Molecular mechanisms of endocytic initiation and cargo selection

内吞起始和货物选择的分子机制

• 批准号: 10715060
• 负责人: Richard Wayne Baker
• 金额: $ 38.88万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-20 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10715060
• 关键词: Address; Area; Automobile Driving; Binding; Biochemical; Cell Survival; Cell membrane; Cell physiology; Clathrin; Clathrin Adaptors; Coated vesicle; Complex; Cryoelectron Microscopy; Cues; Data; Decision Making; Defect; Development; Disease; Disease model; Endocytosis; Environment; Fluorescence Microscopy; G-Protein-Coupled Receptors; Growth Factor; Lead; Malignant Neoplasms; Mediating; Medical; Membrane; Methodology; Modeling; Molecular; Molecular Conformation; Mutation; Nature; Nerve Degeneration; Normal Cell; Nutrient; Phosphorylation; Process; Protein Family; Proteins; Quality Control; Regulation; Research; Role; Route; Signal Transduction; Sorting; Systems Development; Vesicle; drug development; enhancer-binding protein AP-2; experimental study; extracellular; human disease; insight; interdisciplinary approach; live cell imaging; reconstitution; response; single molecule; uptake

Abstract The primary route of internalization from the plasma membrane is clathrin-mediated endocytosis (CME), which is required for vital cellular processes such as signaling, nutrient uptake, and development. While the molecular mechanisms underlying the late stages of vesicle formation are well studied, the mechanisms underpinning the earliest stages of endocytosis, including initiation and cargo selection, are poorly understood. Initiation and cargo binding are largely controlled by the AP2 clathrin adaptor complex, a core component of the vesicle coat that serves as a bridge between the rigid clathrin lattice and membrane-embedded cargo. However, it is unclear how AP2 discriminates between hundreds of potential cargo in a complex membrane environment, while also responding to spatial and temporal regulatory cues. While >50 proteins are proposed to regulate or be required for CME, and many physically interact with AP2, we have little mechanistic and structural data for how they are regulated during the earliest stages of endocytosis. We propose that multiple unique AP2 conformations, driven by association with regulatory factors, control higher-order AP2 functions such as cargo selection. Understanding the nature of the regulatory mechanisms controlling endocytosis is critical, as CME largely controls the localization of many medically-relevant proteins such as RTKs and GPCRs. This proposal therefore seeks to reconstitute and define the molecular mechanisms of AP2-mediated endocytic initiation and cargo sorting. Current models of endocytosis largely rely on biochemical experiments performed with soluble components and live cell imaging. Importantly, our methodology is focused on modifying all experimental approaches — cryo-EM, biochemical reconstitution, and single molecule fluorescence microscopy — to include a membrane, thereby addressing a critical need to develop mechanistic models in a near-native membrane environment. This approach is poised to provide an understanding of the role of membrane-induced allostery in driving regulatory decision-making during endocytic initiation. This proposal will focus on two broad areas of endocytic regulation — cargo selection mediated by the conserved Muniscin family proteins, and a quality control checkpoint controlled by a single phosphorylation mark on the μ2 subunit of AP2. As diseases associated with endocytic defects are likely caused by missorting of important trans-membrane cargo, our insights into the mechanisms of endocytic initiation and cargo selection will enable hypothesis-driven research into disease model systems and drug development.

抽象的 质膜内部化的主要途径是网格蛋白介导的内吞作用（CME），它 对于重要的细胞过程，例如信号，养分吸收和发育所必需的 对囊泡形成晚期的后阶段的机制进行了充分的研究，其基础的机制是 Lee最早的吞噬作用和货物的最早阶段是较差的启动和货物 结合由AP2网格蛋白适配器配合物控制，这是囊泡的核心成分 用高等的量高达ASA AP2在复杂的膜环境中区分了数百个潜在的货物 响应时空调节线索。 对于CME，并且许多与AP2物理相互作用，我们几乎没有机械和结构数据，因为它们是 在内吞作用的高峰期，我们提出了多个独特的AP2置信 通过与规律性因素相关联，控制较高的AP2功能，例如货物选择。 控制内吞作用的规则性机制的性质至关重要，因为CME在很大程度上控制了 因此，许多与医疗相关的蛋白质（例如RTK和GPCR）的定位。 重建并定义AP2介导的内吞开始和货物分类的分子机制。 当前的内吞作用模型更大的模型依赖于可溶性成分和 活细胞成像。 生化重构和单分子荧光显微镜 - 包括膜，此处 解决在近乎本地的膜环境中开发机械模型的关键需求 方法有索赔，以理解膜诱导的变构的作用 内吞作业期间的决策。 - 由保守的Munscin家族蛋白和质量控制检查站介导的货物选择 由AP2的μ2亚基上的单个磷酸化标记控制。 缺陷很可能是由于对重要的跨膜货物的错过而引起的，这是我们对机制的见解 内吞引发和货物选择将以假设为疾病模型系统和系统的研究 药物开发。