Molecular Mechanisms of Sweet Receptor Function

描述(由申请人提供)：这项提案的长期目标是通过结合实验和计算方法来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的能量来源来开发食物。甜味受体由两个1型味觉受体单体(T1R2+T1R3)组成，显然是异源二聚体。该方案利用甜味受体的突变、在HEK 293细胞中的表达、钙离子成像、生物发光共振能量转移和受体的表面表达来探讨甜味受体与配体的相互作用。目的1使用计算方法对异源二聚体的大胞外域进行同源建模，以mGluRl的胞外域的晶体结构为模板，mGluRl是该受体家族的另一个成员。通过突变T1R2+T1R3中的残基来检验和改进得到的同源模型，预测T1R2+T1R3中的残基将形成二聚界面，然后检测表达的受体对甜味配体的反应和异源二聚体的形成。得到的优化模型将有助于在后续的AIMS中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。目的2a利用TIR S与mGluR1的比对来选择T1R2中潜在的与配体相互作用的残基，然后对它们进行突变，以发现它们对甜味配体受体反应的影响。目的2b利用物种特异性味觉的差异和嵌合人/鼠T1R2受体来追踪负责人类对甜味剂的类似反应的受体部分。AIM 2c使用突变技术扫描表面可达的精氨酸和赖氨酸，这些精氨酸和赖氨酸可能与Brazzein偶极子相互作用。突变的受体被表达，检测对Brazzein的反应丧失，然后测试Brazzein突变的补偿受体突变的能力。目的3继续我们最近的观察，即人类T1R3富含半胱氨酸的连接区的两个残基是对Brazzein的受体反应所必需的。我们建议在这个区域制造额外的突变，以识别和表征那些使人类受体对Brazzein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，从而可能导致设计出更好的人工甜味剂。我们对甜味受体的分子研究可能有助于揭示这一受体家族中其他成员共有的转导机制，如调节钙代谢的钙敏感受体和参与多种神经反应的代谢性谷氨酸受体。

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.