Agonist & Antagonist Activity and Binding on the TMD of hT1R3

由单体T1 R2 + T1 R3组成的人类甜味受体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基因G蛋白共表达时，该异源二聚体受体对人类在适当浓度下感觉到的全部甜味化合物作出响应。甜味受体对一组令人惊讶的不同配体做出反应，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点被激活。利用异源表达、钙离子成像、BRET、诱变和计算机模拟等技术，本实验室和同事们描述了至少4个甜味受体的结合区域：hT 1 R2的venus fly trap module（VFTM）、hT 1 R3的cystine-rich domain（CRD）和hT 1 R3的transmammal domain（TMD）。目前的建议重点和建立在我们最近发现的重叠结合口袋内的TMD的hT 1 R3的激动剂（甜蜜素和类似物）和拮抗剂（lactisole和类似物）。该结构域对于甜味受体活化至关重要，因为人类感知为甜味的所有不同配体都可以通过结合T1 R3 TMD口袋中的lactisole来阻断。这表明该结构域是导致受体活化的最终构象变化的关键元件。由于甜味受体也可以被T1 R3的TMD中结合的配体激活，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提供对甜味受体的基态和活性状态要求之间的差异的了解。我们建议在这里表征的hT 1 R3 TMD负责使用诱变，异源表达和活性测定和配体对接位点的计算建模的受体的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-NMR直接监测T1 R3 TMD的结合环境和配体的结合，STD-NMR是最近适用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（或基态稳定）的分子事件以及G蛋白活化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.