SWEET RECEPTOR BINDING INTERACTION STUDIES

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 由单体T1 R2 + T1 R3组成的人类甜味受体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基因G蛋白共表达时，这种异源二聚体受体对人类在人类品尝的浓度下感知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。我们已经使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。通过使用异源表达，钙成像，BRET和诱变以及我的实验室和我的同事的计算建模，我们已经映射了甜味剂与甜味受体的至少三个结构域的结合：hT 1 R2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），hT 1 R3的富含半胱氨酸的结构域（CRD（植物甜蛋白）和hT 1 R3的跨膜结构域（TMD）（环己基氨基磺酸盐和NHDC）。迄今为止，没有甜味剂已被证明在T1 R2的TMD中结合，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。在这个建议中，我们建议进一步确定的hT 1 R2 TMD的特点，促进变构相互作用与其他领域的受体。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。除了我们已经建立的技术（受体的异源表达，诱变，功能测定和计算建模）用于探索甜味剂与甜味受体的相互作用外，我们的合作者Fariba Assadi-Porter将使用饱和转移差（STD）NMR来跟踪配体与表达全长T1 R2 + T1 R3的细胞的结合，每个单体本身，突变体受体或不表达受体的亲本细胞。这一令人兴奋的新发展将使我们能够独立于受体活性来监测配体结合。它还将使我们能够确定和识别决定配体的灵敏度和选择性的关键配体-受体结合位点，以及通过监测每个配体的NMR光谱变化来确定甜味受体突变对配体结合口袋环境的影响。

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.