SWEET RECEPTOR BINDING INTERACTION STUDIES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 人类甜味受体由单体T1R2和T1R3组成，似乎是解释人类甜味所需的主要(也许是唯一的)受体。当与报告的G蛋白在异源系统中共表达时，这种异二聚体受体对人类感觉到的各种甜味化合物做出反应，其浓度与人类品尝的浓度相同。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。我们利用甜味受体活性的这种多样性作为了解配体-受体相互作用的工具，以及探索导致这种复杂受体激活的分子事件。通过使用异源表达、钙成像、Bret和突变以及我和我的同事的计算模型，我们已经将甜味剂结合到甜味受体的至少三个结构域：hT1R2(各种小分子人工甜味剂、天然糖和二肽甜味剂)的金蝇诱捕模块(Vftm)，hT1R3的半胱氨酸富含结构域(CRD)(Brazzein)，以及hT1R3的跨膜区(TMD)(甜蜜素和NHDC)。到目前为止，还没有发现甜味剂与T1R2的TMD结合，然而，我们最近的发现表明，这个结构域能够变构地调节甜味受体中配体诱导的活性。在这个建议中，我们建议进一步确定hT1R2 TMD的特征，它促进了与受体其他结构域的变构相互作用。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。除了我们为探索甜味剂与甜味剂受体的相互作用而建立的技术(受体的异源表达、突变、功能分析和计算建模)外，我们的合作者Fariba Assadi-Porter还将使用饱和转移差分(STD)核磁共振来追踪配体与共同表达全长T1R2 T1R3的细胞、每个单体本身、突变受体或不表达受体的亲本细胞的结合。这一令人兴奋的新发展将使我们能够监测与受体活性分开的配体结合。它还将使我们能够通过监测每个配体的核磁共振光谱的变化，确定决定配体的敏感性和选择性的关键配体-受体结合位点，以及甜味受体突变对配体结合口袋环境的影响(S)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 and mutagenesis and computational modeling in my laboratory and those of my colleagues, we have mapped sweetener binding to at least three domains of the sweet receptor: the venus fly trap module (VFTM) of hT1R2 (various small molecule artificial sweeteners, natural sugars and dipeptide sweeteners), the cysteine-rich domain (CRD) of hT1R3 (brazzein), and the transmembrane domain (TMD) of hT1R3 (cyclamate and NHDC). To date, no sweeteners have been shown to bind in 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 propose 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, 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, mutants receptors or parent cells not expressing receptors. This exciting new development will allow us to monitor ligand binding separate from receptor activity. It will also allow us to determine and identify the critical ligand-receptor binding sites 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.