Mechanisms of spatiotemporal signaling by GPCRs

GPCR 的时空信号传导机制

基本信息

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
项目类别：
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

项目摘要

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（GPCR）包括动物中最大的信号受体家族，因此，它们代表着重要的治疗靶标。遵循配体结合和G蛋白在质膜，GPCR会经历调节的内吞作用，并分类以进行回收或降解。传统上，配体依赖性GPCR信号的研究集中在血浆处的受体-G蛋白偶联上膜（PM）。但是，现在很明显GPCR可以继续从内部膜位置发出信号并且从细胞内信号引起的下游响应与PM引起的反应不同信号。虽然GPCR激活的生物化学已详细研究，但组织和监管活细胞中的GPCR信号传导仍然是一个未充满活力的边界。拟议的研究将调查 GPCR信号的时空调节基础的基本机制，将提供关键训练使Blythe博士成为分子和细胞的新兴领域的独立领导者生理。该项目的修订阶段将在加利福尼亚大学旧金山分校进行在GPCR贩运和信号传导领域的领导者Mark von Zastrow博士的主要心态下。这项工作的长期目标是了解（1）GPCR的亚细胞定位如何相关的蛋白质响应信号转导和（2）该细胞重组如何调节其独特的下游反应。前两个目标将集中于受体运输和信号，定义独特的内吞（AIM 1）和回收（AIM 2）途径雕刻信号传导的机制在HEK293细胞模型中内源表达的GPCR。在进行这些实验时，Blythe博士将获得高级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通过询问如何以更广泛的角度构建生物学其他蛋白质的动态亚细胞定位有助于GPCR信号的时空调节。在内凡·克罗根（Nevan Krogan）博士（Co-Incertor）和露丝（Ruth）的心态下，使用新颖的接近标签方法 Hüttenhain（合作者），Blythe博士将绘制特定蜂窝室蛋白质组的变化在激活同一模型GPCR并探讨这些变化如何决定信号传导的过程中。提议工作将实现对GPCR信号的系统级分析，而当前方法和为基于质谱的蛋白质组学中的Blythe博士提供宝贵的培训机会。总之，该项目将利用多元化团队和世界一流资源的专业知识以及 UCSF的设施解决GPCR生物学中的基本问题。