Molecular Mechanisms of Sweet Receptor Function

甜味受体功能的分子机制

• 批准号: 7077619
• 负责人: MARIANNA MAX
• 金额: $ 33.1万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7077619
• 关键词: carbohydrates; cell surface receptors; chemoreceptors; computational biology; computer simulation; electrophysiology; fluorescence resonance energy transfer; food flavor; hybrid cells; molecular biology; molecular dynamics; nuclear magnetic resonance spectroscopy; physical model; receptor expression; receptor sensitivity; stimulus /response; taste

DESCRIPTION (provided by applicant): The long term goal of this proposal is to develop a molecular understanding of the mechanisms of sweet receptor activation by combining experimental and computational approaches. The taste preference for sweet compounds allows animals to seek out high carbohydrate energy sources to exploit for food. The sweet receptor is composed of two type 1 taste receptor monomers (T1R2 plus T1R3), apparently as a heterodimer. This proposal uses mutagenesis of the sweet receptor, expression in HEK 293 cells, calcium imaging, bioluminescent-resonance-energy-transfer and surface expression of receptors, to probe the sweet receptor's interaction with ligands. Aim 1 uses computational approaches to homology model the large extracellular domain of the heterodimer, using as template the crystal structure of the extracellular domain of mGluRl , another member of this family of receptors. The resulting homology models are tested and refined by mutagenesis of residues in T1R2+T1R3 predicted to form the dimerization interface, and then the expressed receptors are assayed for responses to sweet ligands and formation of heterodimers. The resulting optimized models will be useful to explain effects of mutations on ligand-induced activity in subsequent Aims. Aim 2 seeks to discover T1R2 residues that influence ligand-receptor interaction and receptor activation. Aim 2a uses the alignment of the TIR s with mGluRl to choose potential ligand-interacting residues in T1R2, then mutate them to discover their effects on receptor responses to sweet ligands. Aim 2b employs differences in species-specific taste perception, and chimeric human/mouse T1R2 receptors to track portions of the receptor responsible for human-like responses to sweeteners. Aim 2c uses mutagenesis to scan the surface-accessible arginines and lysines that might interact with the brazzein dipole. Mutated receptors are expressed, assayed for loss of responsiveness toward brazzein, then brazzein mutants are tested for the ability to compensate for receptor mutations. Aim 3 follows up on our recent observation that two residues in the cysteine-rich linker region of human T1R3 are essential for receptor responses to brazzein. We have proposed makin g additional mutations in this region to identify and characterize those residues that enable the human receptor to respond to brazzein. The knowledge gained from these studies will provide a working model for sweet receptor activity that may lead to the design of superior artificial sweeteners. Our molecular studies of the sweet receptor may shed light on transduction mechanisms common to other members of this family of receptors, such as the calcium-sensing receptor, which regulates calcium metabolism, and the metabotropic glutamate receptors, which are involved in multiple neurological responses.

描述（由申请人提供）：该提案的长期目标是通过结合实验和计算方法来对甜味受体激活机制的机制进行分子理解。对甜化合物的口味偏好使动物可以寻找高碳水化合物的能源来利用食物。甜受体由两个1型味道受体单体（T1R2 Plus T1R3）组成，显然是异二聚体。该建议使用甜味受体的诱变，在HEK 293细胞中的表达，钙成像，生物发光 - 函数 - 能源转移和受体的表面表达，以探测甜味受体与配体的相互作用。 AIM 1使用计算方法来模拟异二聚体的大细胞外结构域，作为模板，MGLURL的细胞外域的晶体结构，Mglurl是该受体家族的另一个成员。通过预测形成二聚化界面的T1R2+T1R3中残基的诱变来测试和完善所得的同源模型，然后分析表达的受体以响应甜味配体和异二聚体的形成。所得的优化模型将有助于解释突变对随后目标中配体诱导活性的影响。 AIM 2试图发现影响配体 - 受体相互作用和受体激活的T1R2残基。 AIM 2A使用TIR S与MGlurl的比对选择T1R2中潜在的配体相互作用残基，然后将它们突变以发现它们对受体对甜配体的受体反应的影响。 AIM 2B采用物种特异性的味道感知和嵌合人/小鼠T1R2受体的差异来跟踪负责人类对甜味剂类似人类反应的受体的部分。 AIM 2C使用诱变来扫描可能与Brazzein偶极子相互作用的表面可访问精氨酸和赖氨酸。表达突变的受体，分析了对Brazzein的反应性丧失，然后测试了Brazzein突变体的补偿能力。 AIM 3跟进了我们最近的观察结果，即人T1R3的富含半胱氨酸连接器区域中的两个残基对于受体对Brazzein的反应至关重要。我们提出了该区域中的其他突变，以识别和表征那些使人体能够对Brazzein反应的残基。从这些研究中获得的知识将为甜蜜的受体活动提供一个工作模型，这可能导致高级人造甜味剂的设计。我们对甜味受体的分子研究可能会阐明该受体家族的其他成员所共有的转导机制，例如调节代谢钙代谢的钙敏感受体和代谢性谷氨酸受体，这些受体涉及多种神经系统反应。