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

异源三聚体Gαβγ蛋白是控制真核生物信号转导的分子开关。在哺乳动物 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）家族通过转导趋化性G蛋白下游的信号来驱动细胞运动- 偶联受体如CXC趋化因子受体4（CXCR 4）。CXCR 4触发异源三聚体解离 在质膜上，允许βγ和GTP结合的Gαi启动各自的信号级联， 在后受体水平，由许多“辅助蛋白”调节和微调。 已经描述了几百种Gi蛋白α亚基的相互作用物，但它们对细胞增殖的贡献是不稳定的。 人们对移徙问题还不太了解。最佳表征的趋化途径由Gβγ而不是Gαi激活， 长期以来，Gαi被认为只是一种定时释放Gβγ的工具。Gαi现在越来越 作为一种信号转导器，通过其cAMP抑制活性和其他研究较少的 效应器Gαi信号传导的核心方面是它必须在空间上受到限制，在时间上协调 在迁移细胞的不同部分之间。Gαi活性的细胞内梯度及其触发 在不同时间尺度上的促迁移和抗迁移信号级联已经显示出对于 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活性仍然难以捉摸，它们参与的时间方面也是如此。 该提案的目标是建立一个有凝聚力的，时空解决的Gαi贩运，活动， 以及CXCR 4下游活细胞中的调节。通过目标1（映射时空分辨 在用CXCL刺激的CXCR 4表达细胞中Gαi的相互作用组（12），我们将确定何时以及在何处 在细胞中，Gαi在CXCL 12触发的过程中与其已知的和迄今未发现的相互作用物结合。 趋化性信号传导，具有高空间和时间分辨率，使用APEX 2的创新方法 邻近生物素化。在目标2中，我们将通过提出一个计算方法来填补一个重要的方法学空白。 一个框架，用于将任何所需目标的APEX数据集转换为系统的，大规模的，时间上的 解决了贩运模型和在感兴趣的信号过程中对该目标的调节。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贩运的模型 以及在CXCR 4迁移信号传导过程中对这一关键信号转导子的调控。该模型将 有助于合理化观察到的扰动表型效应，为未来的实验提供信息，从长远来看， 有助于炎症和其他疾病中新型联合治疗的靶点选择。 总之，这项工作不仅将以前所未有的速度表征Gαi的运输和细胞迁移的调节， 解决，但也将建立一个框架，在其他细胞类型的Gi介导的信号类似的研究， 组织，并用于解剖多个GPCR下游的组织选择性信号传导模式。

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.