Molecular Mechanisms of Sweet Receptor Function

描述（由申请人提供）：本提案的长期目标是通过结合实验和计算方法，发展对甜味受体激活机制的分子理解。对甜味化合物的偏好使动物能够寻找高碳水化合物能量来源作为食物。甜味受体由两种1型味觉受体单体（T1R2和T1R3）组成，显然是异源二聚体。本研究利用糖受体诱变、HEK 293细胞表达、钙成像、生物发光-共振-能量转移和受体表面表达等方法，探讨糖受体与配体的相互作用。Aim 1使用计算方法对异源二聚体的大胞外结构域进行同源性建模，以该受体家族的另一个成员mGluRl的胞外结构域的晶体结构为模板。通过诱变T1R2+T1R3中预测形成二聚化界面的残基，对得到的同源模型进行测试和改进，然后分析表达的受体对甜配体的反应和异源二聚体的形成。所得到的优化模型将有助于解释突变对配体诱导活性的影响。目的2旨在发现影响配体-受体相互作用和受体激活的T1R2残基。Aim 2a利用TIR s与mGluRl的比对来选择T1R2中潜在的配体相互作用残基，然后对它们进行突变，以发现它们对受体对甜配体的反应的影响。Aim 2b利用物种特异性味觉的差异，以及人类/小鼠嵌合的T1R2受体来追踪负责人类对甜味剂反应的受体部分。Aim 2c使用诱变来扫描可能与铜蛋白偶极子相互作用的表面可接近的精氨酸和赖氨酸。表达突变的受体，检测对铜蛋白的反应性丧失，然后测试铜蛋白突变体补偿受体突变的能力。Aim 3跟进了我们最近的观察结果，即人类T1R3中富含半胱氨酸的连接区域中的两个残基对于受体对brazzein的反应至关重要。我们建议在该区域进行额外的突变，以鉴定和表征那些使人类受体对brazzein作出反应的残基。从这些研究中获得的知识将为甜受体活性提供一个工作模型，这可能会导致设计出优质的人工甜味剂。我们对甜受体的分子研究可能会揭示该受体家族其他成员共同的转导机制，例如调节钙代谢的钙敏感受体和参与多种神经反应的代谢促谷氨酸受体。

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.