Agonist & Antagonist Activity and Binding on the TMD of hT1R3

描述（由申请人提供）：人类甜味受体，由单体T1R2 + T1R3组成，似乎是人类甜味的主要（也许是唯一）受体。当在异源系统中与报告蛋白g蛋白共表达时，这种异二聚体受体在适当浓度下对人类感知的各种甜味化合物作出反应。甜味受体对一组令人惊讶的不同配体做出反应，从小的氨基酸到中等大小的甜味植物蛋白质。没有一个共同的结构可以解释所有这些化合物的甜味。我们和其他实验室的研究表明，甜受体可以通过多种结构域和受体上不同的结合位点被激活。利用异源表达、钙成像、BRET、诱变和计算模型，我的实验室和我的同事们已经描述了至少4个甜味受体的结合区域：hT1R2的捕蝇器模块（VFTM）、hT1R3的富含半胱氨酸的结构域（CRD）和hT1R3的跨膜结构域（TMD）。目前的建议集中并建立在我们最近在hT1R3的TMD中发现的激动剂（甜蜜素和类似物）和拮抗剂（乳酸酯和类似物）的重叠结合口袋上。这个结构域对于甜味受体的激活至关重要，因为所有人类认为是甜味的不同配体都可以通过结合T1R3 TMD口袋中的乳酸酯来阻断。这表明该结构域是导致受体激活的最终构象变化的关键因素。由于甜味受体也可以被结合在T1R3的TMD中的配体激活，并且这些结合袋具有共同的残基，因此在分子水平上对这些结合袋进行表征将有助于深入了解甜味受体的基础状态和活性状态需求之间的差异。我们建议利用诱变、异源表达和活性测定以及配体对接位点的计算建模来表征hT1R3 TMD的环境，该环境负责受体的活性和非活性构象。此外，通过提案中概述的合作努力，我们将使用最近用于监测味觉系统的强大工具STD-NMR直接监测T1R3的结合环境和配体与TMD的结合。我们的长期目标是阐明配体结合和配体诱导活性（或基态稳定）的分子事件以及g蛋白激活所需受体的构象变化。公共卫生相关性：今天在世界富裕国家，肥胖、胰岛素抵抗性糖尿病和与饮食有关的疾病流行。人类进化过程中对高碳水化合物/高能量的甜食的喜爱只会让情况变得更糟。味觉感知和味觉偏好无疑会影响寻甜行为和食物消费。T1R2+T1R3作为甜味受体的鉴定为改变一种行为提供了调解的目标，这种行为由于富裕国家的丰富食物而不适应。在分子水平上理解甜味受体将有助于设计出更好的低热量甜味剂。

DESCRIPTION (provided by applicant): The human sweet receptor, composed of the monomers T1R2 + T1R3, appears to be the main (and perhaps the only) receptor underlying sweet taste in humans. When co-expressed with a reporter G-protein in heterologous systems, this heterodimeric receptor responds to the full range of sweet-tasting compounds sensed by humans at appropriate concentrations. The sweet receptor responds to a surprisingly diverse set of ligands, from small amino acids to moderately sized sweet-tasting plant proteins. No common structure accounts for the sweetness of all of these compounds. Studies from our lab and others indicate that the sweet receptor can be activated by means of a variety of domains and distinct binding sites on the receptor. Using heterologous expression, calcium imaging, BRET, mutagenesis and computational modeling, my lab and those of my colleagues have described at least 4 binding regions of the sweet receptor: the venus fly trap module (VFTM) of hT1R2, the cysteine-rich domain (CRD) of hT1R3 and the transmembrane domain (TMD) of hT1R3. The current proposal focuses and builds on our recent discovery of overlapping binding pockets within the TMD of hT1R3 for agonists (cyclamate and analogs) and antagonists (lactisole and analogs). This domain is of critical importance for sweet receptor activation since all of the diverse ligands that humans perceive as sweet can be blocked by binding lactisole in the T1R3 TMD pocket. This suggests that this domain is the key element in the final conformational change leading to receptor activation. Since the sweet receptor can also be activated by ligands that bind in the TMD of T1R3 and these binding pockets share common residues, characterization of these binding pockets at a molecular level will provide insight into the differences between the ground and active state requirements oft the sweet receptor. We propose here to characterize the environment of the hT1R3 TMD responsible for both active and inactive conformations of the receptor using mutagenesis, heterologous expression and activity assays and computational modeling of ligand docking sites. In addition, through the collaborative effort outlined in the proposal, we will directly monitor the binding environment and binding of ligands to the TMD of T1R3 using STD-NMR, a powerful tool recently adapted to monitor the taste system. Our long-term goal is to elucidate the molecular events that underlie ligand binding and ligand induced activity (or stabilization of the ground state) and the conformational changes of the receptor required for G-protein activation. PUBLIC HEALTH RELEVANCE: There is today in the affluent countries of the world an epidemic of obesity, insulin-resistant diabetes and diet-related disorders. This is only made worse by our species evolutionary love affair with high- carbohydrate/energy-rich sweet foods. Taste perception and taste preference undoubtedly contribute to sweet-seeking behavior and food consumption. The identification of T1R2+T1R3 as the sweet receptor provides the target for intercession in modifying a behavior, which is maladaptive because of the plentiful food in affluent countries. Understanding the sweet receptor at a molecular level will enable the design of better low calorie sweeteners.