptor; scaffold; trafficking; trans-Golgi Network; treatment strategy

DESCRIPTION (provided by applicant): The overall objective of this proposal is to define the molecular machinery that supports receptor-mediated vesicle trafficking processes in hepatocytes. These processes are essential for normal iron homeostasis, growth factor receptor signaling, and cell survival. Further, their disruption can lead to a variety of prevalent liver diseases. Here we focus on the vesicle-based machinery utilized by the hepatocyte at the plasma membrane (PM) and trans-Golgi network (TGN) to sort and traffic distinct receptors and cargo proteins. These include the transferrin receptors (TfRs), the epidermal growth factor receptor (EGFR), and lysosomal hydrolases used in their degradation. We have substantial preliminary data implicating the action of a conserved receptor sorting and vesicle formation machinery in hepatocytes that is centered around the membrane-severing GTPase dynamin 2 (Dyn2). We have found that this contractile polymer associates with the actin cytoskeleton and specific adaptor proteins at both the PM to regulate receptor endocytosis and the TGN to mediate trafficking of nascent hydrolases to the lysosome. Most recently, we have found that this complex can sort cargo through interactions with ubiquitin-associated proteins and is regulated by non- receptor tyrosine kinases. Thus, the CENTRAL HYPOTHESIS of this proposal is that a polymeric Dyn2 contractile scaffold associates with the actin cytoskeleton and specific adaptor proteins to mediate the dual processes of cargo sequestration and vesicle formation from the PM and TGN in hepatocytes. We will use state of the art imaging, biochemical, and molecular methods to explore three related but independent aims. In Aim One we will test how the regulated assembly of a Dyn2-actin endocytic complex mediates differential internalization and trafficking of the two different TfRs from the hepatocyte PM. In Aim Two we will define how the Dyn2 complex interacts with the ubiquitin-based sorting machinery in hepatocytes to mediate internalization and post-endocytic trafficking of the EGFR, targeting this receptor to lysosomes for degradation. Finally, in Aim Three we will define how the Dyn2 complex interacts with a novel ubiquitin-based sorting machinery at the hepatocyte TGN to maintain Golgi structure and mediate transport of newly synthesized hydrolases to the lysosome for EGFR degradation. This proposal is conceptually and technically innovative in that it provides major advances toward understanding the molecular mechanism of how essential hepatocyte receptors and cargo proteins are differentially sorted into vesicle carriers for subsequent trafficking to distinct organelles. The information gathered by these studies will help identify strategies for the treatment and prevention of diseases that affect basic liver function.

描述（由申请方提供）：本提案的总体目标是定义支持肝细胞中受体介导的囊泡运输过程的分子机制。这些过程对于正常的铁稳态、生长因子受体信号传导和细胞存活是必不可少的。此外，它们的破坏可能导致各种流行的肝脏疾病。在这里，我们专注于囊泡为基础的机械利用肝细胞在质膜（PM）和trans-Golgi网络（TGN）的排序和交通不同的受体和货物蛋白。这些包括转铁蛋白受体（TfR）、表皮生长因子受体（EGFR）和用于其降解的溶酶体水解酶。我们有大量的初步数据暗示的行动保守的受体分选和囊泡形成机制的肝细胞，是围绕膜切割GT3发动蛋白2（Dyn 2）。我们已经发现，这种收缩性聚合物与肌动蛋白细胞骨架和特定的衔接蛋白在PM调节受体内吞作用和TGN介导运输新生的水解酶的溶酶体。最近，我们发现这种复合物可以通过与泛素相关蛋白的相互作用来分选货物，并且受到非受体酪氨酸激酶的调节。因此，该提议的中心假设是聚合Dyn 2收缩支架与肌动蛋白细胞骨架和特异性衔接蛋白相关联，以介导肝细胞中PM和TGN的货物螯合和囊泡形成的双重过程。我们将使用最先进的成像，生物化学和分子方法来探索三个相关但独立的目标。在目的一，我们将测试如何调节组装的Dyn 2-肌动蛋白内吞复合物介导的差异内化和贩运的两个不同的TfR从肝细胞PM。在目标二中，我们将定义Dyn 2复合物如何与肝细胞中基于泛素的分选机制相互作用，以介导EGFR的内化和内吞后运输，将该受体靶向溶酶体降解。最后，在目标三中，我们将定义Dyn 2复合物如何与肝细胞TGN处的新型基于泛素的分选机制相互作用，以维持高尔基体结构并介导新合成的水解酶转运至溶酶体以降解EGFR。这一建议在概念上和技术上都是创新的，因为它提供了重大进展，有助于理解基本肝细胞受体和货物蛋白如何被差异分选成囊泡载体，随后被运输到不同的细胞器的分子机制。这些研究收集的信息将有助于确定治疗和预防影响基本肝功能的疾病的策略。