Mammalian Chemosensory Systems

哺乳动物化学感应系统

• 批准号: 7297972
• 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/7297972
• 关键词: Mammalian; Chemosensory; Systems

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. More recently, we have used the same methodology to study the human T1R taste receptors. The three T1R taste receptors function as heterodimers to mediate umami (T1R1+T1R3) and sweet (T1R2+T1R3) tastes. These receptors like other family 3 G-protein-coupled receptors (GPCRs) have large extracellular binding domains followed by a rhodopsin-like seven transmembrane core domain. We demonstrated that in the absence of their ligand-binding extracellular domains the hT1R1 and hT1R2 core domains constitutively and robustly (up to 40-fold stimulation over background) activate G proteins. In contrast, the core domain of hT1R3, the common subunit of both the sweet and umami receptors, couples relatively poorly to G proteins. These results suggest that in taste cells the hT1R1 or hT1R2 component of the functional heterodimer is responsible for signaling and that the ligand-unbound extracellular domain functions to repress the spontaneous activity of the core domain, a property likely shared by other family 3 GPCRs. The constitutive activities of the core domains of hT1R1 and hT1R2 allowed us to directly assess their abilities to couple to divergent G alpha subunits and to demonstrate that the hT1Rs signal selectively via G alpha i/o pathways. Taken together, these results have important implications for both the mechanism of activation of family 3 GPCRs and the basic biology of taste receptor function and signal transduction. In addition, these findings open the door to the generation of novel ways to screen for allosteric modulators (many of which act directly on the core domains) of taste and possibly other family C GPCRs. In an attempt to identify novel genes involved in taste perception, we previously 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 PKD1L3. PKD1L3 belongs to the TRPP or PKD subfamily of TRP channels, the founding members of which were originally identified as being associated with polycystic kidney disease. We demonstrate that Pkd1L3 is expressed selectively in a subset of taste cells that are distinct from those dedicated to the detection of sweet and bitter tasting compounds, suggesting a role for PKD1L3 in salty or sour taste detection. Our findings provide the first evidence for a role of TRPP channels in taste transduction. Furthermore, we find that PKD1L3 is co-expressed with a second TRPP channel, PKD2L1, in taste cells. Given the precedence for other members of the family to function as heteromultimer, we hypothesize that PKD1L3 and PKD2L1 function as a heteromeric sour or salty taste channel. To test the hypothesis for an involvement in salty/sour taste transduction, a knock-out construct of Pkd1L3 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。最近，我们使用相同的方法来研究人类T1 R味觉受体。三种T1 R味觉受体作为异二聚体发挥功能，以介导鲜味（T1 R1 + T1 R3）和甜味（T1 R2 + T1 R3）。这些受体像其他家族3 G蛋白偶联受体（GPCR）一样具有大的细胞外结合结构域，随后是视紫红质样七跨膜核心结构域。我们证明，在没有其配体结合胞外结构域的hT 1 R1和hT 1 R2核心结构域组成型和鲁棒性（高达40倍的刺激超过背景）激活G蛋白。相比之下，hT 1 R3的核心结构域，甜味和鲜味受体的共同亚基，与G蛋白的偶联相对较差。这些结果表明，在味觉细胞的hT 1 R1或hT 1 R2的功能异源二聚体的组成部分是负责信号和配体未结合的胞外结构域的功能，以抑制核心结构域的自发活动，其他家庭3 GPCR可能共享的属性。hT 1 R1和hT 1 R2的核心结构域的组成活性使我们能够直接评估它们与不同的G α亚基偶联的能力，并证明hT 1 R选择性地通过G α i/o途径进行信号传导。总之，这些结果对家族3 GPCR的激活机制以及味觉受体功能和信号转导的基础生物学都具有重要意义。此外，这些发现为筛选味觉和可能的其他家族C GPCR的变构调节剂（其中许多直接作用于核心结构域）的新方法的产生打开了大门。 在试图确定新的基因参与味觉感知，我们以前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，我们报告了这些基因之一PKD 1 L3的分析。PKD 1 L3属于TRP通道的TRPP或PKD亚家族，其创始成员最初被鉴定为与多囊肾疾病相关。我们证明，Pkd 1 L3选择性地表达在一个亚组的味觉细胞是不同的，从那些专门用于检测甜味和苦味的化合物，这表明PKD 1 L3在咸味或酸味检测的作用。我们的研究结果提供了第一个证据的TRPP通道在味觉转导中的作用。此外，我们发现PKD 1 L3与第二个TRPP通道PKD 2L 1在味觉细胞中共表达。鉴于该家族的其他成员作为异源多聚体发挥作用的优先性，我们假设PKD 1 L3和PKD 2L 1作为异源酸或咸味通道发挥作用。为了检验参与咸味/酸味转导的假设，已经构建了Pkd 1 L3的敲除构建体并转染到ES细胞中，并且正在筛选所选择的ES细胞系中的同源重组体

项目成果

Mammalian chemosensory receptors.

哺乳动物化学感应受体。

• DOI: 10.1097/00001756-200201210-00003
• 发表时间: 2002
• 期刊: Neuroreport
• 影响因子: 1.7
• 作者: Sullivan,SusanL

The G-protein coupling properties of the human sweet and amino acid taste receptors.

人类甜味和氨基酸味觉受体的 G 蛋白偶联特性。

• DOI: 10.1002/dneu.20403
• 发表时间: 2007
• 期刊: Developmental neurobiology
• 影响因子: 3
• 作者: Sainz,Eduardo;Cavenagh,MargaretM;LopezJimenez,NelsonD;Gutierrez,JoanneC;Battey,JamesF;Northup,JohnK;Sullivan,SusanL
• 通讯作者: Sullivan,SusanL