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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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- β-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. [ABSTRACT FROM AUTHOR]