Development & Function Of Mammalian Chemosensory Systems

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。过去一年的研究工作一直致力于建立苦味受体的功能测定，并鉴定和表征在味觉受体细胞中选择性表达的新基因。 为了更好地了解G蛋白偶联苦味受体的特异性和信号转导途径，我们克隆并构建了23个已鉴定的人苦味受体的杆状病毒表达载体。用从感染这些杆状病毒的细胞中分离的膜进行的原位重建实验表明，膜高度富集功能性苦味受体。到目前为止，已经通过筛选这些受体富集的成员对70种苦味化合物的反应鉴定了两种孤儿苦味受体的配体。除了继续筛选受体-配体相互作用和这些相互作用的定量表征，我们正在使用原位重建系统来检查苦味受体的G蛋白选择性。 该实验室正在努力鉴定参与味觉细胞信号转导和功能的基因。我们先前从味觉组织产生了标准化的消减cDNA文库。对该文库中20000个克隆的序列分析表明，该文库高度富集味觉感受器细胞特异性基因。用选定的克隆进行的原位杂交表达研究正在继续进行，并已鉴定出在味觉细胞中特异性表达的几个基因。这些基因中的两个是过去一年研究的焦点，它们编码味觉细胞信号转导途径的推定组分，包括一种新型离子通道和一种新型G蛋白偶联受体。广泛的原位杂交研究和北方分析表明，这两个基因特异性表达在一个不同的味觉受体细胞的子集。对于这些基因中的每一个，全长cDNA克隆已被分离，并且基于细胞的异源表达系统以及敲除小鼠模型正在开发中。

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 bitter taste receptors and identifying and characterizing novel genes selectively expressed in taste receptor cells. To better understand the specificities and signal transduction pathways of the G-protein coupled bitter receptors, we have cloned and constructed baculoviral expression vectors with the 23 identified human bitter receptors. In situ re-constitution experiments with membranes isolated from cells infected with these baculoviruses indicate that the membranes are highly enriched in functional bitter receptors. Thus far, ligands to two orphan bitter receptors have been identified by screening responses of these receptor-enriched members to a panel of 70 bitter-tasting compounds. In addition to the continued screening for receptor-ligand interactions and the quantitative characterization of these interactions, we are using the in situ re-constitution system to examine the G protein selectivities of bitter receptors. An ongoing effort of the laboratory is to identify genes involved in taste cell signal transduction and function. We previously generated a normalized, subtracted cDNA library from taste tissue. Sequence analyses of 20000 clones from this library indicate that it is highly enriched in taste receptor cell specific genes. In situ hybridization expression studies with selected clones are continuing and have led to the identification of several genes specifically expressed in taste cells. Two of these genes, which have been the focus of study this past year, encode putative components of the taste cell signal transduction pathway including a novel ion channel and a novel G-protein coupled receptor. Extensive in situ hybridization studies and Northern analyses indicate that both of these genes are specifically expressed in a distinct subset of taste receptor cells. For each of these genes, full length cDNAs clones have been isolated, and cell-based heterologous expression systems, as well as knock-out mouse models, are being developed.