Mammalian Chemosensory Systems

分子神经科学部分的研究目标是定义哺乳动物化学感觉系统发育和功能的分子机制。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，以及识别和表征在味觉细胞中选择性表达的新基因。

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