Analysis of sweet receptor ligand binding and activation

甜味受体配体结合和激活分析

• 批准号: 7624581
• 负责人: MARIANNA MAX
• 金额: $ 33.08万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7624581
• 关键词: Address; Affinity; Agonist; Artificial Sweeteners; Aspartame; Bacteria; Behavior; Binding; Binding Sites; Biological Assay; Carbohydrates; Chimera organism; Complex; Consumption; Country; Diabetes Mellitus; Diet; Dipeptides; Disease; Docking; Engineering; Epidemic; Event; Food; Future; Goals; Human; In Vitro; Insulin Resistance; Life; Ligand Binding; Ligands; Measures; Mediating; Methods; Modeling; Molecular; Molecular Conformation; Molecular Models; Monitor; Mus; Mutagenesis; Mutation; Obesity; Receptor Activation; Research; Research Personnel; Signal Transduction; Site; Sweetening Agents; Techniques; Testing; Venus; alitame; craving; fly; food consumption; in vivo; interest; molecular modeling; monomer; mutant; neotame; pharmacophore; programs; receptor; receptor binding; receptor function; sedentary; small molecule; sweet receptor; sweet taste perception

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.

描述（由申请人提供）：我们研究计划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。甜味受体是一种作用非常广泛的受体，能够对天然和人造甜味剂做出反应。体内和体外研究表明，这种单一异二聚体受体是主要或唯一的甜味受体。我们感兴趣的是这么多化学上不同的配体如何与甜味受体结合，不同位点的结合如何导致受体激活，以及每个 T1R 单体的结构域如何有助于结合、激活和信号转导。为了实现这些目标，我们开发了配体结合和活性测定，并将这些技术与诱变和分子建模结合使用，以开始了解这种复杂的受体。本提案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、纽甜和阿力甜（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) 内。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测定，来识别 T1R2 的 VFTM 内参与与二肽甜味剂相互作用的关键残基。在目标 2 中，我们将使用 T1R2 的定向诱变、异源测定和分子建模来物理和化学表征二肽甜味剂与 T1R2 VFTM 的相互作用。在目标 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。如今，肥胖、胰岛素抵抗性糖尿病和饮食相关疾病在世界富裕国家盛行。在我们的进化历史中，消耗高碳水化合物/富含能量的食物的强烈动力对生存是有利的。如今，我们久坐的生活和现成的食物使这种寻求甜食的行为成为一种负担，可能会严重导致肥胖。由异二聚体甜味受体介导的甜味感知无疑有助于寻求甜味的行为和食物消耗。该提案中的研究将增强我们对甜味受体功能的分子水平的理解，希望未来能够找到控制我们对甜食的渴望和过度消费带来的疾病的方法。