RESUMO
The peptidergic system is the most abundant network of ligand-receptor-mediated signaling in humans. However, the physiological roles remain elusive for numerous peptides and more than 100 G protein-coupled receptors (GPCRs). Here we report the pairing of cognate peptides and receptors. Integrating comparative genomics across 313 species and bioinformatics on all protein sequences and structures of human class A GPCRs, we identify universal characteristics that uncover additional potential peptidergic signaling systems. Using three orthogonal biochemical assays, we pair 17 proposed endogenous ligands with five orphan GPCRs that are associated with diseases, including genetic, neoplastic, nervous and reproductive system disorders. We also identify additional peptides for nine receptors with recognized ligands and pathophysiological roles. This integrated computational and multifaceted experimental approach expands the peptide-GPCR network and opens the way for studies to elucidate the roles of these signaling systems in human physiology and disease. VIDEO ABSTRACT.
Assuntos
Genômica , Peptídeos/genética , Conformação Proteica , Receptores Acoplados a Proteínas G/genética , Sequência de Aminoácidos/genética , Biologia Computacional , Redes Reguladoras de Genes/genética , Genitália/metabolismo , Genitália/patologia , Humanos , Ligantes , Neoplasias/genética , Neoplasias/patologia , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/patologia , Transdução de Sinais/genéticaRESUMO
Catecholamine-stimulated ß2-adrenergic receptor (ß2AR) signaling via the canonical Gs-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous ß-agonists in the treatment of airway disease. ß2AR signaling is tightly regulated by GRKs and ß-arrestins, which together promote ß2AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias ß2AR signaling toward the Gs pathway while avoiding ß-arrestin-mediated effects may provide a strategy to improve the functional consequences of ß2AR activation. Since attempts to develop Gs-biased agonists and allosteric modulators for the ß2AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit ß-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of ß-arrestin recruitment to the ß2AR while having no effect on ß2AR coupling to Gs. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the ß2AR and protects against the functional desensitization of ß-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the ß2AR with minimal effects on the ß1AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the ß2AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the ß2AR.
Assuntos
Arrestina , Transdução de Sinais , beta-Arrestinas/metabolismo , Arrestina/metabolismo , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismo , Receptores Adrenérgicos/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismoRESUMO
G-protein-coupled receptors (GPCRs) belonging to the type 2 taste receptors (TAS2Rs) family are predominantly present in taste cells to allow the perception of bitter-tasting compounds. TAS2Rs have also been shown to be expressed in human airway smooth muscle (ASM), and TAS2R agonists relax ASM cells and bronchodilate airways despite elevating intracellular calcium. This calcium "paradox" (calcium mediates contraction by pro-contractile Gq-coupled GPCRs) and the mechanisms by which TAS2R agonists relax ASM remain poorly understood. To gain insight into pro-relaxant mechanisms effected by TAS2Rs, we employed an unbiased phosphoproteomic approach involving dual-mass spectrometry to determine differences in the phosphorylation of contractile-related proteins in ASM following the stimulation of cells with TAS2R agonists, histamine (an agonist of the Gq-coupled H1 histamine receptor) or isoproterenol (an agonist of the Gs-coupled ß2-adrenoceptor) alone or in combination. Our study identified differential phosphorylation of proteins regulating contraction, including A-kinase anchoring protein (AKAP)2, AKAP12, and RhoA guanine nucleotide exchange factor (ARHGEF)12. Subsequent signaling analyses revealed RhoA and the T853 residue on myosin light chain phosphatase (MYPT)1 as points of mechanistic divergence between TAS2R and Gs-coupled GPCR pathways. Unlike Gs-coupled receptor signaling, which inhibits histamine-induced myosin light chain (MLC)20 phosphorylation via protein kinase A (PKA)-dependent inhibition of intracellular calcium mobilization, HSP20 and ERK1/2 activity, TAS2Rs are shown to inhibit histamine-induced pMLC20 via inhibition of RhoA activity and MYPT1 phosphorylation at the T853 residue. These findings provide insight into the TAS2R signaling in ASM by defining a distinct signaling mechanism modulating inhibition of pMLC20 to relax contracted ASM.
Assuntos
Músculo Liso , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Músculo Liso/metabolismo , Músculo Liso/efeitos dos fármacos , Fosforilação , Relaxamento Muscular/efeitos dos fármacos , Histamina/metabolismo , Histamina/farmacologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Isoproterenol/farmacologia , Cálcio/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Paladar/fisiologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Transdução de Sinais , Células CultivadasRESUMO
Activation of ß2-adrenoceptors (ß2ARs) causes airway smooth muscle (ASM) relaxation and bronchodilation, and ß2AR agonists (ß-agonists) are front-line treatments for asthma and other obstructive lung diseases. However, the therapeutic efficacy of ß-agonists is limited by agonist-induced ß2AR desensitization and noncanonical ß2AR signaling involving ß-arrestin that is shown to promote asthma pathophysiology. Accordingly, we undertook the identification of an allosteric site on ß2AR that could modulate the activity of ß-agonists to overcome these limitations. We employed the site identification by ligand competitive saturation (SILCS) computational method to comprehensively map the entire 3D structure of in silico-generated ß2AR intermediate conformations and identified a putative allosteric binding site. Subsequent database screening using SILCS identified drug-like molecules with the potential to bind to the site. Experimental assays in HEK293 cells (expressing recombinant wild-type human ß2AR) and human ASM cells (expressing endogenous ß2AR) identified positive and negative allosteric modulators (PAMs and NAMs) of ß2AR as assessed by regulation of ß-agonist-stimulation of cyclic AMP generation. PAMs/NAMs had no effect on ß-agonist-induced recruitment of ß-arrestin to ß2AR- or ß-agonist-induced loss of cell surface expression in HEK293 cells expressing ß2AR. Mutagenesis analysis of ß2AR confirmed the SILCS identified site based on mutants of amino acids R131, Y219, and F282. Finally, functional studies revealed augmentation of ß-agonist-induced relaxation of contracted human ASM cells and bronchodilation of contracted airways. These findings identify a allosteric binding site on the ß2AR, whose activation selectively augments ß-agonist-induced Gs signaling, and increases relaxation of ASM cells, the principal therapeutic effect of ß-agonists.
Assuntos
Asma , Receptores Adrenérgicos beta 2 , Humanos , Sítio Alostérico , Células HEK293 , beta-Arrestinas , beta-Arrestina 1 , Receptores Adrenérgicos beta 2/genéticaRESUMO
A-kinase-anchoring proteins (AKAPs) act as scaffold proteins that anchor the regulatory subunits of the cAMP-dependent protein kinase A (PKA) to coordinate and compartmentalize signaling elements and signals downstream of Gs-coupled G protein-coupled receptors (GPCRs). The beta-2-adrenoceptor (ß2AR), as well as the Gs-coupled EP2 and EP4 receptor subtypes of the E-prostanoid (EP) receptor subfamily, are effective regulators of multiple airway smooth muscle (ASM) cell functions whose dysregulation contributes of asthma pathobiology. Here, we identify specific roles of the AKAPs Ezrin and Gravin, in differentially regulating PKA substrates downstream of the ß2AR, EP2 receptor (EP2R) and EP4 receptor (EP4R). Knockdown of Ezrin, Gravin, or both in primary human ASM cells caused differential phosphorylation of the PKA substrates vasodilator-stimulated phosphoprotein (VASP) and heat shock protein 20 (HSP20). Ezrin knockdown, as well as combined Ezrin + Gravin knockdown significantly reduced the induction of phospho-VASP and phospho-HSP20 by ß2AR, EP2R, and EP4R agonists. Gravin knockdown inhibited the induction of phospho-HSP20 by ß2AR, EP2R, and EP4R agonists. Knockdown of Ezrin, Gravin, or both also attenuated histamine-induced phosphorylation of MLC20. Moreover, knockdown of Ezrin, Gravin or both suppressed the inhibitory effects of Gs-coupled receptor agonists on cell migration in ASM cells. These findings demonstrate the role of AKAPs in regulating Gs-coupled GPCR signaling and function in ASM, and suggest the therapeutic utility of targeting specific AKAP family members in the management of asthma.
RESUMO
Airway smooth muscle (ASM) cells attain a hypercontractile phenotype during obstructive airway diseases. We recently identified a biased M3 muscarinic acetylcholine receptor (mAChR) ligand, PD 102807, that induces GRK-/arrestin-dependent AMP-activated protein kinase (AMPK) activation to inhibit transforming growth factor-ß-induced hypercontractile ASM phenotype. Conversely, the balanced mAChR agonist, methacholine (MCh), activates AMPK yet does not regulate ASM phenotype. In the current study, we demonstrate that PD 102807- and MCh-induced AMPK activation both depend on Ca2+/calmodulin-dependent kinase kinases (CaMKKs). However, MCh-induced AMPK activation is calcium-dependent and mediated by CaMKK1 and CaMKK2 isoforms. In contrast, PD 102807-induced signaling is calcium-independent and mediated by the atypical subtype protein kinase C-iota and the CaMKK1 (but not CaMKK2) isoform. Both MCh- and PD 102807-induced AMPK activation involve the AMPK α1 isoform. PD 102807-induced AMPK α1 (but not AMPK α2) isoform activation mediates inhibition of the mammalian target of rapamycin complex 1 (mTORC1) in ASM cells, as demonstrated by increased Raptor (regulatory-associated protein of mTOR) phosphorylation as well as inhibition of phospho-S6 protein and serum response element-luciferase activity. The mTORC1 inhibitor rapamycin and the AMPK activator metformin both mimic the ability of PD 102807 to attenuate transforming growth factor-ß-induced α-smooth muscle actin expression (a marker of hypercontractile ASM). These data indicate that PD 102807 transduces a signaling pathway (AMPK-mediated mTORC1 inhibition) qualitatively distinct from canonical M3 mAChR signaling to prevent pathogenic remodeling of ASM, thus demonstrating PD 102807 is a biased M3 mAChR ligand with therapeutic potential for the management of obstructive airway disease.
RESUMO
Rationale: Gastroesophageal reflux disease (GERD) is commonly associated with atopic disorders, but cause-effect relationships remain unclear. Objectives: We applied Mendelian randomization analysis to explore whether GERD is causally related to atopic disorders of the lung (asthma) and/or skin (atopic dermatitis [AD]). Methods: We conducted two-sample bidirectional Mendelian randomization to infer the magnitude and direction of causality between asthma and GERD, using summary statistics from the largest genome-wide association studies conducted on asthma (Ncases = 56,167) and GERD (Ncases = 71,522). In addition, we generated instrumental variables for AD from the latest population-level genome-wide association study meta-analysis (Ncases = 22,474) and assessed their fidelity and confidence of predicting the likely causal pathway(s) leading to asthma and/or GERD. Measurements and Main Results: Applying three different methods, each method revealed similar magnitude of causal estimates that were directionally consistent across the sensitivity analyses. Using an inverse variance-weighted method, the largest effect size was detected for asthma predisposition to AD (odds ratio [OR], 1.46; 95% confidence interval [CI], 1.34-1.59), followed by AD to asthma (OR, 1.34; 95% CI, 1.24-1.45). A significant association was detected for genetically determined asthma on risk of GERD (OR, 1.06; 95% CI, 1.03-1.09) but not genetically determined AD on GERD. In contrast, GERD equally increased risks of asthma (OR, 1.21; 95% CI, 1.09-1.35) and AD (OR, 1.21; 95% CI, 1.07-1.37). Conclusions: This study uncovers previously unrecognized causal pathways that have clinical implications in European-ancestry populations: 1) asthma is a causal risk for AD, and 2) the predisposition to AD, including asthma, can arise from specific pathogenic mechanisms manifested by GERD.
Assuntos
Asma , Dermatite Atópica , Refluxo Gastroesofágico , Humanos , Dermatite Atópica/epidemiologia , Dermatite Atópica/genética , Análise da Randomização Mendeliana , Estudo de Associação Genômica Ampla , Asma/epidemiologia , Asma/genética , Refluxo Gastroesofágico/epidemiologia , Refluxo Gastroesofágico/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Prostaglandin E2 imparts diverse physiological effects on multiple airway cells through its actions on four distinct E-type prostanoid (EP) receptor subtypes (EP1-EP4). Gs-coupled EP2 and EP4 receptors are expressed on airway smooth muscle (ASM), yet their capacity to regulate the ASM contractile state remains subject to debate. We used EP2 and EP4 subtype-specific agonists (ONO-259 and ONO-329, respectively) in cell- and tissue-based models of human ASM contraction-magnetic twisting cytometry (MTC), and precision-cut lung slices (PCLSs), respectively-to study the EP2 and EP4 regulation of ASM contraction and signaling under conditions of histamine or methacholine (MCh) stimulation. ONO-329 was superior (<0.05) to ONO-259 in relaxing MCh-contracted PCLSs (log half maximal effective concentration [logEC50]: 4.9 × 10-7 vs. 2.2 × 10-6; maximal bronchodilation ± SE, 35 ± 2% vs. 15 ± 2%). However, ONO-259 and ONO-329 were similarly efficacious in relaxing histamine-contracted PCLSs. Similar differential effects were observed in MTC studies. Signaling analyses revealed only modest differences in ONO-329- and ONO-259-induced phosphorylation of the protein kinase A substrates VASP and HSP20, with concomitant stimulation with MCh or histamine. Conversely, ONO-259 failed to inhibit MCh-induced phosphorylation of the regulatory myosin light chain (pMLC20) and the F-actin/G-actin ratio (F/G-actin ratio) while effectively inhibiting their induction by histamine. ONO-329 was effective in reversing induced pMLC20 and the F/G-actin ratio with both MCh and histamine. Thus, the contractile-agonist-dependent differential effects are not explained by changes in the global levels of phosphorylated protein kinase A substrates but are reflected in the regulation of pMLC20 (cross-bridge cycling) and F/G-actin ratio (actin cytoskeleton integrity, force transmission), implicating a role for compartmentalized signaling involving muscarinic, histamine, and EP receptor subtypes.
Assuntos
Actinas , Receptores de Prostaglandina E Subtipo EP2 , Humanos , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Histamina/farmacologia , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Dinoprostona , Músculo Liso/metabolismo , Pulmão/metabolismo , Proteínas Quinases Dependentes de AMP CíclicoRESUMO
BACKGROUND: Diacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that DGK inhibition attenuates airway smooth muscle (ASM) cell proliferation, however, the mechanisms mediating this effect are not well established. Given the capacity of protein kinase A (PKA) to effect inhibition of ASM cells growth in response to mitogens, we employed multiple molecular and pharmacological approaches to examine the putative role of PKA in the inhibition of mitogen-induced ASM cell proliferation by the small molecular DGK inhibitor I (DGK I). METHODS: We assayed cell proliferation using CyQUANT™ NF assay, protein expression and phosphorylation using immunoblotting, and prostaglandin E2 (PGE2) secretion by ELISA. ASM cells stably expressing GFP or PKI-GFP (PKA inhibitory peptide-GFP chimera) were stimulated with platelet-derived growth factor (PDGF), or PDGF + DGK I, and cell proliferation was assessed. RESULTS: DGK inhibition reduced ASM cell proliferation in cells expressing GFP, but not in cells expressing PKI-GFP. DGK inhibition increased cyclooxygenase II (COXII) expression and PGE2 secretion over time to promote PKA activation as demonstrated by increased phosphorylation of (PKA substrates) VASP and CREB. COXII expression and PKA activation were significantly decreased in cells pre-treated with pan-PKC (Bis I), MEK (U0126), or ERK2 (Vx11e) inhibitors suggesting a role for PKC and ERK in the COXII-PGE2-mediated activation of PKA signaling by DGK inhibition. CONCLUSIONS: Our study provides insight into the molecular pathway (DAG-PKC/ERK-COXII-PGE2-PKA) regulated by DGK in ASM cells and identifies DGK as a potential therapeutic target for mitigating ASM cell proliferation that contributes to airway remodeling in asthma.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Diacilglicerol Quinase , Diacilglicerol Quinase/metabolismo , Diacilglicerol Quinase/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Células Cultivadas , Proliferação de Células , Miócitos de Músculo Liso/metabolismoRESUMO
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.
Assuntos
Anoctamina-1/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Músculo Liso/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores Odorantes/metabolismo , Adenilil Ciclases/metabolismo , Brônquios/metabolismo , Cálcio/metabolismo , Células Cultivadas , Humanos , Pulmão/metabolismo , Contração Muscular/fisiologia , Relaxamento Muscular , Miócitos de Músculo Liso/metabolismo , Receptores Odorantes/genéticaRESUMO
G protein-coupled receptors (GPCRs) not only are turned on or off to control canonical G protein signaling but also may be fine-tuned to promote qualitative/biased signaling. Qualitative signaling by M3 muscarinic acetylcholine receptors (mAChRs) has been proposed, but its impact on physiologic systems remains unclear, and currently no biased M3 mAChR ligands have been described. Herein, we identify PD 102807 as a biased M3 ligand and delineate its signaling and function in human airway smooth muscle (ASM) cells. PD 102807 induced M3-mediated ß-arrestin recruitment but not calcium mobilization. PD 102807 inhibited methacholine (MCh)-induced calcium mobilization in (M3-expressing) ASM cells. PD 102807 induced phosphorylation of AMP-activated protein kinase (AMPK) and the downstream effector acetyl-coenzyme A carboxylase (ACC). PD 102807- induced phosphorylated (p)-AMPK levels were greatly reduced in ASM cells with minimal M3 expression and were not inhibited by the Gq inhibitor YM-254890. Induction of p-AMPK and p-ACC was inhibited by ß-arrestin 1 or GRK2/3 knockdown. Similarly, MCh induced phosphorylation of AMPK/ACC, but these effects were Gq dependent and unaffected by GRK2/3 knockdown. Consistent with the known ability of AMPK to inhibit transforming growth factor ß (TGF-ß)-mediated functions, PD 102807 inhibited TGF-ß-induced SMAD-Luc activity, sm-α-actin expression, actin stress fiber formation, and ASM cell hypercontractility. These findings reveal that PD 102807 is a biased M3 ligand that inhibits M3-transduced Gq signaling but promotes Gq protein-independent, GRK-/arrestin-dependent, M3-mediated AMPK signaling, which in turn regulates ASM phenotype and contractile function. Consequently, biased M3 ligands hold significant promise as therapeutic agents capable of exploiting the pleiotropic nature of M3 signaling.
Assuntos
Proteínas Quinases Ativadas por AMP , Arrestina , Humanos , Arrestina/genética , Arrestina/metabolismo , Arrestina/farmacologia , Ligantes , Proteínas Quinases Ativadas por AMP/metabolismo , Miócitos de Músculo Liso/metabolismo , beta-Arrestina 1/metabolismo , Actinas/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Diacylglycerol kinase (DGK), a lipid kinase, catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid, thereby terminating DAG-mediated signaling by Gq-coupled receptors that regulate contraction of airway smooth muscle (ASM). A previous study from our laboratory demonstrated that DGK inhibition or genetic ablation leads to reduced ASM contraction and provides protection for allergen-induced airway hyperresponsiveness. However, the mechanism by which DGK regulates contractile signaling in ASM is not well established. Herein, we investigated the role of prorelaxant cAMP-protein kinase A (PKA) signaling in DGK-mediated regulation of ASM contraction. Pretreatment of human ASM cells with DGK inhibitor I activated PKA as demonstrated by the phosphorylation of PKA substrates, VASP, Hsp20, and CREB, which was abrogated when PKA was inhibited pharmacologically or molecularly using overexpression of the PKA inhibitor peptide, PKI. Furthermore, inhibition of DGK resulted in induction of cyclooxygenase (COX) and generation of prostaglandin E2 (PGE2 ) with concomitant activation of Gs-cAMP-PKA signaling in ASM cells in an autocrine/paracrine fashion. Inhibition of protein kinase C (PKC) or extracellular-signal-regulated kinase (ERK) attenuated DGK-mediated production of PGE2 and activation of cAMP-PKA signaling in human ASM cells, suggesting that inhibition of DGK activates the COX-PGE2 pathway in a PKC-ERK-dependent manner. Finally, DGK inhibition-mediated attenuation of contractile agonist-induced phosphorylation of myosin light chain 20 (MLC-20), a marker of ASM contraction, involves COX-mediated cAMP production and PKA activation in ASM cells. Collectively these findings establish a novel mechanism by which DGK regulates ASM contraction and further advances DGK as a potential therapeutic target to provide effective bronchoprotection in asthma.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Diacilglicerol Quinase , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Diacilglicerol Quinase/genética , Dinoprostona/farmacologia , Humanos , Contração Muscular , Proteína Quinase CRESUMO
Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on ß2 adrenoceptor agonists (ß-agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with ß-agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c-Abl). The cooperative effect of ß-agonists (isoproterenol) and c-Abl inhibitors (GNF-5, or imatinib) on contractile agonist (methacholine, or histamine) -induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c-Abl inhibitors and ß-agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c-Abl inhibitors and ß-agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by ß-agonists is near optimal and is not increased by c-Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both ß-agonists and c-Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Sinergismo Farmacológico , Contração Muscular , Relaxamento Muscular , Músculo Liso/fisiologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Traqueia/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Humanos , Mesilato de Imatinib/farmacologia , Isoproterenol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Pirimidinas/farmacologia , Transdução de Sinais , Traqueia/citologia , Traqueia/efeitos dos fármacosRESUMO
Airway remodeling in asthma involves the hyperproliferation of airway smooth muscle (ASM) cells. However, the molecular signals that regulate ASM growth are not completely understood. Gq-coupled G protein-coupled receptor and receptor tyrosine kinase signaling regulate ASM cell proliferation via activation of phospholipase C, generation of inositol triphosphate (IP3) and diacylglycerol (DAG). Diacylglycerol kinase (DGK) converts DAG into phosphatidic acid (PA) and terminates DAG signaling while promoting PA-mediated signaling and function. Herein, we hypothesized that PA is a pro-mitogenic second messenger in ASM, and DGK inhibition reduces the conversion of DAG into PA resulting in inhibition of ASM cell proliferation. We assessed the effect of pharmacological inhibition of DGK on pro-mitogenic signaling and proliferation in primary human ASM cells. Pretreatment with DGK inhibitor I (DGKI) significantly inhibited platelet-derived growth factor-stimulated ASM cell proliferation. Anti-mitogenic effect of DGKI was associated with decreased mTOR signaling and expression of cyclin D1. Exogenous PA promoted pro-mitogenic signaling and rescued DGKI-induced attenuation of ASM cell proliferation. Finally, house dust mite (HDM) challenge in wild type mice promoted airway remodeling features, which were attenuated in DGKζ-/- mice. We propose that DGK serves as a potential drug target for mitigating airway remodeling in asthma.
Assuntos
Remodelação das Vias Aéreas , Asma , Animais , Asma/metabolismo , Proliferação de Células , Ciclina D1/metabolismo , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Diglicerídeos/metabolismo , Humanos , Inositol/farmacologia , Camundongos , Mitógenos/farmacologia , Miócitos de Músculo Liso/metabolismo , Ácidos Fosfatídicos/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
Exaggerated airway smooth muscle (ASM) contraction regulated by the Gq family of G protein-coupled receptors causes airway hyperresponsiveness in asthma. Activation of Gq-coupled G protein-coupled receptors leads to phospholipase C (PLC)-mediated generation of inositol triphosphate (IP3) and diacylglycerol (DAG). DAG signaling is terminated by the action of DAG kinase (DGK) that converts DAG into phosphatidic acid (PA). Our previous study demonstrated that DGKζ and α isoform knockout mice are protected from the development of allergen-induced airway hyperresponsiveness. Here we aimed to determine the mechanism by which DGK regulates ASM contraction. Activity of DGK isoforms was inhibited in human ASM cells by siRNA-mediated knockdown of DGKα and ζ, whereas pharmacological inhibition was achieved by pan DGK inhibitor I (R59022). Effects of DGK inhibition on contractile agonist-induced activation of PLC and myosin light chain (MLC) kinase, elevation of IP3, and calcium levels were assessed. Furthermore, we used precision-cut human lung slices and assessed the role of DGK in agonist-induced bronchoconstriction. DGK inhibitor I attenuated histamine- and methacholine-induced bronchoconstriction. DGKα and ζ knockdown or pretreatment with DGK inhibitor I resulted in attenuated agonist-induced phosphorylation of MLC and MLC phosphatase in ASM cells. Furthermore, DGK inhibition decreased Gq agonist-induced calcium elevation and generation of IP3 and increased histamine-induced production of PA. Finally, DGK inhibition or treatment with DAG analog resulted in attenuation of activation of PLC in human ASM cells. Our findings suggest that DGK inhibition perturbed the DAG:PA ratio, resulting in inhibition of Gq-PLC activation in a negative feedback manner, resulting in protection against ASM contraction.
Assuntos
Broncoconstrição/efeitos dos fármacos , Diacilglicerol Quinase/antagonistas & inibidores , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Liso/enzimologia , Pirimidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Broncoconstrição/genética , Células Cultivadas , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Técnicas de Silenciamento de Genes , Humanos , Contração Muscular/genética , Transdução de Sinais/genéticaRESUMO
Phosphorylation of specific residues in the activation loops of AGC kinase group (protein kinase A, G, and C families) is required for activity of most of these kinases, including the catalytic subunit of PKA (PKAc). Although many phosphorylated AGC kinases are sensitive to phosphatase-mediated dephosphorylation, the PKAc activation loop uniquely resists dephosphorylation, rendering it "constitutively" phosphorylated in cells. Previous biophysical experiments and structural modeling have suggested that the N-terminal myristoylation signal and the C-terminal FXXF motif in PKAc regulate its thermal stability and catalysis. Here, using site-directed mutagenesis, molecular modeling, and in cell-free and cell-based systems, we demonstrate that substitutions of either the PKAc myristoylation signal or the FXXF motif only modestly reduce phosphorylation and fail to affect PKAc function in cells. However, we observed that these two sites cooperate with an N-terminal FXXW motif to cooperatively establish phosphatase resistance of PKAc while not affecting kinase-dependent phosphorylation of the activation loop. We noted that this tripartite cooperative mechanism of phosphatase resistance is functionally relevant, as demonstrated by changes in morphology, adhesion, and migration of human airway smooth muscle cells transfected with PKAc variants containing amino acid substitutions in these three sites. These findings establish that three allosteric sites located at the PKAc N and C termini coordinately regulate the phosphatase sensitivity of this enzyme. This cooperative mechanism of phosphatase resistance of AGC kinase opens new perspectives toward therapeutic manipulation of kinase signaling in disease.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Adesão Celular , Linhagem Celular , Movimento Celular , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citosol/metabolismo , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Transdução de SinaisRESUMO
The proton-sensing receptor, ovarian cancer G protein-coupled receptor (OGR1), has been shown to be expressed in airway smooth muscle (ASM) cells and is capable of promoting ASM contraction in response to decreased extracellular pH. OGR1 knockout (OGR1KO) mice are reported to be resistant to the asthma features induced by inhaled allergen. We recently described certain benzodiazepines as OGR1 activators capable of mediating both procontractile and prorelaxant signaling in ASM cells. Here we assess the effect of treatment with the benzodiazepines lorazepam or sulazepam on the asthma phenotype in wild-type (WT) and OGR1KO mice subjected to inhaled house dust mite (HDM; Dermatophagoides pteronyssius) challenge for 3 wk. In contrast to previously published reports, both WT and OGR1KO mice developed significant allergen-induced lung inflammation and airway hyperresponsiveness (AHR). In WT mice, treatment with sulazepam (a Gs-biased OGR1 agonist), but not lorazepam (a balanced OGR1 agonist), prevented allergen-induced AHR, although neither drug inhibited lung inflammation. The protection from development of AHR conferred by sulazepam was absent in OGR1KO mice. Treatment of WT mice with sulazepam also resulted in significant inhibition of HDM-induced collagen accumulation in the lung tissue. These findings suggest that OGR1 expression is not a requirement for development of the allergen-induced asthma phenotype, but OGR1 can be targeted by the Gs-biased OGR1 agonist sulazepam (but not the balanced agonist lorazepam) to protect from allergen-induced AHR, possibly mediated via suppression of chronic bronchoconstriction and airway remodeling in the absence of effects on airway inflammation.
Assuntos
Alérgenos/toxicidade , Asma/patologia , Hiper-Reatividade Brônquica/patologia , Broncoconstrição , Citocinas/metabolismo , Pneumonia/patologia , Receptores Acoplados a Proteínas G/fisiologia , Animais , Ansiolíticos/farmacologia , Asma/etiologia , Asma/metabolismo , Benzodiazepinas/farmacologia , Hiper-Reatividade Brônquica/etiologia , Hiper-Reatividade Brônquica/metabolismo , Feminino , Lorazepam/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Pneumonia/etiologia , Pneumonia/metabolismo , PyroglyphidaeRESUMO
Bladder dysfunction is associated with the overexpression of the intermediate filament (IF) proteins desmin and vimentin in obstructed bladder smooth muscle (BSM). However, the mechanisms by which these proteins contribute to BSM dysfunction are not known. Previous studies have shown that desmin and vimentin directly participate in signal transduction. In this study, we hypothesized that BSM dysfunction associated with overexpression of desmin or vimentin is mediated via c-Jun N-terminal kinase (JNK). We employed a model of murine BSM tissue in which increased expression of desmin or vimentin was induced by adenoviral transduction to examine the sufficiency of increased IF protein expression to reduce BSM contraction. Murine BSM strips overexpressing desmin or vimentin generated less force in response to KCl and carbachol relative to the levels in control murine BSM strips, an effect associated with increased JNK2 phosphorylation and reduced myosin light chain (MLC20 ) phosphorylation. Furthermore, desmin and vimentin overexpressions did not alter BSM contractility and MLC20 phosphorylation in strips isolated from JNK2 knockout mice. Pharmacological JNK2 inhibition produced results qualitatively similar to those caused by JNK2 knockout. These findings suggest that inhibition of JNK2 may improve diminished BSM contractility associated with obstructive bladder disease.
Assuntos
Desmina/biossíntese , Sistema de Sinalização das MAP Quinases , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Contração Muscular , Músculo Liso/metabolismo , Bexiga Urinária/metabolismo , Vimentina/biossíntese , Animais , Desmina/genética , Camundongos , Camundongos Knockout , Proteína Quinase 9 Ativada por Mitógeno/genética , Músculo Liso/citologia , Bexiga Urinária/citologia , Vimentina/genéticaRESUMO
At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.
Assuntos
Álcoois Benzílicos/química , Álcoois Benzílicos/farmacologia , Descoberta de Drogas , Lorazepam/química , Lorazepam/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Triazinas/química , Triazinas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico , Animais , Ansiolíticos/análise , Ansiolíticos/química , Ansiolíticos/metabolismo , Ansiolíticos/farmacologia , Álcoois Benzílicos/análise , Álcoois Benzílicos/metabolismo , Condicionamento Clássico , Medo , Feminino , Células HEK293 , Humanos , Ligantes , Lorazepam/análise , Lorazepam/metabolismo , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Knockout , Modelos Moleculares , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/deficiência , Transdução de Sinais/efeitos dos fármacos , Triazinas/análise , Triazinas/metabolismoRESUMO
Historically, the drugs used to manage obstructive lung diseases (OLDs), asthma, and chronic obstructive pulmonary disease (COPD) either (1) directly regulate airway contraction by blocking or relaxing airway smooth muscle (ASM) contraction or (2) indirectly regulate ASM contraction by inhibiting the principal cause of ASM contraction/bronchoconstriction and airway inflammation. To date, these tasks have been respectively assigned to two diverse drug types: agonists/antagonists of G protein-coupled receptors (GPCRs) and inhaled or systemic steroids. These two types of drugs "stay in their lane" with respect to their actions and consequently require the addition of the other drug to effectively manage both inflammation and bronchoconstriction in OLDs. Indeed, it has been speculated that safety issues historically associated with beta-agonist use (beta-agonists activate the beta-2-adrenoceptor (ß2AR) on airway smooth muscle (ASM) to provide bronchoprotection/bronchorelaxation) are a function of pro-inflammatory actions of ß2AR agonism. Recently, however, previously unappreciated roles of various GPCRs on ASM contractility and on airway inflammation have been elucidated, raising the possibility that novel GPCR ligands targeting these GPCRs can be developed as anti-inflammatory therapeutics. Moreover, we now know that many GPCRs can be "tuned" and not just turned "off" or "on" to specifically activate the beneficial therapeutic signaling a receptor can transduce while avoiding detrimental signaling. Thus, the fledging field of biased agonism pharmacology has the potential to turn the ß2AR into an anti-inflammatory facilitator in asthma, possibly reducing or eliminating the need for steroids.