Molecular Mechanisms Underlying Odorant Recognition

描述（由申请人提供）：我们的长期目标是了解如何在外周感觉器官中检测到化学物质，如何在大脑中处理信息以识别化学物质，以及大脑如何指导适当的行为反应。这些过程对动物生存和延续物种至关重要。如果没有功能性的嗅觉系统，动物将很难发现食物来源，捕食者，猎物，交配伴侣或环境中的有毒化学物质。对于人类来说，嗅觉是很重要的，原因有很多，从享受美食到检测有害气体泄漏。尽管哺乳动物的气味受体在10年前就被克隆出来了，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。在这个拟议的研究项目中，我们打算确定一组识别特定气味的气味受体，并确定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确定一组气味受体识别特定的气味在小鼠中，并确定一组气味受体识别特定的气味。(2)目的：建立小鼠嗅上皮和嗅球嗅觉受体功能的模型，并对其进行体内分析。(3)目的：通过观察b-arrestin 2的定位，建立一种检测气味受体激活的方法，并鉴定特异性结构域和氨基酸残基，指导气味与气味受体结合。 我们将首先开发和使用一种新的方法来识别多种气味受体，识别特定的脂肪族气味在小鼠中，通过结合光学成像在嗅球，逆行追踪，和克隆的气味受体从单个嗅觉感觉神经元使用改进的单细胞RT-PCR方法。利用特定目标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用绿色荧光蛋白（GFP）起源于它们的轴突。我们将使用这些小鼠来分析气味受体的功能，通过在嗅球以及单个嗅觉感觉神经元中进行功能成像实验。最后，我们计划开发一种替代方法来测量气味受体激活通过观察本地化的b-arrestin 2-GFP融合蛋白在HEK 293细胞。通过使用这种异源表达系统或其他方法，我们计划分析确定气味结合的气味受体中的结构域和氨基酸。

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.