Christina Kuhn

The Molecular Receptive Ranges of Human TAS2R Bitter Taste Receptors

Humans perceive thousands of compounds as bitter. In sharp contrast, only approximately 25 taste 2 receptors (TAS2R) bitter taste receptors have been identified, raising the question as to how the vast array of bitter compounds can be detected by such a limited number of sensors. To address this issue, we have challenged 25 human taste 2 receptors (hTAS2Rs) with 104 natural or synthetic bitter chemicals in a heterologous expression system. Thirteen cognate bitter compounds for 5 orphan receptors and 64 new compounds for previously identified receptors were discovered. Whereas some receptors recognized only few agonists, others displayed moderate or extreme tuning broadness. Thus, 3 hTAS2Rs together were able to detect approximately 50% of the substances used. Conversely, though 63 bitter substances activated only 1-3 receptors, 19 compounds stimulated up to 15 hTAS2Rs. Our data suggest that the detection of the numerous bitter chemicals is related to the molecular receptive ranges of hTAS2Rs.

The cells and peripheral representation of sodium taste in mice

Salt taste in mammals can trigger two divergent behavioural responses. In general, concentrated saline solutions elicit robust behavioural aversion, whereas low concentrations of NaCl are typically attractive, particularly after sodium depletion. Notably, the attractive salt pathway is selectively responsive to sodium and inhibited by amiloride, whereas the aversive one functions as a non-selective detector for a wide range of salts. Because amiloride is a potent inhibitor of the epithelial sodium channel (ENaC), ENaC has been proposed to function as a component of the salt-taste-receptor system. Previously, we showed that four of the five basic taste qualities—sweet, sour, bitter and umami—are mediated by separate taste-receptor cells (TRCs) each tuned to a single taste modality, and wired to elicit stereotypical behavioural responses. Here we show that sodium sensing is also mediated by a dedicated population of TRCs. These taste cells express the epithelial sodium channel ENaC, and mediate behavioural attraction to NaCl. We genetically engineered mice lacking ENaCα in TRCs, and produced animals exhibiting a complete loss of salt attraction and sodium taste responses. Together, these studies substantiate independent cellular substrates for all five basic taste qualities, and validate the essential role of ENaC for sodium taste in mice.

Bitter Taste Receptors for Saccharin and Acesulfame K

Weight-conscious subjects and diabetics use the sulfonyl amide sweeteners saccharin and acesulfame K to reduce their calorie and sugar intake. However, the intrinsic bitter aftertaste, which is caused by unknown mechanisms, limits the use of these sweeteners. Here, we show by functional expression experiments in human embryonic kidney cells that saccharin and acesulfame K activate two members of the human TAS2R family (hTAS2R43 and hTAS2R44) at concentrations known to stimulate bitter taste. These receptors are expressed in tongue taste papillae. Moreover, the sweet inhibitor lactisole did not block the responses of cells transfected with TAS2R43 and TAS2R44, whereas it did block the response of cells expressing the sweet taste receptor heteromer hTAS1R2-hTAS1R3. The two receptors were also activated by nanomolar concentrations of aristolochic acid, a purely bitter-tasting compound. Thus, hTAS2R43 and hTAS2R44 function as cognate bitter taste receptors and do not contribute to the sweet taste of saccharin and acesulfame K. Consistent with the in vitro data, cross-adaptation studies in human subjects also support the existence of common receptors for both sulfonyl amide sweeteners.

Members of RTP and REEP Gene Families Influence Functional Bitter Taste Receptor Expression

Functional characterization of chemosensory receptors is usually achieved by heterologous expression in mammalian cell lines. However, many chemoreceptor genes, including bitter taste receptors (TAS2Rs), show only marginal cell surface expression. Usually, these problems are circumvented by using chimeric receptors consisting of “export tags” and the receptor sequence itself. It seems likely that chemoreceptor cells express factors for cell surface targeting of native receptor molecules in vivo. For TAS2Rs, however, such factors are still unknown. The present study investigates the influence of RTP and REEP proteins on the functional expression of human TAS2Rs in heterologous cells. We expressed hTAS2Rs in HEK 293T cells and observed dramatic differences in responsiveness to agonist stimulation. By immunocytochemistry we show accumulation of the bitter β-glucopyranoside receptor hTAS2R16 in the Golgi compartment. Coexpression of RTP and REEP proteins changed the responses of some hTAS2Rs upon agonist stimulation, which is likely due to efficient cell surface localization as demonstrated by cell surface biotinylation experiments. The coimmunoprecipitation of hTAS2R16 and RTP3 or RTP4 suggests that the mechanism by which these cofactors influence hTAS2R16 function might involve direct protein-protein interaction. Finally, expression analyses demonstrate RTP and REEP gene expression in human circumvallate papillae and testis, both of which are sites of TAS2R gene expression.

Oligomerization of TAS2R Bitter Taste Receptors

A family of 25 G protein-coupled receptors, TAS2Rs, mediates bitter taste in humans. Many of the members of this family are coexpressed in a subpopulation of taste receptor cells on the tongue, thereby allowing the possibility of receptor-receptor interactions with potential influences on their function. In this study, we used several experimental approaches to investigate whether TAS2Rs can form oligomers and if this has an effect on receptor function. Coimmunoprecipitations clearly demonstrated that TAS2Rs can physically interact in HEK293T cells. Further bioluminescence resonance energy transfer analysis of all 325 possible binary combinations of TAS2Rs established that the vast majority of TAS2R pairs form oligomers, both homomers and heteromers. Subsequent screenings of coexpressed bitter receptors with 104 different tastants did not reveal any heteromer-specific agonists. Additional studies also showed no obvious influence of TAS2R hetero-oligomerization on plasma membrane localization or pharmacological properties of the receptors. Thus, our results show that receptor oligomerization occurs between TAS2R bitter taste receptors; however, functional consequences of hetero-oligomerization were not obvious.

The Human Bitter Taste Receptor hTAS2R50 Is Activated by the Two Natural Bitter Terpenoids Andrographolide and Amarogentin

Bitterness perception in mammals is mediated through activation of dedicated bitter taste receptors located in the oral cavity. Genomic analyses revealed the existence of orthologous mammalian bitter taste receptor genes, which presumably recognize the same compounds in different species, as well as species-specific receptor gene expansions believed to fulfill a critical role during evolution. In man, 8 of the 25 bitter taste receptors (hTAS2Rs) are closely related members of such an expanded subfamily of receptor genes. This study identified two natural bitter terpenoids, andrographolide and amarogentin, that are agonists for the orphan receptor hTAS2R50, the most distant member of the subfamily. This paper presents the pharmacological characterization of this receptor and analyzes its functional relationship with the previously deorphaned hTAS2R43, hTAS2R44, hTAS2R46, and hTAS2R47. Insights into the general breadth of tuning, functional redundancies, and relationships between pharmacological activation patterns and amino acid homologies for this receptor subfamily are presented.

Oligomerization of Sweet and Bitter Taste Receptors

The superfamily of G protein-coupled receptors (GPCRs) mediates numerous physiological processes, including neurotransmission, cell differentiation and metabolism, and sensory perception. In recent years, it became evident that these receptors might function not only as monomeric receptors but also as homo- or heteromeric receptor complexes. The family of TAS1R taste receptors are prominent examples of GPCR dimerization as they act as obligate functional heteromers: TAS1R1 and TAS1R3 combine to form an umami taste receptor, while the combination of TAS1R2 and TAS1R3 is a sweet taste receptor. So far, TAS2Rs, a second family of ~25 taste receptors in humans that mediates responses to bitter compounds, have been shown to function on their own, but if they do so as receptor monomers or as homomeric receptors still remains unknown. Using two different experimental approaches, we have recently shown that TAS2Rs can indeed form both homomeric and heteromeric receptor complexes. The employed techniques, coimmunoprecipitations and bioluminescence resonance energy transfer (BRET), are based on different principles and complement each other well and therefore provided compelling evidences for TAS2R oligomerization. Furthermore, we have adapted the protocols to include a number of controls and for higher throughput to accommodate the investigation of a large number of receptors and receptor combinations. Here, we present the protocols in detail.