Agonist & Antagonist Activity and Binding on the TMD of hT1R3

人类甜味受体由单体T1R2+T1R3组成，似乎是人类甜味的主要(也可能是唯一的)受体。当与报告G蛋白在异源系统中共表达时，这种异二聚体受体对人类在适当浓度下感觉到的所有甜味化合物都有反应。甜味受体对一系列令人惊讶的不同配基做出反应，从小氨基酸到中等大小的甜味植物蛋白。没有共同的结构可以解释所有这些化合物的甜味。我们实验室和其他实验室的研究表明，甜味受体可以通过受体上的各种结构域和不同的结合位置来激活。利用异源表达、钙离子成像、Bret、突变和计算模型，我的实验室和我的同事已经描述了甜味受体的至少4个结合区：hT1R2的金蝇诱捕模块(Vftm)、hT1R3的半胱氨酸富集区(CRD)和hT1R3的跨膜区(TMD)。目前的建议侧重于并建立在我们最近发现的hT1R3的TMD内与激动剂(甜蜜素和类似物)和拮抗剂(乳糖醇和类似物)的重叠结合口袋。这个结构域对于甜味受体的激活至关重要，因为人类认为甜味的所有不同的配体都可以通过结合T1R3 TMD口袋中的乳糖来阻断。这表明该结构域是导致受体激活的最终构象变化的关键元件。由于甜味受体也可以被结合在T1R3的TMD中的配体激活，并且这些结合口袋共享共同的残基，因此在分子水平上表征这些结合口袋将有助于深入了解甜味受体的基础状态和活性状态要求之间的差异。我们建议通过突变、异源表达和活性分析以及配体对接位点的计算模型来表征hT1R3 TMD负责受体活性和非活性构象的环境。此外，通过提案中概述的合作努力，我们将使用STD-核磁共振直接监测配体与T1R3的TMD的结合环境和结合，STD-核磁共振是最近适应于监测味觉系统的强大工具。我们的长期目标是阐明配体结合和配体诱导活性(或基态的稳定)背后的分子事件，以及G蛋白激活所需受体的构象变化。

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.