Development And Function Of Mammalian Chemosensory Syste

哺乳动物化学感应系统的发育和功能

• 批准号: 7130168
• 负责人: SUSAN L. SULLIVAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7130168
• 关键词: Baculoviridae; cell surface receptors; chemoreceptors; gene expression; genetic recombination; growth /development; intermolecular interaction; olfactions; receptor expression; sensory receptors; taste; taste buds; transfection

The research goals of the Section of Molecular Neuroscience are to define the molecular mechanisms underlying the development and function of mammalian chemosensory systems. Research efforts this past year have been directed towards establishing functional assays for human and mouse chemosensory receptors and identifying and characterizing novel genes selectively expressed in taste cells. To characterize the functional properties of chemosensory receptors, we previously developed an in vitro reconstitution assay to assess receptor activity. To perform these assays, baculoviruses expressing a given receptor are used to infect insect cells, and the membranes from the infected insect cells are purified. These membranes are highly enriched in the expressed receptor, which can be functionally reconstituted by the addition of purified G proteins. Initial studies with 23 members of the human bitter receptors led to the identification of four novel receptor-ligand interactions. We demonstrated that the hT2R14 and hT2R43 receptors respond selectively to micromolar concentrations of aristolochic acid and that the hT2R47 receptor responds selectively to micromolar concentrations of denatonium. In contrast, hT2R7 is more broadly tuned responding to several bitter-tasting compounds. This year, using these defined ligand-receptor interactions, we assayed the abilities of the ligand-activated T2Rs to catalyze GTP binding on members of the G _____ i subfamily including transducin, G a____ i1, and G a____ OA. With the exception of hT2R47, all of the T2Rs tested coupled with each of the three G _____ i members, although with varying affinities. These results suggest that in vivo T2Rs may signal using alternative G _______ proteins. During this reporting period, we have also begun to apply the in vitro reconstitution techniques to study the function and G protein coupling properties of other types of mammalian chemosensory receptors. Thus far, we have cloned and expressed in insect cells 19 putative mouse and human pheromone receptors and 3 mouse sweet/amino acid receptors. Membrane fractions enriched in these receptors are currently being tested for activity. In an attempt to identify novel genes involved in taste perception, we generated a normalized, subtracted cDNA library from mouse taste tissue. Sequence analyses of 20,000 clones from this library indicated that it is highly enriched in taste cell specific genes. In situ hybridization expression studies with selected clones led to the identification of several genes specifically expressed in taste cells. This year we reported the analyses of one of these genes Gpr113. Gpr113 encodes a G-protein-coupled receptor belonging to family 2B, members of which are characterized by having long N-terminal, extracellular domains. The predicted N-terminal extracellular domain of GPR113 contains 696 amino acids with two functional domains, a peptide hormone-binding domain and a G-protein-coupled receptor proteolytic site. Expression analyses indicate that Gpr113 is selectively expressed in sweet responsive cells, suggesting that it may play a role in the modulation of sweet taste. To test this hypothesis, a knock-out construct of Gpr113 has been constructed and transfected into ES cells, and selected ES cell lines are being screened for homologous recombinants.

分子神经科学部分的研究目标是确定哺乳动物化学感觉系统发展和功能的分子机制。在过去的一年里，研究工作一直致力于建立人类和小鼠化学感觉受体的功能分析，并识别和表征在味觉细胞中选择性表达的新基因。 为了表征化学感觉受体的功能特性，我们以前开发了一种体外重建试验来评估受体的活性。为了进行这些分析，表达特定受体的杆状病毒被用来感染昆虫细胞，并从被感染的昆虫细胞中提纯膜。这些膜高度富含表达的受体，可以通过添加纯化的G蛋白来进行功能重组。对23个人类苦味受体成员的初步研究发现了四种新的受体-配体相互作用。我们发现hT2R14和hT2R43受体对微摩尔浓度的马兜铃酸有选择性反应，hT2R47受体对微摩尔浓度的变性有选择性反应。相比之下，hT2R7对几种苦味化合物的反应范围更广。今年，利用这些已定义的配体-受体相互作用，我们分析了配体激活的T2Rs催化GTP结合G_I亚家族成员的能力，包括转导蛋白、G_a_1和G_a_A。除了hT2R47，所有测试的T2R都与G_I的三个成员中的每一个偶联，尽管有不同的亲和力。这些结果表明，在体内，T2Rs可能使用替代的G_蛋白来发出信号。 在此期间，我们还开始应用体外重建技术来研究其他类型哺乳动物化学感觉受体的功能和G蛋白偶联特性。到目前为止，我们已经克隆并在昆虫细胞中表达了19个可能的小鼠和人类信息素受体以及3个小鼠甜味/氨基酸受体。富含这些受体的膜部分目前正在进行活性测试。 为了找出与味觉相关的新基因，我们从小鼠的味觉组织中构建了一个归一化的、差减的cDNA文库。对该文库中20,000个克隆的序列分析表明，该文库具有高度丰富的味觉细胞特异性基因。对选定克隆的原位杂交表达研究导致了几个在味觉细胞中特异表达的基因的鉴定。今年，我们报道了对其中一个基因Gpr113的分析。Gpr113编码一种G蛋白偶联受体，属于2B家族，其成员具有长N末端的胞外结构域。GPR113的N端胞外区由696个氨基酸组成，有两个功能区，一个是肽激素结合区，另一个是G蛋白偶联受体蛋白水解区。表达分析表明，Gpr113选择性地在甜味反应细胞中表达，这表明它可能在甜味的调节中发挥作用。为了验证这一假设，构建了Gpr113的敲除结构，并将其导入ES细胞，并正在筛选选定的ES细胞系以寻找同源重组体。