Peihua Jiang

Afferent neurotransmission mediated by hemichannels in mammalian taste cells

In mammalian taste buds, ionotropic P2X receptors operate in gustatory nerve endings to mediate afferent inputs. Thus, ATP secretion represents a key aspect of taste transduction. Here, we characterized individual vallate taste cells electrophysiologically and assayed their secretion of ATP with a biosensor. Among electrophysiologically distinguishable taste cells, a population was found that released ATP in a manner that was Ca2+ independent but voltage‐dependent. Data from physiological and pharmacological experiments suggested that ATP was released from taste cells via specific channels, likely to be connexin or pannexin hemichannels. A small fraction of ATP‐secreting taste cells responded to bitter compounds, indicating that they express taste receptors, their G‐protein‐coupled and downstream transduction elements. Single cell RT–PCR revealed that ATP‐secreting taste cells expressed gustducin, TRPM5, PLCβ2, multiple connexins and pannexin 1. Altogether, our data indicate that tastant‐responsive taste cells release the neurotransmitter ATP via a non‐exocytotic mechanism dependent upon the generation of an action potential.

T2R38 taste receptor polymorphisms underlie susceptibility to upper respiratory infection

Innate and adaptive defense mechanisms protect the respiratory system from attack by microbes. Here, we present evidence that the bitter taste receptor T2R38 regulates the mucosal innate defense of the human upper airway. Utilizing immunofluorescent and live cell imaging techniques in polarized primary human sinonasal cells, we demonstrate that T2R38 is expressed in human upper respiratory epithelium and is activated in response to acyl-homoserine lactone quorum-sensing molecules secreted by Pseudomonas aeruginosa and other gram-negative bacteria. Receptor activation regulates calcium-dependent NO production, resulting in stimulation of mucociliary clearance and direct antibacterial effects. Moreover, common polymorphisms of the TAS2R38 gene were linked to significant differences in the ability of upper respiratory cells to clear and kill bacteria. Lastly, TAS2R38 genotype correlated with human sinonasal gram-negative bacterial infection. These data suggest that T2R38 is an upper airway sentinel in innate defense and that genetic variation contributes to individual differences in susceptibility to respiratory infection.

Lactisole Interacts with the Transmembrane Domains of Human T1R3 to Inhibit Sweet Taste

The detection of sweet-tasting compounds is mediated in large part by a heterodimeric receptor comprised of T1R2+T1R3. Lactisole, a broad-acting sweet antagonist, suppresses the sweet taste of sugars, protein sweeteners, and artificial sweeteners. Lactisoles inhibitory effect is specific to humans and other primates; lactisole does not affect responses to sweet compounds in rodents. By heterologously expressing interspecies combinations of T1R2+T1R3, we have determined that the target for lactisoles action is human T1R3. From studies with mouse/human chimeras of T1R3, we determined that the molecular basis for sensitivity to lactisole depends on only a few residues within the transmembrane region of human T1R3. Alanine substitution of residues in the transmembrane region of human T1R3 revealed 4 key residues required for sensitivity to lactisole. In our model of T1R3s seven transmembrane helices, lactisole is predicted to dock to a binding pocket within the transmembrane region that includes these 4 key residues.

Identification of the Cyclamate Interaction Site within the Transmembrane Domain of the Human Sweet Taste Receptor Subunit T1R3

The artificial sweetener cyclamate tastes sweet to humans, but not to mice. When expressed in vitro, the human sweet receptor (a heterodimer of two taste receptor subunits: hT1R2 + hT1R3) responds to cyclamate, but the mouse receptor (mT1R2 + mT1R3) does not. Using mixed-species pairings of human and mouse sweet receptor subunits, we determined that responsiveness to cyclamate requires the human form of T1R3. Using chimeras, we determined that it is the transmembrane domain of hT1R3 that is required for the sweet receptor to respond to cyclamate. Using directed mutagenesis, we identified several amino acid residues within the transmembrane domain of T1R3 that determine differential responsiveness to cyclamate of the human versus mouse sweet receptors. Alanine-scanning mutagenesis of residues predicted to line a transmembrane domain binding pocket in hT1R3 identified six residues specifically involved in responsiveness to cyclamate. Using molecular modeling, we docked cyclamate within the transmembrane domain of T1R3. Our model predicts substantial overlap in the hT1R3 binding pockets for the agonist cyclamate and the inverse agonist lactisole. The transmembrane domain of T1R3 is likely to play a critical role in the interconversion of the sweet receptor from the ground state to the active state.

Major taste loss in carnivorous mammals

Mammalian sweet taste is primarily mediated by the type 1 taste receptor Tas1r2/Tas1r3, whereas Tas1r1/Tas1r3 act as the principal umami taste receptor. Bitter taste is mediated by a different group of G protein-coupled receptors, the Tas2rs, numbering 3 to ∼66, depending on the species. We showed previously that the behavioral indifference of cats toward sweet-tasting compounds can be explained by the pseudogenization of the Tas1r2 gene, which encodes the Tas1r2 receptor. To examine the generality of this finding, we sequenced the entire coding region of Tas1r2 from 12 species in the order Carnivora. Seven of these nonfeline species, all of which are exclusive meat eaters, also have independently pseudogenized Tas1r2 caused by ORF-disrupting mutations. Fittingly, the purifying selection pressure is markedly relaxed in these species with a pseudogenized Tas1r2. In behavioral tests, the Asian otter (defective Tas1r2) showed no preference for sweet compounds, but the spectacled bear (intact Tas1r2) did. In addition to the inactivation of Tas1r2, we found that sea lion Tas1r1 and Tas1r3 are also pseudogenized, consistent with their unique feeding behavior, which entails swallowing food whole without chewing. The extensive loss of Tas1r receptor function is not restricted to the sea lion: the bottlenose dolphin, which evolved independently from the sea lion but displays similar feeding behavior, also has all three Tas1rs inactivated, and may also lack functional bitter receptors. These data provide strong support for the view that loss of taste receptor function in mammals is widespread and directly related to feeding specializations.

The Heterodimeric Sweet Taste Receptor has Multiple Potential Ligand Binding Sites

The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes.

Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue.

Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine‐rich repeat‐containing G‐protein‐coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5‐expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate (CV) and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5‐expressing cells in posterior tongue are a subset of K14‐positive epithelial cells. Lineage‐tracing experiments using an inducible Cre knockin allele in combination with Rosa26‐LacZ and Rosa26‐tdTomato reporter strains showed that Lgr5‐expressing cells gave rise to taste cells, perigemmal cells, along with self‐renewing cells at the bottom of trench areas at the base of CV and foliate papillae. Moreover, using subtype‐specific taste markers, we found that Lgr5‐expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue. STEM CELLS 2013;31:992–1000

Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo

Significance Taste tissue regenerates continuously throughout the life span in mammals. Here, using lineage tracing and a culture system, we show that leucine-rich repeat-containing G protein-coupled receptor 5-expressing and leucine-rich repeat-containing G protein-coupled receptor 6-expressing taste stem/progenitor cells generate mature taste cells in vivo and ex vivo. Importantly, our ex vivo studies show that single-progenitor cells can generate all mature taste cell types and that differentiated taste cells form in the absence of innervation. This ex vivo model mimics the development of taste bud cells in taste papillae, recapitulates the process of taste renewal from adult taste stem cells to mature taste cells, and provides a means to study the regulation of taste cell generation and to understand the origins and cell lineage relationships within taste buds. Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5+) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5+ or Lgr6+ cells from taste tissue can generate continuously expanding 3D structures (“organoids”). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2’-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5+ cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6+ cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5+ or Lgr6+ cells, validating the use of this model for the study of taste cell generation.

Molecular Mechanism of Species-Dependent Sweet Taste toward Artificial Sweeteners

The heterodimer of Tas1R2 and Tas1R3 is a broadly acting sweet taste receptor, which mediates mammalian sweet taste toward natural and artificial sweeteners and sweet-tasting proteins. Perception of sweet taste is a species-selective physiological process. For instance, artificial sweeteners aspartame and neotame taste sweet to humans, apes, and Old World monkeys but not to New World monkeys and rodents. Although specific regions determining the activation of the receptors by these sweeteners have been identified, the molecular mechanism of species-dependent sweet taste remains elusive. Using human/squirrel monkey chimeras, mutagenesis, and molecular modeling, we reveal that the different responses of mammalian species toward the artificial sweeteners aspartame and neotame are determined by the steric effect of a combination of a few residues in the ligand binding pocket. Residues S40 and D142 in the human Tas1R2, which correspond to residues T40 and E142 in the squirrel monkey Tas1R2, were found to be the critical residues for the species-dependent difference in sweet taste. In addition, human Tas1R2 residue I67, which corresponds to S67 in squirrel monkey receptor, modulates the higher affinity of neotame than of aspartame. Our studies not only shed light on the molecular mechanism of species-dependent sweet taste toward artificial sweeteners, but also provide guidance for designing novel effective artificial sweet compounds.

Bacterial d-amino acids suppress sinonasal innate immunity through sweet taste receptors in solitary chemosensory cells

Staphylococcus-derived d-amino acids reduce innate immune responses in the nasal epithelium by stimulating the sweet taste receptor. The sweet taste of bacteria Stimulation of the sweet taste receptor (T1R) in solitary chemosensory cells of the upper respiratory epithelium inhibits the release of antimicrobial peptides by neighboring epithelial cells. In addition to being activated by various sugars, T1R can also be activated by some d-amino acids. Lee et al. found that Staphylococcus species in the nasal cavities of chronic rhinosinusitis patients produced d-Phe and d-Leu, both of which can activate T1R. Treatment of primary human sinonasal epithelial cultures with d-Phe and d-Leu inhibited the release of antimicrobial peptides and increased cell death in response to infection with methicillin-resistant S. aureus. d-Phe and d-Leu, as well as medium conditioned by respiratory isolates of Staphylococcus, inhibited the formation of Pseudomonas aeruginosa biofilms. These findings demonstrate that d-amino acids produced by nasal flora can inhibit innate immune responses through T1R and may shape the microbial community of the airways. In the upper respiratory epithelium, bitter and sweet taste receptors present in solitary chemosensory cells influence antimicrobial innate immune defense responses. Whereas activation of bitter taste receptors (T2Rs) stimulates surrounding epithelial cells to release antimicrobial peptides, activation of the sweet taste receptor (T1R) in the same cells inhibits this response. This mechanism is thought to control the magnitude of antimicrobial peptide release based on the sugar content of airway surface liquid. We hypothesized that d-amino acids, which are produced by various bacteria and activate T1R in taste receptor cells in the mouth, may also activate T1R in the airway. We showed that both the T1R2 and T1R3 subunits of the sweet taste receptor (T1R2/3) were present in the same chemosensory cells of primary human sinonasal epithelial cultures. Respiratory isolates of Staphylococcus species, but not Pseudomonas aeruginosa, produced at least two d-amino acids that activate the sweet taste receptor. In addition to inhibiting P. aeruginosa biofilm formation, d-amino acids derived from Staphylococcus inhibited T2R-mediated signaling and defensin secretion in sinonasal cells by activating T1R2/3. d-Amino acid–mediated activation of T1R2/3 also enhanced epithelial cell death during challenge with Staphylococcus aureus in the presence of the bitter receptor–activating compound denatonium benzoate. These data establish a potential mechanism for interkingdom signaling in the airway mediated by bacterial d-amino acids and the mammalian sweet taste receptor in airway chemosensory cells.