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.
