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由于未能内在化细胞表面受体而导致细胞表面受体的受体摄取和运输的机制可能导致心脏病，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在化的主要机制。质膜处的网格蛋白组件驱动受体摄取。但是，它与受体和其他内吞成分的耦合需要辅助蛋白，例如ARH，DAB2和NUMB。这些蛋白是控制受体传输机械的时空组织的含有PTB（磷酸酪氨酸结合）含有域的内吞转移。它们具有不同的蛋白质相互作用平台模块化，使它们能够促进蛋白质复合物的组装。尽管遗传和细胞生物学研究揭示了PTB适配器的重要性，但调节其在受体转运中动态活性的机制仍然未知。例如，哪些在适当的时间和地点控制内吞机械组件？哪些机制调节货物识别和衔接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未解决的问题。我们的初步观察支持了衔接蛋白磷酸化控制受体转运的观念。实际上，与AP2相关的激酶AAK1L通过靶向包括AP2，NumB和ARH在内的不同内吞转移剂，在多个传输步骤中发挥重要作用。我们假设AAK1L调节适配器的动态脚手架活动，以影响内吞机械的何时何时组装。在此应用程序中，我们将追求三个特定的目标来检验这一假设。 1）我们将解决受体回收中AAK1L作用的分子基础。这将使用AAK1L缺失细胞中的转铁蛋白受体（TFNR）回收的救援方法来完成。我们还将使用生化相互作用筛选来识别AAK1L内体底物。 2）我们将通过体外结合测定法控制磷酸化如何控制涂层坑组装中的麻木活性，以测试与EPS15和AP2相互作用的磷酸化的后果。另外，定量内部化测定将与免疫定位实验结合使用，以剖析麻木磷光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）内吞作用，使用体外测定来确定ARH磷酸化是否会影响与LDLR或核心内吞核的结合。体内实验将验证体外研究，我们将分析ARH磷酸化在LDLR聚类中的作用，并募集到粘蛋白涂层的坑中。总的来说，这些研究的结果不仅将提供重要的机械洞察力，了解如何维持LDLR和TFNR等营养受体的运输效率和保真度，而且还可以作为了解控制信号受体下调的机制的范式。 公共卫生相关性：该项目试图通过了解控制摄取机械的关键事件来更好地定义肝脏如何从血液中清除胆固醇。