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: 9764313
• 负责人: Trang Huyen Duong
• 金额: $ 5万
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9764313
• 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; public health relevance; response; side effect; therapy development

 DESCRIPTION (provided by applicant): 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.

 描述(由申请人提供)：目前批准的药物中有相当一部分针对G蛋白偶联受体(GPCRs)，但大多数具有非靶向作用，其中许多包括牙科后遗症。当一个细胞中不同的GPCR通过类似的第二信使发出信号时，就会出现这种副作用。因此，一个GPCRs的刺激影响下游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，但它们如何在不交叉适应它们的信号的情况下独立地发出信号。了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的能力。我的首要目标是了解GPCR途径的适应如何能够被独立调节。这个问题将在线虫模型系统中进行研究，在该模型系统中，可以在单个嗅觉神经元中研究来自多个GPCR的信号。我们的实验室最近发现，一条内源RNAi(Endo-siRNA)途径促进了对气味物质发出的GPCR信号的适应。这一观察为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提供了一个框架。因此，这可以构成一种机制，通过该机制，高度相似的信号转导通路在同一细胞中彼此隔离。这导致我提出一个假设，即嗅觉神经元对G蛋白信号的适应是由不同的内源性RNAi生物合成途径参与的，这些途径作用于嗅觉GPCR刺激的下游。我将在三个目标中检验这一假设：在目标1中，我将使用行为分析来确定作用于GPCRs下游的内源RNAi加工因子，以促进对异戊醇(IAA)或丁酮(BU)的特别适应。初步数据表明，外切酶ERI-1是一个这样的因子，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来确定ERI-1是否事实上是适应IAA所唯一需要的，如果是，是通过什么机制。在目标3中，我将使用IAA或BU适应种群的高分辨率Endo-siRNA测序来确定是否产生特定的siRNA物种来适应每种气味。然后，我将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，这些结果将为深入了解GPCR信号如何在同一细胞内保持分离，以及内源性siRNA如何介导这一过程提供深入的见解。