The Role of Adaptor Protein Phosphorylation in Regulating Receptor Transport

接头蛋白磷酸化在调节受体转运中的作用

基本信息

批准号：
8312578
负责人：
Sean D Conner
金额：
$ 27.03万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8312578
关键词：
Adaptor Signaling Protein Address Binding Biochemical Biological Biological Assay Blood Cell Surface Receptors Cell membrane Cells Cholesterol Clathrin Complement Coupling Down-Regulation Endocytosis Eukaryota Event Excision Failure Genetic Goals Health Heart Diseases In Transferrin In Vitro Liver Low Density Lipoprotein Receptor Mediating Membrane Molecular Nature Nutrient PTB Domain Pathway interactions Phage Display Phosphorylation Phosphotransferases Phosphotyrosine Proteins Receptor Signaling Recruitment Activity Recycling Regulation Role Testing Time Transcription Factor AP-2 Alpha Transferrin Receptor base coated pit combinatorial in vitro Assay in vivo insight leukemia malignant breast neoplasm mutant protein complex receptor receptor coupling receptor internalization receptor recycling research study scaffold uptake

项目摘要

The long term goal of this project is to understand the mechanisms that govern receptor uptake and transport within cells since failure to internalize cell surface receptors can result in heart disease, leukemia, and breast cancer. The clathrin-mediated endocytic pathway is the major mechanism for receptor internalization in eukaryotes. Clathrin assembly at the plasma membrane drives receptor uptake. However, its coupling to receptors and other endocytic components requires adaptor proteins like ARH, Dab2, and Numb. These proteins are PTB (phosphotyrosine binding) domain-containing endocytic adaptors that control the spatio-temporal organization of the receptor transport machinery. They are modular with distinct protein interaction platforms that enable them to promote assembly of protein complexes. While genetic and cell biological studies reveal the importance of PTB adaptors, the mechanisms that regulate their dynamic activities in receptor transport remain unknown. For example, what controls endocytic machinery assembly at the appropriate time and place? What mechanisms regulate cargo recognition and adaptor protein interaction with other endocytic components? Experiments in this proposal will begin to address these unanswered questions. Our preliminary observations support the idea that adaptor protein phosphorylation controls receptor transport. Indeed the AP2-associated kinase, AAK1L, performs essential roles in multiple transport steps by targeting different endocytic adaptors including AP2, Numb, and ARH. We postulate the AAK1L modulates the dynamic scaffolding activities of adaptors to inuence where and when the endocytic machinery is assembled. In this application, we will pursue three specic aims to test this hypothesis. 1) We will resolve the molecular basis for AAK1L action in receptor recycling. This will be accomplished using a rescue approach for transferrin receptor (TfnR) recycling in AAK1L-depleted cells. We will also use biochemical interaction screens to identify AAK1L endosomal substrates. 2) We will determine how phosphorylation controls Numb activity in coated pit assembly with in vitro binding assays to test the consequence of phosphorylation on Numb interaction with Eps15 and AP2. Additionally, quantitative internalization assays will be combined with immunolocalization experiments to dissect how Numb phosphomutants disrupt receptor endocytosis. 3) We will dene how AAK1-mediated ARH phosphorylation regulates low density lipoprotein receptor (LDLR) endocytosis using in vitro assays to determine if ARH phosphorylation impacts binding to the LDLR or the core endocytic machinery. In vitro studies will be validated with in vivo experiments where we will analyze the role of ARH phosphorylation in LDLR clustering and recruitment to clathrin-coated pits. Collectively, results from these studies will not only provide important mechanistic insight into how transport efciency and delity is maintained for nutrient receptors like LDLR and TfnR, but they may also serve as a paradigm for understanding the mechanisms that control the down-regulation of signaling receptors. PUBLIC HEALTH RELEVANCE This project seeks to better dene how the liver regulates cholesterol removal from the blood by understanding the key events that control the uptake machinery.

该项目的长期目标是了解控制受体摄取和细胞内运输，因为细胞表面受体未能内在化可能导致心脏病、白血病和乳腺癌。网格蛋白介导的内吞途径是受体内化的主要机制在真核生物中。质膜上的网格蛋白组装驱动受体摄取。然而，它的耦合受体和其他内吞成分需要 ARH、Dab2 和 Numb 等衔接蛋白。这些蛋白质是含有 PTB（磷酸酪氨酸结合）结构域的内吞接头，可控制受体转运机制的时空组织。它们是具有不同蛋白质的模块化结构相互作用平台使它们能够促进蛋白质复合物的组装。虽然遗传和细胞生物学研究揭示了 PTB 接头的重要性以及调节其动态活动的机制受体转运的作用仍不清楚。例如，什么控制内吞机器的组装合适的时间和地点？什么机制调节货物识别和接头蛋白与其他内吞成分？该提案中的实验将开始解决这些悬而未决的问题。我们的初步观察结果支持接头蛋白磷酸化控制受体的观点运输。事实上，AP2 相关激酶 AAK1L 通过以下方式在多个转运步骤中发挥重要作用：针对不同的内吞接头，包括 AP2、Numb 和 ARH。我们假设 AAK1L 调节适配器的动态支架活动影响内吞机器组装的地点和时间。在此应用中，我们将追求三个特定目标来检验这一假设。 1）我们将解决 AAK1L 在受体回收中作用的分子基础。这将通过救援方法来完成用于 AAK1L 耗尽细胞中转铁蛋白受体 (TfnR) 的再循环。我们还将利用生化相互作用筛选以确定 AAK1L 内体底物。 2) 我们将确定磷酸化如何控制 Numb 通过体外结合测定来测试包被凹坑组装的活性，以测试磷酸化的结果与 Eps15 和 AP2 的互动麻木。此外，定量内化测定将与免疫定位实验剖析 Numb 磷酸突变体如何破坏受体内吞作用。 3）我们将定义 AAK1 介导的 ARH 磷酸化如何调节低密度脂蛋白受体 (LDLR) 使用体外测定的内吞作用来确定 ARH 磷酸化是否影响与 LDLR 或核心内吞机制。体外研究将通过体内实验进行验证，我们将分析 ARH 磷酸化在 LDLR 聚集和募集到网格蛋白包被的凹坑中的作用。总的来说，这些研究的结果不仅将为运输效率和运输效率提供重要的机制见解。 LDLR 和 TfnR 等营养受体的保真度得以维持，但它们也可以作为了解控制信号受体下调的机制。公共卫生相关性该项目旨在更好地定义肝脏如何通过以下方式调节血液中的胆固醇去除：了解控制摄取机制的关键事件。