Susan L. Sullivan

Identification of a novel member of the T1R family of putative taste receptors

In the gustatory system, the recognition of sugars, amino acids and bitter‐tasting compounds is the function of specialized G protein‐coupled receptors. Recently, two members of novel subfamily of G protein‐coupled receptors were proposed to function as taste receptors based on their specific expression in taste receptor cells. Here, we report the identification of a third member, T1R3, of this family of receptors. T1R3 maps near the telomere of mouse chromosome 4 rendering it a candidate for the Sac locus, a primary determinant of sweet preference in mice. Consistent with its candidacy for the Sac locus, T1R3 displays taste receptor cell‐specific expression. In addition, taster and non‐taster strains of mouse harbor different alleles of T1R3.

Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function.

Positional cloning of hereditary deafness genes is a direct approach to identify molecules and mechanisms underlying auditory function. Here we report a locus for dominant deafness, DFNA36, which maps to human chromosome 9q13–21 in a region overlapping the DFNB7/B11 locus for recessive deafness. We identified eight mutations in a new gene, transmembrane cochlear-expressed gene 1 (TMC1), in a DFNA36 family and eleven DFNB7/B11 families. We detected a 1.6-kb genomic deletion encompassing exon 14 of Tmc1 in the recessive deafness (dn) mouse mutant, which lacks auditory responses and has hair-cell degeneration. TMC1 and TMC2 on chromosome 20p13 are members of a gene family predicted to encode transmembrane proteins. Tmc1 mRNA is expressed in hair cells of the postnatal mouse cochlea and vestibular end organs and is required for normal function of cochlear hair cells.

Nucleoside triphosphate diphosphohydrolase-2 is the ecto-ATPase of type I cells in taste buds.

The presence of one or more calcium‐dependent ecto‐ATPases (enzymes that hydrolyze extracellular 5′‐triphosphates) in mammalian taste buds was first shown histochemically. Recent studies have established that dominant ecto‐ATPases consist of enzymes now called nucleoside triphosphate diphosphohydrolases (NTPDases). Massively parallel signature sequencing (MPSS) from murine taste epithelium provided molecular evidence suggesting that NTPDase2 is the most likely member present in mouse taste papillae. Immunocytochemical and enzyme histochemical staining verified the presence of NTPDase2 associated with plasma membranes in a large number of cells within all mouse taste buds. To determine which of the three taste cell types expresses this enzyme, double‐label assays were performed with antisera directed against the glial glutamate/aspartate transporter (GLAST), the transduction pathway proteins phospholipase Cβ2 (PLCβ2) or the G‐protein subunit α‐gustducin, and serotonin (5HT) as markers of type I, II, and III taste cells, respectively. Analysis of the double‐labeled sections indicates that NTPDase2 immunoreactivity is found on cell processes that often envelop other taste cells, reminiscent of type I cells. In agreement with this observation, NTPDase2 was located to the same membrane as GLAST, indicating that this enzyme is present in type I cells. The presence of ecto‐ATPase in taste buds likely reflects the importance of ATP as an intercellular signaling molecule in this system. J. Comp. Neurol. 497:1–12, 2006.

Functional characterization of human bitter taste receptors

The T2Rs belong to a multi-gene family of G-protein-coupled receptors responsible for the detection of ingested bitter-tasting compounds. The T2Rs are conserved among mammals with the human and mouse gene families consisting of about 25 members. In the present study we address the signalling properties of human and mouse T2Rs using an in vitro reconstitution system in which both the ligands and G-proteins being assayed can be manipulated independently and quantitatively assessed. We confirm that the mT2R5, hT2R43 and hT2R47 receptors respond selectively to micromolar concentrations of cycloheximide, aristolochic acid and denatonium respectively. We also demonstrate that hT2R14 is a receptor for aristolochic acid and report the first characterization of the ligand specificities of hT2R7, which is a broadly tuned receptor responding to strychnine, quinacrine, chloroquine and papaverine. Using these defined ligand-receptor interactions, we assayed the ability of the ligand-activated T2Rs to catalyse GTP binding on divergent members of the G(alpha) family including three members of the G(alphai) subfamily (transducin, G(alphai1) and G(alphao)) as well as G(alphas) and G(alphaq). The T2Rs coupled with each of the three G(alphai) members tested. However, none of the T2Rs coupled to either G(alphas) or G(alphaq), suggesting the T2Rs signal primarily through G(alphai)-mediated signal transduction pathways. Furthermore, we observed different G-protein selectivities among the T2Rs with respect to both G(alphai) subunits and G(betagamma) dimers, suggesting that bitter taste is transduced by multiple G-proteins that may differ among the T2Rs.

Taste Function in Mice with a Targeted Mutation of the Pkd1l3 Gene

Recent studies, both in vitro and in vivo, have suggested the involvement of the polycystic kidney disease-1 and -2 like genes, Pkd1l3 and Pkd2l1, in acid taste transduction. In mice, disruption of taste cells expressing PKD2L1 eliminates gustatory neural responses to acids. However, no previous data exist on taste responses in the absence of PKD1L3 or on behavioral responses in mice lacking either of these proteins. In order to assess the function of PKD1L3, we genetically engineered mice with a targeted mutation of the Pkd1l3 gene. We then examined taste responsiveness of mutant and wild-type mice using several different approaches. In separate groups of mice, we measured preference scores in 48-h 2-bottle tests, determined NaCl or citric acid taste thresholds using a conditioned taste aversion technique, and conducted electrophysiological recordings of activity in the chorda tympani and glossopharyngeal nerves. Multiple taste compounds representing all major taste qualities were used in the preference tests and nerve-recording experiments. We found no significant reduction in taste responsiveness in Pkd1l3 mutant mice in behavioral or electrophysiological tests when compared with wild-type controls. Therefore, further studies are needed to elucidate the function of PKD1L3 in taste bud cells.

Characterization and expression of sema4g, a novel member of the semaphorin gene family.

Semaphorins constitute a large and growing gene family, several members of which are axon guidance molecules. We report the characterization of sema4g, a novel class IV member of the semaphorin gene family, located on mouse chromosome 19. sema4g is expressed early in development in the brain, spinal cord, and several sensory organs as well as specific populations of projection neurons, compatible with the well-established function of semaphorins as axon guidance molecules.