Spatiotemporally resolved architecture of G protein signaling downstream of CXCR4, the driver of lymphocyte migration

CXCR4（淋巴细胞迁移的驱动因素）下游 G 蛋白信号传导的时空解析结构

• 批准号: 10380895
• 负责人: Irina Kufareva
• 金额: $ 19.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10380895
• 关键词: Adenylate Cyclase; Agonist; Architecture; Binding; Biochemical; Biotinylation; CXC Chemokines; CXCL12 gene; Cell membrane; Cells; Chemotaxis; Combined Modality Therapy; Computer Analysis; Cyclic AMP; Data; Data Set; Development; Disease; Dissociation; Engineering; Family; Future; G-Protein-Coupled Receptors; GTP Binding; GTP-Binding Proteins; GTPase-Activating Proteins; Generations; Guanine Nucleotide Dissociation Inhibitors; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Hydrogen Peroxide; Immunity; Immunologic Surveillance; Inflammation; Inflammatory; Knowledge; Location; Lymphocyte; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Methodology; Methods; Modeling; Molecular; Motion; Movement; Pathway interactions; Pattern; Phenotype; Physiologic pulse; Play; Process; Protein Isoforms; Proteins; Regulation; Resolution; Role; Signal Transduction; Signaling Protein; Techniques; Time; Tissues; Transducers; Variant; Work; base; candidate validation; cell motility; cell type; cellular transduction; chemokine receptor; cohesion; computer framework; experimental study; inhibitor; innovation; instrument; interest; macrophage; mathematical model; migration; novel; phenomenological models; receptor; spatiotemporal; temporal measurement; tool; trafficking

Heterotrimeric Gαβγ proteins are molecular switches that control eukaryotic signal transduction. In mammalian cell migration - a key process in immunity, inflammation, and development, - trimeric G proteins of the cAMP- inhibitory (Gi) family drive the movement of cells by transducing signals downstream of chemotactic G protein- coupled receptors such as the CXC chemokine receptor 4 (CXCR4). CXCR4 triggers heterotrimer dissociation at the plasma membrane, allowing βγ and GTP-bound Gαi to initiate their respective signaling cascades which, at the post-receptor level, are regulated and fine-tuned by numerous “accessory proteins”. Several hundred interactors have been described for the α-subunits of Gi proteins but their contribution to cell migration is not well understood. Best characterized chemotactic pathways are activated by Gβγ rather than Gαi, and for a long time, Gαi was considered but an instrument for timed release of Gβγ. Gαi is now increasingly appreciated as a signal transducer of its own, both via its cAMP-suppressing activity and via other, less studied effectors. The central aspect of Gαi signaling is that it must be spatially restricted and temporally coordinated between the different parts of the migrating cell. The intracellular gradients of Gαi activity and its ability to trigger both pro-migratory and anti-migratory signaling cascades on different time scales have been shown critical for directed cell motion. Unfortunately, the specific molecular interactions that maintain and interpret such asymmetric Gαi activity remain elusive, and so do the temporal aspects of their engagement. The objective of this proposal is to build a cohesive, spatiotemporally resolved picture of Gαi trafficking, activity, and regulation in live cells downstream of CXCR4. Through Aim 1 (Map the spatiotemporally resolved interactome of Gαi in CXCR4-expressing cells stimulated with CXCL12), we will determine when and where in the cell Gαi engages its known and so far undiscovered interactors in the course of CXCL12-triggered chemotactic signaling, with high spatial and temporal resolution, using the innovative approach of APEX2 proximity biotinylation. In Aim 2, we will fill an important methodological void by presenting a computational framework for converting APEX datasets for any desired target into systematic, large-scale, temporally resolved models of trafficking and regulation of that target in the signaling process of interest. By combining the Gαi APEX data of Aim 1 and tools of Aim 2, we will generate a model describing the trafficking and regulation of this key signal transducer in the process of CXCR4 migratory signaling. The model will help rationalize the observed phenotypic effects of perturbations, inform future experiments and, in the long run, assist target selection for novel combination therapies in inflammatory and other diseases. Altogether, this work will not only characterize Gαi trafficking and regulation in cell migration at an unprecedented resolution, but will also establish a framework for similar studies of Gi-mediated signaling in other cell types and tissues, and for dissecting tissue-selective signaling patterns downstream of multiple GPCRs.

异三聚体G-αβγ蛋白是控制真核细胞信号转导的分子开关。在哺乳动物中 细胞迁移-免疫、炎症和发育的关键过程-cAMP的三聚体G蛋白- 抑制性(GI)家族通过传递趋化G蛋白下游的信号来驱动细胞移动。 偶联受体，如CXC趋化因子受体4(CXCR4)。CXCR4引发杂三聚体解离 在质膜上，允许βγ和GTP结合的GαI启动它们各自的信号级联， 在受体后水平上，受许多“辅助蛋白”的调节和微调。 已经描述了数百个GI蛋白α亚基的相互作用因子，但它们对细胞的贡献 人们对迁徙的理解还不够深入。最具特征性的趋化途径是由Gβγ而不是GαI激活的， 在很长一段时间内，GαI被认为是一种定时释放Gβγ的工具。GαI现在越来越多地 被认为是一种自身的信号转导，既通过抑制cAMP的活性，也通过其他较少研究的 效应器。GαI信令的核心方面是它必须在空间上受到限制并在时间上进行协调 在迁移细胞的不同部分之间。G-αI活性的细胞内梯度及其触发能力 在不同的时间尺度上，支持和反对迁移的信号级联信号都被证明对 定向细胞运动。不幸的是，维持和解释这种现象的特定分子相互作用 不对称的GαI活动仍然难以捉摸，他们参与的时间方面也是如此。 这项提议的目标是建立一个连贯的、时空分辨率的GαI贩运、活动、 和CXCR4下游活细胞的调控。通过目标1(映射时空分辨率 CXCR4表达细胞中GαI的相互作用，我们将确定时间和地点 在细胞G中，αI在CXCL12触发的过程中参与其已知和迄今未被发现的相互作用 使用APEX2的创新方法，具有高空间和时间分辨率的趋化信号 邻位生物素化。在目标2中，我们将通过提出计算方法来填补一个重要的方法论空白 将任何期望目标的APEX数据集转换为系统的、大规模的、临时的框架 已解决的贩运模式和在感兴趣的信号传递过程中对该目标的管制。通过 结合目标1的GαI APEX数据和目标2的工具，我们将生成一个描述人口贩运的模型 以及CXCR4迁移信号转导过程中这个关键信号转导通路的调控。该模型将 帮助使观察到的扰动的表型效应合理化，为未来的实验提供信息，从长远来看， 协助选择炎症性疾病和其他疾病的新型联合疗法的靶点。 综上所述，这项工作不仅将表征GαI在细胞迁移中的贩运和调控处于前所未有的水平 但也将为其他细胞类型中GI介导的信号转导的类似研究建立一个框架 组织，并用于剖析多个GPCRs下游的组织选择性信号模式。