The short third intracellular loop and cytoplasmic tail of bitter taste receptors provide functionally relevant GRK phosphorylation sites in TAS2R14

For most G protein–coupled receptors, the third intracellular loop (IL3) and carboxy-terminal tail (CT) are sites for G protein–coupled receptor kinase (GRK)–mediated phosphorylation, leading to β-arrestin binding and agonist-specific desensitization. These regions of bitter taste receptors (TAS2Rs) are extremely short compared with the superfamily, and their function in desensitization is unknown. TAS2R14 expressed on human airway smooth muscle cells relax the cell, suggesting a novel target for bronchodilators. To assess IL3 and CT in agonist-promoted TAS2R14 desensitization (tachyphylaxis), we generated fusion proteins of both the WT sequence and Ala substituted for Ser/Thr in the IL3 and CT sequences. In vitro, activated GRK2 phosphorylated WT IL3 and WT CT proteins but not Ala-substituted forms. TAS2R14s with mutations in IL3 (IL-5A), CT (CT-5A), and in both regions (IL/CT-10A) were expressed in human embryonic kidney 293T cells. IL/CT-10A and CT-5A failed to undergo desensitization of the intracellular calcium response compared with WT, indicating that functional desensitization by GRK phosphorylation is at residues in the CT. Desensitization of TAS2R14 was blocked by GRK2 knockdown in human airway smooth muscle cells. Receptor:β-arrestin binding was absent in IL/CT-10A and CT-5A and reduced in IL-5A, indicating a role for IL3 phosphorylation in the β-arrestin interaction for this function. Agonist-promoted internalization of IL-5A and CT-5A receptors was impaired, and they failed to colocalize with early endosomes. Thus, agonist-promoted functional desensitization of TAS2R14 occurs by GRK phosphorylation of CT residues and β-arrestin binding. However, β-arrestin function in the internalization and trafficking of the receptor also requires GRK phosphorylation of IL3 residues. For most G protein–coupled receptors, the third intracellular loop (IL3) and carboxy-terminal tail (CT) are sites for G protein–coupled receptor kinase (GRK)–mediated phosphorylation, leading to β-arrestin binding and agonist-specific desensitization. These regions of bitter taste receptors (TAS2Rs) are extremely short compared with the superfamily, and their function in desensitization is unknown. TAS2R14 expressed on human airway smooth muscle cells relax the cell, suggesting a novel target for bronchodilators. To assess IL3 and CT in agonist-promoted TAS2R14 desensitization (tachyphylaxis), we generated fusion proteins of both the WT sequence and Ala substituted for Ser/Thr in the IL3 and CT sequences. In vitro, activated GRK2 phosphorylated WT IL3 and WT CT proteins but not Ala-substituted forms. TAS2R14s with mutations in IL3 (IL-5A), CT (CT-5A), and in both regions (IL/CT-10A) were expressed in human embryonic kidney 293T cells. IL/CT-10A and CT-5A failed to undergo desensitization of the intracellular calcium response compared with WT, indicating that functional desensitization by GRK phosphorylation is at residues in the CT. Desensitization of TAS2R14 was blocked by GRK2 knockdown in human airway smooth muscle cells. Receptor:β-arrestin binding was absent in IL/CT-10A and CT-5A and reduced in IL-5A, indicating a role for IL3 phosphorylation in the β-arrestin interaction for this function. Agonist-promoted internalization of IL-5A and CT-5A receptors was impaired, and they failed to colocalize with early endosomes. Thus, agonist-promoted functional desensitization of TAS2R14 occurs by GRK phosphorylation of CT residues and β-arrestin binding. However, β-arrestin function in the internalization and trafficking of the receptor also requires GRK phosphorylation of IL3 residues. G protein–coupled receptors (GPCRs) are now recognized as multifunctional signaling units, with the capacity to evoke intracellular events mediated by G protein and non-G protein mechanisms (1Shukla A.K. Xiao K. Lefkowitz R.J. Emerging paradigms of beta-arrestin-dependent seven transmembrane receptor signaling.Trends Biochem. Sci. 2011; 36: 457-469Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). These events include mechanisms that act to coarse- and fine-tune their signaling over short- and long-term periods, such that cells expressing hundreds of receptors can achieve integrative physiologic actions in response to changing needs (2Rajagopal S. Shenoy S.K. GPCR desensitization: Acute and prolonged phases.Cell Signal. 2018; 41: 9-16Crossref PubMed Scopus (123) Google Scholar). The attenuation of receptor responsiveness (termed desensitization) may be particularly germane when therapeutic agonists are targeted to GPCRs, since this may lead to a loss of effectiveness (tachyphylaxis). The mechanism of agonist-promoted desensitization for many GPCRs is by phosphorylation by G protein–coupled receptor kinases (GRKs) of intracellular residues, typically in the third intracellular loop (IL3) or the cytoplasmic carboxy-terminal tail (CT) (3Eason M.G. Moreira S.P. Liggett S.B. Four consecutive serines in the third intracellular loop are the sites for bARK-mediated phosphorylation and desensitization of the a2A-adrenergic receptor.J. Biol. Chem. 1995; 270: 4681-4688Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 4Bouvier M. Hausdorff W.P. De Blasi A. O'Dowd B.F. Kobilka B.K. Caron M.G. Lefkowitz R.J. 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Structure of the neurotensin receptor 1 in complex with beta-arrestin 1.Nature. 2020; 579: 303-308Crossref PubMed Scopus (83) Google Scholar, 12Mafi A. Kim S.K. Goddard 3rd., W.A. Mechanism of beta-arrestin recruitment by the mu-opioid G protein-coupled receptor.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 16346-16355Crossref PubMed Scopus (6) Google Scholar). This competition between the β-arrestin and G protein is often termed “uncoupling” because it acts to attenuate the G protein–mediated signal, such as cAMP production from Gs-coupled receptors or intracellular calcium [Ca2+]i release from Gi- and Gq-coupled receptors. Recruited β-arrestins also bind clathrin and other cellular components that lead to receptor internalization to the cell interior via clathrin-coated pit endocytosis (13Wolfe B.L. Trejo J. Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis.Traffic. 2007; 8: 462-470Crossref PubMed Scopus (180) Google Scholar). This internalization may serve to further limit cellular responsiveness and also acts to initiate receptor degradation over prolonged agonist exposure. Paradoxically, these attenuation functions are also accompanied by an activated state of β-arrestin, which leads to de novo signaling that is independent of G protein coupling (1Shukla A.K. Xiao K. Lefkowitz R.J. Emerging paradigms of beta-arrestin-dependent seven transmembrane receptor signaling.Trends Biochem. Sci. 2011; 36: 457-469Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). GRK-mediated phosphorylation appears to be highly sensitive to the conformation of the intracellular loop or tail of GPCRs, and indeed, there is no identified “consensus” sequence for GRK phosphorylation and β-arrestin binding. In fact, some GPCRs with multiple potential phosphorylation sites fail to undergo GRK-mediated phosphorylation and agonist-dependent desensitization (14Liggett S.B. Freedman N.J. Schwinn D.A. Lefkowitz R.J. 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Furthermore, it appears that the interactions of bound arrestins within the receptor core display structural plasticity and are capable of adopting unique conformations depending on the receptor (11Huang W. Masureel M. Qu Q. Janetzko J. Inoue A. Kato H.E. Robertson M.J. Nguyen K.C. Glenn J.S. Skiniotis G. Kobilka B.K. Structure of the neurotensin receptor 1 in complex with beta-arrestin 1.Nature. 2020; 579: 303-308Crossref PubMed Scopus (83) Google Scholar, 17Staus D.P. Hu H. Robertson M.J. Kleinhenz A.L.W. Wingler L.M. Capel W.D. Latorraca N.R. Lefkowitz R.J. Skiniotis G. Structure of the M2 muscarinic receptor-beta-arrestin complex in a lipid nanodisc.Nature. 2020; 579: 297-302Crossref PubMed Scopus (83) Google Scholar, 18Gagnon L. Cao Y. Cho A. Sedki D. Huber T. Sakmar T.P. Laporte S.A. Genetic code expansion and photocross-linking identify different beta-arrestin binding modes to the angiotensin II type 1 receptor.J. Biol. Chem. 2019; 294: 17409-17420Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Thus, for each GPCR of interest, specific studies are necessary to establish the sites and roles of GRK-mediated phosphorylation and β-arrestin engagement in cellular signaling. In the current work, we have undertaken such studies with the bitter taste 2 receptor member 14 (TAS2R14), a widely expressed receptor that may play a role in metabolic and inflammatory diseases, and has been considered a target for a new class of bronchodilators because of its expression on human airway smooth muscle (HASM) cells. In these cells, TAS2R14 agonists stimulate [Ca2+]i, causing hyperpolarization of the HASM cell membrane and marked HASM relaxation (19Deshpande D.A. Wang W.C. McIlmoyle E.L. Robinett K.S. Schillinger R.M. An S.S. Sham J.S. Liggett S.B. Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction.Nat. Med. 2010; 16: 1299-1304Crossref PubMed Scopus (417) Google Scholar). This has brought forth the concept that TAS2R14 agonists might be useful for treating obstructive lung disease where HASM contraction plays an active role in airway obstruction. Understanding mechanisms that may be in play for agonist-mediated regulation of TAS2R14 is thus a necessary component for such a consideration. The TAS2R14 has five Ser/Thr in the IL3 and five Ser in the cytoplasmic CT. These sequences were screened for potential GRK phosphorylation sites by expressing the glutathione-S-transferase (GST)–tagged fusion proteins representing WT TAS2R14 sequence or the analogous sequence with Ser/Thr substituted with Ala. IL3 peptides are denoted GST-IL-WT and GST-IL-5A for WT and mutant, respectively. The CT peptides are denoted GST-CT-WT and GST-CT-5A for the WT and mutant, respectively. In vitro phosphorylation experiments were carried out with the four proteins and active GRK2 or PKB (which acted as a negative control). None of the GST-tagged fusion proteins underwent phosphorylation in the absence of GRK2 (Fig. 1, B and C). In the presence of active GRK2, GST-IL-WT and GST-CT-WT were phosphorylated (Fig. 1, B and C), whereas the Ala-substituted proteins GST-IL-5A and GST-CT-5A were not (Fig. 1, B and C). In experiments where the proteins were electrophoresed on the same gel with equal protein loading, we found that the third loop peptide and the C-tail peptide phosphorylated to the same extent by active GRK2 (Fig. 1, D and E). These peptides do not have a consensus sequence for PKB, and indeed none were phosphorylated by PKB (Fig. S1). To ascertain the functional effects of potential GRK phosphorylation of TAS2R14, three mutant receptor complementary DNAs were constructed substituting Ala in the encoded receptor for all Ser/Thr in the IL3 (IL-5A), the cytoplasmic tail (CT-5A), or both (IL/CT-10A). WT and mutant receptors were expressed in HEK-293 cells, and [Ca2+]i release quantitated in real time for 45 s after exposure of the cells to the TAS2R14 agonist diphenhydramine (DPD). Nontransfected cells had no detectable [Ca2+]i response (Fig. S2). Representative results from the TAS2R14-transfected cells are shown in Figure S2, and mean results from multiple experiments are shown in Figure 2A. WT-expressing cells showed a rapid increase in [Ca2+]i that peaked after ∼10 s and then decreased (Fig. S2), with a mean slope of the decline from 10 s to 50 s being −1.51 ± 0.194 (Fig. 2A and Table 1). In contrast, IL/CT-10A, which lacks all 10 potential phosphoacceptor sites in the third loop and the CT, exhibited very little decrease in signaling, with a slope that was approximately sevenfold lower in absolute value than WT (p < 0.003: Fig. 2A and Table 1). In a similar manner, the CT mutant CT-5A failed to desensitize. Indeed, the slopes of the [Ca2+]i responses of IL/CT-10A and CT-5A were not statistically different (p = 0.14; Fig. 2A). On the other hand, the third loop mutant IL-5A displayed [Ca2+]i responses that were similar to R14-WT, with no statistical difference in the slopes (p = 0.127; Fig. 2A and Table 1). Mean expression levels of each of the receptors were comparable (Fig. 2B shows a representative Western blot). Taken together, these results suggested that despite both regions displaying GRK-mediated phosphorylation in vitro, functional desensitization of TAS2R14 requires only phosphorylation of one or more of the C-terminal Ser residues.Table 1Desensitization of TAS2R14Plasmid/siRNASlope (N = 5)p Versus WT/siCtrlHEK-293T cells WT−1.51 ± 0.19REF IL/CT-10A−0.22 ± 0.23<0.003 CT-5A0.44 ± 0.34<0.002 IL-5A−0.99 ± 0.230.127HASM cells SiCtrl−1.74 ± 0.03REF siGRK2−0.03 ± 0.35<0.001Transfected HEK-293T cells (upper section) expressing the indicated receptors were exposed to agonist and [Ca2+]i monitored in real time. In the lower section, the same experiments were performed using HASM cells, which were transfected with siRNA constructs targeting GRK2 (siGRK2) or scrambled control (siCtrl). The slopes of the responses from 10 to 50 s were determined by linear regression and compared with that of WT in the case of the HEK-293 experiments or siCtrl in the case of HASM cells. REF is the reference for statistical comparison. Open table in a new tab Transfected HEK-293T cells (upper section) expressing the indicated receptors were exposed to agonist and [Ca2+]i monitored in real time. In the lower section, the same experiments were performed using HASM cells, which were transfected with siRNA constructs targeting GRK2 (siGRK2) or scrambled control (siCtrl). The slopes of the responses from 10 to 50 s were determined by linear regression and compared with that of WT in the case of the HEK-293 experiments or siCtrl in the case of HASM cells. REF is the reference for statistical comparison. To further implicate GRK phosphorylation as a mechanism of rapid desensitization, and, to ascertain relevance in a cell type of interest, cultured HASM cells were utilized to ascertain [Ca2+]i responsiveness in siCtrl- and siGRK2-transfected cells (Fig. S3). These cells have been previously shown to endogenously express TAS2R14 and GRK2 (19Deshpande D.A. Wang W.C. McIlmoyle E.L. Robinett K.S. Schillinger R.M. An S.S. Sham J.S. Liggett S.B. Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction.Nat. Med. 2010; 16: 1299-1304Crossref PubMed Scopus (417) Google Scholar, 20Guindon S. Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood.Syst. Biol. 2003; 52: 696-704Crossref PubMed Scopus (13496) Google Scholar). In the siGRK2-transfected cells, GRK expression was decreased by >85% compared with siCtrl-transfected cells (Fig. 3A). The [Ca2+]i response to agonist (Fig. 3B) was markedly altered by the GRK2 knockdown, revealing a pattern consistent with less desensitization, with mean slope of −0.03 ± 0.35 versus −1.74 ± 0.03 for siCtrl-transfected cells (p < 0.001). Agonist-promoted GRK-mediated phosphorylation of most GPCRs results in recruitment of β-arrestins from the cytosol and subsequent binding to the receptor, leading to the uncoupling from the G protein and desensitization. We reasoned that recruitment would be impaired with one or more of the phosphodeficient mutant receptors. Using cells transfected with GFP–β-arrestin2, the translocation of β-arrestins is observed under confocal microscopy as a loss of the intracellular signal and a punctate appearance of GFP–β-arrestin2 at the cell surface (21Wang W.C. Mihlbachler K.A. Brunnett A.C. Liggett S.B. Targeted transgenesis reveals discrete attenuator functions of GRK and PKA in airway b2-adrenergic receptor physiologic signaling.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 15007-15012Crossref PubMed Scopus (29) Google Scholar, 22Woo J.A. Castano M. Goss A. Kim D. Lewandowski E.M. Chen Y. Liggett S.B. Differential long-term regulation of TAS2R14 by structurally distinct agonists.FASEB J. 2019; 33: 12213-12225Crossref PubMed Scopus (4) Google Scholar). As an initial qualitative screen, we assessed whether this characteristic response is altered with any of the mutated receptors. As indicated in Figure 4, WT TAS2R14 displayed the aforementioned phenotype, as we have recently described (22Woo J.A. Castano M. Goss A. Kim D. Lewandowski E.M. Chen Y. Liggett S.B. Differential long-term regulation of TAS2R14 by structurally distinct agonists.FASEB J. 2019; 33: 12213-12225Crossref PubMed Scopus (4) Google Scholar). IL-5A receptors also recruited GFP–β-arrestin2 in an agonist-dependent manner similar to that of WT. However, there was no evidence of redistribution or the appearance of puncta at the cell surface with IL/CT-10A or CT-5A (Fig. 4). To quantitatively assess the binding of β-arrestin2 to the WT and mutant receptors, we utilized a proximity ligation assay (PLA) that we have recently described in detail (see Experimental procedures section) (23Woo J.A. Liu T. Fang C.C. Castano M. Kee T. Yrigoin K. Yan Y. Cazzaro S. Matlack J. Wang X. Kang D. Liggett S.B. β-arrestin2 oligomers impair the clearance of pathological tau and increase tau aggregates.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 5006-5015Crossref PubMed Scopus (10) Google Scholar). A signal in the red spectra is produced when the two proteins of interest (β-arrestin2 and one of the receptors) are within 30 nm. Figure 5A shows a representative PLA with the four receptors, with mean data from multiple experiments found in Figure 5B. Negative controls were experiments that were performed lacking the primary antibody to GFP or FLAG, or the PLA probes, which showed no signals (Fig. S4), as well as experiments with cells expressing WT receptor that emitted no signal in the absence of agonist (Fig. 5A). With the WT receptor, a robust PLA signal upon exposure of the cells to agonist was readily apparent after a 10-min incubation. At the other extreme, IL/CT-10A, which lacks all potential GRK-mediated phosphorylation sites, showed no agonist-promoted PLA signal (no difference from 0% by the one-sample t test; Fig. 5, A and B), consistent with a lack of β-arrestin2 recruitment with this mutated receptor. Similarly, the CT-5A mutant lacking the five Ser displayed no agonist-promoted PLA signal. For the IL-5A receptor, we found a significant agonist-promoted PLA signal, but on average, it was ∼35% lower than that observed for R14-WT (Fig. 5B). This modest decrease observed with IL-5A suggested that the third loop phosphorylation sites may play a role in β-arrestin2 binding and function that is distinct from the rapid uncoupling event.Figure 5Proximity ligation assay (PLA) reveals differential β-arrestin2 binding to WT and mutant receptors. A, representative experiment showing agonist-promoted β-arrestin2 binding (red signal) with the indicated receptors. B, mean ± SD, and individual results, from five PLA experiments. ∗p < 0.01 versus WT; #not significant versus 0% change as determined by the one-sample t test. DAPI, 4′,6-diamidino-2-phenylindole; DPD, diphenhydramine; IL, intracellular loop.View Large Image Figure ViewerDownload Hi-res image Download (PPT) With continuous agonist exposure, many GPCRs (13Wolfe B.L. Trejo J. Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis.Traffic. 2007; 8: 462-470Crossref PubMed Scopus (180) Google Scholar), including TAS2R14 (22Woo J.A. Castano M. Goss A. Kim D. Lewandowski E.M. Chen Y. Liggett S.B. Differential long-term regulation of TAS2R14 by structurally distinct agonists.FASEB J. 2019; 33: 12213-12225Crossref PubMed Scopus (4) Google Scholar), internalize to the cell interior and begin to undergo endosomal degradation. We have shown that TAS2R14 internalizes and associates with the early endosomal marker early endosome antigen 1 (EEA1) (22Woo J.A. Castano M. Goss A. Kim D. Lewandowski E.M. Chen Y. Liggett S.B. Differential long-term regulation of TAS2R14 by structurally distinct agonists.FASEB J. 2019; 33: 12213-12225Crossref PubMed Scopus (4) Google Scholar) under conditions of prolonged agonist exposure. A time course for receptor–EEA1 colocalization shows that steady state was reached after 2 h of agonist exposure (Fig. S5). Based on the partial loss of β-arrestin2 binding with IL-5A, we assessed receptor internalization and EEA1 colocalization with WT and each of the mutated receptors. Cells were treated in culture plates with the agonist DPD or vehicle at 37 °C for 2 h, cooled to 4 °C, washed, and cell membrane fractions derived. These fractions were probed with the FLAG antibody in immunoblots along with the Na+/K+ ATPase as a membrane protein marker (Fig. 6, A and B). WT receptor exhibited a 40% loss of cell surface receptors under these conditions. In contrast, IL-5A, CT-5A, and IL/CT-10A displayed an impairment of internalization, with the latter showing the smallest degree of cell surface loss. We ascertained agonist-promoted colocalization of receptors with EEA1 under the same conditions, using confocal microscopy in permeabilized cells for detection (Fig. 6, C and D). WT showed agonist-promoted colocalization of receptor with EEA1 (yellow signal; Fig. 6C). In contrast, experiments with the IL/CT-10A and CT-5A receptors showed very little colocalization. Importantly, the IL-5A receptor also failed to colocalize, indicating a role for these IL3 GRK phosphorylation sites in internalization and trafficking that are distinct from the uncoupling event. The adapter functions of β-arrestin serve to internalize GPCRs and also promote β-arrestin–dependent extracellular signal–regulated kinase 1/2 (ERK1/2) activation. In our hands, nontransfected HEK-293T cells have a high ERK1/2 off-target response to TAS2R agonists, so we were unable to study the receptors in this cell type. However, HASM cells, which endogenously express this receptor, show a TAS2R14-specific ERK1/2 response (24Kim D. Woo J.A. Geffken E. An S.S. Liggett S.B. Coupling of airway smooth muscle bitter taste receptors to intracellular signaling and relaxation is via Galphai1,2,3.Am. J. Respir. Cell Mol. Biol. 2017; 56: 762-771Crossref PubMed Scopus (26) Google Scholar, 25Kim D. Cho S. Castano M.A. Panettieri R.A. Woo J.A. Liggett S.B. Biased TAS2R bronchodilators inhibit airway smooth muscle growth by downregulating phosphorylated extracellular signal-regulated kinase 1/2.Am. J. Respir. Cell Mol. Biol. 2019; 60: 532-540Crossref PubMed Scopus (12) Google Scholar). We thus studied these cells in the absence and presence of an internalization inhibitor (dynasore). As shown in Figure S6, dynasore inhibited agonist-dependent ERK1/2 activation by ∼50%. By inference from studies shown in Figures 5 and 6, we consider that ERK1/2 activation may also require the phosphorylation sites in both the third loop and CT regions of the receptor. TAS2Rs are GPCRs that were thought to be expressed exclusively on taste cells of the tongue, having evolved to detect toxic bitter substances from plants (26Chandrashekar J. Mueller K.L. Hoon M.A. Adler E. Feng L. Guo W. Zuker C.S. Ryba N.J. T2Rs function as bitter taste receptors.Cell. 2000; 100: 703-711Abstract Full Text Full Text PDF PubMed Scopus (1012) Google Scholar). Recent studies, however, have shown that many of the 25 human TAS2R subtypes are expressed on multiple extragustatory cell types within various organs in the body. Examples include airway smooth muscle, brain, thyroid, pancreas, gastrointestinal tract, white blood cells, and uterus (27Clark A.A. Liggett S.B. Munger S.D. Extraoral bitter taste receptors as mediators of off-target drug effects.FASEB J. 2012; 26: 4827-4831Crossref PubMed Scopus (82) Google Scholar, 28An S.S. Liggett S.B. Taste and smell GPCRs in the lung: Evidence for a previously unrecognized widespread chemosensory system.Cell Signal. 2017; 41: 82-88Crossref PubMed Scopus (36) Google Scholar). Thus, it is now clear that a previously unrecognized TAS2R-based chemosensory system is present in the body, which may act in a regulatory, compensatory, or pathogenic manner in normal homeostasis or in disease. Based on this distribution and functional outcomes from receptor activation, TAS2Rs may represent potential novel drug targets as well (19Deshpande D.A. Wang W.C. McIlmoyle E.L. Robinett K.S. Schillinger R.M. An S.S. Sham J.S. Liggett S.B. Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction.Nat. Med. 2010; 16: 1299-1304Crossref PubMed Scopus (417) Google Scholar). Activation of TAS2R14, which is highly expressed on HASM cells, causes marked relaxation of the cells and dilation of the bronchus, which appears to be of greater magnitude (29Deshpande D.A. Robinett K.S. Wang W.C. Sham J.S. An S.S. Liggett S.B. Bronchodilator activity of bitter tastants in human tissue.Nat. Med. 2011; 17: 776-778Crossref PubMed Scopus (36) Google Scholar) than the only direct bronchodilators (β2-adrenergic receptor [β2AR] agonists) available for treating airway constriction in asthma and chronic obstructive pulmonary disease. The expression and function of TAS2Rs is not altered in HASM cells derived from asthmatic donor lungs compared with nonasthmatic lungs (30Robinett K.S. Koziol-White C.J. Akoluk A. An S.S. Panettieri Jr., R.A. Liggett S.B. Bitter taste receptor function in asthmatic and nonasthmatic human airway smooth muscle cells.Am. J. Respir. Cell Mol. Biol. 2014; 50: 678-683Crossref PubMed Scopus (51) Google Scholar), confirming the availability of the target in the diseased state. TAS2Rs couple to the Gi family of G proteins (24Kim D. Woo J.A. Geffken E. An S.S. Liggett S.B. Coupling of airway smooth muscle bitter taste receptors to intracellular signaling and relaxation is via Galphai1,2,3.Am. J. Respir. Cell Mol. Biol. 2017; 56: 762-771Crossref PubMed Scopus (26) Google Scholar) (Ggust in taste cells and Gi1,2,3 in HASM cells). Upon binding to the α-s