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

Abstract

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.

Taste Receptors: New Players in Sperm Biology

“…Membrane remodelling, a prerequisite for acrosome reaction, is induced by cholesterol efflux, that alters lipid raft stability and distribution, favours specific protein-protein interactions by concentrating certain proteins in specific microdomains while excluding others. The physiological cholesterol efflux seems to play a regulatory role on taste receptors through the CRAC motif, influencing TasRs signalling efficacy, as reported in human airway cells [99].…”

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

“…All the GECs were cultured in serum free KGM‐2 supplemented with 0.6mM CaCl 2 . HEK293T and HEK293T cells overexpressing T2R14 and T2R39 receptor were generated as previously described 24 . The promyeloblast (HL‐60) cells were cultured in IMDM supplemented with 20% FBS.…”

“…The intracellular calcium mobilization (Ca 2+ )i upon agonist stimulation was measured using calcium binding fluorescent dye Fluo-4NW as described previously [17,24]. Briefly, GECs (3x10 /well) and HEK293T cells stably over-expressing T2R14 or T2R39 (1x10 5 /well each) were seeded in a black well 96-well BD optilux plate and grown overnight to full confluency.…”

Cholesterol in Class C GPCRs: Role, Relevance, and Localization

“…A comparison of the dynamics of wild-type T2R14 receptors and mutant T2R14 receptors revealed that the amino acid residues K110, F236, and L239 are required for the receptor to function appropriately when cholesterol is present. Based on this study, it could be suggested that cholesterol influences taste receptors by directly interacting with the receptor [ 165 ].…”