Mechanisms of spatiotemporal signaling by GPCRs

GPCR 的时空信号传导机制

• 批准号: 10722825
• 负责人: Emily Elizabeth Blythe
• 金额: $ 12.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10722825
• 关键词: Adenosine; Adrenergic Receptor; Affect; Agonist; Animals; Area; Ascorbic Acid; Behavior; Biochemical; Biochemistry; Biological Assay; Biology; California; Cardiovascular Physiology; Cell membrane; Cell model; Cell physiology; Cells; Coupled; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Disease; Endocrine; Endocytosis; Endosomes; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Imaging Techniques; In Vitro; Integral Membrane Protein; Knowledge; Label; Ligand Binding; Ligands; Link; Location; Maps; Mass Spectrum Analysis; Membrane; Mentors; Mentorship; Modeling; Molecular; Mutate; Outcome; Output; Pathway interactions; Perception; Peroxidases; Phase; Phosphorylation Site; Physiological Processes; Polypeptide Hormones; Postdoctoral Fellow; Protein Biochemistry; Protein translocation; Proteins; Proteome; Proteomics; Receptor Activation; Receptor Signaling; Recycling; Regulation; Research; Resources; San Francisco; Sensory; Signal Transduction; Signal Transduction Pathway; Sorting; System; Therapeutic; Total Internal Reflection Fluorescent; Training; Universities; VIPR1 gene; Vasoactive Intestinal Peptide Receptors; Work; beta-arrestin; design; experience; experimental study; frontier; insight; novel; protein activation; receptor; receptor internalization; receptor recycling; reconstitution; response; skills; spatiotemporal; therapeutic target; tool; trafficking; training opportunity

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.

项目摘要 G蛋白偶联受体(GPCRs)是动物中最大的信号受体家族，因此， 它们代表了一类重要的治疗靶点。在配体结合和G蛋白激活之后 质膜，GPCRs经历调节的内吞作用和分类回收或降解。传统上， 对配体依赖的GPCR信号的研究主要集中在血浆中受体-G蛋白的偶联 膜(PM)。然而，现在很明显，GPCRs可以继续从内部膜位置发出信号 细胞内信号引起的下游反应与PM引起的下游反应是不同的 发信号。虽然对gpr激活的生物化学已经进行了详细的研究，但它的组织和调控 活细胞中GPCR信号的研究仍然是一个未被探索的前沿。拟议的研究将调查 支持GPCR信号时空调控的基本机制，并将提供关键 培训使布莱斯博士成为这一新兴的分子和细胞领域的独立领导者 生理学。该项目的指导阶段将在加州大学旧金山分校进行 在马克·冯·扎斯特罗博士的主要指导下，他是GPCR贩运和信号领域的领导者。 这项工作的长期目标是了解(1)GPCRs的亚细胞定位及其 相关蛋白质对信号的响应和(2)这种细胞重组如何调节它们的 截然不同的下游响应。前两个目标将集中在受体贩运和信号传递上，定义 独特的内吞(目标1)和循环(目标2)通路塑造信号的机制 在HEK293细胞模型中内源性表达GPCRs。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整体膜蛋白生物化学方面的新经验 Aashish Manglik博士(合作者)的帮助。Aim 3通过询问如何在更广泛的角度来构建生物学 其他蛋白质的动态亚细胞定位有助于GPCR信号的时空调控。 在内万·克罗根博士(共同导师)和露丝博士的指导下使用一种新的邻近标记方法 Hüttenhain(合作者)，Blythe博士将绘制特定细胞隔间蛋白质组的变化图 在相同模型的激活期间，GPCR并探索这些变化如何指示信令。建议数 这项工作将使GPCR信令的系统级分析成为可能，而目前的方法和 为Blythe博士提供了基于质谱学的蛋白质组学方面的宝贵培训机会。总而言之， 该项目将利用多样化的指导团队的专业知识和世界级的资源和 加州大学旧金山分校的设施，以解决GPCR生物学中的基本问题。