The Role of the TM of T1R2 in Sweet Receptor Activation

描述（由申请人提供）：由单体T1R2 + T1R3组成的人类甜味受体似乎是解释人类甜味所需的主要（也可能是唯一）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。我们利用这种甜受体活性的多样性作为理解配体受体相互作用以及探测导致这种复杂受体激活的分子事件的工具。通过异种表达、钙成像、BRET、诱变和计算建模，我和我的同事们绘制了甜味剂与甜味受体至少四个结构域的结合图谱：hT1R2的捕蝇器模块（VFTM）（各种小分子人工甜味剂、天然糖和二肽甜味剂）、hT1R3的VFTM（天然糖）、hT1R3的富含半胱氨酸结构域（CRD） （brazzein）和hT1R3的跨膜结构域（TMD）（环己酸和NHDC）。到目前为止，还没有甜味剂被明确地证明与T1R2的TMD结合，然而，我们最近的发现表明，该结构域能够变构调节配体诱导的甜味受体活性。在本提案中，我们概述了进一步确定hT1R2 TMD促进与受体其他结构域的变构相互作用的特性的计划。我们还将确定是否有任何甜味剂映射到其假定的螺旋内TMD结合位点。除了我们已经建立的技术（受体的异源表达、诱变、功能分析和计算建模），用于探索甜味剂与甜味受体的相互作用，我们的合作者Fariba Assadi-Porter博士将使用饱和转移差（STD） NMR来跟踪配体结合到表达全长T1R2 + T1R3的细胞上，每个单个体本身或突变受体（使用不表达受体的亲本细胞作为对照）。这一令人兴奋的新进展将使我们能够监测与受体活性分离的配体结合。它还将使我们能够识别决定配体敏感性和选择性的关键配体-受体结合残基，以及通过监测每个配体的核磁共振光谱变化来确定甜受体突变对配体结合口袋环境的影响。

DESCRIPTION (provided by applicant): The human sweet receptor composed of the monomers T1R2 + T1R3, appears to be the main (and perhaps the only) receptor required to explain 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 concentrations that humans taste. 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. We have used this diversity in sweet receptor activity as a tool for understanding ligand receptor interactions as well as for probing the molecular events that lead to activation of this complex receptor. By using heterologous expression, calcium imaging, BRET, mutagenesis and computational modeling my colleagues and I have mapped sweetener binding to at least four domains of the sweet receptor: the venus fly trap module (VFTM) of hT1R2 (various small molecule artificial sweeteners, natural sugars and dipeptide sweeteners), the VFTM of hT1R3 (natural sugars), the cysteine-rich domain (CRD) of hT1R3 (brazzein) and the transmembrane domain (TMD) of hT1R3 (cyclamate and NHDC). To date, no sweeteners have definitively been shown to bind within the TMD of T1R2, however, our recent finding suggests that this domain is able to allosterically regulate ligand- induced activity in the sweet receptor. In this proposal, we outline a plan to further determine the characteristics of the hT1R2 TMD that promotes allosteric interactions with other domains of the receptor. We will also determine whether any sweeteners map to its putative intra-helical TMD binding site. In addition to our established techniques (heterologous expression of receptors, mutagenesis, functional assay and computational modeling) for exploring sweetener interactions with the sweet receptor, our collaborator, Dr. Fariba Assadi-Porter will use saturation transfer difference (STD) NMR to track ligand-binding to cells expressing full length T1R2 + T1R3 together, each monomer by itself or mutants receptors (using parent cells not expressing receptors as control). This exciting new development will allow us to monitor ligand binding separate from receptor activity. It will also allow us to identify the critical ligand-receptor binding residues that determine sensitivity and selectivity for ligands, in addition to the effect of sweet receptor mutations on the ligand-binding pocket environment(s) by monitoring changes in the NMR spectra for each ligand.