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

 描述（由申请人提供）：目前批准的药物中有很大一部分靶向G蛋白偶联受体（GPCR），但大多数具有脱靶效应，其中许多包括牙齿后遗症。当一个细胞中不同的GPCR通过相似的第二信使发出信号时，可能会出现这种副作用。因此，一个GPCR的刺激影响下游信号传导途径和细胞的其他GPCR的适应。事实上，该领域的一个关键问题是，不同的GPCR如何激活许多相同的第二信使，独立地发出信号，而不会交叉适应它们的信号。了解这种信号是如何分离的可能会导致开发限制串扰和脱靶效应的干预措施的能力。我的首要目标是了解如何适应GPCR途径可以独立调节。这个问题将在C. elegans模型系统，其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，内源性RNAi（endo-siRNA）途径促进气味剂对GPCR信号的适应。这一观察结果提供了一个框架来解释单个感觉神经元如何独立地适应多种气味。因此，这可能构成了一种机制，通过这种机制，高度相似的信号转导途径在同一细胞中彼此绝缘。这导致我的假设，即嗅觉神经元中G蛋白信号传导的适应特异性是由嗅觉GPCR刺激下游作用的不同内源性RNAi生物合成途径的参与指导的。我将在3个目标中测试这一假设：在目标1中，我将使用行为测定来鉴定作用于GPCR下游的内源性RNAi加工因子，以促进对异戊醇（IAA）或丁酮（BU）的特异性适应。初步数据表明，核酸外切酶ERI-1是一个这样的因素，因为它是所需的适应只有IAA。在目标2中，我将使用生物化学和视觉手段来确定ERI-1是否实际上是适应IAA所必需的，如果是，则通过什么机制。在目标3中，我将使用IAA或BU适应群体的高分辨率内切siRNA测序来确定是否产生了适应每种气味的特定siRNA种类。然后，我将确定这些序列中的任何一个或去除它们的靶点是否足以促进IAA适应。总之，这些结果将提供深入了解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。

 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.