G-Protein Coupled Receptor Signaling: a Role for Small Interfering RNAs in Stimulus Specific Adaptation

G 蛋白偶联受体信号转导：小干扰 RNA 在刺激特异性适应中的作用

• 批准号: 10426999
• 负责人: Trang Huyen Duong
• 金额: $ 1.17万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10426999
• 关键词: Affect; Afferent Neurons; Animals; Behavioral Assay; Benzaldehyde; Biochemical; Biological Models; Butanones; Caenorhabditis elegans; Cells; Chronic; Clinical; Complex; Cyclic GMP-Dependent Protein Kinases; Data; Dental; Drug Design; Drug Targeting; Excision; Exonuclease; Exoribonucleases; Exposure to; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genes; Goals; Hydrocodone; Individual; Intervention; Lead; Libraries; Mechanics; Mediating; Messenger RNA; Methadone; Modeling; Mutation; Nematoda; Neurobiology; Neurons; Odors; Olfactory Pathways; Oxycodone; Pain management; Pathway interactions; Pharmaceutical Preparations; Population; Process; Production; RNA Interference; Resolution; Ribonucleases; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Small Interfering RNA; Specificity; Stimulus; System; Testing; Visual; Work; addiction; cohort; desensitization; drug discovery; drug market; follow-up; insight; isopentyl alcohol; member; mutant; olfactory sensory neurons; response; side effect; therapy development

PROJECT SUMMARY A significant portion of currently approved drugs target G Protein Coupled Receptors (GPCRs) but most have off-target effects and many of these include dental sequelae. Such side effects can arise when distinct GPCRs in one cell signal through similar second messengers. Thus, the stimulation of one GPCR affects the down- stream signaling pathway and adaptation of the cell's other GPCRs. Indeed, one key question in the field is how different GPCRs, though they activate many of the same second messengers, signal independently without cross adaptation of their signals. Understanding how this signaling is segregated may lead to the ability to develop interventions that limit crosstalk and off-target effects. My overarching goal is to understand how adaptation of GPCR pathways can be independently regulated. This question will be studied in the C. elegans model system, where signaling from multiple GPCRs can be studied in individual olfactory neurons. Our lab recently discovered that an endogenous RNAi (endo-siRNA) pathway promotes adaptation to GPCR signaling by odorants. This observation provides a framework to explain how multiple odors sensed by a single sensory neuron are adapted to independently. This may thus constitute one mechanism by which highly similar signal transduction pathways are insulated from each other in the same cell. This leads me to the hypothesis that the specificity of adaptation to G protein signaling in olfactory neurons is directed by the engagement of different endogenous RNAi biosynthetic pathways acting downstream of olfactory GPCR stimulation. I will test this hypothesis in 3 aims: In aim 1, I will use behavioral assays to identify endogenous RNAi processing factors that act downstream of GPCRs to promote adaptation specifically to either isoamyl alcohol (IAA) or butanone (BU). Preliminary data suggests that the exonuclease ERI-1 is one such factor as it is required for adaptation only to IAA. In aim 2, I will use biochemical and visual means to determine whether ERI-1 is, in fact uniquely required for adaptation to IAA and if so, by what mechanism. In aim 3, I will use high-resolution endo-siRNA sequencing of either IAA or BU adapted populations to determine if specific siRNA species are produced in adaptation to each odor. I will then determine whether any of these sequences or removal of their targets are sufficient to promote IAA adaptation. Together, these results will provide insight into how GPCR signaling can remain separated within the same cell, and how endogenous siRNA mediates this process.

项目总结 目前批准的药物中有相当一部分针对G蛋白偶联受体(GPCRs)，但大多数 偏离目标的影响，其中许多包括牙齿后遗症。当不同的GPCR时，可能会出现这种副作用 在一个细胞中，通过相似的第二信使发出信号。因此，一个GPCR的刺激会影响下行- 流信号通路和细胞其他GPCRs的适应。事实上，该领域的一个关键问题是 尽管不同的GPCR激活了许多相同的第二信使，但它们是如何独立发出信号的 而不需要交叉调整它们的信号。理解这种信号是如何分离的可能会导致 开发限制串扰和偏离目标影响的干预措施。我的首要目标是了解 GPCR途径的适应可以独立调节。 这个问题将在线虫模型系统中进行研究，在该模型系统中，来自多个GPCR的信号可以 在单个嗅觉神经元上进行了研究。我们实验室最近发现了一种内源性RNAi(Endo-siRNA) PATH促进气味物质对GPCR信号的适应。这一观察结果提供了一个框架 解释单个感觉神经元感受到的多种气味是如何独立适应的。这可能会因此 构成了高度相似的信号转导通路相互隔离的一种机制 在同一间牢房里。这导致了我的假设，适应G蛋白信号转导的特异性 嗅觉神经元受不同内源性RNAi生物合成途径的影响 作用于嗅觉GPCRs刺激的下游。我将在三个目标中测试这个假设：在目标1中，我将使用 行为分析以确定作用于GPCRs下游的内源性RNAi加工因子促进 对异戊醇(IAA)或丁酮(BU)的特别适应。初步数据显示， 核酸外切酶ERI-1就是这样一个因子，因为它只是适应IAA所必需的。在《目标2》中，我将使用 生化和视觉手段，以确定ERI-1是否确实是适应IAA所唯一需要的 如果是这样的话，是通过什么机制。在目标3中，我将使用IAA或BU的高分辨率Endo-siRNA测序 适应种群以确定是否产生特定的siRNA物种以适应每种气味。那我会的 确定这些序列中的任何一个或其靶标的移除是否足以促进IAA适应。 综上所述，这些结果将有助于深入了解GPCR信号如何保持在相同的 细胞，以及内源性siRNA如何介导这一过程。