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）：我们的研究计划的远程目标是了解异二聚体甜味受体（T1R2+T1R3）的分子事件。甜美受体是一种非常广泛的作用受体，能够对天然和人造甜味剂做出反应。体内和体外研究表明，这种单一的异二聚体受体是原发性或唯一的甜味受体。我们对有许多化学多样的配体如何结合甜蜜受体，不同位点的结合如何导致受体激活以及每个T1R单体的域如何有助于结合，激活和信号转导。为了解决这些目标，我们开发了配体的结合和活性测定，并使用诱变和分子建模协同使用这些技术开始理解这种复杂的受体。本提案应用了这些几种技术来检查T1R2的小分子结合位点如何与Aspartame，Neotame和Alitame（所谓的二肽甜味剂）相互作用。在T1R2的“ Venus Fly Trap模块”（VFTM）中发现了此“规范”结合位点。在AIM 1中，我们将使用人和小鼠甜受体的差异敏感性以及异源表达测定二肽甜味剂，以鉴定与二肽甜味剂相互作用的T1R2 VFTM中的关键残基。在AIM 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的VFTM的相互作用。在AIM 3中，我们将使用光谱和量热法来监测二肽甜味剂与T1R2和T1R2突变体的表达VFTM的结合。如今，世界富裕国家有一种肥胖，耐胰岛素糖尿病和与饮食有关的疾病的流行。在我们的进化中，过去的强大动力消费高碳水化合物/能量富含能量的食物对于生存是有利的。如今，我们更久坐的生活和食物的现成可用性使这种寻求甜蜜的行为成为可能对肥胖产生重大贡献的责任。异二聚体甜味受体介导的甜味感知无疑有助于寻求甜食的行为和食物消费。该提案中的研究将增强我们在甜蜜受体功能的分子水平上的理解，希望将来能够控制我们的甜蜜渴望和过度消费的随之而来的疾病。