Mechanisms of spatiotemporal signaling by GPCRs

项目摘要 G蛋白偶联受体（GPCR）包括动物中最大的信号传导受体家族，因此， 它们代表了一类重要的治疗靶点。在配体结合和G蛋白活化后， 在细胞质膜上，GPCR经历受调节的内吞作用和分选以用于再循环或降解。传统上， 配体依赖性GPCR信号传导的研究集中在血浆中的受体-G蛋白偶联 膜（PM）。然而，现在清楚的是，GPCR可以继续从内膜位置发出信号， 并且胞内信号引起的下游反应与PM引起的下游反应不同 信号虽然已经详细研究了GPCR激活的生物化学，但GPCR的组织和调节仍然存在。 GPCR信号在活细胞中的作用仍然是一个未被探索的前沿。拟议的研究将调查 GPCR信号的时空调控的基础机制，并将提供关键 培训，使博士布莱斯成为一个独立的领导者，在这一新兴领域的分子和细胞 physiology.该项目的辅导阶段将在加州大学旧金山弗朗西斯科进行 在GPCR贩运和信号领域的领导者Mark von Zastrow博士的主要指导下。 这项工作的长期目标是了解（1）GPCR的亚细胞定位及其在细胞内的表达。 相关蛋白质的变化，以响应信号和（2）如何这种细胞重组调节其 不同的下游反应。前两个目标将集中在受体运输和信号传导，定义 独特的内吞（Aim 1）和再循环（Aim 2）途径通过以下机制塑造信号传导： 在HEK293细胞模型中内源性表达的GPCR。在进行这些实验时，布莱斯博士将获得 在先进的成像技术，以及在整体膜蛋白生物化学的新经验， Aashish Manglik博士（合作者）的帮助。目标3从更广泛的角度来构建生物学， 其它蛋白质的动态亚细胞定位有助于GPCR信号传导的时空调节。 在Nevan Krogan博士（共同导师）和Ruth博士的指导下使用一种新的邻近标记方法 Blythe博士将绘制特定细胞区室蛋白质组的变化 在相同模型GPCR的激活过程中，并探索这些变化如何决定信号传导。拟议 这项工作将能够对GPCR信号进行系统级分析，这在目前的方法中是不可行的， 为Blythe博士在基于质谱的蛋白质组学方面提供了宝贵的培训机会。总的来说， 该项目将利用多元化导师团队的专业知识和世界一流的资源， UCSF的设施来解决GPCR生物学中的基本问题。

Project Summary G protein-coupled receptors (GPCRs) comprise the largest family of signaling receptors in animals, and as such, they represent an important class of therapeutic targets. Following ligand binding and G protein activation at the plasma membrane, GPCRs undergo regulated endocytosis and sorting for recycling or degradation. Traditionally, studies of ligand-dependent GPCR signaling have focused on receptor-G protein coupling at the plasma membrane (PM). However, it is now clear that GPCRs can continue to signal from internal membrane locations and that the downstream responses elicited from intracellular signaling are distinct from those elicited from PM signaling. While the biochemistry of GPCR activation has been studied in detail, the organization and regulation of GPCR signaling in living cells remains an underexplored frontier. The proposed studies will investigate fundamental mechanisms underpinning spatiotemporal regulation of GPCR signaling and will provide key training to enable Dr. Blythe to become an independent leader in this emerging area of molecular and cellular physiology. The mentored phase of this project will be carried out at the University of California, San Francisco under the primary mentorship of Dr. Mark von Zastrow, a leader in the field of GPCR trafficking and signaling. The long-term goals of this work are to understand (1) how the subcellular localization of GPCRs and their associated proteins change in response to signaling and (2) how this cellular reorganization regulates their distinct downstream responses. The first two Aims will focus on receptor trafficking and signaling, defining the mechanisms by which unique endocytic (Aim 1) and recycling (Aim 2) pathways sculpt signaling by endogenously expressed GPCRs in a HEK293 cell model. In carrying out these experiments, Dr. Blythe will gain new experience in advanced imaging techniques, as well as in integral membrane protein biochemistry with the help of Dr. Aashish Manglik (collaborator). Aim 3 frames the biology in a broader perspective by asking how the dynamic subcellular localization of other proteins contributes to the spatiotemporal regulation of GPCR signaling. Using a novel proximity labeling approach under the mentorship of Dr. Nevan Krogan (co-Mentor) and Dr. Ruth Hüttenhain (collaborator), Dr. Blythe will map the changes in the proteomes of specific cellular compartments during the activation of the same model GPCRs and explore how these changes dictate signaling. The proposed work will enable a systems-level analysis of GPCR signaling that was not feasible with current approaches and provide an invaluable training opportunity for Dr. Blythe in mass spectrometry-based proteomics. In summary, this project will take advantage of the expertise of a diverse mentorship team and the world-class resources and facilities at UCSF to tackle fundamental questions in GPCR biology.