α1B/D-adrenoceptors regulate chemokine receptor-mediated leukocyte migration via formation of heteromeric receptor complexes.

It is known that catecholamines regulate innate immune functions. The underlying mechanisms, however, are not well understood. Here we show that at least 20 members of the human chemokine receptor (CR) family heteromerize with one or more members of the α1-adrenergic receptor (AR) family in recombinant systems and that such heteromeric complexes are detectable in human monocytes and the monocytic leukemia cell line THP-1. Ligand binding to α1-ARs inhibited migration toward agonists of the CR heteromerization partners of α1B/D-ARs with high potency and 50 to 77% efficacy but did not affect migration induced by a noninteracting CR. Incomplete siRNA knockdown of α1B/D-ARs in THP-1 cells partially inhibited migration toward agonists of their CR heteromerization partners. Complete α1B-AR knockout via CRISPR-Cas9 gene editing in THP-1 cells (THP-1_ADRA1BKO) resulted in 82% reduction of α1D-AR expression and did not affect CR expression. Migration of THP-1_ADRA1BKO cells toward agonists of CR heteromerization partners of α1B/D-ARs was reduced by 82 to 95%. Our findings indicate that CR:α1B/D-AR heteromers are essential for normal function of CR heteromerization partners, provide a mechanism underlying neuroendocrine control of leukocyte trafficking, and offer opportunities to modulate leukocyte and/or cancer cell trafficking in disease processes.

Identification and characterization of an atypical Gαs-biased ß2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis.

G protein-coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For ß2-adrenergic receptors (ß2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and ß-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable ß-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling-a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: ß2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from ß-arrestin, in contrast to albuterol and C5-S C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from ß-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of ß2AR actions favorable for treating obstructive lung disease.

A Par3/LIM Kinase/Cofilin Pathway Mediates Human Airway Smooth Muscle Relaxation by TAS2R14.

TAS2Rs (bitter taste receptors) are GPCRs (G protein-coupled receptors) expressed on human airway smooth muscle (HASM) cells; when activated by receptor agonists they evoke marked airway relaxation. In both taste and HASM cells, TAS2Rs activate a canonical Gßγ-mediated stimulation of Ca2+ release from intracellular stores by activation of PLCß (phospholipase Cß). Alone, this [Ca2+]i signaling does not readily account for relaxation, particularly since bronchoconstrictive agonists acting at Gq-coupled receptors also increase [Ca2+]i. We established that TAS2R14 activation in HASM promotes relaxation through F-actin (filamentous actin) severing. This destabilization of actin was from agonist-promoted activation (dephosphorylation) of cofilin, which was pertussis toxin sensitive. Cofilin dephosphorylation was due to TAS2R-mediated deactivation of LIM domain kinase. The link between early receptor action and the distal cofilin dephosphorylation was found to be the polarity protein partitioning defective 3 (Par3), a known binding partner with PLCß that inhibits LIM kinase. The physiologic relevance of this pathway was assessed using knock-downs of cofilin and Par3 in HASM cells and in human precision-cut lung slices. Relaxation by TAS2R14 agonists was ablated with knock-down of either protein as assessed by magnetic twisting cytometry in isolated cells or intact airways in the slices. Blocking [Ca2+]i release by TAS2R14 inhibited agonist-promoted cofilin dephosphorylation, confirming a role for [Ca2+]i in actin-modifying pathways. These results further elucidate the mechanistic basis of TAS2R-mediated HASM relaxation and point toward nodal points that may act as asthma or chronic obstructive pulmonary disease response modifiers or additional targets for novel bronchodilators.

ß-Arrestin2 oligomers impair the clearance of pathological tau and increase tau aggregates.

Multiple G protein-coupled receptors (GPCRs) are targets in the treatment of dementia, and the arrestins are common to their signaling. ß-Arrestin2 was significantly increased in brains of patients with frontotemporal lobar degeneration (FTLD-tau), a disease second to Alzheimer's as a cause of dementia. Genetic loss and overexpression experiments using genetically encoded reporters and defined mutant constructs in vitro, and in cell lines, primary neurons, and tau P301S mice crossed with ß-arrestin2-/- mice, show that ß-arrestin2 stabilizes pathogenic tau and promotes tau aggregation. Cell and mouse models of FTLD showed this to be maladaptive, fueling a positive feedback cycle of enhanced neuronal tau via non-GPCR mechanisms. Genetic ablation of ß-arrestin2 markedly ablates tau pathology and rescues synaptic plasticity defects in tau P301S transgenic mice. Atomic force microscopy and cellular studies revealed that oligomerized, but not monomeric, ß-arrestin2 increases tau by inhibiting self-interaction of the autophagy cargo receptor p62/SQSTM1, impeding p62 autophagy flux. Hence, reduction of oligomerized ß-arrestin2 with virus encoding ß-arrestin2 mutants acting as dominant-negatives markedly reduces tau-laden neurofibrillary tangles in FTLD mice in vivo. Reducing ß-arrestin2 oligomeric status represents a new strategy to alleviate tau pathology in FTLD and related tauopathies.

The odorant receptor OR2W3 on airway smooth muscle evokes bronchodilation via a cooperative chemosensory tradeoff between TMEM16A and CFTR.

The recent discovery of sensory (tastant and odorant) G protein-coupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca2+]i) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca2+]i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca2+]i mobilization and ASM relaxation required Ca2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca2+-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases.

Clinical and molecular implications of RGS2 promoter genetic variation in severe asthma.

BACKGROUND: Regulator of G protein signaling (RGS) 2 terminates bronchoconstrictive Gαq signaling; murine RGS2 knockout demonstrate airway hyperresponsiveness. While RGS2 promoter variants rs2746071 and rs2746072 associate with a clinical mild asthma phenotype, their impact on human airway smooth muscle (HASM) contractility and asthma severity outcomes is unknown. OBJECTIVE: We sought to determine whether reductions in RGS2 expression seen with these 2 RGS2 promoter variants augment HASM contractility and associate with an asthma severity phenotype. METHODS: We transfected HASM with a range of RGS2-specific small interfering RNA (siRNA) concentrations and determined RGS2 protein expression by Western blot analysis and intracellular calcium flux induced by histamine (a Gαq-coupled H1 receptor bronchoconstrictive agonist). We conducted regression-based genotype association analyses of RGS2 variants from 611 patients from the National Heart, Lung, and Blood Institute Severe Asthma Research Program 3. RESULTS: RGS2-specific siRNA caused dose-dependent increases in histamine-stimulated bronchoconstrictive intracellular calcium signaling (2-way ANOVA, P < .0001) with a concomitant decrease in RGS2 protein expression. RGS2-specific siRNA did not affect Gαq-independent ionomycin-induced intracellular calcium signaling (P = .42). The minor allele frequency of rs2746071 and rs2746072 was 0.46 and 0.28 among African American/non-Hispanic Black patients and was 0.28 and 0.27 among non-Hispanic White patients, among whom these single nucleotide polymorphisms were in stronger linkage disequilibrium (r2 = 0.97). Among non-Hispanic White patients, risk allele homozygotes for rs2746072 and rs2746071 each had nearly 2-fold greater asthma exacerbation rates relative to alternative genotypes with wild-type alleles (Padditive = 2.86 × 10-5/Precessive = 5.22 × 10-6 and Padditive = 3.46 × 10-6/Precessive = 6.74 × 10-7, respectively) at baseline, which was confirmed by prospective longitudinal exacerbation data. CONCLUSION: RGS2 promoter variation associates with a molecular and clinical phenotype characterized by enhanced bronchoconstrictive stimulation in vitro and higher asthma exacerbations rates in non-Hispanic White patients.

Genetic Vulnerability of GPCRs: A Call to Action.

G-protein-coupled receptors (GPCRs) are the targets for many drugs, but the response shows interindividual variability. The 'one-drug-fits-all' approach has been challenged by evidence showing multiple human genetic variants of GPCRs. Identification and characterization of GPCR variants must be undertaken for rational, personalized, and economically sound prescribing practices.

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 ß-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:ß-arrestin binding was absent in IL/CT-10A and CT-5A and reduced in IL-5A, indicating a role for IL3 phosphorylation in the ß-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 ß-arrestin binding. However, ß-arrestin function in the internalization and trafficking of the receptor also requires GRK phosphorylation of IL3 residues.

Rhinovirus C15 Induces Airway Hyperresponsiveness via Calcium Mobilization in Airway Smooth Muscle.

Rhinovirus (RV) exposure evokes exacerbations of asthma that markedly impact morbidity and mortality worldwide. The mechanisms by which RV induces airway hyperresponsiveness (AHR) or by which specific RV serotypes differentially evoke AHR remain unknown. We posit that RV infection evokes AHR and inflammatory mediator release, which correlate with degrees of RV infection. Furthermore, we posit that rhinovirus C-induced AHR requires paracrine or autocrine mediator release from epithelium that modulates agonist-induced calcium mobilization in human airway smooth muscle. In these studies, we used an ex vivo model to measure bronchoconstriction and mediator release from infected airways in human precision cut lung slices to understand how RV exposure alters airway constriction. We found that rhinovirus C15 (RV-C15) infection augmented carbachol-induced airway narrowing and significantly increased release of IP-10 (IFN-γ-induced protein 10) and MIP-1ß (macrophage inflammatory protein-1ß) but not IL-6. RV-C15 infection of human airway epithelial cells augmented agonist-induced intracellular calcium flux and phosphorylation of myosin light chain in co-cultured human airway smooth muscle to carbachol, but not after histamine stimulation. Our data suggest that RV-C15-induced structural cell inflammatory responses are associated with viral load but that inflammatory responses and alterations in agonist-mediated constriction of human small airways are uncoupled from viral load of the tissue.

Tropomyosin pseudo-phosphorylation results in dilated cardiomyopathy.

Phosphorylation of cardiac sarcomeric proteins plays a major role in the regulation of the physiological performance of the heart. Phosphorylation of thin filament proteins, such as troponin I and T, dramatically affects calcium sensitivity of the myofiber and systolic and diastolic functions. Phosphorylation of the regulatory protein tropomyosin (Tpm) results in altered biochemical properties of contraction; however, little is known about the physiological effect of Tpm phosphorylation on cardiac function. To address the in vivo significance of Tpm phosphorylation, here we generated transgenic mouse lines having a phosphomimetic substitution in the phosphorylation site of α-Tpm (S283D). High expression of Tpm S283D variant in one transgenic mouse line resulted in an increased heart:body weight ratio, coupled with a severe dilated cardiomyopathic phenotype resulting in death within 1 month of birth. Moderate Tpm S283D mice expression in other lines caused mild myocyte hypertrophy and fibrosis, did not affect lifespan, and was coupled with decreased expression of extracellular signal-regulated kinase 1/2 kinase signaling. Physiological analysis revealed that the transgenic mice exhibit impaired diastolic function, without changes in systolic performance. Surprisingly, we observed no alterations in calcium sensitivity of the myofibers, cooperativity, or calcium-ATPase activity in the myofibers. Our experiments also disclosed that casein kinase 2 plays an integral role in Tpm phosphorylation. In summary, increased expression of pseudo-phosphorylated Tpm impairs diastolic function in the intact heart, without altering calcium sensitivity or cooperativity of myofibers. Our findings provide the first extensive in vivo assessment of Tpm phosphorylation in the heart and its functional role in cardiac performance.

Differential long-term regulation of TAS2R14 by structurally distinct agonists.

Bitter taste receptor-14 (TAS2R14) is a GPCR also expressed on human airway smooth muscle cells, which signals to intracellular [Ca2+], resulting in relaxation of the airway, and is a novel target for bronchodilators. Here, we examine long-term, agonist-promoted down-regulation of TAS2R14 expression because tachyphylaxis would be an undesirable therapeutic characteristic. Five TAS2R structurally distinct full agonists were studied to ascertain biasing away from down-regulation. Agonist exposure for 18 h caused minimal desensitization by diphenhydramine (DPD) compared with ∼50% desensitization with all other agonists. Agonists evoked ß-arrestin recruitment to TAS2R14, which was not seen with a phosphoacceptor-deficient mutant, TAS2R14-10A. All agonists except for DPD also caused subsequent TAS2R14 internalization and trafficking via early and late endosomes to down-regulation. TAS2R14-10A failed to undergo these events with any agonist. Molecular docking showed that DPD has specific interactions deep within a binding pocket that are not observed with the other agonists, which may lock the receptor in a conformation that does not internalize and therefore does not undergo down-regulation. Thus, TAS2R14 is subject to ß-arrestin-mediated internalization and subsequent down-regulation with chronic exposure to most agonists. However, by manipulating the agonist structure, biasing toward G-protein coupling but away from long-term down-regulation can be achieved.-Woo, J. A., Castaño, M., Goss, A., Kim, D., Lewandowski, E. M., Chen, Y., Liggett, S. B. Differential long-term regulation of TAS2R14 by structurally distinct agonists.

Development of a Rhinovirus Inoculum Using a Reverse Genetics Approach.

BACKGROUND: Experimental inoculation is an important tool for common cold and asthma research. Producing rhinovirus (RV) inocula from nasal secretions has required prolonged observation of the virus donor to exclude extraneous pathogens. We produced a RV-A16 inoculum using reverse genetics and determined the dose necessary to cause moderate colds in seronegative volunteers. METHODS: The consensus sequence of RV-A16 from a previous inoculum was cloned, and inoculum virus was produced using reverse genetics techniques. After safety testing, volunteers were inoculated with either RV-A16 (n = 26) or placebo (n = 10), Jackson cold scores were recorded, and nasal secretions were tested for shedding of RV-A16 ribonucleic acid. RESULTS: The reverse genetics process produced infectious virus that was neutralized by specific antisera and had a mutation rate similar to conventional virus growth techniques. The 1000 median tissue culture infectious dose (TCID50) dose produced moderate colds in most individuals with effects similar to that of a previously tested conventional RV-A16 inoculum. CONCLUSIONS: Reverse genetics techniques produced a RV-A16 inoculum that can cause clinical colds in seronegative volunteers, and they also serve as a stable source of virus for laboratory use. The recombinant production procedures eliminate the need to derive seed virus from nasal secretions, thus precluding introduction of extraneous pathogens through this route.

Biased TAS2R Bronchodilators Inhibit Airway Smooth Muscle Growth by Downregulating Phosphorylated Extracellular Signal-regulated Kinase 1/2.

Bitter taste receptor (TAS2R) agonists dilate airways by receptor-dependent smooth muscle relaxation. Besides their coupling to relaxation, we have found that human airway smooth muscle (HASM) cell TAS2Rs activate (phosphorylate) extracellular signal-related kinase 1/2 (ERK1/2), but the cellular effects are not known. In the present study, we show in HASM cells that TAS2R agonists initially stimulate phosphorylated ERK1/2 (pERK1/2) but by 24 hours cause a marked (50-70%) downregulation of pERK1/2 without a change in total ERK1/2. It was hypothesized that TAS2R agonists suppress cell growth through this pERK1/2 downregulation. Agonist-dependent inhibition of cell proliferation was indeed found in HASM cells derived from normal and asthmatic human lungs, as well as in an immortalized HASM cell line. pERK1/2 downregulation was linked to downregulation of the upstream kinase MEK1/2 (mitogen-activated protein kinase/extracellular signal-regulated kinase). Various structurally diverse TAS2R agonists evoked a range of inhibition of HASM proliferation, the magnitude of which directly correlated with the downregulation of pERK1/2 (R2 = 0.86). Some TAS2R agonists were as effective as pharmacological inhibitors of Raf1 and MEK1/2 in suppressing growth. siRNA silencing of TAS2Rs (subtypes 10, 14, and 31) ablated the pERK1/2 and growth-inhibitory effects of TAS2R agonists. These phenotypes were attenuated by inhibiting the TAS2R G protein Gαi and by knocking down ß-arrestin 1/2, indicating a dual pathway, although there may be additional mechanisms involved in this HASM TAS2R multidimensional signaling. Thus, TAS2R agonist structure can be manipulated to maintain the relaxation response and can be biased toward suppression of HASM growth. The latter response is of potential therapeutic benefit in asthma, in which an increase in smooth muscle mass contributes to airway obstruction.

A CREB-mediated increase in miRNA let-7f during prolonged ß-agonist exposure: a novel mechanism of ß2-adrenergic receptor down-regulation in airway smooth muscle.

ß2-Adrenergic receptors (ß2ARs) desensitize during continuous agonist activation, which manifests clinically as tachyphylaxis. ß-Agonist desensitization of ß2ARs in human airway smooth muscle (HASM) cells is recognized in the treatment of asthma and may be related to poor outcomes. Rapid events in desensitization include receptor phosphorylation and internalization, but mechanisms responsible for the decrease in receptor protein after prolonged agonist exposure (down-regulation) are ill defined. The microRNA (miRNA) let-7f regulates ß2AR expression by translational repression. In cultured HASM cells from nonasthmatic and asthmatic lungs, 18 h of ß-agonist exposure increased let-7f by 2-3-fold, concomitant with a ∼90% decrease in ß2ARs. Inhibition of let-7f attenuated this down-regulation response by ∼50%. The let-7f increase was found to be cAMP/PKA-dependent. The mechanism of the let-7f increase was found by chromatin immunoprecipitation to be from activated cAMP response element-binding protein (CREB) binding to the let-7f promoter, thereby increasing let-7f expression. Knockdown of CREB attenuated agonist-promoted ß2AR down-regulation by ∼50%. Thus, ß2AR down-regulation occurs as a result of not only internalized receptor degradation but also a novel cAMP/PKA/CREB-mediated increase in let-7f, which causes enhanced repression of the ß2AR gene, adrenoreceptor ß2 ( ADRB2) translation and represents ∼50% of the net loss of receptors observed after prolonged agonist exposure. This mechanism is apparent in asthmatic HASM cells, indicating relevance in a disease model.-Kim, D., Cho, S., Woo, J. A., Liggett, S. B. A CREB-mediated increase in miRNA let-7f during prolonged ß-agonist exposure: a novel mechanism of ß2-adrenergic receptor down-regulation in airway smooth muscle.

Coupling of Airway Smooth Muscle Bitter Taste Receptors to Intracellular Signaling and Relaxation Is via Gαi1,2,3.

Bitter taste receptors (TAS2Rs) are expressed on human airway smooth muscle (HASM) and evoke marked relaxation. Agonist interaction with TAS2Rs activates phospholipase C and increases compartmentalized intracellular Ca2+ ([Ca2+]i) via inositol 1,4,5 triphosphate. In taste cells, the G protein gustducin couples TAS2R to phospholipase C; however, we find very low levels of Gαgust mRNA or protein in HASM. We hypothesized that another G protein in HASM transmits TAS2R function. TAS2R signaling to [Ca2+]i, extracellular signal-regulated kinase (ERK) 1/2, and physiologic relaxation was sensitive to pertussis toxin, confirming a role for a member of the Gi family. α subunit expression in HASM was Gαi2 > Gαi1 = Gαi3 > Gαtrans1 ≈ Gαtrans2, with Gαgust and Gαo at the limits of detection (>100-fold lower than Gαi2). Small interfering RNA knockdowns in HASM showed losses of [Ca2+]i and ERK1/2 signaling when Gαi1, Gαi2, or Gαi3 were reduced. Gαtrans1 and Gαtrans2 knockdowns had no effect on [Ca2+]i and a minimal, transient effect on ERK1/2 phosphorylation. Furthermore, Gαgust and Gαo knockdowns did not affect any TAS2R signaling. In overexpression experiments in human embryonic kidney-293T cells, we confirmed an agonist-dependent physical interaction between TAS2R14 and Gαi2. ASM cells from transgenic mice expressing a peptide inhibitor of Gαi2 had attenuated relaxation to TAS2R agonist. These data indicate that, unlike in taste cells, TAS2Rs couple to the prevalent G proteins, Gαi1, Gαi2, and Gαi3, with no evidence for functional coupling to Gαgust. This absence of function for the "canonical" TAS2R G protein in HASM may be due to the very low expression of Gαgust, indicating that TAS2Rs can optionally couple to several G proteins in a cell type-dependent manner contingent upon G protein expression.

ß2-Adrenergic Receptors Chaperone Trapped Bitter Taste Receptor 14 to the Cell Surface as a Heterodimer and Exert Unidirectional Desensitization of Taste Receptor Function.

Bitter taste receptors (TAS2Rs) are G-protein-coupled receptors now recognized to be expressed on extraoral cells, including airway smooth muscle (ASM) where they evoke relaxation. TAS2Rs are difficult to express in heterologous systems, with most receptors being trapped intracellularly. We find, however, that co-expression of ß2-adrenergic receptors (ß2AR) in HEK-293T routes TAS2R14 to the cell surface by forming receptor heterodimers. Cell surface TAS2R14 expression was increased by ∼5-fold when ß2AR was co-expressed. Heterodimer formation was shown by co-immunoprecipitation with tagged receptors, biomolecular fluorescence complementation, and merged confocal images. The dynamic nature of this interaction was shown by: a gene-dose relationship between transfected ß2AR and TAS2R14 expression, enhanced (up to 3-fold) TAS2R14 agonist stimulation of [Ca(2+)]i with ß2AR co-transfection, ∼53% decrease in [Ca(2+)]i signaling with shRNA knockdown of ß2AR in H292 cells, and ∼60% loss of [Ca(2+)]i responsiveness in ßAR knock-out mouse ASM. Once expressed on the surface, we detected unidirectional, conformation-dependent, interaction within the heterodimer, with ß2AR activation rapidly uncoupling TAS2R14 function (∼65% desensitization). Cross-talk was independent of ß2AR internalization and cAMP/PKA, and not accompanied by TAS2R14 internalization. With prolonged ß-agonist exposure, TAS2R14 internalized, consistent with slow recycling of naked TAS2R14 in the absence of the heterodimeric milieu. In studies of ASM mechanics, rapid cross-talk was confirmed at the physiologic level, where relaxation from TAS2R14 agonist was decreased by ∼50% with ß-agonist co-treatment. Thus the ß2AR acts as a double-edged sword: increasing TAS2R14 cell surface expression, but when activated by ß-agonist, partially offsetting the expression phenotype by direct receptor:receptor desensitization of TAS2R14 function.

Future translational applications from the contemporary genomics era: a scientific statement from the American Heart Association.

The field of genetics and genomics has advanced considerably with the achievement of recent milestones encompassing the identification of many loci for cardiovascular disease and variable drug responses. Despite this achievement, a gap exists in the understanding and advancement to meaningful translation that directly affects disease prevention and clinical care. The purpose of this scientific statement is to address the gap between genetic discoveries and their practical application to cardiovascular clinical care. In brief, this scientific statement assesses the current timeline for effective translation of basic discoveries to clinical advances, highlighting past successes. Current discoveries in the area of genetics and genomics are covered next, followed by future expectations, tools, and competencies for achieving the goal of improving clinical care.

Bitter taste receptor function in asthmatic and nonasthmatic human airway smooth muscle cells.

Bitter taste receptors (TAS2Rs) have recently been found to be expressed on human airway smooth muscle (HASM), and their activation results in marked relaxation. These agents have been proposed as a new class of bronchodilators in the treatment of obstructive lung diseases because they act via a different mechanism than ß-agonists. The TAS2R signal transduction pathway in HASM has multiple elements that are potentially subject to regulation by inflammatory, genetic, and epigenetic mechanisms associated with asthma. To address this, expression, signaling, and physiologic functions of the three major TAS2Rs (subtypes 10, 14, and 31) on HASM were studied. Transcript expression of these TAS2Rs was not decreased in HASM cells derived from donors with asthma compared with those without asthma (n = 6 from each group). In addition, intracellular calcium ([Ca(2+)]i) signaling using TAS2R subtype-specific agonists (diphenhydramine, chloroquine, saccharin, and flufenamic acid) was not impaired in the cells derived from donors with asthma, nor was the response to quinine, which activates all three subtypes. HASM cell mechanics measured by magnetic twisting cytometry revealed equivalent TAS2R-mediated relaxation of methacholine-treated cells between the two groups. Human precision-cut lung slices treated with IL-13 caused a decrease in ß-agonist (formoterol)-mediated relaxation of carbachol-contracted airways compared with control slices. In contrast, TAS2R-mediated relaxation was unaffected by IL-13. We conclude that TAS2R expression or function is unaffected in HASM cells derived from patients with asthma or the IL-13 inflammatory environment.

Targeted transgenesis identifies Gαs as the bottleneck in ß2-adrenergic receptor cell signaling and physiological function in airway smooth muscle.

G protein-coupled receptors are the most pervasive signaling superfamily in the body and act as receptors to endogenous agonists and drugs. For ß-agonist-mediated bronchodilation, the receptor-G protein-effector network consists of the ß2-adrenergic receptor (ß2AR), Gs, and adenylyl cyclase, expressed on airway smooth muscle (ASM). Using ASM-targeted transgenesis, we previously explored which of these three early signaling elements represents a limiting factor, or bottleneck, in transmission of the signal from agonist binding to ASM relaxation. Here we overexpressed Gαs in transgenic mice and found that agonist-promoted relaxation of airways was enhanced in direct proportion to the level of Gαs expression. Contraction of ASM from acetylcholine was not affected in Gαs transgenic mice, nor was relaxation by bitter taste receptors. Furthermore, agonist-promoted (but not basal) cAMP production in ASM cells from Gαs-transgenic mice was enhanced compared with ASM from nontransgenic littermates. Agonist-promoted inhibition of platelet-derived growth factor-stimulated ASM proliferation was also enhanced in Gαs mouse ASM. The enhanced maximal ß-agonist response was of similar magnitude for relaxation, cAMP production, and growth inhibition. Taken together, it appears that a limiting factor in ß-agonist responsiveness in ASM is the expression level of Gαs. Gene therapy or pharmacological means of increasing Gαs (or its coupling efficiency to ß2AR) thus represent an interface for development of novel therapeutic agents for improvement of ß-agonist therapy.

Bitter taste receptors in the wrong place: novel airway smooth muscle targets for treating asthma.

There is a need to expand the classes of drugs used to treat obstructive lung diseases to achieve better outcomes. With only one class of direct bronchodilators (ß-agonists), we sought to find receptors on human airway smooth muscle (ASM) that act via a unique mechanism to relax the muscle, have a diverse agonist binding profile to enhance the probability of finding new therapeutics, and relax ASM with equal or greater efficacy than ß-agonists. We have found that human and mouse ASM express six bitter taste receptor (TAS2R) subtypes, previously thought only to exist in taste buds of the tongue. Agonists acting at TAS2Rs evoke profound bronchodilation via a Ca(2+)-dependent mechanism. TAS2R function is not altered in asthma models, undergoes minimal tachyphylaxis upon repetitive dosing, and relaxes even under extreme desensitization of relaxation by ß-agonists. Taken together, TAS2Rs on ASM represent a novel pathway to consider for development of agonists in the treatment of asthma and chronic obstructive lung disease.