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Background: T2Rs are activated by hundreds of bitter compounds; however, only five blockers are known. Results: T2R4 residues involved in binding to agonist quinine and two novel bitter blockers GABA and BCML were identified. Conclusion: Bitter blockers and agonists share the same orthosteric site in T2R4. Significance: Bitter blockers identified in this study have tremendous physiological and nutraceutical importance.

The human bitter taste receptors (T2Rs) are non-Class A members of the G-protein-coupled receptor (GPCR) superfamily, with very limited structural information. Amino acid sequence analysis reveals that most of the important motifs present in the transmembrane helices (TM1-TM7) of the well studied Class A GPCRs are absent in T2Rs, raising fundamental questions regarding the mechanisms of activation and how T2Rs recognize bitter ligands with diverse chemical structures. In this study, the bitter receptor T2R1 was used to systematically investigate the role of 15 transmembrane amino acids in T2Rs, including 13 highly conserved residues, by amino acid replacements guided by molecular modeling. form a putative LXXSL motif which performs predominantly a structural role by stabilizing the helical conformation of TM5 at the cytoplasmic end. This study provides for the first time mechanistic insights into the roles of the conserved transmembrane residues in T2Rs and allows comparison of the activation mechanisms of T2Rs with the Class A GPCRs.The mammalian taste sensation provides valuable information about the nature and quality of food. Taste transduction predominantly involves the interaction of molecules (i.e. tastants) with taste receptor-expressing cells that reside in the taste buds located on the papillae of the tongue. Taste buds relay information to the brain on the nutrient content of food.

Host-pathogen interactions play an important role in defining the outcome of a disease. Recent studies have shown bacterial quorum sensing molecules (QSM) can interact with host cell membrane proteins, mainly G protein-coupled receptors (GPCRs), and induce innate immune responses. However, few studies have examined QSM-GPCR interactions and their influence on oral innate immune responses. In this study, we examined the role of bitter taste receptor T2R14 in sensing competence stimulating peptides (CSPs) secreted by cariogenic bacterium Streptococcus mutans and in mediating innate immune responses in gingival epithelial cells (GECs). Transcriptomic and western blot analyses identify T2R14 to be highly expressed in GECs. Our data shows that only CSP-1 from S. mutans induces robust intracellular calcium mobilization compared to CSP-2 and CSP-3. By using CRISPR-Cas9, we demonstrate that CSP-1 induced calcium signaling and secretion of cytokines CXCL-8/IL-8, TNF-α and IL-6 is mediated through T2R14 in GECs. Interestingly, the NF-kB signaling activated by CSP-1 in GECs was independent of T2R14. CSP-1 primed GECs attracted differentiated HL-60 immune cells (dHL-60) and this effect was abolished in T2R14 knock down GECs and also in cells primed with T2R14 antagonist 6-Methoxyflavone (6-MF). Our findings identify S. mutans CSP-1 as a peptide ligand for the T2R family. Our study establishes a novel host-pathogen interaction between cariogenic S. mutans CSP-1 and T2R14 in GECs leading to an innate immune response. Collectively, these findings suggest T2Rs as potential therapeutic targets to modulate innate immune responses upon oral bacterial infections.

The 25 bitter taste receptors (T2Rs) in humans perform a chemosensory function. However, very little is known about the level of expression of these receptors in different tissues. In this study, using nCounter gene expression we analyzed the expression patterns of human TAS2R transcripts in cystic fibrosis bronchial epithelial (CuFi-1), normal bronchial epithelial (NuLi-1), airway smooth muscle (ASM), pulmonary artery smooth muscle (PASM), mammary epithelial, and breast cancer cells. Our results suggest a specific pattern of TAS2R expression with TAS2R3, 4, 5, 10, 13, 19, and 50 transcripts expressed at moderate levels and TAS2R14 and TAS2R20 (or TASR49) at high levels in the various tissues analyzed. This pattern of expression is mostly independent of tissue origin and the pathological state, except in cancer cells. To elucidate the expression at the protein level, we pursued flow cytometry analysis of select T2Rs from CuFi-1 and NuLi-1 cells. The expression levels observed at the gene level by nCounter analysis correlate with the protein levels for the T2Rs analyzed. Next, to assess the functionality of the expressed T2Rs in these cells, we pursued functional assays measuring intracellular calcium mobilization after stimulation with the bitter compound quinine. Using PLC inhibitor, U-73122, we show that the calcium mobilized in these cells predominantly takes place through the Quinine-T2R-Gαβγ-PLC pathway. This report will accelerate studies aimed at analyzing the pathophysiological function of T2Rs in different extraoral tissues.

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