Mammalian Chemosensory Systems

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。最近，我们用同样的方法研究了人类的T1R味觉受体。三种T1R味觉受体作为异源二聚体调节鲜味(T1R1+T1R3)和甜味(T1R2+T1R3)。这些受体和其他3G蛋白偶联受体(GPCRs)一样，具有较大的胞外结合结构域，其次是视紫红质样的七个跨膜核心区。我们证明，在没有配体结合的胞外区的情况下，hT1R1和hT1R2核心区结构性地和强健地(高达40倍于背景的刺激)激活G蛋白。相比之下，hT1R3的核心区是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，在味觉细胞中，功能异二聚体的hT1R1或hT1R2组件负责信号传递，而未与配体结合的胞外结构域起到抑制核心区自发活性的作用，这一特性可能与其他家族3 GPCR相同。HT1R1和hT1R2核心区的结构活性使我们能够直接评估它们与不同的Gα亚基偶联的能力，并证明hT1Rs通过GαI/O通路选择性地传递信号。综上所述，这些结果对GPCRs家族3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的意义。此外，这些发现为筛选变构调节剂(其中许多直接作用于味觉的核心域)以及可能的其他C家族GPCR的新方法打开了大门。 为了找出与味觉相关的新基因，我们先前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PKD1L3的分析。PKD1L3属于Trp通道的TRPP或PKD亚家族，其创始成员最初被认为与多囊肾病有关。我们证明了Pkd1L3在一组味觉细胞中选择性地表达，这些细胞不同于那些专门检测甜味和苦味化合物的味觉细胞，这表明PKD1L3在咸味或酸味检测中起到了作用。我们的发现为TRPP通道在味觉转导中的作用提供了第一个证据。此外，我们还发现，在味觉细胞中，PKD1L3与第二个TRPP通道PKD2L1共表达。鉴于该家族的其他成员优先作为异多聚体发挥作用，我们假设PKD1L3和PKD2L1作为异构酸或咸味通道发挥作用。为了验证与咸味/酸味转导有关的假设，构建了Pkd1L3的敲除结构，并将其导入ES细胞，并正在筛选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. 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