Maik Behrens

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.

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.

Gustatory Expression Pattern of the Human TAS2R Bitter Receptor Gene Family Reveals a Heterogenous Population of Bitter Responsive Taste Receptor Cells

Human bitter taste is mediated by ∼25 members of the human TAS2 receptor (hTAS2R) gene family. The hTAS2R genes are expressed in taste buds of gustatory papillae on the tongue surface. Because many naturally occurring bitter compounds are toxic, bitter taste receptors are believed to serve as warning sensors against the ingestion of toxic food compounds. An important question is whether bitter taste receptor cells are a homogeneous, broadly tuned population of cells, which uniformly express all bitter taste receptor genes, or not. Gene expression analyses in rodents demonstrated an essentially overlapping expression of TAS2R genes indicating a broad tuning, whereas functional in vivo analyses suggest a narrow tuning. The present study demonstrates the expression of all 25 human TAS2R genes in taste receptor cells of human circumvallate papillae. As shown by in situ hybridization experiments, the expression of hTAS2R genes differs in both the apparent level of expression and the number of taste receptor cells expressing these genes, suggesting a heterogeneous bitter taste receptor cell population. Differences in gene expression levels were verified by quantitative reverse transcription-PCR experiments for a subset of hTAS2R genes. Direct evidence for the heterogeneity of bitter taste receptor cells is provided by dual-labeling in situ hybridizations with selected pairs of hTAS2R gene-specific probes. Functional coexpression experiments in heterologous cells show competition among hTAS2Rs, indicating a possible biological reason for the observed expression pattern. From the data, we conclude that human bitter taste receptor cells are tuned to detect a limited subset of bitter stimuli.

Gustatory and extragustatory functions of mammalian taste receptors.

An ever increasing number of reports about taste receptors in non-gustatory tissues suggest that these molecules must have additional functions apart from taste. Of the extraoral tissues expressing taste receptors, the gastrointestinal tract received particular attention since evidence is mounting that tastants after being ingested might exert important regulatory roles in digestive and metabolic processes. At present, the G protein-coupled taste receptors for sweet, umami and bitter stimuli along with taste-related signaling molecules have been investigated in various parts of the alimentary canal. While the mechanism linking the gastrointestinal activity of sweet compounds via the activation of sweet taste receptors to metabolic adjustments has been worked out in some detail, other taste receptor mediated gastrointestinal activities are less well understood. The present article summarizes current knowledge on mammalian G protein-coupled taste receptors as well as various aspects of their proposed role in gastrointestinal tissues.

Structural requirements of bitter taste receptor activation

An important question in taste research is how 25 receptors of the human TAS2R family detect thousands of structurally diverse compounds. An answer to this question may arise from the observation that TAS2Rs in general are broadly tuned to interact with numerous substances. Ultimately, interaction with chemically diverse agonists requires architectures of binding pockets tailored to combine flexibility with selectivity. The present study determines the structure of hTAS2R binding pockets. We focused on a subfamily of closely related hTAS2Rs exhibiting pronounced amino acid sequence identities but unique agonist activation spectra. The generation of chimeric and mutant receptors followed by calcium imaging analyses identified receptor regions and amino acid residues critical for activation of hTAS2R46, -R43, and -R31. We found that the carboxyl-terminal regions of the investigated receptors are crucial for agonist selectivity. Intriguingly, exchanging two residues located in transmembrane domain seven between hTAS2R46, activated by strychnine, and hTAS2R31, activated by aristolochic acid, was sufficient to invert agonist selectivity. Further mutagenesis revealed additional positions involved in agonist interaction. The transfer of functionally relevant amino acids identified in hTAS2R46 to the corresponding positions of hTAS2R43 and -R31 resulted in pharmacological properties indistinguishable from the parental hTAS2R46. In silico modeling of hTAS2R46 allowed us to visualize the putative mode of interaction between agonists and hTAS2Rs. Detailed structure-function analyses of hTAS2Rs may ultimately pave the way for the development of specific antagonists urgently needed for more sophisticated analyses of human bitter taste perception.

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.

Modulation of bitter taste perception by a small molecule hTAS2R antagonist

Human bitter taste is mediated by the hTAS2R family of G protein-coupled receptors. The discovery of the hTAS2Rs enables the potential to develop specific bitter receptor antagonists that could be beneficial as chemical probes to examine the role of bitter receptor function in gustatory and nongustatory tissues. In addition, they could have widespread utility in food and beverages fortified with vitamins, antioxidants, and other nutraceuticals, because many of these have unwanted bitter aftertastes. We employed a high-throughput screening approach to discover a novel bitter receptor antagonist (GIV3727) that inhibits activation of hTAS2R31 (formerly hTAS2R44) by saccharin and acesulfame K, two common artificial sweeteners. Pharmacological analyses revealed that GIV3727 likely acts as an orthosteric, insurmountable antagonist of hTAS2R31. Surprisingly, we also found that this compound could inhibit five additional hTAS2Rs, including the closely related receptor hTAS2R43. Molecular modeling and site-directed mutagenesis studies suggest that two residues in helix 7 are important for antagonist activity in hTAS2R31 and hTAS2R43. In human sensory trials, GIV3727 significantly reduced the bitterness associated with the two sulfonamide sweeteners, indicating that hTAS2R antagonists are active in vivo. Our results demonstrate that small molecule bitter receptor antagonists can effectively reduce the bitter taste qualities of foods, beverages, and pharmaceuticals.

Receptor Agonism and Antagonism of Dietary Bitter Compounds

Food contains complex blends of structurally diverse bitter compounds that trigger bitterness through activation of one or more of the ∼25 human TAS2 bitter taste receptors. It remains unsolved, however, whether the perceived bitterness of binary bitter-compound mixtures can be considered an additive function of all bitter-inducing chemicals in the mouth, suggesting that little mutual interaction takes place among bitter substances or if mixture suppression and synergism occurs. Here we report on two natural sesquiterpene lactones from edible plants, which stimulate distinct sets of hTAS2Rs in transfected cells. Both chemicals also robustly inhibit different but overlapping subsets of agonist-activated hTAS2Rs. These findings demonstrate that mixtures of bitter compounds, because they normally occur in human foodstuff, likely elicit bitter perception in a complex and not in a merely additive manner. An unexpected implication of this discovery is that, during evolution, the naturally occurring bitter taste receptor antagonists have shaped some of the pharmacological properties of the receptors, such as overlapping recognition profiles and breadth of tuning.

Mammalian Bitter Taste Perception

Bitter taste in mammals is achieved by a family of approximately 30 bitter taste receptor genes. The main function of bitter taste is to protect the organism against the ingestion of, frequently bitter, toxic food metabolites. The field of taste research has advanced rapidly during the last several years. This is especially true for the G-protein-coupled-receptor-mediated taste qualities, sweet, umami, and bitter. This review summarizes current knowledge of bitter taste receptor gene expression, signal transduction, the structure-activity relationship of bitter taste receptor proteins, as well as their variability leading to a high degree of individualization of this taste quality in mammals.

BMP mRNA and protein expression in the developing mouse olfactory system

The bone morphogenetic proteins (BMPs) play fundamental roles during the organization of the central nervous system. The presence of these proteins has also been demonstrated in regions of the adult brain that are characterized by neural plasticity. In this study, we examined the expression of BMP4, 6, and 7 mRNAs and proteins in the murine olfactory system. The olfactory system is a useful model for studying cell proliferation and neural differentiation because both of these processes persist throughout life in the olfactory epithelium (OE) and olfactory bulb (OB). Our results demonstrate a differential expression of BMP4, 6, and 7 in the embryonic, postnatal, and adult olfactory system. In particular, BMP4 and BMP7 showed similar immunostaining patterns, being expressed in the olfactory region from the earliest stages studied (embryonic day 15.5) to adulthood. During development BMPs were expressed in the OE, olfactory bulb nerve layer, glomerular layer (GL), mitral cell layer (MCL), and subventricular zone. During the first postnatal week of life, BMP4 and 7 immunoreactivity (‐ir) was particularly evident in the GL, MCL, and in the subependymal layer (SEL), which originates postnatally from the subventricular zone. In adults, BMP4 and 7 immunostaining was present in the GL and SEL. Within the SEL, BMP4 and 7 proteins were expressed primarily in association with the astrocytic glial compartment. BMP6‐ir was always found in mature olfactory receptor neurons and their axonal projections to the OB. In summary, these data support the hypothesis that BMPs play a role in the morphogenesis of the olfactory system during development and in its plasticity during adulthood. J. Comp. Neurol. 451:267–278, 2002.