Single Molecule Analysis of Olfactory Receptor Assembly

描述（由申请人提供）：该项目的重点是解读控制嗅觉受体从几个亚基组装成同源或异源蛋白的机制，并确定与辅助蛋白的相互作用。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理解嗅觉的接收是如何工作的至关重要。嗅觉是一种重要的感觉，可以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7跨膜结构域g蛋白偶联受体，但其膜拓扑结构和转导机制存在差异。了解不同物种嗅觉受体之间的异同将有助于发现嗅觉的共同原理。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表达依赖于辅助蛋白的共同表达，辅助蛋白帮助受体运输到细胞表面。其中一些蛋白质被认为与受体结合形成多蛋白复合物。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，将分析非洲爪蟾卵母细胞中的两种脊椎动物受体和一种昆虫嗅觉受体复合物。一种基于荧光标记单分子可视化的新方法可以直接观察不同蛋白质之间的关联，并计算复合物中每种蛋白质的亚基数。在K99期后期和R00期，我们建议使用HEK293细胞作为气味受体和辅助蛋白之间相互作用重建的表达系统。这将允许分析在没有辅助蛋白的非洲爪蟾卵母细胞中不能表达的受体。相关性：嗅觉和味觉是任何生物体生存的基本感官。这些感觉是由专门的神经细胞中的嗅觉和味觉受体介导的。通过破译气味受体如何从不同的亚基组装，我们了解了它们如何与细胞中的其他成分相互作用。最终，这将增加我们对我们如何感知气味以及如何处理这些信息的理解。

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.