The Role of the TM of T1R2 in Sweet Receptor Activation

T1R2 TM 在甜味受体激活中的作用

• 批准号: 7535496
• 负责人: MARIANNA MAX
• 金额: $ 19.69万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-03 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7535496
• 关键词: Accounting; Affinity; Agonist; Amino Acids; Artificial Sweeteners; Binding; Binding Sites; Biological Assay; Calcium; Calcium-Sensing Receptors; Cell surface; Cells; Characteristics; Complex; Computer Simulation; Cyclamates; Cysteine; Cysteine-Rich Domain; DNA Sequence Rearrangement; Development; Dipeptides; Environment; Event; Extracellular Domain; Family; GTP-Binding Proteins; Goals; Human; Image; Individual; Lead; Length; Ligand Binding; Ligand Binding Domain; Ligands; Link; Maps; Membrane; Metabotropic Glutamate Receptors; Molecular Probes; Monitor; Mutagenesis; Mutation; Output; Parents; Plant Proteins; Proteins; Receptor Activation; Reporter; Research; Role; Site; Structure; Structure-Activity Relationship; Sweetening Agents; System; Taste Perception; Techniques; Testing; Transmembrane Domain; Venus; Venus Flytrap; Work; base; design; fly; monomer; mutant; olfactory receptor; programs; protein activation; receptor; receptor binding; receptor expression; research study; response; small molecule; sugar; sweet receptor; sweet taste perception; tool; trichlorosucrose

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.

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。通过使用异源表达、钙成像、BRET、诱变和计算模型，我和我的同事已经绘制了甜味剂与甜味受体至少四个结构域的结合图谱：hT1R2 的维纳斯捕蝇模块 (VFTM)（各种小分子人工甜味剂、天然糖和二肽甜味剂）、hT1R3 的 VFTM（天然糖）、 hT1R3（brazzein）的富含半胱氨酸结构域（CRD）和 hT1R3（甜蜜素和 NHDC）的跨膜结构域（TMD）。迄今为止，尚未明确显示甜味剂与 T1R2 的 TMD 内结合，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，我们概述了一项计划，以进一步确定 hT1R2 TMD 的特性，以促进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、功能测定和计算模型）外，我们的合作者 Fariba Assadi-Porter 博士将使用饱和转移差（STD）NMR 来追踪配体与同时表达全长 T1R2 + T1R3 的细胞、每个单体本身或突变体受体（使用不表达 受体作为对照）。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核磁共振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，它还将使我们能够识别决定配体敏感性和选择性的关键配体-受体结合残基。