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

• 批准号: 7903950
• 负责人: Sean D Conner
• 金额: $ 27.37万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7903950
• 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; 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; public health relevance; receptor; receptor coupling; receptor internalization; receptor recycling; research study; scaffold; uptake

DESCRIPTION (provided by applicant): 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 influence where and when the endocytic machinery is assembled. In this application, we will pursue three specific 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 define 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 efficiency and fidelity 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 define how the liver regulates cholesterol removal from the blood by understanding the key events that control the uptake machinery.

描述（由申请人提供）：该项目的长期目标是了解控制细胞内受体摄取和运输的机制，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真核生物中受体内化的主要机制。质膜上的网格蛋白组装驱动受体摄取。然而，它与受体和其他内吞成分的偶联需要 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。它们是模块化的，具有独特的蛋白质相互作用平台，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，但调节其在受体转运中动态活动的机制仍然未知。例如，什么控制内吞机器在适当的时间和地点组装？什么机制调节货物识别和接头蛋白与其他内吞成分的相互作用？该提案中的实验将开始解决这些悬而未决的问题。我们的初步观察支持接头蛋白磷酸化控制受体转运的观点。事实上，AP2 相关激酶 AAK1L 通过针对不同的内吞接头（包括 AP2、Numb 和 ARH）在多个转运步骤中发挥重要作用。我们假设 AAK1L 调节接头的动态支架活动，以影响内吞机制组装的地点和时间。在此应用中，我们将追求三个具体目标来检验这一假设。 1）我们将解决AAK1L在受体循环中作用的分子基础。这将通过在 AAK1L 耗尽的细胞中利用转铁蛋白受体 (TfnR) 回收的救援方法来实现。我们还将使用生化相互作用筛选来鉴定 AAK1L 内体底物。 2) 我们将通过体外结合测定确定磷酸化如何控制包被凹坑组装中的 Numb 活性，以测试磷酸化对 Numb 与 Eps15 和 AP2 相互作用的结果。此外，定量内化测定将与免疫定位实验相结合，以剖析 Numb 磷酸突变体如何破坏受体内吞作用。 3) 我们将使用体外测定来定义 AAK1 介导的 ARH 磷酸化如何调节低密度脂蛋白受体 (LDLR) 内吞作用，以确定 ARH 磷酸化是否影响与 LDLR 或核心内吞机制的结合。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可以为如何维持 LDLR 和 TfnR 等营养受体的转运效率和保真度提供重要的机制见解，而且还可以作为理解控制信号受体下调的机制的范例。 。 公共健康相关性：该项目旨在通过了解控制摄取机制的关键事件，更好地定义肝脏如何调节血液中胆固醇的清除。