Towards molecular mechanisms of invertebrate Gustatory Receptors

项目总结 无脊椎动物味觉受体(GRs)是一个进化上多样化的感觉受体大家族 已知在无脊椎动物的味觉、嗅觉和热传导中起着重要作用。鉴于……的重要性 这些感觉模式在重要的体液和疾病媒介如蚊子中寻找宿主行为 家庭成员是媒介控制媒介潜在的强大目标。然而，人们对此知之甚少。 GR的结构和功能。我们建议对两个GR成员进行生理生化分析 亚科：Gr43a和Gr28bd。这些初步研究将作为后续R01进行的先导 对这些GRs进行了结构和功能分析。 我们建议通过两个目标实现这些目标： 目的#1：鉴定Gr43a和Gr28bD的多个同源基因并鉴定其生理特性。 与大多数GRs不同，Gr43a和Gr28bD同源基因可以在异源细胞中进行功能鉴定。在AIM 1.a.，我们将表达来自其他昆虫物种的这些GR的同源基因，包括病媒和 异种细胞中的极端细菌，并对其生理特性进行表征。这将使 每类受体的序列和功能的比较分析。 目的#2：多个Gr43a和Gr28bD同源基因的生化特征。我们发现Gr43a和 Gr28bD同源蛋白可从异种细胞中部分纯化。在目标2中，我们将把这种方法扩展到 整合目标1中描述的其他同源基因，并优化我们的纯化方案和探索关键 在各种膜模拟物中的性能包括齐聚状态和热稳定性。这将提供 有关GR复合体的重要生化信息，并确定最适合后续研究的同源基因 结构分析。 多个Gr43a和Gr28bD同源基因的生理特性将使直接检查 GR家族功能的进化变异和保守性。多聚体的表达和纯化 家庭成员将提供多个生物化学和结构确定的候选对象，最大限度地 随后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.