Analysis of sweet receptor ligand binding and activation

描述（由申请人提供）：我们研究项目的长期目标是了解异二聚体甜味受体（T1R2+T1R3）功能的分子事件。甜受体是一种作用非常广泛的受体，能够对天然和人工甜味剂做出反应。体内和体外研究表明，这种单一的异二聚体受体是主要的或唯一的甜味受体。我们感兴趣的是这么多化学上不同的配体是如何与甜受体结合的，不同位点的结合是如何导致受体激活的，以及每个T1R单体的结构域是如何参与结合、激活和信号转导的。为了实现这些目标，我们开发了配体结合和活性分析，并将这些技术与诱变和分子建模相结合，开始了解这种复杂的受体。本研究应用这几种技术来研究T1R2的小分子结合位点如何与阿斯巴甜、纽甜和阿利甜（所谓的二肽甜味剂）相互作用。这个“规范”结合位点位于T1R2的“捕蝇器模块”（VFTM）中。在目标1中，我们将利用人类和小鼠甜味受体对二肽甜味剂的不同敏感性，以及异源表达试验，确定T1R2 VFTM中与二肽甜味剂相互作用相关的关键残基。在目标2中，我们将使用T1R2的定向诱变，异种测定和分子模型来物理和化学表征二肽甜味剂与T1R2的VFTM的相互作用。在Aim 3中，我们将使用光谱和量热技术来监测二肽甜味剂与T1R2和T1R2突变体表达的VFTM的结合。今天，在世界上的富裕国家，肥胖、胰岛素抵抗性糖尿病和与饮食有关的疾病普遍存在。在我们的进化过程中，消耗高碳水化合物/高能量食物的强烈欲望有利于生存。如今，我们久坐不动的生活和现成的食物使得这种追求甜食的行为成为一种负担，这可能是导致肥胖的重要原因。异二聚体甜味受体介导的甜味感知无疑对寻甜行为和食物消费起着重要作用。本研究将提高我们在分子水平上对甜味受体功能的理解，并希望未来能够控制我们对甜味的渴望和伴随的过度食用疾病。

DESCRIPTION (provided by applicant): The long-range goal of our research program is to understand the molecular events underlying function of the heterodimeric sweet taste receptor (T1R2+T1R3). The sweet receptor is a remarkably broadly acting receptor, capable of responding to native and artificial sweeteners. In vivo and in vitro studies suggest that this single heterodimeric receptor is the primary or only sweet taste receptor. We are interested in how so many chemically diverse ligands can bind to the sweet receptor, how binding at different sites leads to receptor activation, and how the domains of each T1R monomer contribute to binding, activation and signal transduction. To address these goals we have developed ligand binding and activity assays, and used these techniques in concert with mutagenesis and molecular modeling to begin to understand this complex receptor. The present proposal applies these several techniques to examine how the small molecule-binding site of T1R2 interacts with aspartame, neotame and alitame (so-called dipeptide sweeteners). This "canonical" binding site is found within the "venus fly trap module" (VFTM) of T1R2. In Aim 1 we will use the differential sensitivity of the human and mouse sweet receptors to dipeptide sweeteners, along with heterologous expression assays, to identify key residues within the VFTM of T1R2 involved in the interactions with dipeptide sweeteners. In Aim 2 we will use directed mutagenesis of T1R2, heterologous assays and molecular modeling to physically and chemically characterize the interaction of dipeptide sweeteners with the VFTM of T1R2. In Aim 3 we will use spectroscopic and calorimetric techniques to monitor binding of dipeptide sweeteners to the expressed VFTM of T1R2 and T1R2 mutants. There is today in the affluent countries of the world an epidemic of obesity, insulin-resistant diabetes and diet-related disorders. In our evolutionary past a strong drive to consume high-carbohydrate/energy-rich foods was advantageous for survival. Today, our more sedentary lives and the ready availability of food makes this sweet-seeking behavior a liability that may contribute significantly to obesity. Sweet taste perception mediated by the heterodimeric sweet taste receptor undoubtedly contributes to sweet-seeking behavior and food consumption. The studies in this proposal will enhance our understanding at the molecular level of sweet receptor function with the hope of future means to control our sweet cravings and the attendant diseases of over-consumption.