Towards molecular mechanisms of invertebrate Gustatory Receptors

项目摘要 无脊椎动物味觉受体（Gustatory Receptors，GRs）是一个庞大的、进化上多样的感觉受体家族 已知在无脊椎动物的味觉、嗅觉和热传导中起重要作用。鉴于必须 这些感觉方式在重要的人类和疾病媒介，如蚊子，GR 家族成员作为载体控制剂的潜在强有力的靶标。然而，人们对 GR结构和功能。我们提出了两个GR的成员的生理和生化分析 亚家族：Gr43a和Gr28bD。这些初步研究将作为后续R01的先驱， 这些GR的结构和功能分析。 我们建议通过两个目标实现这些目标： 目的#1：鉴定和生理学表征Gr43a和Gr28bD的多种直系同源物。 与大多数GR不同，Gr43a和Gr28bD直系同源物可以在异源细胞中进行功能表征。在aim中 1.a.，我们将表达来自其他昆虫物种的这些GR的直系同源物，包括疾病载体， 极端微生物，在异源细胞和表征其生理特性。这将使 对各受体的序列和功能进行比较分析。 目的#2：生物化学表征多种Gr43a和Gr28bD直系同源物。我们找到了Gr43a， Gr28bD直系同源物可以从异源细胞中部分纯化。在目标2中，我们将把这种方法扩展到 并优化我们的纯化方案和探索关键 性质包括低聚状态和各种膜模拟物的热稳定性。这将提供 关于GR复合物的重要生物化学信息，并鉴定最适合用于后续研究的直系同源物。 结构分析 多种Gr43a和Gr28bD直系同源物的生理学表征将使得能够直接检查 GR家族功能的进化变异和保守性。多聚体的表达和纯化 家族成员将为生物化学和结构测定提供多种候选物， 后续GR结构测定成功的可能性。

PROJECT SUMMARY Invertebrate Gustatory Receptors (GRs) are a large and evolutionarily diverse family of sensory receptors known to play important roles in invertebrate taste, smell and thermotransduction. Given the importance of these sensory modalities in host-seeking behavior in important humand disease vectors like mosquitoes, GR family members serve as potentially powerful targets for vector control agents. However, little is known about GR structure and function. We propose a physiological and biochemical analysis of members of two GR subfamilies: Gr43a and Gr28bD. These initial studies will serve as a precursor for a subsequent R01 to carry out structural and functional analyses of these GRs. We propose to achieve these goals in two aims: Aim #1: Identify and physiologically characterize multiple orthologs of Gr43a and Gr28bD. Unlike most GRs, Gr43a and Gr28bD orthologs can be functionally characterized in heterologous cells. In aim 1.a., we will express orthologs of these GRs from additional insect species, including disease vectors and extremophiles, in heterologous cells and characterize their physiological properties. This will enable a comparative analysis of sequence and function among each receptor class. Aim #2: Biochemically characterize multiple Gr43a and Gr28bD orthologs. We find Gr43a and Gr28bD orthologs can be partially purified from heterologous cells. In aim 2, we will expand this approach to incorporate additional orthologs characterized in aim 1 and optimize our purification protocol and explore key properties including oligomeric state and thermal stability in various membrane mimics. This will provide important biochemical information about GR complexes and identify orthologs best suited for subsequent structural analysis. The physiological characterization of multiple Gr43a and Gr28bD orthologs will enable direct examination of evolutionary variation and conservation in GR family function. The expression and purification of multiple family members will provide multiple candidates for biochemistry and structural determination, maximizing the likelihood of success of subsequent GR structural determinations.