SWEET RECEPTOR BINDING INTERACTION STUDIES

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 人类甜味受体由单体 T1R2 T1R3 组成，似乎是解释人类甜味所需的主要（也许是唯一）受体。当在异源系统中与报告 G 蛋白共表达时，这种异二聚体受体会对人类在人类尝到的浓度下感受到的所有甜味化合物做出反应。甜味受体对一组令人惊讶的多样化配体做出反应，从小型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点来激活。我们利用甜味受体活性的这种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。通过在我和我的同事的实验室中使用异源表达、钙成像、BRET、诱变和计算模型，我们绘制了甜味剂与甜味受体至少三个结构域的结合图谱：hT1R2 的金星捕蝇模块 (VFTM)（各种小分子人工甜味剂、天然糖和二肽甜味剂）、hT1R3 的富含半胱氨酸结构域 (CRD)（brazzein）和跨膜结构域(TMD) hT1R3（甜蜜素和 NHDC）。迄今为止，还没有甜味剂被证明与 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甜味受体中配体诱导的活性。在本提案中，我们建议进一步确定 hT1R2 TMD 促进与受体其他结构域变构相互作用的特征。我们还将确定是否有任何甜味剂映射到其推定的螺旋内 TMD 结合位点。除了我们已建立的用于探索甜味剂与甜味受体相互作用的技术（受体的异源表达、诱变、功能测定和计算建模）之外，我们的合作者 Fariba Assadi-Porter 将使用饱和转移差（STD）NMR 来追踪配体与同时表达全长 T1R2 T1R3 的细胞、每个单体本身、突变体受体或不表达受体的亲代细胞的结合。这一令人兴奋的新进展将使我们能够独立于受体活性来监测配体结合。除了通过监测每个配体 NMR 谱的变化来确定甜味受体突变对配体结合口袋环境的影响之外，它还将使我们能够确定和识别决定配体敏感性和选择性的关键配体-受体结合位点。

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.