Appalaraju Jaggupilli

Bitter taste receptors: Novel insights into the biochemistry and pharmacology

Bitter taste receptors (T2Rs) belong to the super family of G protein-coupled receptors (GPCRs). There are 25 T2Rs expressed in humans, and these interact with a large and diverse group of bitter ligands. T2Rs are expressed in many extra-oral tissues and can perform diverse physiological roles. Structure-function studies led to the identification of similarities and dissimilarities between T2Rs and Class A GPCRs including amino acid conservation and novel motifs. However, the efficacy of most of the T2R ligands is not yet elucidated and the biochemical pharmacology of T2Rs is poorly understood. Recent studies on T2Rs characterized novel ligands including blockers for these receptors that include inverse agonist and antagonists. In this review we discuss the techniques used for elucidating bitter blockers, concept of ligand bias, generic amino acid numbering, the role of cholesterol, and conserved water molecules in the biochemistry and pharmacology of T2Rs.

Abscisic Acid Acts as a Blocker of the Bitter Taste G Protein-Coupled Receptor T2R4

Bitter taste receptors (T2Rs) belong to the G protein-coupled receptor superfamily. In humans, 25 T2Rs mediate bitter taste sensation. In addition to the oral cavity, T2Rs are expressed in many extraoral tissues, including the central nervous system, respiratory system, and reproductive system. To understand the mechanistic roles of the T2Rs in oral and extraoral tissues, novel blockers or antagonists are urgently needed. Recently, we elucidated the binding pocket of T2R4 for its agonist quinine, and an antagonist and inhibitory neurotransmitter, γ-aminobutyric acid. This structure-function information about T2R4 led us to screen the plant hormone abscisic acid (ABA), its precursor (xanthoxin), and catabolite phaseic acid for their ability to bind and activate or inhibit T2R4. Molecular docking studies followed by functional assays involving calcium imaging confirmed that ABA is an antagonist with an IC50 value of 34.4 ± 1.1 μM. However, ABA precursor xanthoxin acts as an agonist on T2R4. Interestingly, molecular model-guided site-directed mutagenesis suggests that the T2R4 residues involved in quinine binding are also predominantly involved in binding to the novel antagonist, ABA. The antagonist ability of ABA was tested using another T2R4 agonist, yohimbine. Our results suggest that ABA does not inhibit yohimbine-induced T2R4 activity. The discovery of natural bitter blockers has immense nutraceutical and physiological significance and will help in dissecting the T2R molecular pathways in various tissues.

The Pharmacochaperone Activity of Quinine on Bitter Taste Receptors

Bitter taste is one of the five basic taste sensations which is mediated by 25 bitter taste receptors (T2Rs) in humans. The mechanism of bitter taste signal transduction is not yet elucidated. The cellular processes underlying T2R desensitization including receptor internalization, trafficking and degradation are yet to be studied. Here, using a combination of molecular and pharmacological techniques we show that T2R4 is not internalized upon agonist treatment. Pretreatment with bitter agonist quinine led to a reduction in subsequent quinine-mediated calcium responses to 35 ± 5% compared to the control untreated cells. Interestingly, treatment with different bitter agonists did not cause internalization of T2R4. Instead, quinine treatment led to a 2-fold increase in T2R4 cell surface expression which was sensitive to Brefeldin A, suggesting a novel pharmacochaperone activity of quinine. This phenomenon of chaperone activity of quinine was also observed for T2R7, T2R10, T2R39 and T2R46. Our results suggest that the observed action of quinine for these T2Rs is independent of its agonist activity. This study provides novel insights into the pharmacochaperone activity of quinine and possible mechanism of T2R desensitization, which is of fundamental importance in understanding the mechanism of bitter taste signal transduction.

Analysis of the expression of human bitter taste receptors in extraoral tissues

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.

Plasticity of the ligand binding pocket in the bitter taste receptor T2R7

Bitter taste receptors (T2Rs) are a group of 25 G protein-coupled receptors (GPCRs) in humans. The cognate agonists and the mechanism of ligand binding to the majority of the T2Rs remain unknown. Here we report the first structure-function analysis of T2R7 and study the ability of this receptor to bind to different agonists by site-directed mutagenesis. Screening of ligands for T2R7 in calcium based assays lead to the identification of novel compounds that activate this receptor. Quinine, diphenidol, dextromethorphan and diphenhydramine showed substantial activation of T2R7. Interestingly, these bitter compounds showed different pharmacological characteristics. To investigate the structural features in T2R7 that might contribute to the observed differences in agonist specificities, molecular model guided ligand docking and site-directed mutagenesis was pursued. Amino acids D65, D86, W89, N167, T169, W170, S181, T255 and E271 in the ligand-binding pocket were replaced and the mutants characterized pharmacologically. Our results suggest D86, S181 and W170 present on the extracellular side of transmembrane 3 (TM3), TM5 and in extracellular loop 2 (ECL2) are essential for agonist binding in T2R7. Mutations of these amino acids lead to loss-of-function. We also identified gain-of-function residues that are agonist specific. These results suggest that agonists bind at an extracellular site rather than deep within the TM core involving residues present in both ECL2 and TM helices in T2R7. Similar to majority of the Class A GPCRs, ECL2 in T2R7 plays a significant role in agonist binding and activation.

Structural commonality of C1q TNF‐related proteins and their potential to activate relaxin/insulin‐like family peptide receptor 1 signalling pathways in cancer cells

We established the role of the GPCR relaxin/insulin‐like family peptide receptor 1 (RXFP1 receptor) as a novel active receptor in human glioblastoma (GB), a fatal brain tumour. We identified C1q/TNF‐related protein 8 (CTRP8) as a novel agonist of the RXFP1 receptor. CTRP8 enhanced the motility and matrix invasion of GB, and this involved PKC‐mediated up‐regulation of cathepsin B, a marker for poor prognosis in GB patients. We conclude that the absence of relaxin isoforms does not preclude the activation of the RXFP1 receptor, as the least known member of the CTRP family, CTRP8, can effectively target and activate RXFP1 receptors.

Structural commonality of C1q Tumor Necrosis Factor‐related proteins and their potential to activate RXFP1 signaling pathways in cancer cells

We established the role of the GPCR relaxin/insulin‐like family peptide receptor 1 (RXFP1 receptor) as a novel active receptor in human glioblastoma (GB), a fatal brain tumour. We identified C1q/TNF‐related protein 8 (CTRP8) as a novel agonist of the RXFP1 receptor. CTRP8 enhanced the motility and matrix invasion of GB, and this involved PKC‐mediated up‐regulation of cathepsin B, a marker for poor prognosis in GB patients. We conclude that the absence of relaxin isoforms does not preclude the activation of the RXFP1 receptor, as the least known member of the CTRP family, CTRP8, can effectively target and activate RXFP1 receptors.

Study of adenylyl cyclase-GαS interactions and identification of novel AC ligands

Adenylyl cyclases (ACs) are membrane bound enzymes that catalyze the production of cAMP from ATP in response to the activation by G-protein Gαs. Different isoforms of ACs are ubiquitously expressed in different tissues involved in regulatory mechanisms in response to specific stimulants. There are 9 AC isoforms present in humans, with AC5 and AC6 proposed to play a vital role in cardiac functions. The activity of AC6 is sensitive to nitric oxide, such that nitrosylation of the protein might regulate its function. However, the information on structural determinants of nitrosylation in ACs and how they interact with Gαs is limited. Here we used homology modeling to build a molecular model of human AC6 bound to Gαs. Based on this 3D model, we predict the nitrosylation amenable cysteines, and identify potential novel ligands of AC6 using virtual ligand screening. Our model suggests Cys1004 in AC6 (subunit C2) and Cys174 in Gαs present at the AC-Gαs interface as the possible residues that might undergo reversible nitrosylation. Docking analysis predicted novel ligands of AC6 that include forskolin-based compounds and its derivatives. Further work involving site-directed mutagenesis of the predicted residues will allow manipulation of AC activity using novel ligands, and crucial insights on the role of nitrosylation of these proteins in pathophysiological conditions.

Regulation of Rac1 GTPase activity by quinine through G-protein and bitter taste receptor T2R4

Rac1 belongs to the Rho family of small GTPases and regulates actin cytoskeleton reorganization. T2R4 is a bitter taste receptor belonging to the G protein-coupled receptor family of proteins. In addition to mediating bitter taste perception from the tongue, T2R4s are found in extra-oral tissues, e.g., nasal epithelium, airways, brain, testis suggesting a much broader physiological function for these receptors. Anti-malarial drug and a bitter tasting compound, quinine, is a known agonist for T2R4, whereas BCML (Nα,Nα-Bis(carboxymethyl)-l-lysine) acts as an inverse agonist. Using western blot and Ca++ mobilization assays, the effects of quinine on Rac1 activity in HEK293T cells stably expressing T2R4/Gα16/44, T2R4, or Gα16/44 and transiently transfected with HA-Rac1 were investigated. Quinine treatment caused a significant reduction in the amount of active Rac1, whereas in the presence of BCML, quinine failed to cause any significant change in active Rac1. No significant change in Rac1 activity was observed in BAPTA-AM plus quinine-treated Gα16/44 cells, suggesting possibility of a pathway in addition to the canonical Ca++-dependent pathway. A noticeable role for Gα16/44 independent of T2R4 is observed in quinine-mediated Rac1 inactivation. Further, a significant difference in quinine-induced Ca++ response in T2R4/Gα16/44 or T2R4 cells was observed validating the partial role of calcium and importance of Gα16/44. This study is the first to show an inhibitory downstream action of a T2R4 agonist on Rac1 function. Further investigation will help in better understanding the downstream signal transduction network of T2R4 and its extra-oral physiological roles.

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. FASEB J. 33, 501–517 (2019). www.fasebj.org