Characterization of Bitter Taste Receptor-Dependent Autophagy in Oral Epithelial Cells.

Microbial dysbiosis is an important trigger in the development of oral diseases. Oral keratinocytes or gingival epithelial cells (GECs) offer protection against various microbial insults. Recent studies suggest that GECs expressed higher level of bitter taste receptor 14 (T2R14) compared to other taste receptors and toll-like receptors and act as innate immune sentinels. Macroautophagy or autophagy is a cellular conserved process involved in the regulation of host innate immune responses against microbial infection. Here, we describe a robust method for evaluation of T2R14-dependent autophagy flux in GECs. Autophagy flux was detected using Western blot analysis in GECs and further was confirmed using Acridine Orange-dependent flow cytometry analysis.

Bitter Taste Receptor T2R14 and Autophagy Flux in Gingival Epithelial Cells.

Macroautophagy (hereafter autophagy) is a lysosomal degradation pathway that functions in nutrient recycling and as a mechanism of innate immunity. Previously, we reported a novel host-bacteria interaction between cariogenic S. mutans and bitter taste receptor (T2R14) in gingival epithelial cells (GECs), leading to an innate immune response. Further, S. mutans might be using the host immune system to inhibit other Gram-positive bacteria, such as S. aureus. To determine whether these bacteria exploit the autophagic machinery of GEC, it is first necessary to evaluate the role of T2R14 in modulating autophagic flux. So far, the role of T2R14 in the regulation of autophagy is not well characterized. Therefore, in this study, for the first time, we report that T2R14 downregulates autophagy flux in GECs, and T2R14 knockout increases acidic vacuoles. However, the treatments of GEC WT with a T2R14 agonist and antagonist did not lead to a significant change in acidic vacuole formation. Transmission electron microscopy morphometric results also suggested an increased number of autophagic vesicles in T2R14-knockout GEC. Further, our results suggest that S. mutans competence stimulating peptide CSP-1 showed robust intracellular calcium release and this effect is both T2R14- and autophagy protein 7-dependent. In this study, we provide the first evidence that T2R14 modulates autophagy flux in GEC. The results of the current study could help in identifying the impact of T2R in regulation of the immuno-microenvironment of GEC and subsequently oral health.

Characterization of Adenylyl Cyclase Isoform 6 Residues Interacting with Forskolin.

BACKGROUND: The adenylyl cyclase (AC) pathway, crucial for pulmonary vasodilation, is inhibited by hypoxia. Forskolin (FSK) binds allosterically to AC, stimulating ATP catalysis. As AC6 is the primary AC isoform in the pulmonary artery, selective reactivation of AC6 could provide targeted reinstatement of hypoxic AC activity. This requires elucidation of the FSK binding site in AC6. METHODS: HEK293T cells stably overexpressing AC 5, 6, or 7 were incubated in normoxia (21% O2) or hypoxia (10% O2) or exposed to s-nitrosocysteine (CSNO). AC activity was measured using terbium norfloxacin assay; AC6 structure built by homology modeling; ligand docking to examine FSK-interacting amino acids; roles of selected residues determined by site-directed mutagenesis; FSK-dependent cAMP generation measured in wild-type and FSK-site mutants by biosensor-based live cell assay. RESULTS: Only AC6 is inhibited by hypoxia and nitrosylation. Homology modeling and docking revealed residues T500, N503, and S1035 interacting with FSK. Mutation of T500, N503, or S1035 decreased FSK-stimulated AC activity. FSK site mutants were not further inhibited by hypoxia or CSNO; however, mutation of any of these residues prevented AC6 activation by FSK following hypoxia or CSNO treatment. CONCLUSIONS: FSK-interacting amino acids are not involved in the hypoxic inhibition mechanism. This study provides direction to design FSK derivatives for selective activation of hypoxic AC6.

Genetic variants in taste genes play a role in oral microbial composition and severe early childhood caries.

Severe early childhood caries (S-ECC) is a multifactorial disease with strong evidence of genetic inheritance. Previous studies suggest that variants in taste genes are associated with dental caries due to the role of taste proteins in mediating taste preferences, oral innate immunity, and important host-microbial interactions. However, few taste genes have been investigated in caries studies. Therefore, the associations of genetic variants in sweet, bitter, umami, salt, sour, carbonation, and fat taste-related genes with S-ECC and plaque microbial composition (16S and ITS1 rRNA sequencing) were evaluated. The results showed that sixteen variants in seven taste genes (SCNN1D, CA6, TAS2R3, OTOP1, TAS2R5, TAS2R60, and TAS2R4) were associated with S-ECC. Twenty-one variants in twelve taste genes were correlated with relative abundances of bacteria or fungi. These results suggest that S-ECC risk and composition of the plaque microbiome can be partially influenced by genetic variants in genes related to taste sensation.

Extraoral expression and characterization of bitter taste receptors in Astyanax mexicanus (Mexican tetra fish).

The chemical senses of olfaction and taste are well developed in fish and play a vital role in its various activities such as navigation, mate recognition, and food detection. The small teleost fish Astyanax mexicanus consists of interfertile river-dwelling and cave-dwelling populations, referred to as "surface fish" and "cavefish" respectively. An important anatomical feature of cavefish is the lack of eyes leading them to be referred to as blind fish and suggesting an enhanced functional role for other senses such as taste. In this study, we characterize the expression of bitter taste receptors (T2Rs or Tas2Rs) in A. mexicanus and investigate their functionality in a heterologous expression system. The genome database of A. mexicanus (ensemble and NCBI) showed 7 Tas2Rs, among these Tas2R1, Tas2R3, Tas2R4, and Tas2R114 are well characterized in humans and mice but not in A. mexicanus. Therefore, the 4 Tas2Rs were selected for further analysis and their expression in A. mexicanus was confirmed by in situ hybridization and RT-PCR in early developmental stages. These Tas2Rs are expressed in various oral and extraoral organs (liver, fins, jaws, and gills) in A. mexicanus, and Tas2R1 has maximum expression and is localized throughout the fish body. Using the heterologous expression of A. mexicanus T2Rs in HEK293T cells coupled with cell-based calcium mobilization assays, we show that A. mexicanus T2Rs are activated by commonly used fish food and known bitter agonists, including quinine. This study provides novel insights into the extraoral expression of T2Rs in A. mexicanus and suggests their importance in extraoral food detection.

Critical cysteines in the functional interaction of adenylyl cyclase isoform 6 with Gαs.

Activation of adenylyl cyclases (ACs) by G-protein Gαs catalyzes the production of cyclic adenosine monophosphate (cAMP), a key second messenger that regulates diverse physiological responses. There are 10 AC isoforms present in humans, with AC5 and AC6 proposed to play vital roles in cardiac function. We have previously shown that under hypoxic conditions, AC6 is amenable to post-translational modification by nitrosylation, resulting in decreased AC catalytic activity. Using a computational model of the AC6-Gαs complex, we predicted key nitrosylation-amenable cysteine residues involved in the interaction of AC6 with Gαs and pursued a structure-function analysis of these cysteine residues in both AC6 and Gαs. Our results based on site-directed mutagenesis of AC6 and Gαs, a constitutively active Gαs, AC activity, and live cell intracellular cAMP assays suggest that Cys1004 in AC6 (subunit C2) and Cys237 in Gαs are present at the AC-Gαs interface and are important for the activation of AC6 by Gαs. We further provide mechanistic evidence to show that mutating Cys 1004 in the second catalytic domain of AC6 makes it amenable to inhibition by Gαi, which may account for decreased functional activity of AC6 when this residue is unavailable.

Association between Downstream Taste Signaling Genes, Oral Microbiome, and Severe Early Childhood Caries.

Polymorphisms in taste receptor genes have been shown to play a role in early childhood caries (ECC), a multifactorial, biofilm-mediated disease. This study aimed to evaluate associations between severe-ECC (S-ECC), the oral microbiome, and variants in genes that encode components of the G protein-coupled receptor (GPCR) signaling cascade involved in taste sensation. A total of 176 children (88 caries-free; 88 with S-ECC) were recruited. Analyses of 16S and ITS1 rRNA microbial genes and seven (GNAQ, GNAS, GNAT3, GNAI2, RAC1, RALB, and PLCB2) human genes were pursued using next-generation sequencing. Regression analyses were performed to evaluate associations between genetic variants, S-ECC, and the supragingival plaque microbiome. Results suggest that PLCB2 rs2305645 (T), rs1869901 (G), and rs2305649 (G) alleles had a protective effect on S-ECC (rs2305645, odds ratio (OR) = 0.27 (95% confidence interval (CI): 0.14-0.51); rs1869901, OR = 0.34 (95% CI: 0.20-0.58); and rs2305649, OR = 0.43 (95% CI: 0.26-0.71)). Variants in GNAQ, GNAS, GNAT3, PLCB2, RALB, and RAC1 were associated with oral fungal and bacterial community composition. This study revealed that three loci at PLCB2 are significantly associated with S-ECC. Variants in multiple genes were associated with the composition of dental biofilm. These findings contribute to the current knowledge about the role of genetics in S-ECC.

Pharmacology of T2R Mediated Host-Microbe Interactions.

Bitter taste receptors (T2Rs) belong to the G protein-coupled receptor superfamily. Humans express 25 T2Rs that are known to detect several bitter compounds including bacterial quorum sensing molecules (QSM). Primarily found to be key receptors for bitter sensation T2Rs are known to play an important role in mediating innate immune responses in oral and extraoral tissues. Several studies have led to identification of Gram-negative and Gram-positive bacterial QSMs as agonists for T2Rs in airway epithelial cells and immune cells. However, the pharmacological characterization for many of the QSM-T2R interactions remains poorly defined. In this chapter, we discuss the extraoral roles including localization of T2Rs in extracellular vesicles, molecular pharmacology of QSM-T2R interactions, role of T2Rs in mediating innate immune responses, and some of the challenges in understanding T2R pharmacology.

Association of Bitter Taste Receptor T2R38 Polymorphisms, Oral Microbiota, and Rheumatoid Arthritis.

The association of taste genetics and the oral microbiome in autoimmune diseases such as rheumatoid arthritis (RA) has not been reported. We explored a novel oral mucosal innate immune pathway involving the bitter taste G protein-coupled receptor T2R38. This case-control study aimed to evaluate whether T2R38 polymorphisms associate with the buccal microbial composition in RA. Genomic DNA was obtained from buccal swabs of 35 RA patients and 64 non-RA controls. TAS2R38 genotypes were determined by Sanger sequencing. The buccal microbiome was assessed by Illumina MiSeq sequencing of the V4-16S rRNA gene. Bacterial community differences were analyzed with alpha and beta diversity measures. Linear discriminant analysis effect size identified taxa discriminating between RA versus non-RA and across TAS2R38 genotypes. TAS2R38 genotype frequency was similar between RA and non-RA controls (PAV/PAV; PAV/AVI; AVI/AVI: RA 42.9%; 45.7%; 11.4% versus controls 32.8%; 48.4%; 18.8%, chi-square (2, N = 99) = 2.1, p = 0.35). The relative abundance of Porphyromonas, among others, differed between RA and non-RA controls. The relative abundance of several bacterial species also differed across TAS2R38 genotypes. These findings suggest an association between T2R38 polymorphisms and RA buccal microbial composition. However, further research is needed to understand the impact of T2R38 in oral health and RA development.

Bitter Taste Receptor T2R14 Modulates Gram-Positive Bacterial Internalization and Survival in Gingival Epithelial Cells.

Bitter-taste receptors (T2Rs) have emerged as key players in host-pathogen interactions and important modulators of oral innate immunity. Previously, we reported that T2R14 is expressed in gingival epithelial cells (GECs) and interacts with competence stimulating peptides (CSPs) secreted by the cariogenic Streptococcus mutans. The underlying mechanisms of the innate immune responses and physiological effects of T2R14 on Gram-positive bacteria are not well characterized. In this study, we examined the role of T2R14 in internalization and growth inhibitory effects on Gram-positive bacteria, namely Staphylococcus aureus and S. mutans. We utilized CRISPR-Cas9 T2R14 knockdown (KD) GECs as the study model to address these key physiological mechanisms. Our data reveal that the internalization of S. aureus is significantly decreased, while the internalization of S. mutans remains unaffected upon knockdown of T2R14 in GECs. Surprisingly, GECs primed with S. mutans CSP-1 resulted in an inhibition of growth for S. aureus, but not for S. mutans. The GECs infected with S. aureus induced T2R14-dependent human ß-defensin-2 (hBD-2) secretion; however, S. mutans-infected GECs did not induce hBD-2 secretion, but induced T2R14 dependent IL-8 secretion. Interestingly, our results show that T2R14 KD affects the cytoskeletal reorganization in GECs, thereby inhibiting S. aureus internalization. Our study highlights the distinct mechanisms and a direct role of T2R14 in influencing physiological responses to Gram-positive bacteria in the oral cavity.

Characterization of Supragingival Plaque and Oral Swab Microbiomes in Children With Severe Early Childhood Caries.

The human oral cavity harbors one of the most diverse microbial communities with different oral microenvironments allowing the colonization of unique microbial species. This study aimed to determine which of two commonly used sampling sites (dental plaque vs. oral swab) would provide a better prediction model for caries-free vs. severe early childhood caries (S-ECC) using next generation sequencing and machine learning (ML). In this cross-sectional study, a total of 80 children (40 S-ECC and 40 caries-free) < 72 months of age were recruited. Supragingival plaque and oral swab samples were used for the amplicon sequencing of the V4-16S rRNA and ITS1 rRNA genes. The results showed significant differences in alpha and beta diversity between dental plaque and oral swab bacterial and fungal microbiomes. Differential abundance analyses showed that, among others, the cariogenic species Streptococcus mutans was enriched in the dental plaque, compared to oral swabs, of children with S-ECC. The fungal species Candida dubliniensis and C. tropicalis were more abundant in the oral swab samples of children with S-ECC compared to caries-free controls. They were also among the top 20 most important features for the classification of S-ECC vs. caries-free in oral swabs and for the classification of dental plaque vs. oral swab in the S-ECC group. ML approaches revealed the possibility of classifying samples according to both caries status and sampling sites. The tested site of sample collection did not change the predictability of the disease. However, the species considered to be important for the classification of disease in each sampling site were slightly different. Being able to determine the origin of the samples could be very useful during the design of oral microbiome studies. This study provides important insights into the differences between the dental plaque and oral swab bacteriome and mycobiome of children with S-ECC and those caries-free.

Bitter taste receptor T2R14 detects quorum sensing molecules from cariogenic Streptococcus mutans and mediates innate immune responses in gingival epithelial cells.

Host-pathogen interactions play an important role in defining the outcome of a disease. Recent studies have shown that the 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 show 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.

Chemosensory bitter taste receptors T2R4 and T2R14 activation attenuates proliferation and migration of breast cancer cells.

The emerging significance of the bitter taste receptors (T2Rs) role in the extraoral tissues alludes to their potential role in many pathophysiological conditions. The dysregulation of T2R expression and function in disease conditions has now been demonstrated in airways diseases, neurological disorders, and in some cancers. However, the role of T2Rs in the pathophysiology of breast cancer is unexplored thus far. Previously, we demonstrated differential expression of the 25 T2Rs in breast cancer (BC) cells. Based on our previous findings we selected two T2Rs, T2R4 and T2R14 for this work. The objective of the current study is to investigate the expression of T2R4 and T2R14 in BC clinical samples and to examine their physiological role using highly metastatic BC and non-cancerous cell lines. Using approaches, which involve receptor knockdown, pharmacological activation and biochemical assays we report that (i) T2R4 and T2R14 expression patterns are dissimilar, with decreased levels of T2R4 and increased levels of T2R14 in BC clinical samples compared to non-cancerous controls. (ii) Activation of T2Rs with their respective agonist elicited physiological responses in metastatic breast cancer cells, and no responses were seen in non-tumorigenic breast epithelial cells. (iii) Agonist activation of T2Rs (irrespective of T2R subtype) induced anti-proliferative, pro-apoptotic, and anti-migratory responses in highly metastatic breast cancer cells. Taken together, our findings demonstrate that the chemosensory T2R signaling network is involved in evoking physiological responses in the metastatic breast cancer cell line.

Differential effects of membrane sphingomyelin and cholesterol on agonist-induced bitter taste receptor T2R14 signaling.

Membrane lipids regulate the structure and function of G protein-coupled receptors (GPCRs). Previously we have shown that membrane cholesterol regulates the signaling of two human bitter taste receptors (T2Rs), T2R4 and T2R14. Another major plasma membrane lipid known to influence the function of membrane proteins including GPCRs is sphingomyelin. The role of sphingomyelin in T2R function is unexplored thus far. In this work, we examined the significance of sphingomyelin in T2R14 signaling. Results suggest that unavailability of membrane sphingomyelin did not affect the agonist-promoted T2R14 Ca2+ signaling in heterologous expression system and also in primary airway smooth muscle cells (HASM cells). In addition, T2R14 mediated downstream AMPK activation was also unaffected in sphingomyelin-depleted condition; however, cholesterol depletion impaired the T2R14-mediated AMPK activation. Angiotensin II type1A receptor (AT1R) expressed in HASM cells and signals through Ca2+ and AMPK was used as a control. Results suggest that similar to T2R14, membrane sphingomyelin depletion did not affect AT1R signaling. However, membrane cholesterol depletion impaired AT1R mediated Ca2+ signaling and AMPK activation. Interestingly, amino acid sequence analysis revealed the presence of putative sphingolipid binding motif in both T2R14 and AT1R suggesting that the presence of a motif alone might not be suggestive of sphingomyelin sensitivity. In conclusion, these results demonstrate that in contrast to membrane cholesterol, sphingomyelin does not affect the agonist-induced T2R14 signaling, however it may play a role in other aspects of T2R14 function.

Glycated Beef Protein Hydrolysates as Sources of Bitter Taste Modifiers.

Being averse to bitter taste is a common phenomenon for humans and other animals, which requires the pharmaceutical and food industries to source compounds that can block bitterness intensity and increase consumer acceptability. In this work, beef protein alcalase hydrolysates (BPAH) and chymotrypsin hydrolysates (BPCH) were reacted with glucose to initiate Maillard reactions that led to the formation of glycated or advanced glycation end products (AGEs), BPAH-AGEs and BPCH-AGEs, respectively. The degree of glycation was higher for the BPAH-AGEs (47-55%) than the BPCH-AGEs (30-38%). Analysis by an electronic tongue instrument showed that BPAH-AGEs and BPCH-AGEs had bitterness scores that were significantly (p < 0.05) less than quinine. The addition of BPAH-AGEs or BPCH-AGEs to quinine led to significant (p < 0.05) reductions (up to 38%) in bitterness intensity of quinine. The use of 3% hydrolysate to react with glucose yielded glycated peptides with a stronger ability to reduce quinine bitterness than when 1% was used. Calcium release from HEK293T cells stably expressing the T2R4 human bitter taste receptor was significantly (p < 0.05) attenuated by BPAH-AGEs (up to 96%) and BPCH-AGEs (up to 92%) when compared to the BPAH (62%) and BPCH (3%) or quinine (0%). We concluded that BPAH-AGEs and BPCH-AGEs may be used as bitter taste blockers to formulate better tasting foods.

Highly conserved intracellular H208 residue influences agonist selectivity in bitter taste receptor T2R14.

Bitter taste receptors (T2Rs) are a specialized class of cell membrane receptors of the G protein-coupled receptor family and perform a crucial role in chemosensation. The 25 T2Rs in humans are activated by structurally diverse ligands of plant, animal and microbial origin. The mechanisms of activation of these receptors are poorly understood. Therefore, identification of structural determinants of T2Rs that regulate its efficacy could be beneficial in understanding the molecular mechanisms of T2R activation. In this work, we characterized a highly conserved histidine (H208), present at TM5-ICL3 region of T2R14 and its role in agonist-induced T2R14 signaling. Surprisingly, mutation of the conserved H208 (H208A) did not result in increased basal activity of T2R14, in contrast to similar H206A mutation in T2R4 that showed constitutive or basal activity. However, H208A mutation in T2R14 resulted in an increase in agonist-induced efficacy for Flufenamic acid (FFA). Interestingly, H208A did not affect the potency of another T2R14 agonist Diphenhydramine (DPH). The H208R compensatory mutation showed FFA response similar to wild-type T2R14. Molecular modeling suggests a FFA-induced shift in TM3 and TM5 helices of H208A, which changes the network of interactions connecting TM5-ICL3-TM6. This report identifies a crucial residue on the intracellular surface of T2Rs that is involved in bitter ligand selectivity. It also highlights the varied roles carried out by some conserved residues in different T2Rs.

Advanced Glycation End-Products Can Activate or Block Bitter Taste Receptors.

Bitter taste receptors (T2Rs) are expressed in several tissues of the body and are involved in a variety of roles apart from bitter taste perception. Advanced glycation end-products (AGEs) are produced by glycation of amino acids in proteins. There are varying sources of AGEs, including dietary food products, as well as endogenous reactions within our body. Whether these AGEs are T2R ligands remains to be characterized. In this study, we selected two AGEs, namely, glyoxal-derived lysine dimer (GOLD) and carboxymethyllysine (CML), based on their predicted interaction with the well-studied T2R4, and its physiochemical properties. Results showed predicted binding affinities (Kd) for GOLD and CML towards T2R4 in the nM and µM range, respectively. Calcium mobilization assays showed that GOLD inhibited quinine activation of T2R4 with IC50 10.52 ± 4.7 µM, whilst CML was less effective with IC50 32.62 ± 9.5 µM. To characterize whether this antagonism was specific to quinine activated T2R4 or applicable to other T2Rs, we selected T2R14 and T2R20, which are expressed at significant levels in different human tissues. A similar effect of GOLD was observed with T2R14; and in contrast, GOLD and CML activated T2R20 with an EC50 of 79.35 ± 29.16 µM and 65.31 ± 17.79 µM, respectively. In this study, we identified AGEs as novel T2R ligands that caused either activation or inhibition of different T2Rs.

Hen protein-derived peptides as the blockers of human bitter taste receptors T2R4, T2R7 and T2R14.

Bitter sensation is mediated by various bitter taste receptors (T2Rs), thus T2R antagonists are actively explored. Our objective was to look for novel T2R blockers in hen protein hydrolysate (HPH). We screened the least bitter HPH fractions using electronic tongue, and analyzed their peptide sequences and calcium mobilization in HEK293T cells expressing T2Rs. The results showed that the HPH fractions with higher bitterness intensity had higher hydrophobicity, more hydrophobic amino acids, and more positively charged peptides, but fewer known umami peptides. The peptide fractions from the least bitter HPH fraction significantly inhibited quinine bitterness (P < 0.05), and also significantly inhibited quinine- or diphenhydramine-dependent calcium mobilization of HEK293T cells expressing human T2R4, T2R7, or T2R14 (P < 0.05). Among them, the first eluted (least bitter) peptide fraction showed the strongest bitter-inhibitory effect. In conclusion, HPH peptides are the blockers of T2R4, T2R7, and T2R14.

Chemosensory bitter taste receptors (T2Rs) are activated by multiple antibiotics.

Many medications including antibiotics taste bitter. The potency of these antibiotics on the 25 bitter taste receptors (T2Rs) in humans remains poorly understood. Here we characterize by sensory and structure-function analyses how antibiotics frequently used to treat airway infections in cystic fibrosis activate multiple human T2Rs. The potency of the broad-spectrum antibiotics, tobramycin, levofloxacin, and azithromycin on the highly expressed T2Rs in airways, T2R4, T2R14, and T2R20 was pursued. The amino acids and structural features of T2R4, T2R14, and T2R20 important for antibiotic binding were characterized by mutational analysis in heterologous cell-based assays. Strikingly, extracellular loop 2 in T2Rs performs a key function in binding to antibiotics with contribution from residues in transmembrane helices. Our results suggest that different antibiotics activate multiple T2Rs with different potencies. An understanding of the nonantibiotic and physiologic effects mediated through T2Rs on the host cells is much needed.-Jaggupilli, A., Singh, N., De Jesus, V. C., Gounni, M. S., Dhanaraj, P., Chelikani, P. Chemosensory bitter taste receptors (T2Rs) are activated by multiple antibiotics.

Cholesterol modulates the signaling of chemosensory bitter taste receptor T2R14 in human airway cells.

Bitter taste receptors (T2Rs) are a group of 25 chemosensory receptors expressed at significant levels in the human airways. In human airways, bitter taste receptor 14 (T2R14)-mediated physiological response in ameliorating obstructive airway disorders is an active area of investigation. Therefore, understanding various factors regulating the structure and function of T2R14 will be beneficial. We hypothesize that membrane lipids like cholesterol play a regulatory role in T2R14 signaling in airway cells. We confirmed the expression and signaling of T2R14 in primary human airway smooth muscle (HASM) cells and the human airway epithelial cell line (NuLi-1) using immunoblot analysis and intracellular calcium concentration mobilization experiments, respectively. Next, T2R14 signaling was examined in membrane cholesterol-altered environments by methyl-ß-cyclodextrin or cholesterol oxidase treatments. In the cells analyzed, cholesterol depletion affected the agonist-induced T2R14 signaling, and cholesterol replenishment rescued its efficacy. An alternative approach for cholesterol depletion (with cholesterol oxidase pretreatment) also negatively affected the agonist potency at T2R14 in HASM cells. To understand the molecular mechanism of interaction between cholesterol and T2R14, we used site-directed mutagenesis coupled with functional assays and examined the role of putative cholesterol-binding motifs (CRAC and CARC) in T2R14. Functional characterization of wild-type and mutant T2R14 receptors suggests that amino acid residues K110, F236, and L239 are crucial in T2R14-cholesterol functional interaction. In conclusion, our results show that cholesterol influences the T2R14 signaling efficacy by forming direct interactions with the receptor and consequently plays a regulatory role in T2R14-mediated signaling in human airway cells.