Opsins as a new class of evolutionarily conserved taste receptors: Diversity Supplement for Pre-Doctoral Training

摘要 视蛋白是一种经典的G蛋白偶联受体，世纪以来一直被认为是视蛋白的功能受体 专门用作光探测器。然而，利用果蝇，我们已经发现了多种不依赖于光的作用， 用于视蛋白，包括检测苦味促味剂。这一发现提出了令人兴奋的问题，我们建议 地址在这里。视蛋白只对苦味化合物有反应，还是在味觉上有更广泛的功能 感觉？特别令人感兴趣的是，视蛋白在其他动物中是否充当味觉受体，包括蚊子， 伊蚊Aedes（Ae.）埃及？这种病媒传播的病毒可引起登革热、黄热病和其他疾病， 每年折磨着数百万人无论是雄性还是雌性斑蚊，都是用味觉来辨别花蜜，避免有毒 化学物质，而女性使用许多感官来识别人的血餐。然而，受体需要 对蚊子的味道是未知的。另一个令人着迷的问题是视蛋白是否作为味觉 哺乳动物的受体为了解决这些问题，我们建议采用一套广泛的方法 包括电生理学、Ca2+成像、行为测定、细胞生物学、体外受体活化测定， 和最先进的分子遗传学我们还将利用我们在果蝇味觉、视蛋白和 我们最近的经验进行分子遗传学和行为在Ae。埃及人和老鼠。目标1将 定义了果蝇视蛋白在氨基酸味觉中的作用，这在果蝇中是一种知之甚少的味觉形态。 如果果蝇的饮食中缺乏氨基酸，那么对氨基酸的吸引力就会增加。我们概述实验 以确定视蛋白对氨基酸吸引力的动态变化的贡献。我们还将测试 这表明视蛋白直接被氨基酸激活。目标2将建立在我们的初步数据，两个Ae。 埃及视蛋白在主要味觉器官中表达，并且是感受类黄酮所需的。的目标 目的是揭示这些视蛋白在体外和体内的化学特异性， 独立地或一起，并鉴定其中这些视蛋白起作用的味觉受体神经元。目标3 将描述视蛋白在哺乳动物味觉中的作用。初步数据显示， 多种小鼠视蛋白在小鼠舌头中表达，并在体外被苦味化合物激活， 包括一种没有已知受体的类黄酮。此外，我们提出的初步数据， 在视蛋白突变体中对这种类黄酮的反应受损。我们建议测试的假设，这种视蛋白 功能广泛的感觉类黄酮，确定味觉受体细胞，表达这个和其他 “味觉视蛋白”，并进行行为测试与突变体，以表征每一个视蛋白的要求 用于检测苦味化合物。该项目的结果将视蛋白作为一类新的味觉 从昆虫到哺乳动物的保守受体。我们建议，视蛋白作为第一个苦味受体， 蚊子提供了利用这些信息来操纵蚊子行为的可能性， 减少它们的数量，从而减少昆虫传播的疾病。

Abstract Opsins are classical G-protein coupled receptors, which for more than a century have been thought to function exclusively as light detectors. However, using Drosophila, we have uncovered multiple, light-independent roles for opsins including detection of bitter tastants. This discovery raises exciting questions, which we propose to address here. Do opsins only respond to bitter compounds, or do they function more broadly in taste sensation? Of particular interest, do opsins serve as taste receptors in other animals, including the mosquito, Aedes (Ae.) aegypti? This vector spreads viruses that cause dengue, yellow fever, and other diseases that afflict millions of people each year. Both male and female Aedes use taste to identify nectar and avoid toxic chemicals, while females use many senses to identify people for blood meals. However, the receptors required for mosquito taste are largely unknown. Another fascinating question is whether opsins serve as taste receptors in mammals. To address these questions, we propose to employ an extensive suite of approaches including electrophysiology, Ca2+ imaging, behavioral assays, cell biology, an in vitro receptor activation assay, and state-of-the art molecular genetics. We will also bring to bear our expertise in Drosophila taste, opsins, and our recent experience performing molecular genetics and behavior in Ae. aegypti and in the mouse. Aim 1 will define the role of a Drosophila opsin in amino acid taste, which in flies is a poorly understood taste modality. Attraction to amino acids increases if the flies are kept on an amino acid deficient diet. We outline experiments to define the contribution of the opsin to this dynamic change in amino acid appeal. We will also test the proposal that the opsin is activated directly by amino acids. Aim 2 will build on our preliminary data that two Ae. aegypti opsins are expressed in the major taste organ, and are required for sensing a flavonoid. The goals of this aim are to reveal the chemical specificity of these opsins in vitro and in vivo, determine whether they act independently or together, and identify the gustatory receptor neurons in which these opsins function. Aim 3 will characterize the roles of opsins in mammalian taste. This aim is supported by preliminary data showing that multiple mouse opsins are expressed in the mouse tongue, and are activated in vitro by bitter compounds, including a flavonoid for which there is no known receptor. In addition, we present preliminary data that the response to this flavonoid is impaired in the opsin mutant. We propose to test the hypothesis that this opsin functions broadly in sensing flavonoids, identify the taste receptor cells that express this and the other “gustatory opsins,” and perform behavioral tests with mutants to characterize the requirements for each opsin for sensing bitter compounds. The results from this project would establish opsins as a new class of taste receptor conserved from insects to mammals. We propose that defining opsins as the first bitter receptors in mosquitoes offers the potential to harness this information to manipulate mosquito behavior and thereby reduce their populations and thus insect-borne disease.