Mechanisms of odor detection and transduction

气味检测和传导机制

• 批准号: 8490342
• 负责人: Steven D Munger
• 金额: $ 18.23万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8490342
• 关键词: Address; Afferent Neurons; Aging; Amino Acids; Antibodies; Area; Arrestins; Binding; Biological; Biology; Brain; Calmodulin; Cardiovascular Diseases; Cell membrane; Cells; Cilia; Co-Immunoprecipitations; Complex; Consumption; Cyclic AMP; Dendritic Spines; Desire for food; Detection; Development; Diabetes Mellitus; Disease; Drosophila genus; Elements; Enzymes; Family member; Food; Food Processing; Gene Targeting; Goals; Health; Human; Hygiene; Hypertension; Immune Sera; Immunohistochemistry; Immunoprecipitation; In Situ Hybridization; In Vitro; Incidence; Injury; Intervention; Kinetics; Label; Lead; Life; Liquid Chromatography; Macromolecular Complexes; Mass Spectrum Analysis; Membrane; Membrane Lipids; Mental Depression; Molecular; Molecular Chaperones; Mus; NGFRAP1 gene; Neurons; Obesity; Odorant Receptors; Odors; Olfactory Epithelium; Peripheral; Phenotype; Phosphotransferases; Procedures; Process; Protein Family; Proteins; Proteomics; Quality of life; Research; Role; Scaffolding Protein; Signal Transduction; Specificity; Stable Isotope Labeling; Stimulus; System; Translating; Validation; Withdrawal; Yang; adenylyl cyclase III; base; biological systems; cell type; gel electrophoresis; human RIPK1 protein; in vivo; insight; interdisciplinary approach; novel; novel strategies; olfactory marker protein; programs; protein protein interaction; receptor; relating to nervous system; response; scaffold; social; tandem mass spectrometry; trafficking

DESCRIPTION (provided by applicant): Many of the molecules that are critical for the detection and transduction of odors by olfactory sensory neurons (OSNs) have been identified, and their basic roles in these processes defined. However, significant gaps in our understanding of the olfactory transduction process remain. For example, signal transduction cascades found in many neurons function within signaling complexes composed of receptors, effector enzymes, channels, scaffolding elements and other molecules. These "signalplexes" may enhance both the efficiency and specificity of signaling by increasing the local concentration of signaling elements (e.g., receptors, enzymes and soluble messengers such as cAMP or Ca2+), restricting proteins to functionally important cellular domains (e.g., dendritic spines or dendritic cilia), orby regulating access of "modulatory" proteins (e.g., receptor kinases, ¿-arrestin). However, little is known about how olfactory transduction proteins interact to impact olfactory function. Our proposed studies will address this important yet understudied area of olfactory biology by using a cutting-edge, multidisciplinary approach to define protein-protein interactions for key components of the olfactory transduction cascade. Our studies will focus on identifying proteins that directly interact with two key olfactory transduction molecules: olfactory marker protein (OMP) and canonical odorant receptors (OR). While both OMP and the ORs have been shown to interact with other OSN proteins (Bex proteins in the case of OMP and receptor trafficking protein (RTP) family members in the case of ORs), functional studies suggests that other partners exist for both proteins. For example, OMP influences cAMP kinetics and Ca2+ dynamics in OSNs, while native ORs are hypothesized to require additional OSN-specific co-receptors or chaperones to efficiently target the plasma membrane. Recent advances in proteomics now offer a unique opportunity to both validate previously implicated OMP and OR interactors as well as to identify novel proteins that associate with these key transduction molecules. In this proposal, the P.I.s will take advantage of our complementary expertise in olfactory transduction and state-of-the-art proteomics approaches to complete two parallel Specific Aims focused on identifying protein interactors for two "baits:" OMP (Aim 1) and the heptanal-responsive OR I7 (Aim 2). We will use stable isotope labeling of mice expressing different levels of the either bait, followed by immunoprecipitation of interacting complexes from native olfactory epithelium and subsequent liquid chromatography-tandem mass spectroscopy to quantitatively isolate specific interactors with high sensitivity. These studies will result in significant advances in our understanding of odor detection and transduction, and will establish an important new approach for characterizing the interactions of rare proteins in nearly any biological system.

描述(申请人提供)：许多对嗅觉感觉神经元(OSN)的气味检测和转导至关重要的分子已经被识别，并确定了它们在这些过程中的基本作用。然而，我们对嗅觉转导过程的理解仍然存在重大差距。例如，在许多神经元中发现的信号转导级联在由受体、效应酶、通道、支架元件和其他分子组成的信号复合体中发挥作用。这些“信号丛”可通过增加信号元件(如受体、酶和可溶性信使，如cAMP或Ca~(2+))的局部浓度，将蛋白质限制在具有重要功能的细胞域(如树突棘或树突纤毛)，或通过调节“调节”蛋白质的通路(如受体激酶、β-arrestin)来提高信号传递的效率和特异性。然而，几乎没有什么是 了解嗅觉转导蛋白如何相互作用影响嗅觉功能。我们建议的研究将通过使用前沿的、多学科的方法来定义嗅觉转导级联的关键组件的蛋白质-蛋白质相互作用，来解决这一重要但研究不足的嗅觉生物学领域。我们的研究将集中在识别直接与两个关键的嗅觉转导分子相互作用的蛋白质：嗅觉标记蛋白(OMP)和典型气味受体(OR)。虽然OMP和ORS都被证明与其他OSN蛋白(OMP中的Bex蛋白和ORs中的受体运输蛋白(RTP)家族成员)相互作用，但功能研究表明，这两种蛋白都存在其他伴侣。例如，OMP影响OSN中的cAMP动力学和钙动力学，而天然ORs被认为需要额外的OSN特异性辅助受体或伴侣来有效靶向质膜。蛋白质组学的最新进展现在提供了一个独特的机会，既可以验证先前涉及的OMP和OR相互作用因子，也可以识别与这些关键转导分子相关的新蛋白质。在这项提案中，P.I.s将利用我们在嗅觉转导和最先进的蛋白质组学方法方面的互补专业知识来完成两个平行的特定目标，重点是确定两个“诱饵”的蛋白质相互作用：OMP(Aim 1)和庚醛反应OR I7(Aim 2)。我们将使用稳定同位素标记表达不同水平的这两种诱饵的小鼠，然后从天然嗅觉上皮中免疫沉淀相互作用的复合体，并随后进行液相色谱-串联质谱分析，以高灵敏度定量分离特定的相互作用。这些研究将在我们对气味检测和转导的理解方面取得重大进展，并将建立一种重要的新方法来表征几乎任何生物系统中稀有蛋白质的相互作用。