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，对甜味受体激活机制进行分子理解。对甜味化合物的味觉偏好使动物能够寻找高碳水化合物能量来源来利用食物。甜味受体由两个1型味觉受体单体（T1 R2加T1 R3）组成，显然是异二聚体。该提议使用甜味受体的诱变、在HEK 293细胞中的表达、钙成像、生物发光共振能量转移和受体的表面表达来探测甜味受体与配体的相互作用。目的1使用计算方法同源建模的大胞外结构域的异二聚体，使用作为模板的晶体结构的胞外结构域的mGluR 1，该家族的受体的另一个成员。通过诱变预测形成二聚化界面的T1 R2 + T1 R3中的残基来测试和改进所得的同源性模型，然后测定表达的受体对甜味配体的响应和异源二聚体的形成。由此产生的优化模型将有助于解释突变对配体诱导的活性在随后的目标的影响。目的2旨在发现影响配体-受体相互作用和受体活化的T1 R2残基。目的2a使用TIR与mGluRl的比对来选择T1 R2中潜在的配体相互作用残基，然后突变它们以发现它们对受体对甜味配体的反应的影响。目标2b利用物种特异性味觉的差异，以及嵌合的人/小鼠T1 R2受体来跟踪负责对甜味剂的人类样反应的受体部分。目的2c使用诱变来扫描可能与植物甜蛋白偶极相互作用的表面可及的甘氨酸和赖氨酸。表达突变的受体，测定对植物甜蛋白的响应性的丧失，然后测试植物甜蛋白突变体补偿受体突变的能力。目的3跟进我们最近的观察，在人T1 R3的富含半胱氨酸的连接区的两个残基是必不可少的受体应答植物甜蛋白。我们已经提出在这个区域进行额外的突变，以鉴定和表征那些能够使人类受体对植物甜蛋白产生反应的残基。从这些研究中获得的知识将为甜味受体活性提供一个工作模型，这可能导致设计出上级人工甜味剂。我们对甜味受体的分子研究可能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型谷氨酸受体。

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.