Development And Function Of Mammalian Chemosensory Syste

分子神经科学的研究目标是确定哺乳动物化学感受系统的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受器的功能测定，并确定和表征在味觉细胞中选择性表达的新基因。 为了表征化学感受受体的功能特性，我们以前开发了一种体外重建试验来评估受体活性。为了进行这些测定，使用表达给定受体的杆状病毒感染昆虫细胞，并纯化来自感染的昆虫细胞的膜。这些膜在表达的受体中高度富集，其可以通过添加纯化的G蛋白而功能性地重建。对23个人类苦味受体成员的初步研究鉴定了四种新的受体-配体相互作用。我们证明了hT 2 R14和hT 2 R43受体选择性地响应于微摩尔浓度的马兜铃酸，hT 2 R47受体选择性地响应于微摩尔浓度的地那铵。相比之下，hT 2 R7更广泛地响应于几种苦味化合物。今年，利用这些确定的配体-受体相互作用，我们分析了配体激活的T2 R催化GTP结合G _ i亚家族成员的能力，包括transducin、G a_i 1和G a_ OA。除了hT 2 R47之外，所有测试的T2 R都与三个G _ i成员中的每一个偶联，尽管亲和力不同。这些结果表明，在体内T2受体可能使用替代G __蛋白信号。 在本报告期间，我们也开始应用体外重建技术来研究其他类型的哺乳动物化学感受器的功能和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个推定的小鼠和人类信息素受体和3个小鼠甜味/氨基酸受体。目前正在测试富含这些受体的膜组分的活性。 在试图确定新的基因参与味觉，我们产生了一个标准化的，减去cDNA文库从小鼠味觉组织。对来自该文库的20，000个克隆的序列分析表明其高度富集味觉细胞特异性基因。用选定的克隆进行原位杂交表达研究，鉴定了在味觉细胞中特异性表达的几个基因。今年，我们报告了对其中一个基因Gpr 113的分析。Gpr 113编码属于家族2B的G蛋白偶联受体，其成员的特征在于具有长的N-末端胞外结构域。GPR 113的N端胞外结构域由696个氨基酸组成，具有两个功能结构域，一个是肽结合结构域，一个是G蛋白偶联受体蛋白水解位点。表达分析表明，Gpr 113选择性地表达在甜味反应细胞中，这表明它可能在甜味调节中发挥作用。为了验证这一假设，构建了Gpr 113的敲除构建体并转染到ES细胞中，并筛选所选ES细胞系中的同源重组体。

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.