Dextromethorphan Mediated Bitter Taste Receptor Activation in the Pulmonary Circuit Causes Vasoconstriction

Activation of bitter taste receptors (T2Rs) in human airway smooth muscle cells leads to muscle relaxation and bronchodilation. This finding led to our hypothesis that T2Rs are expressed in human pulmonary artery smooth muscle cells and might be involved in regulating the vascular tone. RT-PCR was performed to reveal the expression of T2Rs in human pulmonary artery smooth muscle cells. Of the 25 T2Rs, 21 were expressed in these cells. Functional characterization was done by calcium imaging after stimulating the cells with different bitter agonists. Increased calcium responses were observed with most of the agonists, the largest increase seen for dextromethorphan. Previously in site-directed mutational studies, we have characterized the response of T2R1 to dextromethorphan, therefore, T2R1 was selected for further analysis in this study. Knockdown with T2R1 specific shRNA decreased mRNA levels, protein levels and dextromethorphan-induced calcium responses in pulmonary artery smooth muscle cells by up to 50%. To analyze if T2Rs are involved in regulating the pulmonary vascular tone, ex vivo studies using pulmonary arterial and airway rings were pursued. Myographic studies using porcine pulmonary arterial and airway rings showed that stimulation with dextromethorphan led to contraction of the pulmonary arterial and relaxation of the airway rings. This study shows that dextromethorphan, acting through T2R1, causes vasoconstrictor responses in the pulmonary circuit and relaxation in the airways.     

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.     

Constitutively active mutant gives novel insights into the mechanism of bitter taste receptor activation

The human bitter taste receptors (T2Rs) belong to the G-protein coupled receptor (GPCR) superfamily. T2Rs share little homology with the large subfamily of Class A G-protein coupled receptors, and their mechanisms of activation are poorly understood. Guided by biochemical and molecular approaches, we identified two conserved amino acids Gly281·?? and Ser285?·?? present on transmembrane (TM) helices, TM1 and TM7, which might play important roles in T2R activation. Previously, it was shown that naturally occurring Gly511·?? mutations in the dim light receptor, rhodopsin, cause autosomal dominant retinitis pigmentosa, with the mutants severely defective in signal transduction. We mutated Gly281·?? and Ser285?·?? in T2R4 to G28A, G28L, S285A, S285T, and S285P, and carried out pharmacological characterization of the mutants. No major changes in signaling were observed upon mutation of Gly281·?? in T2R4. Interestingly, S285A mutant displayed agonist-independent activity (approximately threefold over basal wild-type T2R4 or S285T or S285P). We propose that Ser285?·?? stabilizes the inactive state of T2R4 by a network of hydrogen-bonds connecting important residues on TM1-TM2-TM7. We compare and contrast this hydrogen-bond network with that present in rhodopsin. Thus far, S285A is the first constitutively active T2R mutant reported, and gives novel insights into T2R activation.     

Structural basis of activation of bitter taste receptor T2R1 and comparison with Class A G-protein-coupled receptors (GPCRs)

The human bitter taste receptors (T2Rs) are non-Class A members of the G-protein-coupled receptor (GPCR) superfamily, with very limited structural information. Amino acid sequence analysis reveals that most of the important motifs present in the transmembrane helices (TM1-TM7) of the well studied Class A GPCRs are absent in T2Rs, raising fundamental questions regarding the mechanisms of activation and how T2Rs recognize bitter ligands with diverse chemical structures. In this study, the bitter receptor T2R1 was used to systematically investigate the role of 15 transmembrane amino acids in T2Rs, including 13 highly conserved residues, by amino acid replacements guided by molecular modeling. Functional analysis of the mutants by calcium imaging analysis revealed that replacement of Asn-66(2.65) and the highly conserved Asn-24(1.50) resulted in greater than 90% loss of agonist-induced signaling. Our results show that Asn-24(1.50) plays a crucial role in receptor activation by mediating an hydrogen bond network connecting TM1-TM2-TM7, whereas Asn-66(2.65) is essential for binding to the agonist dextromethorphan. The interhelical hydrogen bond between Asn-24(1.50) and Arg-55(2.54) restrains T2R receptor activity because loss of this bond in I27A and R55A mutants results in hyperactive receptor. The conserved amino acids Leu-197(5.50), Ser-200(5.53), and Leu-201(5.54) form a putative LXXSL motif which performs predominantly a structural role by stabilizing the helical conformation of TM5 at the cytoplasmic end. This study provides for the first time mechanistic insights into the roles of the conserved transmembrane residues in T2Rs and allows comparison of the activation mechanisms of T2Rs with the Class A GPCRs.     

Role of rhodopsin N-terminus in structure and function of rhodopsin-bitter taste receptor chimeras

The bitter taste receptors (T2Rs) belong to the G protein-coupled receptor (GPCR) superfamily. In humans, bitter taste sensation is mediated by 25 T2Rs. Structure–function studies on T2Rs are impeded by the low-level expression of these receptors. Different lengths of rhodopsin N-terminal sequence inserted at the N-terminal region of T2Rs are commonly used to express these receptors in heterologous systems. While the additional sequences were reported, to enhance the expression of the T2Rs, the local structural perturbations caused by these sequences and its effect on receptor function or allosteric ligand binding were not characterized. In this study, we elucidated how different lengths of rhodopsin N-terminal sequence effect the structure and function of the bitter taste receptor, T2R4. Guided by molecular models of T2R4 built using a rhodopsin crystal structure as template, we constructed chimeric T2R4 receptors containing the rhodopsin N-terminal 33 and 38 amino acids. The chimeras were functionally characterized using calcium imaging, and receptor expression was determined by flow cytometry. Our results show that rhodopsin N-terminal 33 amino acids enhance expression of T2R4 by 2.5-fold and do not cause perturbations in the receptor structure.     

Site-directed mutations and the polymorphic variant Ala160Thr in the human thromboxane receptor uncover a structural role for transmembrane helix 4

The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala160(4.53) is highly conserved as is Gly164(4.57). Here we target Ala160(4.53) and Gly164(4.57) in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala160(4.53) and Gly164(4.57) in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions.     

Marinobacterium alkalitolerans sp. nov., with nitrate reductase and urease activity isolated from green algal mat collected from a solar saltern

Antonie van Leeuwenhoek - A novel Gram-staining-negative, rod-shaped, 0.6–0.8 µm wide and 2.0–3.0 µm in length, motile bacterium designated strain AK62T, was...