Single Molecule Analysis of Olfactory Receptor Assembly

嗅觉感受器组装的单分子分析

• 批准号: 7643506
• 负责人: Maximilian H Ulbrich
• 金额: $ 8.92万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7643506
• 关键词: Afferent Neurons; Architecture; Biology; Cell Line; Cell membrane; Cell physiology; Cell surface; Cells; Chemicals; Complex; Dimerization; Drosophila genus; Environment; Eugenol; G-Protein-Coupled Receptors; Genes; Goals; Goldfish; Homo; Imagery; Insecta; Investments; Ion Channel; Knowledge; Label; Mammalian Cell; Mediating; Membrane; Methods; Molecular; Mus; N-terminal; Neurons; Nutrient; Odorant Receptors; Oocytes; Organism; Phase; Poison; Process; Protein Family; Proteins; Receptor Gene; Research Personnel; Rhodopsin; Signal Transduction; Smell Perception; Staging; System; Systems Analysis; Taste Perception; Techniques; Testing; Transmembrane Domain; Work; Xenopus laevis; Xenopus oocyte; base; dimer; insight; interest; mammalian genome; monomer; novel; olfactory receptor; programs; protein complex; receptor; receptor coupling; receptor function; reconstitution; research study; single molecule; stoichiometry

DESCRIPTION (provided by applicant): The focus of this project is to decipher the mechanisms that govern the assembly of olfactory receptors from several subunits into homo- or heteromeric proteins and identify interactions with accessory proteins. Gaining insight into these assembly mechanisms and the analysis of the composition of olfactory receptors is crucial for a deeper understanding of how the reception of smell works. Smell is an important sense that helps us discriminate a myriad of nutrients and toxic substances. Mammalian and insect olfactory receptors are both seven-transmembrane-domain G-protein coupled receptors, but the membrane topology and the transduction mechanism have been shown to diverge. To understand the similarities and differences between olfactory receptors from different species will help to find common principles underlying olfaction. Olfactory receptors are thought to require dimerization to function. However, functional expression in heterologous cells depends on the co-expression of accessory proteins which help transport of the receptors to the cell surface. Some of these proteins are proposed to associate with the receptors to form multi-protein complexes. The experiments proposed intend to determine the number of subunits of the different receptor components, and the associations between them. At the beginning of the K99 phase, two vertebrate receptors and one insect olfactory receptor complex will be analyzed in Xenopus laevis oocytes. A novel method based on the visualization of fluorescently labeled single molecules allows the direct observation of associations between different proteins and counting of the subunit number of each protein in the complex. In the later stages of the K99 phase and in the R00 phase, we propose to use HEK293 cells as an expression system for the reconstitution of interactions between odorant receptors and accessory proteins. This will allow the analysis of receptors which cannot be expressed in Xenopus laevis oocytes without accessory proteins. RELEVANCE: Smell and taste are fundamental senses for the survival of any organism. These senses are mediated by odorant and taste receptors in specialized nerve cells. By deciphering how odorant receptors assemble from different subunits, we gain insight on how they interact with other components in the cell. Eventually, this will increase our understanding of how we sense smell and how this information is processed.

描述（由申请人提供）：该项目的重点是破译控制嗅觉受体从几个亚基组装成同源或异源蛋白质的机制，并鉴定与辅助蛋白质的相互作用。深入了解这些组装机制和分析嗅觉受体的组成对于更深入地了解气味的接收是至关重要的。它是一种重要的感觉，帮助我们区分无数的营养物质和有毒物质。哺乳动物和昆虫的嗅觉受体都是七跨膜结构域的G蛋白偶联受体，但膜拓扑结构和转导机制已被证明是不同的。了解不同物种的嗅觉感受器之间的相似性和差异性将有助于找到嗅觉的共同原理。嗅觉受体被认为需要二聚化才能发挥作用。然而，在异源细胞中的功能性表达依赖于辅助蛋白的共表达，辅助蛋白有助于受体转运至细胞表面。这些蛋白质中的一些被提议与受体结合以形成多蛋白质复合物。提出的实验旨在确定不同受体成分的亚基数量以及它们之间的关联。在K99期开始时，将在非洲爪蟾卵母细胞中分析两种脊椎动物受体和一种昆虫嗅觉受体复合物。基于荧光标记的单分子可视化的新方法允许直接观察不同蛋白质之间的关联，并计算复合物中每个蛋白质的亚基数目。在K99阶段的后期和R00阶段，我们建议使用HEK293细胞作为表达系统，用于重建气味受体和辅助蛋白之间的相互作用。这将允许受体的分析，不能表达在非洲爪蟾卵母细胞没有辅助蛋白。相关性：嗅觉和味觉是任何生物生存的基本感官。这些感觉是由特殊神经细胞中的气味和味觉受体介导的。通过破译气味受体如何从不同的亚基组装，我们可以深入了解它们如何与细胞中的其他成分相互作用。最终，这将增加我们对如何感知气味以及如何处理这些信息的理解。