Amino acid derivatives as bitter taste receptor (T2R) blockers.

In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only five antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis, we elucidated the ligand-binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn-173 and Thr-174 are essential for quinine binding. Guided by a molecular model of T2R4, a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays that led to identification of γ-aminobutryic acid (GABA) and Nα,Nα-bis(carboxymethyl)-L-lysine (BCML) as competitive inhibitors of quinine-activated T2R4 with an IC50 of 3.2 ± 0.3 μM and 59 ± 18 nM, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, whereas BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala-90 and Lys-270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understanding the roles of T2Rs in extraoral tissues.

Pydi SP ，Sobotkiewicz T ，Billakanti R ，Bhullar RP ，Loewen MC ，Chelikani P ... -  《-》

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

Pydi SP ，Jaggupilli A ，Nelson KM ，Abrams SR ，Bhullar RP ，Loewen MC ，Chelikani P ... -  《-》

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

Sidhu C ，Jaggupilli A ，Chelikani P ，Bhullar RP ... -  《-》

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.

Liu K ，Jaggupilli A ，Premnath D ，Chelikani P ... -  《BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES》

The third intracellular loop plays a critical role in bitter taste receptor activation.

Bitter taste receptors (T2Rs) belong to the superfamily of G protein-coupled receptors (GPCRs). T2Rs are chemosensory receptors with important therapeutic potential. In humans, bitter taste is perceived by 25 T2Rs, which are distinct from the well-studied Class A GPCRs. The activation mechanism of T2Rs is poorly understood and none of the structure-function studies are focused on the role of the important third intracellular loop (ICL3). T2Rs have a unique signature sequence at the cytoplasmic end of fifth transmembrane helix (TM5), a highly conserved LxxSL motif. Here, we pursue an alanine scan mutagenesis of the ICL3 of T2R4 and characterize the functionality of 23 alanine mutants. We identify four mutants, H214A, Q216A, V234A and M237A, that exhibit constitutive activity. To our surprise, the H214A mutant showed very high constitutive activity over wild type T2R4. Interestingly, His214 is highly conserved (96%) in T2Rs and is present two amino acids below the LxxSL motif in TM5. Molecular modeling shows a dynamic network of interactions involving residues in TM5-ICL3-TM6 that restrain the movement of the helices. Changes in this network, as in the case of H214A, Q216A, V234A and M237A mutants, cause the receptor to adopt an active conformation. The conserved LxxSL motif in TM5 performs both structural and functional roles in this process. These results provide insight into the activation mechanism of T2Rs, and emphasize the unique functional role of ICL3 even within the GPCR subfamilies.

Pydi SP ，Singh N ，Upadhyaya J ，Bhullar RP ，Chelikani P ... -  《-》