Molecular Mechanisms Underlying Odorant Recognition

描述(由申请者提供)：我们的长期目标是了解化学物质是如何在外周感觉器官中被检测到的，信息是如何在大脑中处理以识别化学物质的，以及大脑是如何引导适当的行为反应的。这些过程是动物生存和延续其物种所必需的。如果没有功能性的嗅觉系统，动物将很难检测到食物来源、捕食者、猎物、交配伙伴或环境中的有毒化学物质。对于人类来说，嗅觉很重要，原因有很多，从享用食物到检测有害气体泄漏。虽然哺乳动物的气味受体在10多年前就被克隆了，但人们对不同的气味分子如何与气味受体相互作用知之甚少。在这项拟议的研究项目中，我们打算识别一组识别特定气味的气味受体，并确定气味受体中决定气味结合的氨基酸和基序。 我们提出了三个具体的目标：(1)建立一种新的方法来识别一组识别特定气味的小鼠气味受体，并识别一组识别特定气味的气味受体。(2)：建立和分析用于体内分析嗅觉上皮和大脑嗅球气味感受器功能的模型鼠。(3)：建立一种通过观察b-arrestin2的定位来测量气味受体激活的方法，并鉴定与气味受体直接结合的特定结构域和氨基酸残基。 我们将首先开发并使用一种新的方法来识别小鼠身上识别特定脂肪气味的多个气味受体，方法是结合嗅球的光学成像、逆行追踪以及使用改进的单细胞RT-PCR方法克隆单个嗅觉神经元的气味受体。利用在特定目标1中获得的信息，我们接下来将创建转基因小鼠系，以遗传标记表达特定气味受体的特定嗅觉感觉神经元以及来自它们的轴突，并使用绿色荧光蛋白(GFP)。我们将利用这些小鼠，通过在嗅球和单个嗅觉神经元中进行功能成像实验，来分析气味感受器的功能。最后，我们计划开发一种替代方法来测量气味受体的激活，通过观察b-arrestin2-GFP融合蛋白在HEK293细胞中的定位。通过使用这个异源表达系统或其他方法，我们计划分析气味受体中决定气味结合的结构域和氨基酸。

DESCRIPTION (provided by applicant): Our long-term objective is to understand how chemicals are detected in the peripheral sense organ, how the information is processed in the brain to recognize chemicals, and how the brain directs appropriate behavioral responses. These processes are essential for animals to survive and continue their species. Without a functional olfactory system, animals would have difficulty detecting food sources, predators, prey, mating partners, or noxious chemicals in the environment. In humans, a sense of smell is important for a variety of reasons, from enjoying a meal, to detecting harmful gas leakage. Although mammalian odorant receptors were cloned over 10 years ago, little is known how different odorant molecules interact with odorant receptors. In this proposed research project, we intend to identify a set of odorant receptors that recognize particular odorants and to identify amino acids and motifs in the odorant receptors that determine odorant binding. We propose three specific aims: (1): To establish a novel method to identify a set of odorant receptors that recognize particular odorants in mice, and to identify a set of odorant receptors that recognize particular odorants. (2): To create and analyze model mice for in vivo analysis of odorant receptor functions in the olfactory epithelium and in the olfactory bulb of the brain. (3): To develop a method to measure odorant receptor activation by observing b-arrestin2 localization and to identify of specific domains and amino acid residues that direct odorant binding to odorant receptors. We will first develop and use a novel method to identify multiple odorant receptors that recognize particular aliphatic odorants in mice, by combining optical imaging in the olfactory bulb, retrograde tracing, and cloning of odorant receptors from single olfactory sensory neurons using an improved single-cell RT-PCR method. Using the information gained in Specific Aim 1, we will next create transgenic mouse lines to genetically mark specific olfactory sensory neurons that express particular odorant receptors as well as axons that originate from them with green fluorescent protein (GFP). We will use these mice to analyze functions of odorant receptors by performing functional imaging experiments in the olfactory bulb as well as in single olfactory sensory neurons. Finally, we plan to develop an alternative method to measure odorant receptor activation by observing localization of b-arrestin2-GFP fusion proteins in HEK293 cells. By using this heterologous expression system or other methods, we plan to analyze domains and amino acids in the odorant receptors that determine odorant binding.