Opsins as a new class of evolutionarily conserved taste receptors

摘要 视黄素是经典的G蛋白偶联受体，一个多世纪以来一直被认为具有功能 完全作为光探测器。然而，使用果蝇，我们发现了多个与光无关的角色 对于包括苦味检测在内的眼镜蛇。这一发现提出了令人兴奋的问题，我们建议 地址在这里。视黄素是只对苦味化合物起反应，还是对味道起更广泛的作用？ 轰动一时？特别有趣的是，视蛋白在包括蚊子在内的其他动物中是否作为味觉感受器， 伊蚊(Aedes(Ae.)埃及伊蚊？这种媒介传播导致登革热、黄热病和其他疾病的病毒 每年折磨着数百万人。雄性和雌性伊蚊都用味道来辨别花蜜并避免有毒 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 对于蚊子的味道在很大程度上是未知的。另一个令人着迷的问题是视蛋白是否起到味觉的作用 哺乳动物中的受体。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、钙离子成像、行为分析、细胞生物学、体外受体激活分析、 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 我们最近在Ae中进行分子遗传学和行为研究的经验。埃及伊蚊和老鼠体内。目标1将 确定果蝇视蛋白在氨基酸味道中的作用，氨基酸味道在果蝇中是一种鲜为人知的味道形式。 如果果蝇保持缺乏氨基酸的饮食，对氨基酸的吸引力就会增加。我们勾勒出实验大纲 以确定视蛋白对氨基酸吸引力的这种动态变化的贡献。我们还将测试 提出视蛋白是由氨基酸直接激活的。目标2将建立在我们的初步数据基础上，即两个Ae。 埃及伊蚊视蛋白在主要的味觉器官中表达，是感觉类黄酮类化合物所必需的。的目标 本研究的目的是揭示这些视蛋白在体外和体内的化学特异性，确定它们是否起作用 独立地或一起，并识别这些视蛋白在其中发挥作用的味觉感受器神经元。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，这一目标得到了支持 多个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， 包括一种尚不清楚受体的类黄酮类化合物。此外，我们提供的初步数据显示， 在视蛋白突变体中，对这种类黄酮的反应受损。我们建议检验这样一个假设，即这个视盘 在感知类黄酮方面具有广泛的功能，识别表达这个和那个的味觉感受器细胞 “味觉视蛋白”，并对突变体进行行为测试，以确定每种视蛋白的要求。 用来感测苦味化合物。这个项目的结果将使Opsins成为一种新的品味 受体从昆虫到哺乳动物都是保守的。我们建议将视黄素定义为第一个苦味受体 蚊子提供了利用这些信息来操纵蚊子行为的可能性，从而 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.