Agonist-induced conformational changes in the G-protein-coupling domain of the β2 adrenergic receptor

PubMed Central

Ghanouni, Pejman; Steenhuis, Jacqueline J.; Farrens, David L.; Kobilka, Brian K.

2001-01-01

The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the β2 adrenergic receptor (β2AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-β2AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and/or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the β2AR, which may reflect the different energetics of activation by diffusible ligands. PMID:11353823

Forskolin-free cAMP assay for Gi-coupled receptors.

PubMed

Gilissen, Julie; Geubelle, Pierre; Dupuis, Nadine; Laschet, Céline; Pirotte, Bernard; Hanson, Julien

2015-12-01

G protein-coupled receptors (GPCRs) represent the most successful receptor family for treating human diseases. Many are poorly characterized with few ligands reported or remain completely orphans. Therefore, there is a growing need for screening-compatible and sensitive assays. Measurement of intracellular cyclic AMP (cAMP) levels is a validated strategy for measuring GPCRs activation. However, agonist ligands for Gi-coupled receptors are difficult to track because inducers such as forskolin (FSK) must be used and are sources of variations and errors. We developed a method based on the GloSensor system, a kinetic assay that consists in a luciferase fused with cAMP binding domain. As a proof of concept, we selected the succinate receptor 1 (SUCNR1 or GPR91) which could be an attractive drug target. It has never been validated as such because very few ligands have been described. Following analyses of SUCNR1 signaling pathways, we show that the GloSensor system allows real time, FSK-free detection of an agonist effect. This FSK-free agonist signal was confirmed on other Gi-coupled receptors such as CXCR4. In a test screening on SUCNR1, we compared the results obtained with a FSK vs FSK-free protocol and were able to identify agonists with both methods but with fewer false positives when measuring the basal levels. In this report, we validate a cAMP-inducer free method for the detection of Gi-coupled receptors agonists compatible with high-throughput screening. This method will facilitate the study and screening of Gi-coupled receptors for active ligands. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of the Role of G Protein-Coupled Receptor Kinase 3 in Desensitization of Mouse Odorant Receptors in a Mammalian Cell Line and in Olfactory Sensory Neurons

PubMed Central

Kato, Aya; Reisert, Johannes; Ihara, Sayoko; Yoshikawa, Keiichi

2014-01-01

Thousands of odors are sensed and discriminated by G protein-coupled odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs). G protein-coupled receptor kinases (GRKs) may have a role in desensitization of ORs. However, whether ORs are susceptible to agonist-dependent desensitization and whether GRKs affect odorant responsiveness of OSNs are currently unknown. Here we show that GRK3 attenuated the agonist responsiveness of a specific mouse odorant receptor for eugenol (mOR-EG) upon agonist pretreatment in HEK293 cells, but GRK3 did not affect the response amplitude or the recovery kinetics upon repeated agonist stimulation. We performed electrophysiological recordings of single OSNs which expressed mOR-EG and green fluorescent protein (GFP) in the presence or absence of GRK3. The kinetics and amplitude of agonist responsiveness of individual GFP-labeled mOR-EG neurons were not significantly affected by the absence of GRK3. These results indicate that the role of GRK3 in attenuating ORs responsiveness in OSNs may have been overestimated. PMID:25313015

Aromatic interactions impact ligand binding and function at serotonin 5-HT2C G protein-coupled receptors: receptor homology modelling, ligand docking, and molecular dynamics results validated by experimental studies

NASA Astrophysics Data System (ADS)

Córdova-Sintjago, Tania; Villa, Nancy; Fang, Lijuan; Booth, Raymond G.

2014-02-01

The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ∼75% transmembrane (TM) sequence identity. Agonists for 5-HT2C receptors are under development for psychoses; whereas, at 5-HT2A receptors, antipsychotic effects are associated with antagonists - in fact, 5-HT2A agonists can cause hallucinations and 5-HT2B agonists cause cardiotoxicity. It is known that 5-HT2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function; however, ligand interactions with these residues at the 5-HT2C receptor have not been reported. To predict and validate molecular determinants for 5-HT2C-specific activation, results from receptor homology modelling, ligand docking, and molecular dynamics simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT2C receptors.

Synthesis and pharmacological profile of a new selective G protein-coupled receptor 119 agonist; 6-((2-fluoro-3-(1-(3-isopropyl-1,2,4-oxadiazol-5-yl)piperidin-4-yl)propyl)amino)-2,3-dihydro-1H-inden-1-one.

PubMed

Sakairi, Masao; Kogami, Masakazu; Torii, Masafumi; Makino, Mitsuhiro; Kataoka, Daisuke; Okamoto, Ryuji; Miyazawa, Toshiyuki; Inoue, Megumi; Takahashi, Naoki; Harada, Satoko; Watanabe, Nobuhide

2012-01-01

6-((2-Fluoro-3-(1-(3-isopropyl-1,2,4-oxadiazol-5-yl)piperidin-4-yl)propyl)amino)-2,3-dihydro-1H-inden-1-one is a potent drug-like G protein-coupled receptor 119 (GPR119) agonist. It is hoped that this compound would be instrumental in probing the pharmacological potential of GPR119 agonists.

Heterologous desensitization of cardiac β-adrenergic signal via hormone-induced βAR/arrestin/PDE4 complexes

PubMed Central

Shi, Qian; Li, Minghui; Mika, Delphine; Fu, Qin; Kim, Sungjin; Phan, Jason; Shen, Ao; Vandecasteele, Gregoire; Xiang, Yang K.

2017-01-01

Aims Cardiac β-adrenergic receptor (βAR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between βARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists. Methods and results Rat ventricular cardiomyocytes were used to determine heterologous phosphorylation of βARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled α1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of β1AR and β2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled α2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote β2AR phosphorylation effectively inhibit βAR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced β2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated β2AR in a β-arrestin 2 dependent manner without promoting β2AR endocytosis. The increased binding between β2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in β2AR, inhibition of PDE4, or genetic ablation of PDE4D or β-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of β-arrestin 2 or PDE4D gene also rescues β-adrenergic stimuli-induced myocyte contractile function. Conclusions These data reveal essential roles of β-arrestin 2 and PDE4D in a common mechanism for heterologous desensitization of cardiac βARs under hormonal stimulation, which is associated with impaired cardiac function during the development of pathophysiological conditions. PMID:28339772

Heterologous desensitization of cardiac β-adrenergic signal via hormone-induced βAR/arrestin/PDE4 complexes.

PubMed

Shi, Qian; Li, Minghui; Mika, Delphine; Fu, Qin; Kim, Sungjin; Phan, Jason; Shen, Ao; Vandecasteele, Gregoire; Xiang, Yang K

2017-05-01

Cardiac β-adrenergic receptor (βAR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between βARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists. Rat ventricular cardiomyocytes were used to determine heterologous phosphorylation of βARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled α1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of β1AR and β2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled α2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote β2AR phosphorylation effectively inhibit βAR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced β2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated β2AR in a β-arrestin 2 dependent manner without promoting β2AR endocytosis. The increased binding between β2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in β2AR, inhibition of PDE4, or genetic ablation of PDE4D or β-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of β-arrestin 2 or PDE4D gene also rescues β-adrenergic stimuli-induced myocyte contractile function. These data reveal essential roles of β-arrestin 2 and PDE4D in a common mechanism for heterologous desensitization of cardiac βARs under hormonal stimulation, which is associated with impaired cardiac function during the development of pathophysiological conditions. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions please email: journals.permissions@oup.com.

β2-Adrenergic Receptor Agonists Inhibit the Proliferation of 1321N1 Astrocytoma CellsS⃞

PubMed Central

Toll, L.; Jimenez, L.; Waleh, N.; Jozwiak, K.; Woo, A.Y.-H.; Xiao, R.-P.; Bernier, M.

2011-01-01

Astrocytomas and glioblastomas have been particularly difficult to treat and refractory to chemotherapy. However, significant evidence has been presented that demonstrates a decrease in astrocytoma cell proliferation subsequent to an increase in cAMP levels. The 1321N1 astrocytoma cell line, as well as other astrocytomas and glioblastomas, expresses β2-adrenergic receptors (β2-ARs) that are coupled to Gs activation and consequent cAMP production. Experiments were conducted to determine whether the β2-AR agonist (R,R′)-fenoterol and other β2-AR agonists could attenuate mitogenesis and, if so, by what mechanism. Receptor binding studies were conducted to characterize β2-AR found in 1321N1 and U118 cell membranes. In addition, cells were incubated with (R,R′)-fenoterol and analogs to determine their ability to stimulate intracellular cAMP accumulation and inhibit [3H]thymidine incorporation into the cells. 1321N1 cells contain significant levels of β2-AR as determined by receptor binding. (R,R′)-fenoterol and other β2-AR agonists, as well as forskolin, stimulated cAMP accumulation in a dose-dependent manner. Accumulation of cAMP induced a decrease in [3H]thymidine incorporation. There was a correlation between concentration required to stimulate cAMP accumulation and inhibit [3H]thymidine incorporation. U118 cells have a reduced number of β2-ARs and a concomitant reduction in the ability of β2-AR agonists to inhibit cell proliferation. These studies demonstrate the efficacy of β2-AR agonists for inhibition of growth of the astrocytoma cell lines. Because a significant portion of brain tumors contain β2-ARs to a greater extent than whole brain, (R,R′)-fenoterol, or some analog, may be useful in the treatment of brain tumors after biopsy to determine β2-AR expression. PMID:21071556

Molecular interactions of agonist and inverse agonist ligands at serotonin 5-HT2C G protein-coupled receptors: computational ligand docking and molecular dynamics studies validated by experimental mutagenesis results

NASA Astrophysics Data System (ADS)

Córdova-Sintjago, Tania C.; Liu, Yue; Booth, Raymond G.

2015-02-01

To understand molecular determinants for ligand activation of the serotonin 5-HT2C G protein-coupled receptor (GPCR), a drug target for obesity and neuropsychiatric disorders, a 5-HT2C homology model was built according to an adrenergic β2 GPCR (β2AR) structure and validated using a 5-HT2B GPCR crystal structure. The models were equilibrated in a simulated phosphatidyl choline membrane for ligand docking and molecular dynamics studies. Ligands included (2S, 4R)-(-)-trans-4-(3'-bromo- and trifluoro-phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalene-2-amine (3'-Br-PAT and 3'-CF3-PAT), a 5-HT2C agonist and inverse agonist, respectively. Distinct interactions of 3'-Br-PAT and 3'-CF3-PAT at the wild-type (WT) 5-HT2C receptor model were observed and experimental 5-HT2C receptor mutagenesis studies were undertaken to validate the modelling results. For example, the inverse agonist 3'-CF3-PAT docked deeper in the WT 5-HT2C binding pocket and altered the orientation of transmembrane helices (TM) 6 in comparison to the agonist 3'-Br-PAT, suggesting that changes in TM orientation that result from ligand binding impact function. For both PATs, mutation of 5-HT2C residues S3.36, T3.37, and F5.47 to alanine resulted in significantly decreased affinity, as predicted from modelling results. It was concluded that upon PAT binding, 5-HT2C residues T3.37 and F5.47 in TMs 3 and 5, respectively, engage in inter-helical interactions with TMs 4 and 6, respectively. The movement of TMs 5 and 6 upon agonist and inverse agonist ligand binding observed in the 5-HT2C receptor modelling studies was similar to movements reported for the activation and deactivation of the β2AR, suggesting common mechanisms among aminergic neurotransmitter GPCRs.

Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor.

PubMed

Weiss, Dahlia R; Ahn, SeungKirl; Sassano, Maria F; Kleist, Andrew; Zhu, Xiao; Strachan, Ryan; Roth, Bryan L; Lefkowitz, Robert J; Shoichet, Brian K

2013-05-17

A prospective, large library virtual screen against an activated β2-adrenergic receptor (β2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated β2AR structure returned few hits of only marginal potency.

Probing receptor structure/function with chimeric G-protein-coupled receptors.

PubMed

Yin, Dezhong; Gavi, Shai; Wang, Hsien-yu; Malbon, Craig C

2004-06-01

Owing its name to an image borrowed from Greek mythology, a chimera is seen to represent a new entity created as a composite from existing creatures or, in this case, molecules. Making use of various combinations of three basic domains of the receptors (i.e., exofacial, transmembrane, and cytoplasmic segments) that couple agonist binding into activation of effectors through heterotrimeric G-proteins, molecular pharmacology has probed the basic organization, structure/function relationships of this superfamily of heptahelical receptors. Chimeric G-protein-coupled receptors obviate the need for a particular agonist ligand when the ligand is resistant to purification or, in the case of orphan receptors, is not known. Chimeric receptors created from distant members of the heptahelical receptors enable new strategies in understanding how these receptors transduce agonist binding into receptor activation and may be able to offer insights into the evolution of G-protein-coupled receptors from yeast to humans.

Effect of adrenaline and alpha-agonists on net rate of liquid absorption from the pleural space of rabbits.

PubMed

Zocchi, L; Raffaini, A; Agostoni, E

1997-05-01

Indirect evidence supporting a solute-coupled liquid absorption from the pleural space of rabbits has recently been provided; moreover, the beta 2-adrenoceptor agonist terbutaline has been found to increase this absorption. In this study the effect of adrenaline and alpha-adrenoceptor agonists on net rate of liquid absorption (Jnet) from albumin Ringer hydrothoraces of various sizes has been determined in anaesthetized rabbits. In hydrothoraces with adrenaline (5 x 10(-6) M) the relationship between Jnet and volume of liquid injected was displaced upwards by 0.09 ml h-1 relative to that in control hydrothoraces (P < 0.01). This displacement did not occur with lower adrenaline concentrations or after pretreatment with the beta-blocker propranolol. Hence, this increase in Jnet is mediated by stimulation of beta-receptors. It seems to be caused by an increase in solute-coupled liquid absorption, since beta-agonists inhibit lymphatic activity while, at relatively high concentrations, they may increase active transport. Conversely, the strong stimulation of lymphatic alpha-receptors that should occur with adrenaline after beta-blockade may fail to increase lymphatic drainage, because it has been shown that the increase in contraction frequency of lymphatics may be balanced by the decrease in their stroke volume. Arterial blood pressure during the hydrothoraces with adrenaline was unchanged. In hydrothoraces with the alpha 2-agonist clonidine (5 x 10(-6) M; a less potent agent than adrenaline) the slope of the relationship between Jnet and volume injected increased by 26% (P < 0.01), while its origin did not change. This increase in slope did not occur with a lower clonidine concentration or after pretreatment with the alpha-blocker phentolamine. Hence, it is caused by stimulation of alpha 2-receptors, which probably lead to an increase in lymphatic drainage related to liquid load. In hydrothoraces with the alpha 1-agonist phenylephrine (5 x 10(-6) or 10(-7) M) Jnet was simlar to control values.

Agonist-induced internalization of the platelet-activating factor receptor is dependent on arrestins but independent of G-protein activation. Role of the C terminus and the (D/N)PXXY motif.

PubMed

Chen, Zhangguo; Dupré, Denis J; Le Gouill, Christian; Rola-Pleszczynski, Marek; Stanková, Jana

2002-03-01

As with most G-protein-coupled receptors, repeated agonist stimulation of the platelet-activating factor receptor (PAFR) results in its desensitization, sequestration, and internalization. In this report, we show that agonist-induced PAFR internalization is independent of G-protein activation but is dependent on arrestins and involves the interaction of arrestins with a limited region of the PAFR C terminus. In cotransfected COS-7 cells, both arrestin-2 and arrestin-3 could be coimmunoprecipitated with PAFR, and agonist stimulation of PAFR induced the translocation of both arrestin-2 and arrestin-3. Furthermore, coexpression of arrestin-2 with PAFR potentiated receptor internalization, whereas agonist-induced PAFR internalization was inhibited by a dominant negative mutant of arrestin-2. The coexpression of a minigene encoding the C-terminal segment of the receptor abolished PAF-induced arrestin translocation and inhibited PAFR internalization. Using C terminus deletion mutants, we determined that the association of arrestin-2 with the receptor was dependent on the region between threonine 305 and valine 330 because arrestin-2 could be immunoprecipitated with the mutant PAFRstop330 but not PAFRstop305. Consistently, stop330 could mediate agonist-induced arrestin-2 translocation, whereas stop305 could not. Two other deletion mutants with slightly longer regions of the C terminus, PAFRstop311 and PAFRstop317, also failed to induce arrestin-2 translocation. Finally, the PAFR mutant Y293A, containing a single substitution in the putative internalization motif DPXXY in the seventh transmembrane domain (which we had shown to be able to internalize but not to couple to G-proteins) could efficiently induce arrestin translocation. Taken together, our results indicate that ligand-induced PAFR internalization is dependent on arrestins, that PAFR can associate with both arrestin-2 and -3, and that their translocation involves interaction with the region of residues 318-330 in the PAFR C terminus but is independent of G-protein activation.

Effects of hand configuration on muscle force coordination, co-contraction and concomitant intermuscular coupling during maximal isometric flexion of the fingers.

PubMed

Charissou, Camille; Amarantini, David; Baurès, Robin; Berton, Eric; Vigouroux, Laurent

2017-11-01

The mechanisms governing the control of musculoskeletal redundancy remain to be fully understood. The hand is highly redundant, and shows different functional role of extensors according to its configuration for a same functional task of finger flexion. Through intermuscular coherence analysis combined with hand musculoskeletal modelling during maximal isometric hand contractions, our aim was to better understand the neural mechanisms underlying the control of muscle force coordination and agonist-antagonist co-contraction. Thirteen participants performed maximal isometric flexions of the fingers in two configurations: power grip (Power) and finger-pressing on a surface (Press). Hand kinematics and force/moment measurements were used as inputs in a musculoskeletal model of the hand to determine muscular tensions and co-contraction. EMG-EMG coherence analysis was performed between wrist and finger flexors and extensor muscle pairs in alpha, beta and gamma frequency bands. Concomitantly with tailored muscle force coordination and increased co-contraction between Press and Power (mean difference: 48.08%; p < 0.05), our results showed muscle-pair-specific modulation of intermuscular coupling, characterized by pair-specific modulation of EMG-EMG coherence between Power and Press (p < 0.05), and a negative linear association between co-contraction and intermuscular coupling for the ECR/FCR agonist-antagonist muscle pair (r = - 0.65; p < 0.05). This study brings new evidence that pair-specific modulation of EMG-EMG coherence is related to modulation of muscle force coordination during hand contractions. Our results highlight the functional importance of intermuscular coupling as a mechanism contributing to the control of muscle force synergies and agonist-antagonist co-contraction.

Reverse Induced Fit-Driven MAS-Downstream Transduction: Looking for Metabotropic Agonists.

PubMed

Pernomian, Larissa; Gomes, Mayara S; de Paula da Silva, Carlos H Tomich; Rosa, Joaquin M C

2017-01-01

Protective effects of MAS activation have spurred clinical interests in developing MAS agonists. However, current bases that drive this process preclude that physiological concentrations of peptide MAS agonists induce an atypical signaling that does not reach the metabotropic efficacy of constitutive activation. Canonical activation of MAS-coupled G proteins is only achieved by supraphysiological concentrations of peptide MAS agonists or physiological concentrations of chemically modified analogues. These pleiotropic differences are because of two overlapped binding domains: one non-metabotropic site that recognizes peptide agonists and one metabotropic domain that recognizes modified analogues. It is feasible that supraphysiological concentrations of peptide MAS agonists undergo to chemical modifications required for binding to metabotropic domain. Receptor oligomerization enhances pharmacological parameters coupled to metabotropic signaling. The formation of receptor-signalosome complex makes the transduction of agonists more adaptive. Considering the recent identification of MAS-signalosome, we aimed to postulate the reverse induced fit hypothesis in which MAS-signalosome would trigger chemical modifications required for agonists bind to MAS metabotropic domain. Here we cover rational perspectives for developing novel metabotropic MAS agonists in the view of the reverse induced-fit hypothesis. Predicting a 3D model of MAS metabotropic domain may guide the screening of chemical modifications required for metabotropic efficacy. Pharmacophore-based virtual screening would select potential metabotropic MAS agonists from virtual libraries from human proteome. Rational perspectives that consider reverse induced fit hypothesis during MAS activation for developing metabotropic MAS agonists represents the best approach in providing MAS ligands with constitutive efficacy at physiological concentrations. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

G protein-coupled estrogen receptor 1 agonist G-1 induces cell cycle arrest in the mitotic phase, leading to apoptosis in endometriosis.

PubMed

Mori, Taisuke; Ito, Fumitake; Matsushima, Hiroshi; Takaoka, Osamu; Tanaka, Yukiko; Koshiba, Akemi; Kusuki, Izumi; Kitawaki, Jo

2015-05-01

To demonstrate the effects of the selective G protein-coupled estrogen receptor 1 (GPER) agonist G-1 in human ovarian endometriotic stromal cells (ESCs). Experimental in vitro study. University hospital. A total of 33 patients with ovarian endometrioma. Endometriotic stromal cells from ovarian chocolate cysts were treated with the GPER agonist G-1. The primary outcomes were cell proliferation, measured using the WST-8 assay; cell cycle, as analyzed using flow cytometry, fluorescent immunocytochemistry, and cytotoxicity; caspase activity, as measured by fluorescent and luminescent enzyme assays; and protein expression levels, as determined by Western blot analysis. G-1 suppressed ESC proliferation in a concentration-dependent manner. The inhibitory effect was not blocked when GPER signaling pathways, including the GPER itself, were inhibited. G-1 induced cell cycle arrest and accumulation in the sub-G1 phase in ESCs. Immunofluorescence analysis demonstrated that G-1 interrupted microtubule assembly at the mitotic phase. G-1 also induced caspase-3-dependent apoptosis without significant cytotoxicity. G-1 suppressed proliferation and induced apoptosis in ESCs, suggesting the potential use of this compound as a therapeutic drug for the treatment of endometriosis. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Activation of G protein-coupled estrogen receptor 1 (GPER-1) ameliorates blood-brain barrier permeability after global cerebral ischemia in ovariectomized rats.

PubMed

Lu, Dan; Qu, Yan; Shi, Fei; Feng, Dayun; Tao, Kai; Gao, Guodong; He, Shiming; Zhao, Tianzhi

2016-08-19

G protein-coupled estrogen receptor 1 (GPER-1) plays important roles in estrogen-mediated neuroprotection. However, protective effects of GPER-1 on blood-brain barrier (BBB) after ischemic stroke have not been determined. The aim of present study was to determine whether GPER-1 activation ameliorates BBB permeability in ovariectomized rats with induced global cerebral ischemia (GCI). GCI was induced by 4-vessel occlusion for 20 min followed by 24 h reperfusion period. The GPER-1 agonist (G1) was bilaterally administered immediately upon reperfusion by intracerebroventricular (icv) injection. We found that the GPER-1 agonist could significantly decrease immunoglobulin G (IgG) extravasation and increase the levels of tight junctions (occludin and claudin-5) in the CA1 at 24 h of reperfusion after GCI. Further, protein levels of vascular endothelial growth factor A (VEGF-A) was significantly decreased in the ischemic CA1 by G1. Our results suggest that GPER-1 activation reduce tight junctions disruption via inhibition of VEGF-A expression after ischemic injury. Copyright © 2016 Elsevier Inc. All rights reserved.

Distinct loops in arrestin differentially regulate ligand binding within the GPCR opsin

PubMed Central

Sommer, Martha E.; Hofmann, Klaus Peter; Heck, Martin

2012-01-01

G-protein-coupled receptors are universally regulated by arrestin binding. Here we show that rod arrestin induces uptake of the agonist all-trans-retinol in only half the population of phosphorylated opsin in the native membrane. Agonist uptake blocks subsequent entry of the inverse agonist 11-cis-retinal (that is, regeneration of rhodopsin), but regeneration is not blocked in the other half of aporeceptors. Environmentally sensitive fluorophores attached to arrestin reported that conformational changes in loopV−VI (N-domain) are coupled to the entry of agonist, while loopXVIII−XIX (C-domain) engages the aporeceptor even before agonist is added. The data are most consistent with a model in which each domain of arrestin engages its own aporeceptor, and the different binding preferences of the domains lead to asymmetric ligand binding by the aporeceptors. Such a mechanism would protect the rod cell in bright light by concurrently sequestering toxic all-trans-retinol and allowing regeneration with 11-cis-retinal. PMID:22871814

Distinct loops in arrestin differentially regulate ligand binding within the GPCR opsin.

PubMed

Sommer, Martha E; Hofmann, Klaus Peter; Heck, Martin

2012-01-01

G-protein-coupled receptors are universally regulated by arrestin binding. Here we show that rod arrestin induces uptake of the agonist all-trans-retinal [corrected] in only half the population of phosphorylated opsin in the native membrane. Agonist uptake blocks subsequent entry of the inverse agonist 11-cis-retinal (that is, regeneration of rhodopsin), but regeneration is not blocked in the other half of aporeceptors. Environmentally sensitive fluorophores attached to arrestin reported that conformational changes in loop(V-VI) (N-domain) are coupled to the entry of agonist, while loop(XVIII-XIX) (C-domain) engages the aporeceptor even before agonist is added. The data are most consistent with a model in which each domain of arrestin engages its own aporeceptor, and the different binding preferences of the domains lead to asymmetric ligand binding by the aporeceptors. Such a mechanism would protect the rod cell in bright light by concurrently sequestering toxic all-trans-retinal [corrected] and allowing regeneration with 11-cis-retinal.

The human bitter taste receptor TAS2R10 is tailored to accommodate numerous diverse ligands.

PubMed

Born, Stephan; Levit, Anat; Niv, Masha Y; Meyerhof, Wolfgang; Behrens, Maik

2013-01-02

Bitter taste is a basic taste modality, required to safeguard animals against consuming toxic substances. Bitter compounds are recognized by G-protein-coupled bitter taste receptors (TAS2Rs). The human TAS2R10 responds to the toxic strychnine and numerous other compounds. The mechanism underlying the development of the broad tuning of some TAS2Rs is not understood. Using comparative modeling, site-directed mutagenesis, and functional assays, we identified residues involved in agonist-induced activation of TAS2R10, and investigated the effects of different substitutions on the receptor's response profile. Most interestingly, mutations in S85(3.29) and Q175(5.40) have differential impact on stimulation with different agonists. The fact that single point mutations lead to improved responses for some agonists and to decreased activation by others indicates that the binding site has evolved to optimally accommodate multiple agonists at the expense of reduced potency. TAS2R10 shares the agonist strychnine with TAS2R46, another broadly tuned receptor. Engineering the key determinants for TAS2R46 activation by strychnine in TAS2R10 caused a loss of response to strychnine, indicating that these paralog receptors display different strychnine-binding modes, which suggests independent acquisition of agonist specificities. This implies that the gene duplication event preceding primate speciation was accompanied by independent evolution of the strychnine-binding sites.

Dynamical Binding Modes Determine Agonistic and Antagonistic Ligand Effects in the Prostate-Specific G-Protein Coupled Receptor (PSGR).

PubMed

Wolf, Steffen; Jovancevic, Nikolina; Gelis, Lian; Pietsch, Sebastian; Hatt, Hanns; Gerwert, Klaus

2017-11-22

We analysed the ligand-based activation mechanism of the prostate-specific G-protein coupled receptor (PSGR), which is an olfactory receptor that mediates cellular growth in prostate cancer cells. Furthermore, it is an olfactory receptor with a known chemically near identic antagonist/agonist pair, α- and β-ionone. Using a combined theoretical and experimental approach, we propose that this receptor is activated by a ligand-induced rearrangement of a protein-internal hydrogen bond network. Surprisingly, this rearrangement is not induced by interaction of the ligand with the network, but by dynamic van der Waals contacts of the ligand with the involved amino acid side chains, altering their conformations and intraprotein connectivity. Ligand recognition in this GPCR is therefore highly stereo selective, but seemingly lacks any ligand recognition via polar contacts. A putative olfactory receptor-based drug design scheme will have to take this unique mode of protein/ligand action into account.

Unravelling intrinsic efficacy and ligand bias at G protein coupled receptors: A practical guide to assessing functional data.

PubMed

Stott, Lisa A; Hall, David A; Holliday, Nicholas D

2016-02-01

Stephenson's empirical definition of an agonist, as a ligand with binding affinity and intrinsic efficacy (the ability to activate the receptor once bound), underpins classical receptor pharmacology. Quantifying intrinsic efficacy using functional concentration response relationships has always presented an experimental challenge. The requirement for realistic determination of efficacy is emphasised by recent developments in our understanding of G protein coupled receptor (GPCR) agonists, with recognition that some ligands stabilise different active conformations of the receptor, leading to pathway-selective, or biased agonism. Biased ligands have potential as therapeutics with improved selectivity and clinical efficacy, but there are also pitfalls to the identification of pathway selective effects. Here we explore the basics of concentration response curve analysis, beginning with the need to distinguish ligand bias from other influences of the functional system under study. We consider the different approaches that have been used to quantify and compare biased ligands, many of which are based on the Black and Leff operational model of agonism. Some of the practical issues that accompany these analyses are highlighted, with opportunities to improve estimates in future, particularly in the separation of true agonist intrinsic efficacy from the contributions of system dependent coupling efficiency. Such methods are by their nature practical approaches, and all rely on Stephenson's separation of affinity and efficacy parameters, which are interdependent at the mechanistic level. Nevertheless, operational analysis methods can be justified by mechanistic models of GPCR activation, and if used wisely are key elements to biased ligand identification. Copyright © 2015 Elsevier Inc. All rights reserved.

Design, Synthesis, and Evaluation of N- and C-Terminal Protein Bioconjugates as G Protein-Coupled Receptor Agonists.

PubMed

Healey, Robert D; Wojciechowski, Jonathan P; Monserrat-Martinez, Ana; Tan, Susan L; Marquis, Christopher P; Sierecki, Emma; Gambin, Yann; Finch, Angela M; Thordarson, Pall

2018-02-21

A G protein-coupled receptor (GPCR) agonist protein, thaumatin, was site-specifically conjugated at the N- or C-terminus with a fluorophore for visualization of GPCR:agonist interactions. The N-terminus was specifically conjugated using a synthetic 2-pyridinecarboxyaldehyde reagent. The interaction profiles observed for N- and C-terminal conjugates were varied; N-terminal conjugates interacted very weakly with the GPCR of interest, whereas C-terminal conjugates bound to the receptor. These chemical biology tools allow interactions of therapeutic proteins:GPCR to be monitored and visualized. The methodology used for site-specific bioconjugation represents an advance in application of 2-pyridinecarboxyaldehydes for N-terminal specific bioconjugations.

Agonist-specific coupling of a cloned Drosophila octopamine/tyramine receptor to multiple second messenger systems.

PubMed Central

Robb, S; Cheek, T R; Hannan, F L; Hall, L M; Midgley, J M; Evans, P D

1994-01-01

A cloned seven transmembrane-spanning Drosophila octopamine/tyramine receptor, permanently expressed in a Chinese hamster ovary cell line, both inhibits adenylate cyclase activity and leads to the elevation of intracellular Ca2+ levels by separate G-protein-coupled pathways. Agonists of this receptor (octopamine and tyramine), differing by only a single hydroxyl group in their side chain, may be capable of differentially coupling it to different second messenger systems. Thus, a single receptor may have a different pharmacological profile depending on which second messenger system is used to assay its efficacy. PMID:8137817

Somatostatin receptor subtypes SSTR2 and SSTR5 couple negatively to an L-type Ca2+ current in the pituitary cell line AtT-20.

PubMed

Tallent, M; Liapakis, G; O'Carroll, A M; Lolait, S J; Dichter, M; Reisine, T

1996-04-01

The somatostatin receptor subtypes SSTR2 and SSTR5 mediate distinct endocrine and exocrine functions of somatostatin and may also be involved in mediating the neuromodulatory actions of somatostatin in the brain. To investigate whether these receptors couple to voltage-sensitive Ca2+ channels, SSTR2 and SSTR5 selective agonists were tested for their effects on AtT-20 cells using whole cell patch clamp techniques. The SSTR2 selective agonist MK 678 inhibited Ca2+ currents in AtT-20 cells. The effects of MK 678 were reversible and blocked by pertussis toxin pretreatment, suggesting that SSTR2 couples to the L-type Ca2+ channels via G proteins. Other SSTR2-selective agonists, including BIM 23027 and NC8-12, were able to inhibit the Ca2+ currents in these cells. The SSTR5 selective agonist BIM 23052 also inhibited the Ca2+ currents in these cells and this effect was reversible and blocked by pertussis toxin treatment. The ability of SSTR5 to mediate inhibition of the Ca2+ current was greatly attenuated by pretreatment with the SSTR5-selective agonist BIM 23052, whereas SSTR2-mediated inhibition of the Ca2+ current was not altered by pretreatment with the SSTR2-selective agonist MK 678. Thus, the SSTR2 and SSTR5 couplings to the Ca2+ current are differentially regulated. The peptide L362,855, which we previously have shown to have high affinity for the cloned SSTR5, had minimal effects on Ca2+ currents in AtT-20 cells at concentrations up to 100 nM and did not alter the ability of MK 678 to inhibit Ca2+ currents. However, it completely antagonized the effects of the SSTR5-selective agonist BIM 23052 on the Ca2+ currents. L362,855 is an antagonist/partial agonist at SSTR5 since it can reduce Ca2+ currents in these cells at concentrations above 100 nM. L362,855 is also an antagonist/partial agonist at the cloned rat SSTR5 expressed in CHO cells since it is able to block the inhibition of cAMP accumulation induced by somatostatin at concentrations below 100 nM but at higher concentrations can inhibit cAMP formation itself. Structural analysis of L362,855 reveals that only a single hydroxyl group at residue seven in the peptide is needed to convert the compound from an antagonist/partial agonist to a full agonist at SSTR5. These studies reveal that two different somatostatin receptor subtypes, SSTR2 and SSTR5, can mediate the inhibition of an L-type Ca2+ channel in AtT-20 cells by somatostatin. The receptor subtype responses can be distinguished by selective agonists and antagonists and are regulated differently by agonist pretreatment. The inhibition of Ca2+ influx into endocrine cells and neurons may be a major cellular mechanism by which somatostatin modulates hormone and neurotransmitter release. Our results reveal that at least two receptor subtypes can mediate this cellular response.

Molecular Signature That Determines the Acute Tolerance of G Protein-Coupled Receptors

PubMed Central

Min, Chengchun; Zhang, Xiaohan; Zheng, Mei; Sun, Ningning; Acharya, Srijan; Zhang, Xiaowei; Kim, Kyeong-Man

2017-01-01

Desensitization and acute tolerance are terms used to describe the attenuation of receptor responsiveness by prolonged or intermittent exposure to an agonist. Unlike desensitization of G protein-coupled receptors (GPCRs), which is commonly explained by steric hindrance caused by the β-arrestins that are translocated to the activated receptors, molecular mechanisms involved in the acute tolerance of GPCRs remain unclear. Our studies with several GPCRs and related mutants showed that the acute tolerance of GPCRs could occur independently of agonist-induced β-arrestin translocation. A series of co-immunoprecipitation experiments revealed a correlation between receptor tolerance and interactions among receptors, β-arrestin2, and Gβγ. Gβγ displayed a stable interaction with receptors and β-arrestin2 in cells expressing GPCRs that were prone to undergo tolerance compared to the GPCRs that were resistant to acute tolerance. Strengthening the interaction between Gβγ and β-arrestin rendered the GPCRs to acquire the tendency of acute tolerance. Overall, stable interaction between the receptor and Gβγ complex is required for the formation of a complex with β-arrestin, and determines the potential of a particular GPCR to undergo acute tolerance. Rather than turning off the signal, β-arrestins seem to contribute on continuous signaling when they are in the context of complex with receptor and Gβγ. PMID:27956717

Activation of G-protein-coupled receptor 30 is sufficient to enhance spatial recognition memory in ovariectomized rats.

PubMed

Hawley, Wayne R; Grissom, Elin M; Moody, Nicole M; Dohanich, Gary P; Vasudevan, Nandini

2014-04-01

In ovariectomized rats, administration of estradiol, or selective estrogen receptor agonists that activate either the α or β isoforms, have been shown to enhance spatial cognition on a variety of learning and memory tasks, including those that capitalize on the preference of rats to seek out novelty. Although the effects of the putative estrogen G-protein-coupled receptor 30 (GPR30) on hippocampus-based tasks have been reported using food-motivated tasks, the effects of activation of GPR30 receptors on tasks that depend on the preference of rats to seek out spatial novelty remain to be determined. Therefore, the aim of the current study was to determine if short-term treatment of ovariectomized rats with G-1, an agonist for GPR30, would mimic the effects on spatial recognition memory observed following short-term estradiol treatment. In Experiment 1, ovariectomized rats treated with a low dose (1 μg) of estradiol 48 h and 24 h prior to the information trial of a Y-maze task exhibited a preference for the arm associated with the novel environment on the retention trial conducted 48 h later. In Experiment 2, treatment of ovariectomized rats with G-1 (25 μg) 48 h and 24 h prior to the information trial of a Y-maze task resulted in a greater preference for the arm associated with the novel environment on the retention trial. Collectively, the results indicated that short-term treatment of ovariectomized rats with a GPR30 agonist was sufficient to enhance spatial recognition memory, an effect that also occurred following short-term treatment with a low dose of estradiol. Copyright © 2014 Elsevier B.V. All rights reserved.

Gene transfer of heterologous G protein-coupled receptors to cardiomyocytes: differential effects on contractility.

PubMed

Laugwitz, K L; Weig, H J; Moretti, A; Hoffmann, E; Ueblacker, P; Pragst, I; Rosport, K; Schömig, A; Ungerer, M

2001-04-13

In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to beta-adrenergic stimulation. Parathyroid hormone (PTH)-related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations. To bypass the defective beta-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V(2) vasopressin receptors (rV(2)-Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant beta(2)-adrenergic receptors (rbeta(2)-ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V(2)-Rs are uniquely coupled to Gs, PTH1-Rs and beta(2)-ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or rbeta(2)-ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV(2)-R-expressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of beta(2)-ARS: In the absence of receptor agonists, rPTH1-Rs and rbeta(2)-ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV(2)-Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V(2)-Rs might be more suited to test the effects of cAMP-stimulating receptors in heart failure than that of PTH1-Rs or beta(2)-ARS:

The pharmacology of Ro 64-6198, a systemically active, nonpeptide NOP receptor (opiate receptor-like 1, ORL-1) agonist with diverse preclinical therapeutic activity.

PubMed

Shoblock, James R

2007-01-01

The NOP receptor (formerly referred to as opiate receptor-like 1, ORL-1, LC132, OP(4), or NOP(1)) is a G protein-coupled receptor that shares high homology to the classic opioid MOP, DOP, and KOP (mu, delta, and kappa, respectively) receptors and was first cloned in 1994 by several groups. The NOP receptor remained an orphan receptor until 1995, when the endogenous neuropeptide agonist, known as nociceptin or orphanin FQ (N/OFQ) was isolated. Five years later, a group at Hoffmann-La Roche reported on the selective, nonpeptide NOP agonist Ro 64-6198, which became the most extensively published nonpeptide NOP agonist and a valuable pharmacological tool in determining the potential of the NOP receptor as a therapeutic target. Ro 64-6198 is systemically active and achieves high brain penetration. It has subnanomolar affinity for the NOP receptor and is at least 100 times more selective for the NOP receptor over the classic opioid receptors. Ro 64-6198 ranges from partial to full agonist, depending on the assay. Preclinical data indicate that Ro 64-6198 may have broad clinical uses, such as in treating stress and anxiety, addiction, neuropathic pain, cough, and anorexia. This review summarizes the pharmacology and preclinical data of Ro 64-6198.

Interactions of ligands with active and inactive conformations of the dopamine D2 receptor.

PubMed

Malmberg, A; Mohell, N; Backlund Höök, B; Johansson, A M; Hacksell, U; Nordvall, G

1998-04-10

The affinities of 19 pharmacologically diverse dopamine D2 receptor ligands were determined for the active and inactive conformations of cloned human dopamine D2 receptors expressed in Ltk cells. The agonist [3H]quinpirole was used to selectively label the guanine nucleotide-binding protein-coupled, active receptor conformation. The antagonist [3H]raclopride, in the presence of the non-hydrolysable GTP-analogue Gpp(NH)p and sodium ions and in the absence of magnesium ions, was used to label the free inactive receptor conformation. The intrinsic activities of the ligands were determined in a forskolin-stimulated cyclic AMP assay using the same cells. An excellent correlation was shown between the affinity ratios (KR/KRG) of the ligands for the two receptor conformations and their intrinsic activity (r=0.96). The ligands included eight structurally related and enantiopure 2-aminotetralin derivatives; the enantiomers of 5-hydroxy-2-(dipropylamino)tetralin, 5-methoxy-2-(dipropylamino)tetralin, 5-fluoro-2-(dipropylamino)tetralin and 2-(dipropylamino)tetralin. The (S)-enantiomers behaved as full agonists in the cyclic AMP assay and displayed a large KR/KRG ratio. The (R)-enantiomers were classified as partial agonists and had lower ratios. The structure-affinity relationships of these compounds at the active and the inactive receptor conformations were analysed separately, and used in conjunction with a homology based receptor model of the dopamine D2 receptor. This led to proposed binding modes for agonists, antagonists and partial agonists in the 2-aminotetralin series. The concepts used in this study should be of value in the design of ligands with predetermined affinity and intrinsic activity.

Analysis of molecular determinants of affinity and relative efficacy of a series of R- and S-2-(dipropylamino)tetralins at the 5-HT1A serotonin receptor

PubMed Central

Alder, J Tracy; Hacksell, Uli; Strange, Philip G

2003-01-01

Factors influencing agonist affinity and relative efficacy have been studied for the 5-HT1A serotonin receptor using membranes of CHO cells expressing the human form of the receptor and a series of R-and S-2-(dipropylamino)tetralins (nonhydroxylated and monohydroxylated (5-OH, 6-OH, 7-OH, 8-OH) species). Ligand binding studies were used to determine dissociation constants for agonist binding to the 5-HT1A receptor: Ki values for agonists were determined in competition versus the binding of the agonist [3H]-8-OH DPAT. Competition data were all fitted best by a one-binding site model.Ki values for agonists were also determined in competition versus the binding of the antagonist [3H]-NAD-199. Competition data were all fitted best by a two-binding site model, and agonist affinities for the higher (Kh) and lower affinity (Kl) sites were determined. The ability of the agonists to activate the 5-HT1A receptor was determined using stimulation of [35S]-GTPγS binding. Maximal effects of agonists (Emax) and their potencies (EC50) were determined from concentration/response curves for stimulation of [35S]-GTPγS binding. Kl/Kh determined from ligand binding assays correlated with the relative efficacy (relative Emax) of agonists determined in [35S]-GTPγS binding assays. There was also a correlation between Kl/Kh and Kl/EC50 for agonists determined from ligand binding and [35S]-GTPγS binding assays. Simulations of agonist binding and effect data were performed using the Ternary Complex Model in order to assess the use of Kl/Kh for predicting the relative efficacy of agonists. PMID:12684269

Simultaneous detection of residues of 25 β₂-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass spectrometry.

PubMed

Sai, Fan; Hong, Miao; Yunfeng, Zhao; Huijing, Chen; Yongning, Wu

2012-02-29

A sensitive method has been developed for the simultaneous determination of residues of 25 β₂-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass spectrometry (HPLC-LIT-MS). This method is based on a new procedure of hydrolysis and extraction by 5% trichloracetic acid, and then cleaned up by mixed strong cation exchange (MCX) cartridges coupled with a novelty cleanup step by methanol. Methanol and 0.1% formic acid were used as mobile phases for gradient elution, while a Supelco Ascentis Express Rp-Amide column was used for LC separation. ESI positive ion scan mode was used with consecutive reaction monitoring (CRM, MS³). Nine β₂-agonists labeled by the deuterium isotope were used as internal standards for quantification. The linear ranges of 48 analytes were from 5 to 200 μg/L; the coefficient of correlation was not less than 0.995. Blank pork muscle, blank liver, and blank kidney were selected as representative matrix for spiked standard recovery test. The recoveries of each compound were in the range of 46.6-118.9%, and the relative standard deviations were in the range of 1.9-28.2%. Decision limits (CCα, α = 0.01) of 48 analytes in muscles, liver, and kidney samples ranged from 0.05 to 0.49 μg/kg, and the detection capability (CCβ, β = 0.05) ranged from 0.13 to 1.64 μg/kg. This method was successfully applied to 110 real animal origin food samples including meat, liver, and kidney of pig and chicken samples.

Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill.

PubMed

González, Alfredo; Crittenden, Elizabeth L; García, Dana M

2004-07-13

In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism.

PubMed

Wootten, Denise; Reynolds, Christopher A; Smith, Kevin J; Mobarec, Juan C; Koole, Cassandra; Savage, Emilia E; Pabreja, Kavita; Simms, John; Sridhar, Rohan; Furness, Sebastian G B; Liu, Mengjie; Thompson, Philip E; Miller, Laurence J; Christopoulos, Arthur; Sexton, Patrick M

2016-06-16

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Agonist-Specific Recruitment of Arrestin Isoforms Differentially Modify Delta Opioid Receptor Function

PubMed Central

Perroy, Julie; Walwyn, Wendy M.; Smith, Monique L.; Vicente-Sanchez, Ana; Segura, Laura; Bana, Alia; Kieffer, Brigitte L.; Evans, Christopher J.

2016-01-01

Ligand-specific recruitment of arrestins facilitates functional selectivity of G-protein-coupled receptor signaling. Here, we describe agonist-selective recruitment of different arrestin isoforms to the delta opioid receptor in mice. A high-internalizing delta opioid receptor agonist (SNC80) preferentially recruited arrestin 2 and, in arrestin 2 knock-outs (KOs), we observed a significant increase in the potency of SNC80 to inhibit mechanical hyperalgesia and decreased acute tolerance. In contrast, the low-internalizing delta agonists (ARM390, JNJ20788560) preferentially recruited arrestin 3 with unaltered behavioral effects in arrestin 2 KOs. Surprisingly, arrestin 3 KO revealed an acute tolerance to these low-internalizing agonists, an effect never observed in wild-type animals. Furthermore, we examined delta opioid receptor–Ca2+ channel coupling in dorsal root ganglia desensitized by ARM390 and the rate of resensitization was correspondingly decreased in arrestin 3 KOs. Live-cell imaging in HEK293 cells revealed that delta opioid receptors are in pre-engaged complexes with arrestin 3 at the cell membrane and that ARM390 strengthens this membrane interaction. The disruption of these complexes in arrestin 3 KOs likely accounts for the altered responses to low-internalizing agonists. Together, our results show agonist-selective recruitment of arrestin isoforms and reveal a novel endogenous role of arrestin 3 as a facilitator of resensitization and an inhibitor of tolerance mechanisms. SIGNIFICANCE STATEMENT Agonists that bind to the same receptor can produce highly distinct signaling events and arrestins are a major mediator of this ligand bias. Here, we demonstrate that delta opioid receptor agonists differentially recruit arrestin isoforms. We found that the high-internalizing agonist SNC80 preferentially recruits arrestin 2 and knock-out (KO) of this protein results in increased efficacy of SNC80. In contrast, low-internalizing agonists (ARM390 and JNJ20788560) preferentially recruit arrestin 3 and, surprisingly, KO of arrestin 3 produces acute tolerance and impaired receptor resensitization to these agonists. Arrestin 3 is in pre-engaged complexes with the delta opioid receptor at the cell membrane and low-internalizing agonists promote this interaction. This study reveals a novel role for arrestin 3 as a facilitator of receptor resensitization. PMID:27013682

Engineered Context-Sensitive Agonism: Tissue-Selective Drug Signaling through a G Protein-Coupled Receptor.

PubMed

Seemann, Wiebke K; Wenzel, Daniela; Schrage, Ramona; Etscheid, Justine; Bödefeld, Theresa; Bartol, Anna; Warnken, Mareille; Sasse, Philipp; Klöckner, Jessica; Holzgrabe, Ulrike; DeAmici, Marco; Schlicker, Eberhard; Racké, Kurt; Kostenis, Evi; Meyer, Rainer; Fleischmann, Bernd K; Mohr, Klaus

2017-02-01

Drug discovery strives for selective ligands to achieve targeted modulation of tissue function. Here we introduce engineered context-sensitive agonism as a postreceptor mechanism for tissue-selective drug action through a G protein-coupled receptor. Acetylcholine M 2 -receptor activation is known to mediate, among other actions, potentially dangerous slowing of the heart rate. This unwanted side effect is one of the main reasons that limit clinical application of muscarinic agonists. Herein we show that dualsteric (orthosteric/allosteric) agonists induce less cardiac depression ex vivo and in vivo than conventional full agonists. Exploration of the underlying mechanism in living cells employing cellular dynamic mass redistribution identified context-sensitive agonism of these dualsteric agonists. They translate elevation of intracellular cAMP into a switch from full to partial agonism. Designed context-sensitive agonism opens an avenue toward postreceptor pharmacologic selectivity, which even works in target tissues operated by the same subtype of pharmacologic receptor. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

A Novel Method for Analyzing Extremely Biased Agonism at G Protein–Coupled Receptors

PubMed Central

Zhou, Lei; Ehlert, Frederick J.; Bohn, Laura M.

2015-01-01

Seven transmembrane receptors were originally named and characterized based on their ability to couple to heterotrimeric G proteins. The assortment of coupling partners for G protein–coupled receptors has subsequently expanded to include other effectors (most notably the βarrestins). This diversity of partners available to the receptor has prompted the pursuit of ligands that selectively activate only a subset of the available partners. A biased or functionally selective ligand may be able to distinguish between different active states of the receptor, and this would result in the preferential activation of one signaling cascade more than another. Although application of the “standard” operational model for analyzing ligand bias is useful and suitable in most cases, there are limitations that arise when the biased agonist fails to induce a significant response in one of the assays being compared. In this article, we describe a quantitative method for measuring ligand bias that is particularly useful for such cases of extreme bias. Using simulations and experimental evidence from several κ opioid receptor agonists, we illustrate a “competitive” model for quantitating the degree and direction of bias. By comparing the results obtained from the competitive model with the standard model, we demonstrate that the competitive model expands the potential for evaluating the bias of very partial agonists. We conclude the competitive model provides a useful mechanism for analyzing the bias of partial agonists that exhibit extreme bias. PMID:25680753

Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

PubMed Central

Copik, Alicja. J.; Baldys, Aleksander; Nguyen, Khanh; Sahdeo, Sunil; Ho, Hoangdung; Kosaka, Alan; Dietrich, Paul J.; Fitch, Bill; Raymond, John R.; Ford, Anthony P. D. W.; Button, Donald; Milla, Marcos E.

2015-01-01

The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gαq. PMID:25606852

Synthesis of the PPARbeta/delta-selective agonist GW501516 and C4-thiazole-substituted analogs.

PubMed

Pereira, Raquel; Gaudon, Claudine; Iglesias, Beatriz; Germain, Pierre; Gronemeyer, Hinrich; de Lera, Angel R

2006-01-01

Sequential, position-selective, Pd-catalyzed cross-coupling reactions of 2,4-dibromo-5-hydroxymethylthiazole provided the scaffold for the synthesis of GW501516, the most potent PPARbeta/delta agonist yet described, and equally selective analogs at the thiazole-C4 position.

Differential profile of typical, atypical and third generation antipsychotics at human 5-HT7a receptors coupled to adenylyl cyclase: detection of agonist and inverse agonist properties.

PubMed

Rauly-Lestienne, Isabelle; Boutet-Robinet, Elisa; Ailhaud, Marie-Christine; Newman-Tancredi, Adrian; Cussac, Didier

2007-10-01

5-HT(7) receptors are present in thalamus and limbic structures, and a possible role of these receptors in the pathology of schizophrenia has been evoked. In this study, we examined binding affinity and agonist/antagonist/inverse agonist properties at these receptors of a large series of antipsychotics, i.e., typical, atypical, and third generation compounds preferentially targeting D(2) and 5-HT(1A) sites. Adenylyl cyclase (AC) activity was measured in HEK293 cells stably expressing the human (h) 5-HT(7a) receptor isoform. 5-HT and 5-CT increased cyclic adenosine monophosphate level by about 20-fold whereas (+)-8-OH-DPAT, the antidyskinetic agent sarizotan, and the novel antipsychotic compound bifeprunox exhibited partial agonist properties at h5-HT(7a) receptors stimulating AC. Other compounds antagonized 5-HT-induced AC activity with pK (B) values which correlated with their pK (i) as determined by competition binding vs [(3)H]5-CT. The selective 5-HT(7) receptor ligand, SB269970, was the most potent antagonist. For antipsychotic compounds, the following rank order of antagonism potency (pK (B)) was ziprasidone > tiospirone > SSR181507 > or = clozapine > or = olanzapine > SLV-314 > SLV-313 > or = aripiprazole > or = chlorpromazine > nemonapride > haloperidol. Interestingly, pretreatment of HEK293-h5-HT(7a) cells with forskolin enhanced basal AC activity and revealed inverse agonist properties for both typical and atypical antipsychotics as well as for aripiprazole. In contrast, other novel antipsychotics exhibited diverse 5-HT(7a) properties; SLV-313 and SLV-314 behaved as quasi-neutral antagonists, SSR181507 acted as an inverse agonist, and bifeprunox as a partial agonist, as mentioned above. In conclusion, the differential properties of third generation antipsychotics at 5-HT(7) receptors may influence their antipsychotic profile.

Principal component analysis of binding energies for single-point mutants of hT2R16 bound to an agonist correlate with experimental mutant cell response.

PubMed

Chen, Derek E; Willick, Darryl L; Ruckel, Joseph B; Floriano, Wely B

2015-01-01

Directed evolution is a technique that enables the identification of mutants of a particular protein that carry a desired property by successive rounds of random mutagenesis, screening, and selection. This technique has many applications, including the development of G protein-coupled receptor-based biosensors and designer drugs for personalized medicine. Although effective, directed evolution is not without challenges and can greatly benefit from the development of computational techniques to predict the functional outcome of single-point amino acid substitutions. In this article, we describe a molecular dynamics-based approach to predict the effects of single amino acid substitutions on agonist binding (salicin) to a human bitter taste receptor (hT2R16). An experimentally determined functional map of single-point amino acid substitutions was used to validate the whole-protein molecular dynamics-based predictive functions. Molecular docking was used to construct a wild-type agonist-receptor complex, providing a starting structure for single-point substitution simulations. The effects of each single amino acid substitution in the functional response of the receptor to its agonist were estimated using three binding energy schemes with increasing inclusion of solvation effects. We show that molecular docking combined with molecular mechanics simulations of single-point mutants of the agonist-receptor complex accurately predicts the functional outcome of single amino acid substitutions in a human bitter taste receptor.

GPR40 partial agonist MK-2305 lower fasting glucose in the Goto Kakizaki rat via suppression of endogenous glucose production

PubMed Central

Kirkland, Melissa E.; Kosinski, Daniel T.; Mane, Joel; Bunzel, Michelle; Cao, Jin; Souza, Sarah; Thomas-Fowlkes, Brande; Di Salvo, Jerry; Weinglass, Adam B.; Li, Xiaoyan; Myers, Robert W.; Knagge, Kevin; Carrington, Paul E.; Hagmann, William K.

2017-01-01

GPR40 (FFA1) is a fatty acid receptor whose activation results in potent glucose lowering and insulinotropic effects in vivo. Several reports illustrate that GPR40 agonists exert glucose lowering in diabetic humans. To assess the mechanisms by which GPR40 partial agonists improve glucose homeostasis, we evaluated the effects of MK-2305, a potent and selective partial GPR40 agonist, in diabetic Goto Kakizaki rats. MK-2305 decreased fasting glucose after acute and chronic treatment. MK-2305-mediated changes in glucose were coupled with increases in plasma insulin during hyperglycemia and glucose challenges but not during fasting, when glucose was normalized. To determine the mechanism(s) mediating these changes in glucose metabolism, we measured the absolute contribution of precursors to glucose production in the presence or absence of MK-2305. MK-2305 treatment resulted in decreased endogenous glucose production (EGP) driven primarily through changes in gluconeogenesis from substrates entering at the TCA cycle. The decrease in EGP was not likely due to a direct effect on the liver, as isolated perfused liver studies showed no effect of MK-2305 ex vivo and GPR40 is not expressed in the liver. Taken together, our results suggest MK-2305 treatment increases glucose stimulated insulin secretion (GSIS), resulting in changes to hepatic substrate handling that improve glucose homeostasis in the diabetic state. Importantly, these data extend our understanding of the underlying mechanisms by which GPR40 partial agonists reduce hyperglycemia. PMID:28542610

Conserved Binding Mode of Human [beta subscript 2] Adrenergic Receptor Inverse Agonists and Antagonist Revealed by X-ray Crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wacker, Daniel; Fenalti, Gustavo; Brown, Monica A.

2010-11-15

G protein-coupled receptors (GPCRs) represent a large fraction of current pharmaceutical targets, and of the GPCRs, the {beta}{sub 2} adrenergic receptor ({beta}{sub 2}AR) is one of the most extensively studied. Previously, the X-ray crystal structure of {beta}{sub 2}AR has been determined in complex with two partial inverse agonists, but the global impact of additional ligands on the structure or local impacts on the binding site are not well-understood. To assess the extent of such ligand-induced conformational differences, we determined the crystal structures of a previously described engineered {beta}{sub 2}AR construct in complex with two inverse agonists: ICI 118,551 (2.8 {angstrom}),more » a recently described compound (2.8 {angstrom}) (Kolb et al, 2009), and the antagonist alprenolol (3.1 {angstrom}). The structures show the same overall fold observed for the previous {beta}{sub 2}AR structures and demonstrate that the ligand binding site can accommodate compounds of different chemical and pharmacological properties with only minor local structural rearrangements. All three compounds contain a hydroxy-amine motif that establishes a conserved hydrogen bond network with the receptor and chemically diverse aromatic moieties that form distinct interactions with {beta}{sub 2}AR. Furthermore, receptor ligand cross-docking experiments revealed that a single {beta}{sub 2}AR complex can be suitable for docking of a range of antagonists and inverse agonists but also indicate that additional ligand-receptor structures may be useful to further improve performance for in-silico docking or lead-optimization in drug design.« less

Structural insight into the activation of a class B G-protein-coupled receptor by peptide hormones in live human cells

PubMed Central

Seidel, Lisa; Zarzycka, Barbara; Zaidi, Saheem A; Katritch, Vsevolod; Coin, Irene

2017-01-01

The activation mechanism of class B G-protein-coupled receptors (GPCRs) remains largely unknown. To characterize conformational changes induced by peptide hormones, we investigated interactions of the class B corticotropin-releasing factor receptor type 1 (CRF1R) with two peptide agonists and three peptide antagonists obtained by N-truncation of the agonists. Surface mapping with genetically encoded photo-crosslinkers and pair-wise crosslinking revealed distinct footprints of agonists and antagonists on the transmembrane domain (TMD) of CRF1R and identified numerous ligand-receptor contact sites, directly from the intact receptor in live human cells. The data enabled generating atomistic models of CRF- and CRF(12-41)-bound CRF1R, further explored by molecular dynamics simulations. We show that bound agonist and antagonist adopt different folds and stabilize distinct TMD conformations, which involves bending of helices VI and VII around flexible glycine hinges. Conservation of these glycine hinges among all class B GPCRs suggests their general role in activation of these receptors. DOI: http://dx.doi.org/10.7554/eLife.27711.001 PMID:28771403

G Protein-Coupled Estrogen Receptor (GPER) Agonist Dual Binding Mode Analyses toward Understanding of its Activation Mechanism: A Comparative Homology Modeling Approach.

PubMed

Arnatt, Christopher K; Zhang, Yan

2013-07-01

G protein-coupled estrogen receptor (GPER) has been shown to be important in several disease states such as estrogen sensitive cancers. While several selective ligands have been identified for the receptor, little is known about how they interact with GPER and how their structures influence their activity. Specifically, within one series of ligands, whose structure varied only at one position, the replacement of a hydrogen atom with an acetyl group changed a potent antagonist into a potent agonist. In this study, two GPER homology models were constructed based on the x-ray crystal structures of both the active and inactive β 2 -adrenergic receptors (β 2 AR) in an effort to characterize the differences of binding modes between agonists and antagonists to the receptor, and to understand their activity in relation to their structures. The knowledge attained in this study is expected to provide valuable information on GPER ligands structure activity relationship to benefit future rational design of potent agonists and antagonists of the receptor for potential therapeutic applications.

G Protein-Coupled Estrogen Receptor (GPER) Agonist Dual Binding Mode Analyses toward Understanding of its Activation Mechanism: A Comparative Homology Modeling Approach

PubMed Central

Arnatt, Christopher K.; Zhang, Yan

2015-01-01

G protein-coupled estrogen receptor (GPER) has been shown to be important in several disease states such as estrogen sensitive cancers. While several selective ligands have been identified for the receptor, little is known about how they interact with GPER and how their structures influence their activity. Specifically, within one series of ligands, whose structure varied only at one position, the replacement of a hydrogen atom with an acetyl group changed a potent antagonist into a potent agonist. In this study, two GPER homology models were constructed based on the x-ray crystal structures of both the active and inactive β2-adrenergic receptors (β2AR) in an effort to characterize the differences of binding modes between agonists and antagonists to the receptor, and to understand their activity in relation to their structures. The knowledge attained in this study is expected to provide valuable information on GPER ligands structure activity relationship to benefit future rational design of potent agonists and antagonists of the receptor for potential therapeutic applications. PMID:26229572

Bile alcohols function as the ligands of membrane-type bile acid-activated G protein-coupled receptor

PubMed Central

Iguchi, Yusuke; Yamaguchi, Masafumi; Sato, Hiroyuki; Kihira, Kenji; Nishimaki-Mogami, Tomoko; Une, Mizuho

2010-01-01

TGR5 is a G protein-coupled receptor that is activated by bile acids, resulting in an increase in cAMP levels and the subsequent modulation of energy expenditure in brown adipose tissue and muscle. Therefore, the development of a TGR5-specific agonist could lead to the prevention and treatment of various metabolic disorders related to obesity. In the present study, we evaluated the ability of bile alcohols, which are structurally and physiologically similar to bile acids and are produced as the end products of cholesterol catabolism in evolutionarily primitive vertebrates, to act as TGR5 agonists. In a cell-based reporter assay and a cAMP production assay performed in vitro, most bile alcohols with a side chain containing hydroxyl group(s) were highly efficacious agonists for TGR5 comparable to its most potent ligand in the naturally occurring bile acid, lithocholic acid. However, the abilities of the bile alcohols to activate TGR5 varied with the position and number of the hydroxyl substituent in the side chain. Additionally, the conformation of the steroidal nucleus of bile alcohols is also important for its activity as a TGR5 agonist. Thus, we have provided new insights into the structure-activity relationships of bile alcohols as TGR5 agonists. PMID:20023205

Toll-like receptor-2 agonist-allergen coupling efficiently redirects Th2 cell responses and inhibits allergic airway eosinophilia.

PubMed

Krishnaswamy, Jayendra Kumar; Jirmo, Adan Chari; Baru, Abdul Mannan; Ebensen, Thomas; Guzmán, Carlos A; Sparwasser, Tim; Behrens, Georg M N

2012-12-01

Toll-like receptor (TLR) agonists beneficially modulate allergic airway inflammation. However, the efficiency of TLR agonists varies considerably, and their exact cellular mechanisms (especially of TLR 2/6 agonists) are incompletely understood. We investigated at a cellular level whether the administration of the pharmacologically improved TLR2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPP) conjugated to antigenic peptide (BPP-OVA) could divert an existing Th2 response and influence airway eosinophilia. The effects of BPP-OVA on airway inflammation were assessed in a classic murine sensitization/challenge model and an adoptive transfer model, which involved the adoptive transfer of in vitro differentiated ovalbumin (OVA)-specific Th2 cells. Functional T-cell stimulation by lung dendritic cells (DCs) was determined both in vitro and in vivo, combined with a cytokine secretion analysis. A single mucosal application of BPP-OVA efficiently delivered antigen, led to TLR2-mediated DC activation, and resulted in OVA-specific T-cell proliferation via lung DCs in vivo. In alternative models of allergic airway disease, a single administration of BPP-OVA before OVA challenge (but not BPP alone) significantly reduced airway eosinophilia, most likely through altered antigen-specific T-cell stimulation via DCs. Analyses of adoptively transferred Th2-biased cells after BPP-OVA administration in vivo suggested that BPP-OVA guides antigen-specific Th2 cells to produce significantly higher amounts of IFN-γ upon allergen challenge. In conclusion, our data show for the first time that a single mucosal administration of a TLR 2/6 agonist-allergen conjugate can provoke IFN-γ responses in Th2-biased cells and alleviate allergic airway inflammation.

Arrestin–dependent but G–protein coupled receptor kinase–independent uncoupling of D2–dopamine receptors

PubMed Central

Celver, Jeremy; Sharma, Meenakshi; Thanawala, Vaidehi; Octeau, J. Christopher; Kovoor, Abraham

2016-01-01

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G-protein gated inwardly rectifying potassium channels (Kir3) and directly compared the effects of co-expression of G-protein coupled receptor kinase (GRK) and arrestin on agonist-dependent desensitization of the receptor response. We found, as described previously, that co-expression of a GRK and an arrestin synergistically increased the rate of agonist-dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin-dependent GRK-independent desensitization of D2R-Kir3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin-dependent GRK-independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist-dependent desensitization even after GRK co-expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor. PMID:23815307

Arrestin-dependent but G-protein coupled receptor kinase-independent uncoupling of D2-dopamine receptors.

PubMed

Celver, Jeremy; Sharma, Meenakshi; Thanawala, Vaidehi; Christopher Octeau, J; Kovoor, Abraham

2013-10-01

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G-protein gated inwardly rectifying potassium channels (K(ir)3) and directly compared the effects of co-expression of G-protein coupled receptor kinase (GRK) and arrestin on agonist-dependent desensitization of the receptor response. We found, as described previously, that co-expression of a GRK and an arrestin synergistically increased the rate of agonist-dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin-dependent GRK-independent desensitization of D2R-K(ir)3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin-dependent GRK-independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist-dependent desensitization even after GRK co-expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor. © 2013 International Society for Neurochemistry.

Graded activation and free energy landscapes of a muscarinic G-protein-coupled receptor.

PubMed

Miao, Yinglong; McCammon, J Andrew

2016-10-25

G-protein-coupled receptors (GPCRs) recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. However, the mechanism of such graded activation remains unclear. Using the Gaussian accelerated molecular dynamics (GaMD) method that enables both unconstrained enhanced sampling and free energy calculation, we have performed extensive GaMD simulations (∼19 μs in total) to investigate structural dynamics of the M 2 muscarinic GPCR that is bound by the full agonist iperoxo (IXO), the partial agonist arecoline (ARC), and the inverse agonist 3-quinuclidinyl-benzilate (QNB), in the presence or absence of the G-protein mimetic nanobody. In the receptor-nanobody complex, IXO binding leads to higher fluctuations in the protein-coupling interface than ARC, especially in the receptor transmembrane helix 5 (TM5), TM6, and TM7 intracellular domains that are essential elements for GPCR activation, but less flexibility in the receptor extracellular region due to stronger binding compared with ARC. Two different binding poses are revealed for ARC in the orthosteric pocket. Removal of the nanobody leads to GPCR deactivation that is characterized by inward movement of the TM6 intracellular end. Distinct low-energy intermediate conformational states are identified for the IXO- and ARC-bound M 2 receptor. Both dissociation and binding of an orthosteric ligand are observed in a single all-atom GPCR simulation in the case of partial agonist ARC binding to the M 2 receptor. This study demonstrates the applicability of GaMD for exploring free energy landscapes of large biomolecules and the simulations provide important insights into the GPCR functional mechanism.

Synthesis and biological evaluation of bivalent cannabinoid receptor ligands based on hCB₂R selective benzimidazoles reveal unexpected intrinsic properties.

PubMed

Nimczick, Martin; Pemp, Daniela; Darras, Fouad H; Chen, Xinyu; Heilmann, Jörg; Decker, Michael

2014-08-01

The design of bivalent ligands targeting G protein-coupled receptors (GPCRs) often leads to the development of new, highly selective and potent compounds. To date, no bivalent ligands for the human cannabinoid receptor type 2 (hCB₂R) of the endocannabinoid system (ECS) are described. Therefore, two sets of homobivalent ligands containing as parent structure the hCB2R selective agonist 13a and coupled at different attachment positions were synthesized. Changes of the parent structure at these positions have a crucial effect on the potency and efficacy of the ligands. However, we discovered that bivalency has an influence on the effect at both cannabinoid receptors. Moreover, we found out that the spacer length and the attachment position altered the efficacy of the bivalent ligands at the receptors by turning agonists into antagonists and inverse agonists. Copyright © 2014 Elsevier Ltd. All rights reserved.

An intracellular loop 2 amino acid residue determines differential binding of arrestin to the dopamine D2 and D3 receptors.

PubMed

Lan, Hongxiang; Teeter, Martha M; Gurevich, Vsevolod V; Neve, Kim A

2009-01-01

Dopamine D(2) and D(3) receptors are similar subtypes with distinct interactions with arrestins; the D(3) receptor mediates less agonist-induced translocation of arrestins than the D(2) receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D(2) and D(3) receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin 3 bound to glutathione transferase (GST) fusion proteins of the D(2) receptor IC2 more avidly than to the D(3) receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin 3 to D(2)-IC2; arrestin binding to D(2)-IC2-K149C was greatly decreased compared with wild-type D(2)-IC2, whereas binding to the reciprocal mutant D(3)-IC2-C147K was enhanced compared with wild-type D(3)-IC2. Mutating this lysine in the full-length D(2) receptor to cysteine decreased the ability of the D(2) receptor to mediate agonist-induced arrestin 3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D(3) receptor increased receptor-mediated translocation of arrestin 3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D(2) receptor over the D(3) receptor is due in part to Lys149, which could be exposed as a result of receptor activation.

Discovery of 3-aryl-4-isoxazolecarboxamides as TGR5 receptor agonists.

PubMed

Evans, Karen A; Budzik, Brian W; Ross, Sean A; Wisnoski, David D; Jin, Jian; Rivero, Ralph A; Vimal, Mythily; Szewczyk, George R; Jayawickreme, Channa; Moncol, David L; Rimele, Thomas J; Armour, Susan L; Weaver, Susan P; Griffin, Robert J; Tadepalli, Sarva M; Jeune, Michael R; Shearer, Todd W; Chen, Zibin B; Chen, Lihong; Anderson, Donald L; Becherer, J David; De Los Frailes, Maite; Colilla, Francisco Javier

2009-12-24

A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent small molecule agonists of the human TGR5 G-protein coupled receptor is described. Subsequent optimization resulted in the rapid identification of potent exemplars 6 and 7 which demonstrated improved GLP-1 secretion in vivo via an intracolonic dose coadministered with glucose challenge in a canine model. These novel TGR5 receptor agonists are potentially useful therapeutics for metabolic disorders such as type II diabetes and its associated complications.

Assessment of cell line competence for studies of pharmacological GPR30 modulation.

PubMed

Sousa, Cátia; Ribeiro, Madalena; Rufino, Ana Teresa; Leitão, Alcino Jorge; Mendes, Alexandrina Ferreira

2017-04-01

Cell lines used to study the role of the G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor (GPER) as a mediator of estrogen responses have yielded conflicting results. This work identified a simple assay to predict cell line competence for pharmacological studies of GPR30. The phosphorylation or expression levels of ERK1/2, Akt, c-Fos and eNOS were evaluated to assess GPR30 activation in response to known agonists (17β-estradiol and G-1) in MCF-7 and T-47D breast cancer cell lines and in bovine aortic endothelial cells. GPR30 expression was analyzed by qRT-PCR and Western blot with two distinct antibodies directed at its carboxy and amino terminals. None of the agonists, at any of the concentrations tested, activated any of those target proteins. Additional experiments excluded the disruption of the signaling pathway, interference of phenol red in the culture medium and constitutive proteasome degradation of GPR30 as possible causes for the lack of response of the three cell lines. Analysis of receptor expression showed the absence of clearly detectable GPR30 species of 44 and 50-55 kDa previously identified in cell lines that respond to 17β-estradiol and G-1. Cells that do not express the 44 and 50-55 kDa species do not respond to GPR30 agonists. Thus, the presence or absence of these GPR30 species is a simple and rapid manner to determine whether a given cell line is suitable for pharmacological or molecular studies of GPR30 modulation.

Improving virtual screening of G protein-coupled receptors via ligand-directed modeling

PubMed Central

Simms, John; Christopoulos, Arthur; Wootten, Denise

2017-01-01

G protein-coupled receptors (GPCRs) play crucial roles in cell physiology and pathophysiology. There is increasing interest in using structural information for virtual screening (VS) of libraries and for structure-based drug design to identify novel agonist or antagonist leads. However, the sparse availability of experimentally determined GPCR/ligand complex structures with diverse ligands impedes the application of structure-based drug design (SBDD) programs directed to identifying new molecules with a select pharmacology. In this study, we apply ligand-directed modeling (LDM) to available GPCR X-ray structures to improve VS performance and selectivity towards molecules of specific pharmacological profile. The described method refines a GPCR binding pocket conformation using a single known ligand for that GPCR. The LDM method is a computationally efficient, iterative workflow consisting of protein sampling and ligand docking. We developed an extensive benchmark comparing LDM-refined binding pockets to GPCR X-ray crystal structures across seven different GPCRs bound to a range of ligands of different chemotypes and pharmacological profiles. LDM-refined models showed improvement in VS performance over origin X-ray crystal structures in 21 out of 24 cases. In all cases, the LDM-refined models had superior performance in enriching for the chemotype of the refinement ligand. This likely contributes to the LDM success in all cases of inhibitor-bound to agonist-bound binding pocket refinement, a key task for GPCR SBDD programs. Indeed, agonist ligands are required for a plethora of GPCRs for therapeutic intervention, however GPCR X-ray structures are mostly restricted to their inactive inhibitor-bound state. PMID:29131821

Cannabis agonist injection effect on the coupling architecture in cortex of WAG/Rij rats during absence seizures

NASA Astrophysics Data System (ADS)

Sysoeva, Marina V.; Kuznetsova, Galina D.; van Rijn, Clementina M.; Sysoev, Ilya V.

2016-04-01

WAG/Rij rats are well known genetic model of absence epilepsy, which is traditionally considered as a nonconvulsive generalised epilepsy of unknown aetiology. In current study the effect of (R)-(+)-WIN 55,212-2 (cannabis agonist) injection on the coupling between different parts of cortex was studied on 27 male 8 month old rats using local field potentials. Recently developed non-linear adapted Granger causality approach was used as a primary method. It was shown that first 2 hours after the injection the coupling between most channel pairs rises in comparison with the spontaneous activity, whilst long after the injection (2-6 hours) it drops down. The coupling increase corresponds to the mentioned before treatment effect, when the number and the longitude of seizures significantly decreases. However the subsequent decrease of the coupling in the cortex is accompanied by the dramatic increase of the longitude and the number of seizures. This assumes the hypothesis that a relatively higher coupling in the cortical network can prevent the seizure propagation and generalisation.

Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor

PubMed Central

Thompson, Dawn; McArthur, Simon; Hislop, James N.; Flower, Roderick J.; Perretti, Mauro

2014-01-01

Formyl-peptide receptor type 2 (FPR2; also called ALX because it is the receptor for lipoxin A4) sustains a variety of biological responses relevant to the development and control of inflammation, yet the cellular regulation of this G-protein-coupled receptor remains unexplored. Here we report that, in response to peptide agonist activation, FPR2/ALX undergoes β-arrestin-mediated endocytosis followed by rapid recycling to the plasma membrane. We identify a transplantable recycling sequence that is both necessary and sufficient for efficient receptor recycling. Furthermore, removal of this C-terminal recycling sequence alters the endocytic fate of FPR2/ALX and evokes pro-apoptotic effects in response to agonist activation. This study demonstrates the importance of endocytic recycling in the anti-apoptotic properties of FPR2/ALX and identifies the molecular determinant required for modulation of this process fundamental for the control of inflammation. PMID:25326384

C-terminal of human histamine H1 receptors regulates their agonist-induced clathrin-mediated internalization and G-protein signaling.

PubMed

Hishinuma, Shigeru; Nozawa, Hiroki; Akatsu, Chizuru; Shoji, Masaru

2016-11-01

It has been suggested that the agonist-induced internalization of G-protein-coupled receptors from the cell surface into intracellular compartments regulates cellular responsiveness. We previously reported that G q/11 -protein-coupled human histamine H 1 receptors internalized via clathrin-dependent mechanisms upon stimulation with histamine. However, the molecular determinants of H 1 receptors responsible for agonist-induced internalization remain unclear. In this study, we evaluated the roles of the intracellular C-terminal of human histamine H 1 receptors tagged with hemagglutinin (HA) at the N-terminal in histamine-induced internalization in Chinese hamster ovary cells. The histamine-induced internalization was evaluated by the receptor binding assay with [ 3 H]mepyramine and confocal immunofluorescence microscopy with an anti-HA antibody. We found that histamine-induced internalization was inhibited under hypertonic conditions or by pitstop, a clathrin terminal domain inhibitor, but not by filipin or nystatin, disruptors of the caveolar structure and function. The histamine-induced internalization was also inhibited by truncation of a single amino acid, Ser487, located at the end of the intracellular C-terminal of H 1 receptors, but not by its mutation to alanine. In contrast, the receptor-G-protein coupling, which was evaluated by histamine-induced accumulation of [ 3 H]inositol phosphates, was potentiated by truncation of Ser487, but was lost by its mutation to alanine. These results suggest that the intracellular C-terminal of human H 1 receptors, which only comprises 17 amino acids (Cys471-Ser487), plays crucial roles in both clathrin-dependent internalization of H 1 receptors and G-protein signaling, in which truncation of Ser487 and its mutation to alanine are revealed to result in biased signaling toward activation of G-proteins and clathrin-mediated internalization, respectively. © 2016 International Society for Neurochemistry.

Phosphorylation state of mu-opioid receptor determines the alternative recycling of receptor via Rab4 or Rab11 pathway.

PubMed

Wang, Feifei; Chen, Xiaoqing; Zhang, Xiaoqing; Ma, Lan

2008-08-01

Agonist-induced phosphorylation, internalization, and intracellular trafficking of G protein-coupled receptors are critical in regulating both cellular responsiveness and signal transduction. The current study investigated the role of receptor phosphorylation state in regulation of agonist-induced internalization and intracellular trafficking of mu-opioid receptor (MOR). Our results showed that after agonist stimulation, the recycle of a mutant MOR that lacks the C-terminal residues after Asn(362) (MOR362T) was greatly decreased, whereas a C-terminal phosphorylation sites-mutated MOR (MOR3A), which is deficient in agonist-induced phosphorylation recycled back to the membrane at a level comparable to that of the wild-type receptor, however, interestingly at a slower rate. Inhibition of functions of either Rab4 or Rab11 by dominant-negative mutants and small interfering RNA both significantly impaired the recycling of the wild-type MOR, whereas the recycling of the phosphorylation-deficient mutant was only inhibited by the dominant-negative mutant and small interfering RNA of Rab11, suggesting that the recycling of nonphosphorylated MOR is exclusively via Rab11-mediated pathway. Furthermore, phosphorylated MOR was observed accumulated in Rab5- and Rab4-, but not Rab11-positive vesicles. Our data indicate that both phosphorylated and nonphosphorylated MOR internalize via Rab5-dependent pathway after agonist stimulation, and the phosphorylated and nonphosphorylated MORs recycle through distinct vesicular trafficking pathways mediated by Rab4 and Rab11, respectively, which may ultimately lead to differential cellular responsiveness or downstream signaling.

Structure and biological activity of endogenous and synthetic agonists of GPR119

NASA Astrophysics Data System (ADS)

Tyurenkov, I. N.; Ozerov, A. A.; Kurkin, D. V.; Logvinova, E. O.; Bakulin, D. A.; Volotova, E. V.; Borodin, D. D.

2018-02-01

A G-protein-coupled receptor, GPR119, is a promising pharmacological target for a new class of hypoglycaemic drugs with an original mechanism of action, namely, increase in the glucose-dependent incretin and insulin secretion. In 2005, the first ligands were found and in the subsequent years, a large number of GPR119 agonists were synthesized in laboratories in various countries; the safest and most promising agonists have entered phase I and II clinical trials as agents for the treatment of type 2 diabetes mellitus and obesity. The review describes the major endogenous GPR119 agonists and the main trends in the design and modification of synthetic structures for increasing the hypoglycaemic activity. The data on synthetic agonists are arranged according to the type of the central core of the molecules. The bibliography includes 104 references.

Adenosine receptor desensitization and trafficking.

PubMed

Mundell, Stuart; Kelly, Eamonn

2011-05-01

As with the majority of G-protein-coupled receptors, all four of the adenosine receptor subtypes are known to undergo agonist-induced regulation in the form of desensitization and trafficking. These processes can limit the ability of adenosine receptors to couple to intracellular signalling pathways and thus reduce the ability of adenosine receptor agonists as well as endogenous adenosine to produce cellular responses. In addition, since adenosine receptors couple to multiple signalling pathways, these pathways may desensitize differentially, while the desensitization of one pathway could even trigger signalling via another. Thus, the overall picture of adenosine receptor regulation can be complex. For all adenosine receptor subtypes, there is evidence to implicate arrestins in agonist-induced desensitization and trafficking, but there is also evidence for other possible forms of regulation, including second messenger-dependent kinase regulation, heterologous effects involving G proteins, and the involvement of non-clathrin trafficking pathways such as caveolae. In this review, the evidence implicating these mechanisms is summarized for each adenosine receptor subtype, and we also discuss those issues of adenosine receptor regulation that remain to be resolved as well as likely directions for future research in this field. Copyright © 2010 Elsevier B.V. All rights reserved.

Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by XMetA, an allosteric partial agonist antibody

USDA-ARS?s Scientific Manuscript database

XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by xmeta, an allosteric partial agonist antibody

USDA-ARS?s Scientific Manuscript database

XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

Modelling the Interaction of Catecholamines with the α1A Adrenoceptor Towards a Ligand-induced Receptor Structure

NASA Astrophysics Data System (ADS)

Kinsella, Gemma K.; Rozas, Isabel; Watson, Graeme W.

2005-06-01

Adrenoceptors are members of the important G protein coupled receptor family for which the detailed mechanism of activation remains unclear. In this study, we have combined docking and molecular dynamics simulations to model the ligand induced effect on an homology derived human α1A adrenoceptor. Analysis of agonist/α1A adrenoceptor complex interactions focused on the role of the charged amine group, the aromatic ring, the N-methyl group of adrenaline, the beta hydroxyl group and the catechol meta and para hydroxyl groups of the catecholamines. The most critical interactions for the binding of the agonists are consistent with many earlier reports and our study suggests new residues possibly involved in the agonist-binding site, namely Thr-174 and Cys-176. We further observe a number of structural changes that occur upon agonist binding including a movement of TM-V away from TM-III and a change in the interactions of Asp-123 of the conserved DRY motif. This may cause Arg-124 to move out of the TM helical bundle and change the orientation of residues in IC-II and IC-III, allowing for increased affinity of coupling to the G-protein.

Structure-Activity Analysis of Biased Agonism at the Human Adenosine A3 Receptor

PubMed Central

Baltos, Jo-Anne; Paoletta, Silvia; Nguyen, Anh T. N.; Gregory, Karen J.; Tosh, Dilip K.; Christopoulos, Arthur; Jacobson, Kenneth A.

2016-01-01

Biased agonism at G protein–coupled receptors (GPCRs) has significant implications for current drug discovery, but molecular determinants that govern ligand bias remain largely unknown. The adenosine A3 GPCR (A3AR) is a potential therapeutic target for various conditions, including cancer, inflammation, and ischemia, but for which biased agonism remains largely unexplored. We now report the generation of bias “fingerprints” for prototypical ribose containing A3AR agonists and rigidified (N)-methanocarba 5′-N-methyluronamide nucleoside derivatives with regard to their ability to mediate different signaling pathways. Relative to the reference prototypical agonist IB-MECA, (N)-methanocarba 5′-N-methyluronamide nucleoside derivatives with significant N6 or C2 modifications, including elongated aryl-ethynyl groups, exhibited biased agonism. Significant positive correlation was observed between the C2 substituent length (in Å) and bias toward cell survival. Molecular modeling suggests that extended C2 substituents on (N)-methanocarba 5′-N-methyluronamide nucleosides promote a progressive outward shift of the A3AR transmembrane domain 2, which may contribute to the subset of A3AR conformations stabilized on biased agonist binding. PMID:27136943

Cannabinoids reduce cAMP levels in the striatum of freely moving rats: an in vivo microdialysis study.

PubMed

Wade, Mark R; Tzavara, Eleni T; Nomikos, George G

2004-04-16

The cannabinoid receptor subtype 1 (CB1R) is a member of the G(i)-protein-coupled receptor family and cannabinoid signaling is largely dependent on the suppression of adenylyl cyclase-catalyzed cAMP production. In cell lines transfected with the CB1R or in native tissue preparations, treatment with cannabinoid agonists reduces both basal and forskolin-stimulated cAMP synthesis. We measured extracellular cAMP concentrations in the striatum of freely moving rats utilizing microdialysis to determine if changes in cAMP concentrations in response to CB1R agonists can be monitored in vivo. Striatal infusion of the CB1R agonist WIN55,212-2 (100 microM or 1 mM), dose-dependently decreased basal and forskolin-stimulated extracellular cAMP. These effects were reversed by co-infusion of the CB1R antagonist SR141716A (30 microM), which alone had no effect up to the highest concentration tested (300 microM). These data indicate that changes in extracellular cAMP concentrations in response to CB1R stimulation can be monitored in vivo allowing the study of cannabinoid signaling in the whole animal.

Histamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Notcovich, Cintia; Laboratorio de Farmacologia de Receptores, Catedra de Quimica Medicinal, Departamento de Farmacologia, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires; Diez, Federico

2010-02-01

It is well established that histamine modulates cell proliferation through the activation of the histamine H1 receptor (H1R), a G protein-coupled receptor (GPCR) that is known to couple to phospholipase C (PLC) activation via Gq. In the present study, we aimed to determine whether H1R activation modulates Rho GTPases, well-known effectors of Gq/G{sub 11}-coupled receptors, and whether such modulation influences cell proliferation. Experiments were carried out in CHO cells stably expressing H1R (CHO-H1R). By using pull-down assays, we found that both histamine and a selective H1R agonist activated Rac and RhoA in a time- and dose-dependent manner without significant changesmore » in the activation of Cdc42. Histamine response was abolished by the H1R antagonist mepyramine, RGS2 and the PLC inhibitor U73122, suggesting that Rac and RhoA activation is mediated by H1R via Gq coupling to PLC stimulation. Histamine caused a marked activation of serum response factor activity via the H1R, as determined with a serum-responsive element (SRE) luciferase reporter, and this response was inhibited by RhoA inactivation with C3 toxin. Histamine also caused a significant activation of JNK which was inhibited by expression of the Rac-GAP {beta}2-chimaerin. On the other hand, H1R-induced ERK1/2 activation was inhibited by U73122 but not affected by C3 or {beta}2-chimaerin, suggesting that ERK1/2 activation was dependent on PLC and independent of RhoA or Rac. [{sup 3}H]-Thymidine incorporation assays showed that both histamine and the H1R agonist inhibited cell proliferation in a dose-dependent manner and that the effect was independent of RhoA but partially dependent on JNK and Rac. Our results reveal that functional coupling of the H1R to Gq-PLC leads to the activation of RhoA and Rac small GTPases and suggest distinct roles for Rho GTPases in the control of cell proliferation by histamine.« less

Rapid desensitization and resensitization of 5-HT sub 2 receptor mediated phosphatidyl inositol hydrolysis by serotonin agonists in quiescent calf aortic smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pauwels, P.J.; Van Gompel, P.; Leysen, J.E.

1990-01-01

Agonist regulation of 5-hydroxytryptamine{sub 2} (5-HT{sub 2}) receptors was studied in calf aortic smooth muscle cultures incubated in a quiescent, defined synthetic medium that does not stimulate cell proliferation, but that provides cells with supplements that maintain cell viability. In these cells, 5-hydroxytryptamine (5-HT)-induced ({sup 3}H)inositol phosphates accumulation showed the characteristics of a 5-HT{sub 2} receptor coupled transducing system according to the inhibition of the response by 5-HT{sub 2} antagonists at nanomolar concentrations. The 5-HT{sub 2} receptor coupled response became rapidly desensitized during continued incubation with 5-HT and 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM); nearly full desensitization was obtained in two hours with 10more » {mu}M 5-HT and DOM pretreatment. The recovery of the response had a half-live of 5 hours after 2 hours pretreatment and of 9.5 to 12.5 hours after 24 to 96 hours agonist pretreatment. The DOM-induced desensitization of the 5-HT{sub 2} receptor coupled response was fully blocked by 0.1 {mu}M cinanserin. Cinanserin alone did not induce desensitization or up-regulation of the 5-HT{sub 2} receptor coupled response at 0.1 {mu}M.« less

Diversity in arrestin function.

PubMed

Kendall, Ryan T; Luttrell, Louis M

2009-09-01

The termination of heptahelical receptor signaling is a multilevel process coordinated, in large part, by members of the arrestin family of proteins. Arrestin binding to agonist-occupied receptors promotes desensitization by interrupting receptor-G protein coupling, while simultaneously recruiting machinery for receptor endocytosis, vesicular trafficking, and receptor fate determination. By simultaneously binding other proteins, arrestins also act as ligand-regulated scaffolds that recruit protein and lipid kinase, phosphatase, phosphodiesterase, and ubiquitin ligase activity into receptor-based multiprotein 'signalsome' complexes. Arrestin-binding thus 'switches' receptors from a transient G protein-coupled state to a persistent arrestin-coupled state that continues to signal as the receptor transits intracellular compartments. While it is clear that signalsome assembly has profound effects on the duration and spatial characteristics of heptahelical receptor signals, the physiologic functions of this novel signaling mechanism are poorly understood. Growing evidence suggests that signalsomes regulate such diverse processes as endocytosis and exocytosis, cell migration, survival, and contractility.

Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment

PubMed Central

Bagley, Elena E.

2014-01-01

Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector. PMID:25009497

Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

PubMed

Bagley, Elena E

2014-01-01

Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than E k . Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector.

Agonist-dependent modulation of G-protein coupling and transduction of 5-HT1A receptors in rat dorsal raphe nucleus.

PubMed

Valdizán, Elsa Maria; Castro, Elena; Pazos, Angel

2010-08-01

5-HT1A receptors couple to different Go/Gi proteins in order to mediate a wide range of physiological actions. While activation of post-synaptic 5-HT1A receptors is mainly related to inhibition of adenylyl cyclase activity, functionality of autoreceptors located in raphe nuclei has been classically ascribed to modifications of the activity of potassium and calcium channels. In order to evaluate the possible existence of agonist-directed trafficking for 5-HT1A autoreceptors in the rat dorsal raphe nucleus, we studied their activation by two agonists with a different profile of efficacy [(+)8-OH-DPAT and buspirone], addressing simultaneously the identification of the specific Galpha subtypes ([35S]GTPgammaS labelling and immunoprecipitation) involved and the subsequent changes in cAMP formation. A significant increase (32%, p<0.05) in (+)8-OH-DPAT-induced [35S]GTPgammaS labelling of immunoprecipitates was obtained with anti-Galphai3 antibodies but not with anti-Galphao, anti-Galphai1, anti-Galphai2, anti-Galphaz or anti-Galphas antibodies. In contrast, in the presence of buspirone, significant [35S]GTPgammaS labelling of immunoprecipitates was obtained with anti-Galphai3 (50%, p<0.01), anti-Galphao (32%, p<0.01) and anti-Galphai2 (29%, p<0.05) antibodies, without any labelling with anti-Galphai1, anti-Galphaz or anti-Galphas. The selective 5-HT1A antagonist WAY 100635 blocked the labelling induced by both agonists. Furthermore, (+)8-OH-DPAT failed to modify forskolin-stimulated cAMP accumulation, while buspirone induced a dose-dependent, WAY 100635-sensitive, inhibition of this response (Imax 30.8+/-4.9, pIC50 5.95+/-0.46). These results demonstrate the existence of an agonist-dependency pattern of G-protein coupling and transduction for 5-HT1A autoreceptors in native brain tissue. These data also open new perspectives for the understanding of the differential profiles of agonist efficacy in pre- vs. post-synaptic 5-HT1A receptor-associated responses.

Involvement of estradiol-17beta and its membrane receptor, G protein coupled receptor 30 (GPR30) in regulation of oocyte maturation in zebrafish, Danio rario.

PubMed

Pang, Yefei; Thomas, Peter

2009-03-01

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20beta-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17beta, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.

A dopamine D2 receptor mutant capable of G protein-mediated signaling but deficient in arrestin binding.

PubMed

Lan, Hongxiang; Liu, Yong; Bell, Michal I; Gurevich, Vsevolod V; Neve, Kim A

2009-01-01

Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D(2) and D(3) receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D(2) and D(3) receptors other than phosphorylation sites and to create a D(2) receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D(2) and D(3) receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D(2) receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D(2)-IC3 and D(3)-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D(2) and D(3) receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D(2) receptor is a key element of the arrestin binding site.

SB265610 is an allosteric, inverse agonist at the human CXCR2 receptor

PubMed Central

Bradley, ME; Bond, ME; Manini, J; Brown, Z; Charlton, SJ

2009-01-01

Background and purpose: In several previous studies, the C-X-C chemokine receptor (CXCR)2 antagonist 1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea (SB265610) has been described as binding competitively with the endogenous agonist. This is in contrast to many other chemokine receptor antagonists, where the mechanism of antagonism has been described as allosteric. Experimental approach: To determine whether it displays a unique mechanism among the chemokine receptor antagonists, the mode of action of SB265610 was investigated at the CXCR2 receptor using radioligand and [35S]-GTPγS binding approaches in addition to chemotaxis of human neutrophils. Key results: In equilibrium saturation binding studies, SB265610 depressed the maximal binding of [125I]-interleukin-8 ([125I]-IL-8) without affecting the Kd. In contrast, IL-8 was unable to prevent binding of [3H]-SB265610. Kinetic binding experiments demonstrated that this was not an artefact of irreversible or slowly reversible binding. In functional experiments, SB265610 caused a rightward shift of the concentration-response curves to IL-8 and growth-related oncogene α, but also a reduction in maximal response elicited by each agonist. Fitting these data to an operational allosteric ternary complex model suggested that, once bound, SB265610 completely blocks receptor activation. SB265610 also inhibited basal [35S]-GTPγS binding in this preparation. Conclusions and implications: Taken together, these data suggest that SB265610 behaves as an allosteric inverse agonist at the CXCR2 receptor, binding at a region distinct from the agonist binding site to prevent receptor activation, possibly by interfering with G protein coupling. PMID:19422399

Angiogenesis in the Primate Ovulatory Follicle Is Stimulated by Luteinizing Hormone via Prostaglandin E21

PubMed Central

Trau, Heidi A.; Davis, John S.; Duffy, Diane M.

2014-01-01

ABSTRACT Rapid angiogenesis occurs as the ovulatory follicle is transformed into the corpus luteum. To determine if luteinizing hormone (LH)-stimulated prostaglandin E2 (PGE2) regulates angiogenesis in the ovulatory follicle, cynomolgus macaques received gonadotropins to stimulate multiple follicular development and chorionic gonadotropin (hCG) substituted for the LH surge to initiate ovulatory events. Before hCG, vascular endothelial cells were present in the perifollicular stroma but not amongst granulosa cells. Endothelial cells entered the granulosa cell layer 24–36 h after hCG, concomitant with the rise in follicular PGE2 and prior to ovulation, which occurs about 40 h after hCG. Intrafollicular administration of the PG synthesis inhibitor indomethacin was coupled with PGE2 replacement to demonstrate that indomethacin blocked and PGE2 restored follicular angiogenesis in a single, naturally developed monkey follicle in vivo. Intrafollicular administration of indomethacin plus an agonist selective for a single PGE2 receptor showed that PTGER1 and PTGER2 agonists most effectively stimulated angiogenesis within the granulosa cell layer. Endothelial cell tracing and three-dimensional reconstruction indicated that these capillary networks form via branching angiogenesis. To further explore how PGE2 mediates follicular angiogenesis, monkey ovarian microvascular endothelial cells (mOMECs) were isolated from ovulatory follicles. The mOMECs expressed all four PGE2 receptors in vitro. PGE2 and all PTGER agonists increased mOMEC migration. PTGER1 and PTGER2 agonists promoted sprout formation while the PTGER3 agonist inhibited sprouting in vitro. While PTGER1 and PTGER2 likely promote the formation of new capillaries, each PGE2 receptor may mediate aspects of PGE2's actions and, therefore, LH's ability to regulate angiogenesis in the primate ovulatory follicle. PMID:25376231

The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors.

PubMed

Oakley, Robert H; Hudson, Christine C; Cruickshank, Rachael D; Meyers, Diane M; Payne, Richard E; Rhem, Shay M; Loomis, Carson R

2002-11-01

G protein-coupled receptors (GPCRs) have proven to be a rich source of therapeutic targets; therefore, finding compounds that regulate these receptors is a critical goal in drug discovery. The Transfluor technology utilizes the redistribution of fluorescently labeled arrestins from the cytoplasm to agonist-occupied receptors at the plasma membrane to monitor quantitatively the activation or inactivation of GPCRs. Here, we show that the Transfluor technology can be quantitated on the INCell Analyzer system (INCAS) using the vasopressin V(2) receptor (V(2)R), which binds arrestin with high affinity, and the beta(2)-adrenergic receptor (beta(2)AR), which binds arrestin with low affinity. U2OS cells stably expressing an arrestin-green fluorescent protein conjugate and either the V(2)R or the beta(2)AR were plated in 96-well plastic plates and analyzed by the INCAS at a screening rate of 5 min per plate. Agonist dose-response and antagonist dose-inhibition curves revealed signal-to-background ratios of approximately 25:1 and 8:1 for the V(2)R and beta(2)AR, respectively. EC(50) values agreed closely with K(d) values reported in the literature for the different receptor agonists. In addition, small amounts of arrestin translocation induced by sub-EC(50) doses of agonist were distinguished from the background noise of untreated cells. Furthermore, differences in the magnitude of arrestin translocation distinguished partial agonists from full agonists, and Z' values for these ligands were >0.5. These data show that the Transfluor technology, combined with an automated image analysis system, provides a direct, robust, and universal assay for high throughput screening of known and orphan GPCRs.

A unique role of RGS9-2 in the striatum as a positive or negative regulator of opiate analgesia.

PubMed

Psifogeorgou, Kassi; Psigfogeorgou, Kassi; Terzi, Dimitra; Papachatzaki, Maria Martha; Varidaki, Artemis; Ferguson, Deveroux; Gold, Stephen J; Zachariou, Venetia

2011-04-13

The signaling molecule RGS9-2 is a potent modulator of G-protein-coupled receptor function in striatum. Our earlier work revealed a critical role for RGS9-2 in the actions of the μ-opioid receptor (MOR) agonist morphine. In this study, we demonstrate that RGS9-2 may act as a positive or negative modulator of MOR-mediated behavioral responses in mice depending on the agonist administered. Paralleling these findings we use coimmunoprecipitation assays to show that the signaling complexes formed between RGS9-2 and Gα subunits in striatum are determined by the MOR agonist, and we identify RGS9-2 containing complexes associated with analgesic tolerance. In striatum, MOR activation promotes the formation of complexes between RGS9-2 and several Gα subunits, but morphine uniquely promotes an association between RGS9-2 and Gαi3. In contrast, RGS9-2/Gαq complexes assemble after acute application of several MOR agonists but not after morphine application. Repeated morphine administration leads to the formation of distinct complexes, which contain RGS9-2, Gβ5, and Gαq. Finally, we use simple pharmacological manipulations to disrupt RGS9-2 complexes formed during repeated MOR activation to delay the development of analgesic tolerance to morphine. Our data provide a better understanding of the brain-region-specific signaling events associated with opiate analgesia and tolerance and point to pharmacological approaches that can be readily tested for improving chronic analgesic responsiveness.

Fast Modulation of μ-Opioid Receptor (MOR) Recycling Is Mediated by Receptor Agonists*

PubMed Central

Roman-Vendrell, Cristina; Yu, Y. Joy; Yudowski, Guillermo Ariel

2012-01-01

The μ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca2+-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance. PMID:22378794

Human uterine and placental arteries exhibit tissue-specific acute responses to 17β-estradiol and estrogen-receptor-specific agonists.

PubMed

Corcoran, Jemma J; Nicholson, Christopher; Sweeney, Michèle; Charnock, Jayne C; Robson, Stephen C; Westwood, Melissa; Taggart, Michael J

2014-05-01

The discrete regulation of vascular tone in the human uterine and placental circulations is a key determinant of appropriate uteroplacental blood perfusion and pregnancy success. Humoral factors such as estrogen, which increases in the placenta and maternal circulation throughout human pregnancy, may regulate these vascular beds as studies of animal arteries have shown that 17β-estradiol, or agonists of estrogen receptors (ER), can exert acute vasodilatory actions. The aim of this study was to compare how acute exposure to ER-specific agonists, and 17β-estradiol, altered human placental and uterine arterial tone in vitro. Uterine and placental arteries were isolated from biopsies obtained from women with uncomplicated pregnancy delivering a singleton infant at term. Vessels were mounted on a wire myograph, exposed to the thromboxane receptor agonist U46619 (10(-6) M), and then incubated with incremental doses (5 min, 0.03-30 µM) of either 17β-estradiol or agonists specific for the ERs ERα (PPT), ERβ (DPN) or the G-protein-coupled estrogen receptor GPER-1 (G1). ERα and ERβ mRNA expression was assessed. 17β-estradiol, PPT and DPN each relaxed myometrial arteries (P < 0.05) in a manner that was partly endothelium-dependent. In contrast, 17β-estradiol or DPN relaxed placental arteries (maximum relaxation to 42 ± 1.1 or 47.6 ± 6.53% of preconstriction, respectively) to a lesser extent than myometrial arteries (to 0.03 ± 0.03 or 8.0 ± 1.0%) and in an endothelial-independent manner whereas PPT was without effect. G1 exposure did not inhibit the constriction of myometrial nor placenta arteries. mRNA expression of ERα and ERβ was greater in myometrial arteries than placental arteries. ER-specific agonists, and 17β-estradiol, differentially modulate the tone of uterine versus placental arteries highlighting that estrogen may regulate human uteroplacental blood flow in a tissue-specific manner.

Expression and agonist responsiveness of CXCR3 variants in human T lymphocytes.

PubMed

Korniejewska, Anna; McKnight, Andrew J; Johnson, Zoë; Watson, Malcolm L; Ward, Stephen G

2011-04-01

The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time-courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gα(i) protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists. © 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.

Expression and agonist responsiveness of CXCR3 variants in human T lymphocytes

PubMed Central

Korniejewska, Anna; McKnight, Andrew J; Johnson, Zoë; Watson, Malcolm L; Ward, Stephen G

2011-01-01

The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time–courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gαi protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists. PMID:21255008

Ligands raise the constraint that limits constitutive activation in G protein-coupled opioid receptors.

PubMed

Vezzi, Vanessa; Onaran, H Ongun; Molinari, Paola; Guerrini, Remo; Balboni, Gianfranco; Calò, Girolamo; Costa, Tommaso

2013-08-16

Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and μ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4-5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the "two state" extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form.

Assessments of cellular melatonin receptor signaling pathways: β-arrestin recruitment, receptor internalization, and impedance variations.

PubMed

Dupré, Clémence; Bruno, Olivier; Bonnaud, Anne; Giganti, Adeline; Nosjean, Olivier; Legros, Céline; Boutin, Jean A

2018-01-05

Melatonin receptors belong to the family of G-protein coupled receptors. Agonist-induced receptor activation is terminated with the recruitment of β-arrestin, which leads to receptor internalization. Furthermore, agonist binding induces a shift in cellular shape that translates into a change in the electric impedance of the cell. In the present study, we employed engineered cells to study these internalization-related processes in the context of the two melatonin receptors, MT 1 and MT 2 . To assess these three receptor internalization-related functions and validate the results, we employed four classical ligands of melatonin receptors: the natural agonist melatonin; the super-agonist 2-iodo-melatonin and the two antagonists luzindole and 4-phenyl-2-propionamidotetralin. The assessments confirmed the nature of the agonistic ligands but showed that 4-phenyl-2-propionamidotetralin, a described antagonist, is a biased partial agonist at MT 2 with poorer affinity for MT 1 . The methods are now available to be applied to any receptor system for which multiple signaling pathways must be evaluated for new molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Highly sensitive electrochemiluminescent immunosensor based on gold nanoparticles-functionalized zinc oxide nanorod and poly(amidoamine)-graphene for detecting brombuterol.

PubMed

Zhu, Qing; Cai, Fudong; Zhang, Jing; Zhao, Kang; Deng, Anping; Li, Jianguo

2016-12-15

β-adrenergic agonists (β-agonists) recognized as a growth promoter will reflect the health of human. Sensitive detection of β-agonists in foodstuff is valuable for the health of animals and human. A novel ultrasensitive competition-type electrochemiluminescent (ECL) immunosensor was developed for detecting brombuterol (Brom) based on CdTe Quantum dot (QDs) and polyamidoamine dendrimer (PAMAM, G2) modified graphene oxide (GO) (CdTe QDs-PAMAM-GO composite) as bioprobe for the first time. The surface of glassy carbon electrode (GCE) was coated with AuNPs-ZnO NRs composite film as the platform, which facilitated the electronic transmission rate to enhance the ECL intensity and provide enough active sites for capturing antibody. The resulting ECL immunosensor enabled the real samples detection of Brom with a lower detection limit of 0.3pgmL(-1) (S/N=3) and a wider linear range from 0.001 to 500ngmL(-1). The proposed immunosensor coupled with the excellent advantages of CdTe QDs-PAMAM-GO and AuNPs-ZnO NRs composite displayed high sensitivity and long-term stability, and provided an approach for determining other important biomarkers. Copyright © 2016 Elsevier B.V. All rights reserved.

Serum stability of selected decapeptide agonists of KISS1R using pseudopeptides.

PubMed

Asami, Taiji; Nishizawa, Naoki; Ishibashi, Yoshihiro; Nishibori, Kimiko; Nakayama, Masaharu; Horikoshi, Yasuko; Matsumoto, Shin-ichi; Yamaguchi, Masashi; Matsumoto, Hirokazu; Tarui, Naoki; Ohtaki, Tetsuya; Kitada, Chieko

2012-10-15

Metastin/kisspeptin, a 54-amino acid peptide, is the ligand of the G-protein-coupled receptor KISS1R which plays a key role in pathways that regulate reproduction and cell migration in many endocrine and gonadal tissues. The N-terminally truncated decapeptide, metastin(45-54), has 3-10 times higher receptor affinity and intracellular calcium ion-mobilizing activity but is rapidly inactivated in serum. In this study we designed and synthesized stable KISS1R agonistic decapeptide analogs with selected substitutions at positions 47, 50, and 51. Replacement of glycine with azaglycine (azaGly) in which the α-carbon is replaced with a nitrogen atom at position 51 improved the stability of amide bonds between Phe(50)-Gly(51) and Gly(51)-Leu(52) as determined by in vitro mouse serum stability studies. Substitution for tryptophan at position 47 with other amino acids such as serine, threonine, β-(3-pyridyl)alanine, and D-tryptophan (D-Trp), produced analogs that were highly stable in mouse serum. D-Trp(47) analog 13 showed not only high metabolic stability but also excellent KISS1R agonistic activity. Other labile peptides may have increased serum stability using amino acid substitution. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells.

PubMed

Wan, Qingwen; Okashah, Najeah; Inoue, Asuka; Nehmé, Rony; Carpenter, Byron; Tate, Christopher G; Lambert, Nevin A

2018-05-11

G protein-coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase-mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype-biased ligands. © 2018 Wan et al.

Aging and cholinergic responses in bovine trachealis muscle.

PubMed Central

Wills, M.; Douglas, J. S.

1988-01-01

1. The relative potencies of muscarinic agonists on bovine tracheal smooth muscle were unchanged as a consequence of aging and were carbachol greater than oxotremorine greater than muscarine greater than pilocarpine greater than McNeil A-343. 2. During aging, the potencies of carbachol, oxotremorine, McNeil A-343 and pilocarpine, but not muscarine, were reduced. 3. Maximal induced tensions to all the agents studied were reduced as a consequence of age. 4. Irreversible antagonism with benzilylcholine mustard showed that agonist efficacy was significantly reduced during aging. 5. Estimated receptor occupancy at the EC50 was significantly greater in tracheal tissues from the mature versus immature cows for every agonist studied. 6. The dissociation constants for full agonists (carbachol, oxotremorine and methacholine) were decreased with maturation while the converse was observed with partial agonists (McNeil A-343, pilocarpine). 7. We conclude that there are significant changes in the properties and coupling of muscarinic receptors during aging. These changes may contribute to the reduced airway reactivity seen in vivo. PMID:3390660

Simple semi-automated portable capillary electrophoresis instrument with contactless conductivity detection for the determination of β-agonists in pharmaceutical and pig-feed samples.

PubMed

Nguyen, Thi Anh Huong; Pham, Thi Ngoc Mai; Doan, Thi Tuoi; Ta, Thi Thao; Sáiz, Jorge; Nguyen, Thi Quynh Hoa; Hauser, Peter C; Mai, Thanh Duc

2014-09-19

An inexpensive, robust and easy to use portable capillary electrophoresis instrument with miniaturized high-voltage capacitively coupled contactless conductivity detection was developed. The system utilizes pneumatic operation to manipulate the solutions for all flushing steps. The different operations, i.e. capillary flushing, interface rinsing, and electrophoretic separation, are easily activated by turning an electronic switch. To allow the analysis of samples with limited available volume, and to render the construction less complicated compared to a computer-controlled counterpart, sample injection is carried out hydrodynamically directly from the sample vial into the capillary by manual syphoning. The system is a well performing solution where the financial means for the highly expensive commercial instruments are not available and where the in-house construction of a sophisticated automated instrument is not possible due to limited mechanical and electronic workshop facilities and software programming expertise. For demonstration, the system was employed successfully for the determination of some β-agonists, namely salbutamol, metoprolol and ractopamine down to 0.7ppm in pharmaceutical and pig-feed sample matrices in Vietnam. Copyright © 2014 Elsevier B.V. All rights reserved.

GPR119 agonists: a promising approach for T2DM treatment? A SWOT analysis of GPR119.

PubMed

Kang, Sang-Uk

2013-12-01

Ever since its advent as a promising therapeutic target for type 2 diabetes mellitus (T2DM), G-protein-coupled receptor 119 (GPR119) has received much interest from the pharmaceutical industry. This interest peaked in June 2010, when Sanofi-Aventis agreed to pay Metabolex (Cymabay Therapeutics) US$375 million for MBX-2982, which was a representative orally active GPR119 agonist. However, Sanofi-Aventis opted to terminate the deal in May 2011 and another leading GPR119 agonist, GSK1292263, had a loss of efficacy during its clinical trial. In this review, I discuss the pros and cons of GPR119 through a strengths, weaknesses, opportunities, and threats (SWOT) analysis and propose development strategies for the eventual success of a GPR119 agonist development program. Copyright © 2013 Elsevier Ltd. All rights reserved.

A key agonist-induced conformational change in the cannabinoid receptor CB1 is blocked by the allosteric ligand Org 27569.

PubMed

Fay, Jonathan F; Farrens, David L

2012-09-28

Allosteric ligands that modulate how G protein-coupled receptors respond to traditional orthosteric drugs are an exciting and rapidly expanding field of pharmacology. An allosteric ligand for the cannabinoid receptor CB1, Org 27569, exhibits an intriguing effect; it increases agonist binding, yet blocks agonist-induced CB1 signaling. Here we explored the mechanism behind this behavior, using a site-directed fluorescence labeling approach. Our results show that Org 27569 blocks conformational changes in CB1 that accompany G protein binding and/or activation, and thus inhibit formation of a fully active CB1 structure. The underlying mechanism behind this behavior is that simultaneous binding of Org 27569 produces a unique agonist-bound conformation, one that may resemble an intermediate structure formed on the pathway to full receptor activation.

Constitutive arrestin-mediated desensitization of a human vasopressin receptor mutant associated with nephrogenic diabetes insipidus

PubMed Central

Barak, Larry S.; Oakley, Robert H.; Laporte, Stéphane A.; Caron, Marc G.

2001-01-01

Agonist-dependent desensitization and internalization of G protein-coupled receptors (GPCR) are mediated by the binding of arrestins to phosphorylated receptors. The affinity of arrestins for the phosphorylated GPCR regulates the ability of the internalized receptor to be dephosphorylated and recycled back to the plasma membrane. In this study, we show that the naturally occurring loss of function vasopressin receptor mutation R137H, which is associated with familial nephrogenic diabetes insipidus, induces constitutive arrestin-mediated desensitization. In contrast to the wild-type vasopressin receptor, the nonsignaling R137H receptor is phosphorylated and sequestered in arrestin-associated intracellular vesicles even in the absence of agonist. Eliminating molecular determinants on the receptor that promote high affinity arrestin–receptor interaction reestablishes plasma membrane localization and the ability of the mutated receptors to signal. These findings suggest that unregulated desensitization can contribute to the etiology of a GPCR-based disease, implying that pharmacological targeting of GPCR desensitization may be therapeutically beneficial. PMID:11134505

Constitutive arrestin-mediated desensitization of a human vasopressin receptor mutant associated with nephrogenic diabetes insipidus.

PubMed

Barak, L S; Oakley, R H; Laporte, S A; Caron, M G

2001-01-02

Agonist-dependent desensitization and internalization of G protein-coupled receptors (GPCR) are mediated by the binding of arrestins to phosphorylated receptors. The affinity of arrestins for the phosphorylated GPCR regulates the ability of the internalized receptor to be dephosphorylated and recycled back to the plasma membrane. In this study, we show that the naturally occurring loss of function vasopressin receptor mutation R137H, which is associated with familial nephrogenic diabetes insipidus, induces constitutive arrestin-mediated desensitization. In contrast to the wild-type vasopressin receptor, the nonsignaling R137H receptor is phosphorylated and sequestered in arrestin-associated intracellular vesicles even in the absence of agonist. Eliminating molecular determinants on the receptor that promote high affinity arrestin-receptor interaction reestablishes plasma membrane localization and the ability of the mutated receptors to signal. These findings suggest that unregulated desensitization can contribute to the etiology of a GPCR-based disease, implying that pharmacological targeting of GPCR desensitization may be therapeutically beneficial.

Tethered agonists: a new mechanism underlying adhesion G protein-coupled receptor activation.

PubMed

Schöneberg, Torsten; Liebscher, Ines; Luo, Rong; Monk, Kelly R; Piao, Xianhua

2015-06-01

The family of adhesion G protein-coupled receptors (aGPCRs) comprises 33 members in the human genome, which are subdivided into nine subclasses. Many aGPCRs undergo an autoproteolytic process via their GPCR Autoproteolysis-INducing (GAIN) domain during protein maturation to generate an N- and a C-terminal fragments, NTF and CTF, respectively. The NTF and CTF are non-covalently reassociated on the plasma membrane to form a single receptor unit. How aGPCRs are activated upon ligand binding remains one of the leading questions in the field of aGPCR research. Recent work from our labs and others shows that ligand binding can remove the NTF from the plasma membrane-bound CTF, exposing a tethered agonist which potently activates downstream signaling.

Activation of Adhesion G Protein-coupled Receptors: AGONIST SPECIFICITY OF STACHEL SEQUENCE-DERIVED PEPTIDES.

PubMed

Demberg, Lilian M; Winkler, Jana; Wilde, Caroline; Simon, Kay-Uwe; Schön, Julia; Rothemund, Sven; Schöneberg, Torsten; Prömel, Simone; Liebscher, Ines

2017-03-17

Members of the adhesion G protein-coupled receptor (aGPCR) family carry an agonistic sequence within their large ectodomains. Peptides derived from this region, called the Stachel sequence, can activate the respective receptor. As the conserved core region of the Stachel sequence is highly similar between aGPCRs, the agonist specificity of Stachel sequence-derived peptides was tested between family members using cell culture-based second messenger assays. Stachel peptides derived from aGPCRs of subfamily VI (GPR110/ADGRF1, GPR116/ADGRF5) and subfamily VIII (GPR64/ADGRG2, GPR126/ADGRG6) are able to activate more than one member of the respective subfamily supporting their evolutionary relationship and defining them as pharmacological receptor subtypes. Extended functional analyses of the Stachel sequences and derived peptides revealed agonist promiscuity, not only within, but also between aGPCR subfamilies. For example, the Stachel -derived peptide of GPR110 (subfamily VI) can activate GPR64 and GPR126 (both subfamily VIII). Our results indicate that key residues in the Stachel sequence are very similar between aGPCRs allowing for agonist promiscuity of several Stachel -derived peptides. Therefore, aGPCRs appear to be pharmacologically more closely related than previously thought. Our findings have direct implications for many aGPCR studies, as potential functional overlap has to be considered for in vitro and in vivo studies. However, it also offers the possibility of a broader use of more potent peptides when the original Stachel sequence is less effective. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The G Protein-Coupled Bile Acid Receptor, TGR5, Stimulates Gallbladder Filling

PubMed Central

Li, Tingting; Holmstrom, Sam R.; Kir, Serkan; Umetani, Michihisa; Schmidt, Daniel R.

2011-01-01

TGR5 is a G protein-coupled bile acid receptor present in brown adipose tissue and intestine, where its agonism increases energy expenditure and lowers blood glucose. Thus, it is an attractive drug target for treating human metabolic disease. However, TGR5 is also highly expressed in gallbladder, where its functions are less well characterized. Here, we demonstrate that TGR5 stimulates the filling of the gallbladder with bile. Gallbladder volume was increased in wild-type but not Tgr5−/− mice by administration of either the naturally occurring TGR5 agonist, lithocholic acid, or the synthetic TGR5 agonist, INT-777. These effects were independent of fibroblast growth factor 15, an enteric hormone previously shown to stimulate gallbladder filling. Ex vivo analyses using gallbladder tissue showed that TGR5 activation increased cAMP concentrations and caused smooth muscle relaxation in a TGR5-dependent manner. These data reveal a novel, gallbladder-intrinsic mechanism for regulating gallbladder contractility. They further suggest that TGR5 agonists should be assessed for effects on human gallbladder as they are developed for treating metabolic disease. PMID:21454404

Mechanistic insights into allosteric regulation of the A 2A adenosine G protein-coupled receptor by physiological cations

DOE PAGES

Ye, Libin; Neale, Chris Andrew; Sljoka, Adnan; ...

2018-04-10

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A 2A GPCR. While Na + reinforces an inactive ensemble and a partial-agonist stabilized state, Ca 2+ and Mg 2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridgemore » specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. Lastly, an understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.« less

Hyodeoxycholic acid derivatives as liver X receptor α and G-protein-coupled bile acid receptor agonists

NASA Astrophysics Data System (ADS)

de Marino, Simona; Carino, Adriana; Masullo, Dario; Finamore, Claudia; Marchianò, Silvia; Cipriani, Sabrina; di Leva, Francesco Saverio; Catalanotti, Bruno; Novellino, Ettore; Limongelli, Vittorio; Fiorucci, Stefano; Zampella, Angela

2017-02-01

Bile acids are extensively investigated for their potential in the treatment of human disorders. The liver X receptors (LXRs), activated by oxysterols and by a secondary bile acid named hyodeoxycholic acid (HDCA), have been found essential in the regulation of lipid homeostasis in mammals. Unfortunately, LXRα activates lipogenic enzymes causing accumulation of lipid in the liver. In addition to LXRs, HDCA has been also shown to function as ligand for GPBAR1, a G protein coupled receptor for secondary bile acids whose activation represents a promising approach to liver steatosis. In the present study, we report a library of HDCA derivatives endowed with modulatory activity on the two receptors. The lead optimization of HDCA moiety was rationally driven by the structural information on the binding site of the two targets and results from pharmacological characterization allowed the identification of hyodeoxycholane derivatives with selective agonistic activity toward LXRα and GPBAR1 and notably to the identification of the first example of potent dual LXRα/GPBAR1 agonists. The new chemical entities might hold utility in the treatment of dyslipidemic disorders.

Mechanistic insights into allosteric regulation of the A 2A adenosine G protein-coupled receptor by physiological cations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Libin; Neale, Chris Andrew; Sljoka, Adnan

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A 2A GPCR. While Na + reinforces an inactive ensemble and a partial-agonist stabilized state, Ca 2+ and Mg 2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridgemore » specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. Lastly, an understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.« less

Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site

NASA Astrophysics Data System (ADS)

Wang, Xizhuo; Wang, Youjun; Zhou, Yandong; Hendron, Eunan; Mancarella, Salvatore; Andrake, Mark D.; Rothberg, Brad S.; Soboloff, Jonathan; Gill, Donald L.

2014-02-01

STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca2+ sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca2+ channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.

Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms.

PubMed

Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; El-Fakahany, Esam E; Jakubík, Jan

2015-07-01

We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant xanomeline. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Nucleotides induce chemotaxis and actin polymerization in immature but not mature human dendritic cells via activation of pertussis toxin-sensitive P2y receptors.

PubMed

Idzko, Marco; Dichmann, Stefan; Ferrari, Davide; Di Virgilio, Francesco; la Sala, Andrea; Girolomoni, Giampiero; Panther, Elisabeth; Norgauer, Johannes

2002-08-01

Dendritic cells (DCs) are considered the principal initiators of immune response because of their ability to migrate into peripheral tissues and lymphoid organs, process antigens, and activate naive T cells. There is evidence that extracellular nucleotides regulate certain functions of DCs via G-protein-coupled P2Y receptors (P2YR) and ion-channel-gated P2X receptors (P2XR). Here we investigated the chemotactic activity and analyzed the migration-associated intracellular signaling events such as actin reorganization and Ca(++) transients induced by common P2R agonists such as adenosine 5'-triphosphate (ATP) and 2-methylthioadenosine triphosphate, the P2YR agonists UTP and adenosine 5'-diphosphate (ADP), or the P2XR agonists alphabeta-methylenadenosine-5'-triphosphate and 2',3'-(4-benzoyl)benzoyl-ATP. The common P2R agonists and the selective P2YR agonists turned out to be potent chemotactic stimuli for immature DCs, but not for mature DCs. In contrast, P2XR agonists had only marginal chemotactic activity in both DC types. Chemotaxis was paralleled by a rise in the intracellular Ca(++) concentration and by actin polymerization. Studies with pertussis toxin implicated that intracellular signaling events such as actin polymerization, mobilization of intracellular Ca(++), and migration induced by nucleotides was mediated via G(i/o) protein-coupled P2YR. Moreover, functional studies revealed selective down-regulation of this G(i/o) protein-coupled chemotactic P2YR responsiveness during maturation, although immature and mature DCs expressed similar amounts of mRNA for the P2R subtypes (P2Y(2)R, P2Y(4)R, P2Y(5)R, P2Y(7)R, P2Y(11)R and P2X(1)R, P2X(4)R, P2X(7)R), and no major differences in respect to the mRNA expression of these receptors could be observed by semiquantitative reverse transcription and polymerase chain reaction (RT-PCR). In summary, our data describe a differential chemotactic response of immature and mature DCs to nucleotides, and lend further support to the hypothesis that P2R are a novel class of immunomodulatory plasma membrane receptors suitable for pharmacological intervention.

Simultaneous and confirmative detection of multi-residues of β₂-agonists and β-blockers in urine using LC-MS/MS/MS coupled with β-receptor molecular imprinted polymer SPE clean-up.

PubMed

Fan, S; Zou, J H; Miao, H; Zhao, Y F; Chen, H J; Zhao, R; Wu, Y N

2013-01-01

A liquid chromatography-linear ion-trap spectrometry (LC-MS³) method using β-receptor molecular-imprinted polymer (MIP) solid-phase extraction (SPE) as clean-up was developed to determine simultaneously and confirmatively residues of 25 β₂-agonists and 21 β-blockers in urine samples. Urine samples were subjected to enzymatic hydrolysis by β-glucoronidase/arylsulphatase, and then extracted with perchloric acid. Sample clean-up was performed using β-receptor MIP SPE. A Supelco Ascentis® express Rp-Amide column was used to separate the analytes, and MS³ detection used an electrospray ionisation source in positive-ion mode. Recovery studies were carried out using blank urine samples fortified with the 46 analytes at the levels of 0.5, 1.0 and 2.0 μg l⁻¹. Recoveries were obtained ranging from 60.1% to 109.9% with relative standard deviations (RSDs, n = 7) from 0.5% to 19.4%. The limits of detection (LODs) and limits of quantitation (LOQs) of the 46 analytes in urine were 0.02-0.18 and 0.05-0.60 μg l⁻¹, respectively. As a result of the selective clean-up by MIP SPE and MS³ detection of the target drugs, the sensitivity and accuracy of the present method was high enough for monitoring β₂-agonist and β-blocker residues in urine samples. Satisfactory results were obtained in the process of the determination of positive urine samples.

Minireview: More Than Just a Hammer: Ligand “Bias” and Pharmaceutical Discovery

PubMed Central

2014-01-01

Conventional orthosteric drug development programs targeting G protein-coupled receptors (GPCRs) have focused on the concepts of agonism and antagonism, in which receptor structure determines the nature of the downstream signal and ligand efficacy determines its intensity. Over the past decade, the emerging paradigms of “pluridimensional efficacy” and “functional selectivity” have revealed that GPCR signaling is not monolithic, and that ligand structure can “bias” signal output by stabilizing active receptor states in different proportions than the native ligand. Biased ligands are novel pharmacologic entities that possess the unique ability to qualitatively change GPCR signaling, in effect creating “new receptors” with distinct efficacy profiles driven by ligand structure. The promise of biased agonism lies in this ability to engender “mixed” effects not attainable using conventional agonists or antagonists, promoting therapeutically beneficial signals while antagonizing deleterious ones. Indeed, arrestin pathway-selective agonists for the type 1 parathyroid hormone and angiotensin AT1 receptors, and G protein pathway-selective agonists for the GPR109A nicotinic acid and μ-opioid receptors, have demonstrated unique, and potentially therapeutic, efficacy in cell-based assays and preclinical animal models. Conversely, activating GPCRs in “unnatural” ways may lead to downstream biological consequences that cannot be predicted from prior knowledge of the actions of the native ligand, especially in the case of ligands that selectively activate as-yet poorly characterized G protein-independent signaling networks mediated via arrestins. Although much needs to be done to realize the clinical potential of functional selectivity, biased GPCR ligands nonetheless appear to be important new additions to the pharmacologic toolbox. PMID:24433041

Evaluation of novel TGR5 agonist in combination with Sitagliptin for possible treatment of type 2 diabetes.

PubMed

Agarwal, Sameer; Sasane, Santosh; Kumar, Jeevan; Deshmukh, Prashant; Bhayani, Hitesh; Giri, Poonam; Giri, Suresh; Soman, Shubhangi; Kulkarni, Neelima; Jain, Mukul

2018-06-01

TGR5 is a member of G protein-coupled receptor (GPCR) superfamily, a promising molecular target for metabolic diseases. Activation of TGR5 promotes secretion of glucagon-like peptide-1 (GLP-1), which activates insulin secretion. A series of 2-thio-imidazole derivatives have been identified as novel, potent and orally efficacious TGR5 agonists. Compound 4d, a novel TGR5 agonist, in combination with Sitagliptin, a DPP-4 inhibitor, has demonstrated an adequate GLP-1 secretion and glucose lowering effect in animal models, suggesting a potential clinical option in treatment of type-2 diabetes. Copyright © 2018 Elsevier Ltd. All rights reserved.

G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

PubMed

Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

2014-08-08

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

An integrated mechanism of cardiomyocyte nuclear Ca(2+) signaling.

PubMed

Ibarra, Cristián; Vicencio, Jose Miguel; Varas-Godoy, Manuel; Jaimovich, Enrique; Rothermel, Beverly A; Uhlén, Per; Hill, Joseph A; Lavandero, Sergio

2014-10-01

In cardiomyocytes, Ca(2+) plays a central role in governing both contraction and signaling events that regulate gene expression. Current evidence indicates that discrimination between these two critical functions is achieved by segregating Ca(2+) within subcellular microdomains: transcription is regulated by Ca(2+) release within nuclear microdomains, and excitation-contraction coupling is regulated by cytosolic Ca(2+). Accordingly, a variety of agonists that control cardiomyocyte gene expression, such as endothelin-1, angiotensin-II or insulin-like growth factor-1, share the feature of triggering nuclear Ca(2+) signals. However, signaling pathways coupling surface receptor activation to nuclear Ca(2+) release, and the phenotypic responses to such signals, differ between agonists. According to earlier hypotheses, the selective control of nuclear Ca(2+) signals by activation of plasma membrane receptors relies on the strategic localization of inositol trisphosphate receptors at the nuclear envelope. There, they mediate Ca(2+) release from perinuclear Ca(2+) stores upon binding of inositol trisphosphate generated in the cytosol, which diffuses into the nucleus. More recently, identification of such receptors at nuclear membranes or perinuclear sarcolemmal invaginations has uncovered novel mechanisms whereby agonists control nuclear Ca(2+) release. In this review, we discuss mechanisms for the selective control of nuclear Ca(2+) signals with special focus on emerging models of agonist receptor activation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview, and fundamental considerations for stable and reproducible measurements

PubMed Central

Stolwijk, Judith A.; Matrougui, Khalid; Renken, Christian W.; Trebak, Mohamed

2014-01-01

The past 20 years have seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists, pharmacological and toxicological compounds. Most studies on barrier function use G protein coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance based techniques such as Electric Cell-Substrate Impedance Sensing (ECIS) reside in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine and Sphingosine-1-Phosphate. PMID:25537398

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.

PubMed

Stolwijk, Judith A; Matrougui, Khalid; Renken, Christian W; Trebak, Mohamed

2015-10-01

The past 20 years has seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists and pharmacological and toxicological compounds. Most studies on barrier function use G protein-coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance-based techniques such as electric cell-substrate impedance sensing (ECIS) resides in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications, and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research, little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency, or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling, and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine, and sphingosine-1-phosphate.

Lifetime of muscarinic receptor-G-protein complexes determines coupling efficiency and G-protein subtype selectivity.

PubMed

Ilyaskina, Olga S; Lemoine, Horst; Bünemann, Moritz

2018-05-08

G-protein-coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR-G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of G i/o , G q/11 , G s , and G 12/13 proteins to muscarinic M 1 , M 2 , and M 3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein-M 3 -R interactions but rather the affinities of G q and G o proteins to M 3 -Rs, their GPCR-G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

Oxyntomodulin differentially affects glucagon-like peptide-1 receptor beta-arrestin recruitment and signaling through Galpha(s).

PubMed

Jorgensen, Rasmus; Kubale, Valentina; Vrecl, Milka; Schwartz, Thue W; Elling, Christian E

2007-07-01

The glucagon-like peptide (GLP)-1 receptor is a promising target for the treatment of type 2 diabetes and obesity, and there is great interest in characterizing the pharmacology of the GLP-1 receptor and its ligands. In the present report, we have applied bioluminescence resonance energy transfer assays to measure agonist-induced recruitment of betaarrestins and G-protein-coupled receptor kinase (GRK) 2 to the GLP-1 receptor in addition to traditional measurements of second messenger generation. The peptide hormone oxyntomodulin is described in the literature as a full agonist on the glucagon and GLP-1 receptors. Surprisingly, despite being full agonists in GLP-1 receptor-mediated cAMP accumulation, oxyntomodulin and glucagon were observed to be partial agonists in recruiting betaarrestins and GRK2 to the GLP-1 receptor. We suggest that oxyntomodulin and glucagon are biased ligands on the GLP-1 receptor.

Selective agonists of somatostatin receptor subtype 1 or 2 injected peripherally induce antihyperalgesic effect in two models of visceral hypersensitivity in mice

PubMed Central

Mulak, Agata; Larauche, Muriel; Biraud, Mandy; Million, Mulugeta; Rivier, Jean; Taché, Yvette

2014-01-01

Somatostatin interacts with 5 G-protein-coupled receptor (sst1–5). Octreotide, a stable sst2≫3≥5 agonist, octreotide, exerts a visceral anti-hyperalgesic effect in experimental and clinical studies. Little is known on the receptor subtypes involved. We investigated the influence of the stable sst1–5 agonist, ODT8-SST and selective receptor subtype peptide agonists (3 or 10 μg/mouse) injected intraperitoneally (ip) on visceral hypersensitivity in mice induced by repeated noxious colorectal distensions (4 sets of 3 CRD, each at 55 mmHg) or corticotropin-releasing factor receptor 1 agonist, cortagine given between 2 sets of graded CRD (15, 30, 45, and 60 mmHg, 3 times each pressure). The mean visceromotor response (VMR) was assessed using a non-invasive manometry method and values were expressed as percentage of the VMR to the 1st set of CRD baseline or to the 60 mmHg CRD, respectively. ODT8-SST (10 μg) and the sst2 agonist, S-346-011 (3 and 10 μg) prevented mechanically-induced visceral hypersensitivity in the 3 sets of CRD, the sst1 agonist (10 μg) blocked only the 2nd set and showed a trend at 3 μg while the sst4 agonist had no effect. The selective sst2 antagonist, S-406-028 blocked the sst2 agonist but not the sst1 agonist effect. The sst1 agonist (3 and 10 μg) prevented cortagine-induced hypersensitivity to CRD at each pressure while the sst2 agonist at 10 μg reduced it. These data indicate that in addition to sst2, the sst1 agonist may provide a novel promising target to alleviate visceral hypersensitivity induced by mechanoreceptor sensitization and more prominently, stress-related visceral nociceptive sensitization. PMID:25451334

Selective agonists of somatostatin receptor subtype 1 or 2 injected peripherally induce antihyperalgesic effect in two models of visceral hypersensitivity in mice.

PubMed

Mulak, Agata; Larauche, Muriel; Biraud, Mandy; Million, Mulugeta; Rivier, Jean; Taché, Yvette

2015-01-01

Somatostatin interacts with five G-protein-coupled receptor (sst1-5). Octreotide, a stable sst2≫3≥5 agonist, exerts a visceral anti-hyperalgesic effect in experimental and clinical studies. Little is known on the receptor subtypes involved. We investigated the influence of the stable sst1-5 agonist, ODT8-SST and selective receptor subtype peptide agonists (3 or 10μg/mouse) injected intraperitoneally (ip) on visceral hypersensitivity in mice induced by repeated noxious colorectal distensions (four sets of three CRD, each at 55mmHg) or corticotropin-releasing factor receptor 1 agonist, cortagine given between two sets of graded CRD (15, 30, 45, and 60mmHg, three times each pressure). The mean visceromotor response (VMR) was assessed using a non-invasive manometry method and values were expressed as percentage of the VMR to the 1st set of CRD baseline or to the 60mmHg CRD, respectively. ODT8-SST (10μg) and the sst2 agonist, S-346-011 (3 and 10μg) prevented mechanically induced visceral hypersensitivity in the three sets of CRD, the sst1 agonist (10μg) blocked only the 2nd set and showed a trend at 3μg while the sst4 agonist had no effect. The selective sst2 antagonist, S-406-028 blocked the sst2 agonist but not the sst1 agonist effect. The sst1 agonist (3 and 10μg) prevented cortagine-induced hypersensitivity to CRD at each pressure while the sst2 agonist at 10μg reduced it. These data indicate that in addition to sst2, the sst1 agonist may provide a novel promising target to alleviate visceral hypersensitivity induced by mechanoreceptor sensitization and more prominently, stress-related visceral nociceptive sensitization. Copyright © 2014 Elsevier Inc. All rights reserved.

Cloud-based simulations on Google Exacycle reveal ligand modulation of GPCR activation pathways

NASA Astrophysics Data System (ADS)

Kohlhoff, Kai J.; Shukla, Diwakar; Lawrenz, Morgan; Bowman, Gregory R.; Konerding, David E.; Belov, Dan; Altman, Russ B.; Pande, Vijay S.

2014-01-01

Simulations can provide tremendous insight into the atomistic details of biological mechanisms, but micro- to millisecond timescales are historically only accessible on dedicated supercomputers. We demonstrate that cloud computing is a viable alternative that brings long-timescale processes within reach of a broader community. We used Google's Exacycle cloud-computing platform to simulate two milliseconds of dynamics of a major drug target, the G-protein-coupled receptor β2AR. Markov state models aggregate independent simulations into a single statistical model that is validated by previous computational and experimental results. Moreover, our models provide an atomistic description of the activation of a G-protein-coupled receptor and reveal multiple activation pathways. Agonists and inverse agonists interact differentially with these pathways, with profound implications for drug design.

Phosphorylation and regulation of a Gq/11-coupled receptor by casein kinase 1alpha.

PubMed

Budd, D C; McDonald, J E; Tobin, A B

2000-06-30

Agonist-mediated receptor phosphorylation by one or more of the members of the G-protein receptor kinase (GRK) family is an established model for G-protein-coupled receptor (GPCR) phosphorylation resulting in receptor desensitization. Our recent studies have, however, suggested that an alternative route to GPCR phosphorylation may be an operation involving casein kinase 1alpha (CK1alpha). In the current study we investigate the involvement of CK1alpha in the phosphorylation of the human m3-muscarinic receptor in intact cells. We show that expression of a catalytically inactive mutant of CK1alpha, designed to act in a dominant negative manner, inhibits agonist-mediated receptor phosphorylation by approximately 40% in COS-7 and HEK-293 cells. Furthermore, we present evidence that a peptide corresponding to the third intracellular loop of the m3-muscarinic receptor (Ser(345)-Leu(463)) is an inhibitor of CK1alpha due to its ability to both act as a pseudo-substrate for CK1alpha and form a high affinity complex with CK1alpha. Expression of this peptide was able to reduce both basal and agonist-mediated m3-muscarinic receptor phosphorylation in intact cells. These results support the notion that CK1alpha is able to mediate GPCR phosphorylation in an agonist-dependent manner and that this may provide a novel mechanism for GPCR phosphorylation. The functional role of phosphorylation was investigated using a mutant of the m3-muscarinic receptor that showed an approximately 80% reduction in agonist-mediated phosphorylation. Surprisingly, this mutant underwent agonist-mediated desensitization suggesting that, unlike many GPCRs, desensitization of the m3-muscarinic receptor is not mediated by receptor phosphorylation. The inositol (1,4, 5)-trisphosphate response did, however, appear to be dramatically potentiated in the phosphorylation-deficient mutant indicating that phosphorylation may instead control the magnitude of the initial inositol phosphate response.

A receptor-G protein coupling-independent step in the internalization of the thyrotropin-releasing hormone receptor.

PubMed

Petrou, C; Chen, L; Tashjian, A H

1997-01-24

To determine whether functional receptor-G protein coupling or signaling are required for internalization of the thyrotropin-releasing hormone receptor (TRHR), we compared the endocytosis of Gq-coupled and uncoupled receptors. A hemagglutinin epitope-tagged TRHR (HA-TRHR) was in the Gq-coupled state when bound to the agonist, MeTRH, and in a nonsignaling state when bound to the HA antibody (12CA5). 12CA5 did not induce an increase in [Ca2+]i or inositol phosphates and did not inhibit [3H]MeTRH binding or MeTRH-induced production of second messengers. Both agonist- and antibody-bound HA-TRHRs were rapidly internalized via the same pathway; internalization was sensitive to hypertonic shock, and both types of internalized receptors were sorted into lysosomes. In addition, the amino acid sequence CNC (positions 335-337) in the C-terminal tail of the TRHR, which is important in ligand-induced receptor internalization as determined by deletion mutagenesis (Nussenzveig, D. R., Heinflink, M., and Gershengorn, M. C. (1993) J. Biol. Chem. 268, 2389-2392), was also important for 12CA5-induced internalization. We expressed two truncated receptors, HA-K338STOP and HA-C335STOP, in GH12C1 pituitary cells. Both HA-TRHR and HA-K338STOP were localized at the plasma membrane of untreated cells and were translocated to intracellular vesicles after MeTRH or 12CA5 binding; however, HA-C335STOP was internalized and recycled constitutively. The intracellular localization of HA-C335STOP was not altered by MeTRH; however, 12CA5 binding induced the disappearance of internalized HA-C335STOP and caused its localization at the plasma membrane, indicating that constitutively cycling HA-C335STOP cannot be reinternalized after antibody binding. Thus, amino acids 335-337, which are important for the internalization of Gq-coupled TRHRs, are also required for the sequestration of functionally uncoupled TRHRs, and in addition, they act as an inhibitory signal that prevents constitutive receptor internalization. Specifically, the Cys residues at positions 335 and 337 are important for preventing constitutive TRHR internalization, because a mutant HA-C335S/C337S receptor was sequestered constitutively. We conclude that release from a negative regulatory internalization sequence or domain is important for HA-TRHR internalization and that the role of the CNC sequence in internalization is independent of functional TRHR-Gq coupling.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Jun; Byrne, Noel; Wang, John

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational couplingmore » between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.« less

Small-molecule agonists for the glucagon-like peptide 1 receptor

PubMed Central

Knudsen, Lotte Bjerre; Kiel, Dan; Teng, Min; Behrens, Carsten; Bhumralkar, Dilip; Kodra, János T.; Holst, Jens J.; Jeppesen, Claus B.; Johnson, Michael D.; de Jong, Johannes Cornelis; Jorgensen, Anker Steen; Kercher, Tim; Kostrowicki, Jarek; Madsen, Peter; Olesen, Preben H.; Petersen, Jacob S.; Poulsen, Fritz; Sidelmann, Ulla G.; Sturis, Jeppe; Truesdale, Larry; May, John; Lau, Jesper

2007-01-01

The peptide hormone glucagon-like peptide (GLP)-1 has important actions resulting in glucose lowering along with weight loss in patients with type 2 diabetes. As a peptide hormone, GLP-1 has to be administered by injection. Only a few small-molecule agonists to peptide hormone receptors have been described and none in the B family of the G protein coupled receptors to which the GLP-1 receptor belongs. We have discovered a series of small molecules known as ago-allosteric modulators selective for the human GLP-1 receptor. These compounds act as both allosteric activators of the receptor and independent agonists. Potency of GLP-1 was not changed by the allosteric agonists, but affinity of GLP-1 for the receptor was increased. The most potent compound identified stimulates glucose-dependent insulin release from normal mouse islets but, importantly, not from GLP-1 receptor knockout mice. Also, the compound stimulates insulin release from perfused rat pancreas in a manner additive with GLP-1 itself. These compounds may lead to the identification or design of orally active GLP-1 agonists. PMID:17213325

G protein-coupled estrogen receptor and estrogen receptor ligands regulate colonic motility and visceral pain.

PubMed

Zielińska, M; Fichna, J; Bashashati, M; Habibi, S; Sibaev, A; Timmermans, J-P; Storr, M

2017-07-01

Diarrhea-predominant irritable bowel syndrome (IBS-D) is a functional gastrointestinal (GI) disorder, which occurs more frequently in women than men. The aim of our study was to determine the role of activation of classical estrogen receptors (ER) and novel membrane receptor, G protein-coupled estrogen receptor (GPER) in human and mouse tissue and to assess the possible cross talk between these receptors in the GI tract. Immunohistochemistry was used to determine the expression of GPER in human and mouse intestines. The effect of G-1, a GPER selective agonist, and estradiol, a non-selective ER agonist, on muscle contractility was characterized in isolated preparations of the human and mouse colon. To characterize the effect of G-1 and estradiol in vivo, colonic bead expulsion test was performed. G-1 and estradiol activity on the visceral pain signaling was assessed in the mustard oil-induced abdominal pain model. GPER is expressed in the human colon and in the mouse colon and ileum. G-1 and estradiol inhibited muscle contractility in vitro in human and mouse colon. G-1 or estradiol administered intravenously at the dose of 20 mg/kg significantly prolonged the time to bead expulsion in females. Moreover, G-1 prolonged the time to bead expulsion and inhibited GI hypermotility in both genders. The injection of G-1 or estradiol resulted in a significant reduction in the number of pain-induced behaviors in mice. GPER and ER receptors are involved in the regulation of GI motility and visceral pain. Both may thus constitute an important pharmacological target in the IBS-D therapy. © 2017 John Wiley & Sons Ltd.

Isolated dorsal root ganglion neurones inhibit receptor-dependent adenylyl cyclase activity in associated glial cells

PubMed Central

Ng, KY; Yeung, BHS; Wong, YH; Wise, H

2013-01-01

Background and Purpose Hyper-nociceptive PGE2 EP4 receptors and prostacyclin (IP) receptors are present in adult rat dorsal root ganglion (DRG) neurones and glial cells in culture. The present study has investigated the cell-specific expression of two other Gs-protein coupled hyper-nociceptive receptor systems: β-adrenoceptors and calcitonin gene-related peptide (CGRP) receptors in isolated DRG cells and has examined the influence of neurone–glial cell interactions in regulating adenylyl cyclase (AC) activity. Experimental Approach Agonist-stimulated AC activity was determined in mixed DRG cell cultures from adult rats and compared with activity in DRG neurone-enriched cell cultures and pure DRG glial cell cultures. Key Results Pharmacological analysis showed the presence of Gs-coupled β2-adrenoceptors and CGRP receptors, but not β1-adrenoceptors, in all three DRG cell preparations. Agonist-stimulated AC activity was weakest in DRG neurone-enriched cell cultures. DRG neurones inhibited IP receptor-stimulated glial cell AC activity by a process dependent on both cell–cell contact and neurone-derived soluble factors, but this is unlikely to involve purine or glutamine receptor activation. Conclusions and Implications Gs-coupled hyper-nociceptive receptors are readily expressed on DRG glial cells in isolated cell cultures and the activity of CGRP, EP4 and IP receptors, but not β2-adrenoceptors, in glial cells is inhibited by DRG neurones. Studies using isolated DRG cells should be aware that hyper-nociceptive ligands may stimulate receptors on glial cells in addition to neurones, and that variable numbers of neurones and glial cells will influence absolute measures of AC activity and affect downstream functional responses. PMID:22924655

Ligand-binding pocket shape differences between S1P1 and S1P3 determine efficiency of chemical probe identification by uHTS

PubMed Central

Schürer, Stephan C.; Brown, Steven J.; Cabrera, Pedro Gonzales; Schaeffer, Marie-Therese; Chapman, Jacqueline; Jo, Euijung; Chase, Peter; Spicer, Tim; Hodder, Peter; Rosen, Hugh

2008-01-01

We have studied the Sphingosine 1-phosphate (S1P) receptor system to better understand why certain molecular targets within a closely related family are much more tractable when identifying compelling chemical leads. Five medically important G protein-coupled receptors for S1P regulate heart rate, coronary artery caliber, endothelial barrier integrity, and lymphocyte trafficking. Selective S1P receptor agonist probes would be of great utility to study receptor subtype-specific function. Through systematic screening of the same libraries, we identified novel selective agonists chemotypes for each of the S1P1 and S1P3 receptors. uHTS for S1P1 was more effective than for S1P3, with many selective, low nanomolar hits of proven mechanism emerging for. Receptor structure modeling and ligand docking reveal differences between the receptor binding pockets, which are the basis for sub-type selectivity. Novel selective agonists interact primarily in the hydrophobic pocket of the receptor in the absence of head-group interactions. Chemistry-space and shape-based analysis of the screening libraries in combination with the binding models explain the observed differential hit rates and enhanced efficiency for lead discovery for S1P1 vs. S1P3 in this closely related receptor family. PMID:18590333

Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor*

PubMed Central

Redka, Dar'ya S.; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V.; Ellis, John; Ernst, Oliver P.; Wells, James W.

2014-01-01

G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5′-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[3H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the “ternary complex model”). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. PMID:25023280

Agonists for G-protein-coupled receptor 84 (GPR84) alter cellular morphology and motility but do not induce pro-inflammatory responses in microglia.

PubMed

Wei, Li; Tokizane, Kyohei; Konishi, Hiroyuki; Yu, Hua-Rong; Kiyama, Hiroshi

2017-10-03

Several G-protein-coupled receptors (GPCRs) have been shown to be important signaling mediators between neurons and glia. In our previous screening for identification of nerve injury-associated GPCRs, G-protein-coupled receptor 84 (GPR84) mRNA showed the highest up-regulation by microglia after nerve injury. GPR84 is a pro-inflammatory receptor of macrophages in a neuropathic pain mouse model, yet its function in resident microglia in the central nervous system is poorly understood. We used endogenous, natural, and surrogate agonists for GPR84 (capric acid, embelin, and 6-OAU, respectively) and examined their effect on mouse primary cultured microglia in vitro. 6-n-Octylaminouracil (6-OAU), embelin, and capric acid rapidly induced membrane ruffling and motility in cultured microglia obtained from C57BL/6 mice, although these agonists failed to promote microglial pro-inflammatory cytokine expression. Concomitantly, 6-OAU suppressed forskolin-induced increase of cAMP in cultured microglia. Pertussis toxin, an inhibitor of Gi-coupled signaling, completely suppressed 6-OAU-induced microglial membrane ruffling and motility. In contrast, no 6-OAU-induced microglial membrane ruffling and motility was observed in microglia from DBA/2 mice, a mouse strain that does not express functional GPR84 protein due to endogenous nonsense mutation of the GPR84 gene. GPR84 mediated signaling causes microglial motility and membrane ruffling but does not promote pro-inflammatory responses. As GPR84 is a known receptor for medium-chain fatty acids, those released from damaged brain cells may be involved in the enhancement of microglial motility through GPR84 after neuronal injury.

Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor.

PubMed

Redka, Dar'ya S; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V; Ellis, John; Ernst, Oliver P; Wells, James W

2014-08-29

G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5'-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[(3)H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the "ternary complex model"). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Diindolylmethane Derivatives: Potent Agonists of the Immunostimulatory Orphan G Protein-Coupled Receptor GPR84.

PubMed

Pillaiyar, Thanigaimalai; Köse, Meryem; Sylvester, Katharina; Weighardt, Heike; Thimm, Dominik; Borges, Gleice; Förster, Irmgard; von Kügelgen, Ivar; Müller, Christa E

2017-05-11

The G i protein-coupled receptor GPR84, which is activated by (hydroxy)fatty acids, is highly expressed on immune cells. Recently, 3,3'-diindolylmethane was identified as a heterocyclic, nonlipid-like GPR84 agonist. We synthesized a broad range of diindolylmethane derivatives by condensation of indoles with formaldehyde in water under microwave irradiation. The products were evaluated at the human GPR84 in cAMP and β-arrestin assays. Structure-activity relationships (SARs) were steep. 3,3'-Diindolylmethanes bearing small lipophilic residues at the 5- and/or 7-position of the indole rings displayed the highest activity in cAMP assays, the most potent agonists being di(5-fluoro-1H-indole-3-yl)methane (38, PSB-15160, EC 50 80.0 nM) and di(5,7-difluoro-1H-indole-3-yl)methane (57, PSB-16671, EC 50 41.3 nM). In β-arrestin assays, SARs were different, indicating biased agonism. The new compounds were selective versus related fatty acid receptors and the arylhydrocarbon receptor. Selected compounds were further investigated and found to display an ago-allosteric mechanism of action and increased stability in comparison to the lead structure.

Development of dopamine receptor radiopharmaceuticals for the study of neurological and psychiatric disorders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dr. Jogeshwar Mukherjee

Our goals in this grant application are directed towards the development of radiotracers that may allow the study of the high-affinity state (functional state) of the dopamine receptors. There have been numerous reports on the presence of two inter-convertible states of these (G-protein coupled) receptors in vitro. However, there is no report that establishes the presence of these separate affinity states in vivo. We have made efforts in this direction in order to provide such direct in vivo evidence about the presence of the high affinity state. This understanding of the functional state of the receptors is of critical significancemore » in our overall diagnosis and treatment of diseases that implicate the G-protein coupled receptors. Four specific aims have been listed in the grant application: (1). Design and syntheses of agonists (2). Radiosyntheses of agonists (3). In vitro pharmacology of agonists (4). In vivo distribution and pharmacology of labeled derivatives. We have accomplished the syntheses and radiosyntheses of three agonist radiotracers labeled with carbon-11. In vitro and in vivo pharmacological experiments have been accomplished in rats and preliminary PET studies in non-human primates have been carried out. Various accomplishments during the funded years, briefly outlined in this document, have been disseminated by several publications in various journals and presentations in national and international meetings (Society of Nuclear Medicine, Society for Neuroscience and International Symposium on Radiopharmaceutical Chemistry).« less

A search for presynaptic inhibitory histamine receptors in guinea-pig tissues: Further H3 receptors but no evidence for H4 receptors.

PubMed

Petri, Doris; Schlicker, Eberhard

2016-07-01

The histamine H4 receptor is coupled to Gi/o proteins and expressed on inflammatory cells and lymphoid tissues; it was suggested that this receptor also occurs in the brain or on peripheral neurones. Since many Gi/o protein-coupled receptors, including the H3 receptor, serve as presynaptic inhibitory receptors, we studied whether the sympathetic neurones supplying four peripheral tissues and the cholinergic neurones in the hippocampus from the guinea-pig are equipped with release-modulating H4 and H3 receptors. For this purpose, we preincubated tissue pieces from the aorta, atrium, renal cortex and vas deferens with (3)H-noradrenaline and hippocampal slices with (3)H-choline and determined the electrically evoked tritium overflow. The stimulation-evoked overflow in the five superfused tissues was inhibited by the muscarinic receptor agonist oxotremorine, which served as a positive control, but not affected by the H4 receptor agonist 4-methylhistamine. The H3 receptor agonist R-α-methylhistamine inhibited noradrenaline release in the peripheral tissues without affecting acetylcholine release in the hippocampal slices. Thioperamide shifted the concentration-response curve of histamine in the aorta and the renal cortex to the right, yielding apparent pA2 values of 8.0 and 8.1, respectively, which are close to its affinity at other H3 receptors but higher by one log unit than its pKi at the H4 receptor of the guinea-pig. In conclusion, histamine H4 receptors could not be identified in five experimental models of the guinea-pig that are suited for the detection of presynaptic inhibitory receptors whereas H3 receptors could be shown in the peripheral tissues but not in the hippocampus. This article is part of the Special Issue entitled 'Histamine Receptors'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cat iris sphincter smooth-muscle contraction: comparison of FP-class prostaglandin analog agonist activities.

PubMed

Sharif, Najam A; Kaddour-Djebbar, Ismail; Abdel-Latif, Ata A

2008-04-01

The pharmacologic characteristics of a number of FP-class prostaglandin (PG) analogs were determined by using the cat iris sphincter smooth-muscle-contraction assay. Cumulative concentration-response curves were generated for each compound. The relative agonist potencies (EC(50)) of the compounds were: cloprostenol (0.0012 +/- 0.0004 nM) > travoprost acid (0.46 +/- 0.13 nM) = bimatoprost acid (0.99 +/- 0.19 nM) > (+/-)-fluprostenol (15.8 +/- 2.6 nM) = PGF(2alpha) (18.6 +/- 1.8 nM) > latanoprost acid (29.9 +/- 1.6 nM) > bimatoprost (140 +/- 45 nM) > S-1033 (588 +/- 39 nM) > unoprostone (UF-021; 1280 +/- 50 nM; n = 4-14). The maximum response induced by travoprost acid (122% +/- 2.3% maximum response relative to PGF(2alpha)) was significantly greater than that induced by all the other PG compounds (P < 0.001 - P < 0.02). Interestingly, the FP-receptor antagonist, AL-8810, behaved as a moderate efficacy partial agonist (EC(50) = 2140 +/- 190 nM; 63 +/- 4.3% maximum response relative to PGF(2alpha)), indicating that the cat iris contains an extremely well-coupled FP-receptor population, and/or the tissue contains an extremely high density of the FP-receptor and/or spare receptors. The cat iris contraction data were well correlated with other FP-receptor-mediated signal-transduction processes, including FP-receptor binding in bovine corpus luteum (r = 0.86), FP-receptor binding in human iris (r = 0.61), phosphoinositide (PI) hydrolysis in human ciliary muscle and trabecular meshwork cells (r = 0.77 - 0.86), PI turnover in rat and mouse cells (r = 0.73 - 0.76) and via cloned human FP-receptor (r = 0.9), and rat uterus contraction (r = 0.84). These data confirm the presence of functional FP-receptors in the cat iris sphincter, which are exquisitely well coupled and which respond to a variety of FP-class PG analogs with differing potencies.

Functional interdependence of neurons in a single canine intrinsic cardiac ganglionated plexus

PubMed Central

Thompson, G W; Collier, K; Ardell, J L; Kember, G; Armour, J A

2000-01-01

To determine the activity characteristics displayed by different subpopulations of neurons in a single intrinsic cardiac ganglionated plexus, the behaviour and co-ordination of activity generated by neurons in two loci of the right atrial ganglionated plexus (RAGP) were evaluated in 16 anaesthetized dogs during basal states as well as in response to increasing inputs from ventricular sensory neurites. These sub-populations of right atrial neurons received afferent inputs from sensory neurites in both ventricles that were responsive to local mechanical stimuli and the nitric oxide donor nitroprusside. Neurons in at least one RAGP locus were activated by epicardial application of veratridine, bradykinin, the β1-adrenoceptor agonist prenaterol or glutamate. Epicardial application of angiotensin II, the selective β2-adrenoceptor agonist terbutaline and selective α-adrenoceptor agonists elicited inconsistent neuronal responses. The activity generated by both populations of atrial neurons studied over 5 min periods during basal states displayed periodic coupled behaviour (cross-correlation coefficients of activities that reached, on average, 0·88 ± 0·03; range 0·71–1) for 15–30 s periods of time. These periods of coupled activity occurred every 30–50 s during basal states, as well as when neuronal activity was enhanced by chemical activation of their ventricular sensory inputs. These results indicate that neurons throughout one intrinsic cardiac ganglionated plexus receive inputs from mechano- and chemosensory neurites located in both ventricles. That such neurons respond to multiple chemical stimuli, including those liberated from adjacent adrenergic efferent nerve terminals, indicates the complexity of the integrative processing of information that occurs within the intrinsic cardiac nervous system. It is proposed that the interdependent activity displayed by populations of neurons in different regions of one intrinsic cardiac ganglionated plexus, responding as they do to multiple cardiac sensory inputs, forms the basis for integrated regional cardiac control. PMID:11060132

Magnetic molecularly imprinted polymers for the determination of β-agonist residues in milk by ultra high performance liquid chromatography with tandem mass spectrometry.

PubMed

Liu, Hongcheng; Lin, Xin; Lin, Tao; Zhang, Yulong; Luo, Yinglan; Li, Qiwan

2016-09-01

A simple, accurate, and highly sensitive analytical method was developed in this study for the determination of nine β-agonists in milk. In this method, a new magnetic adsorbent of molecularly imprinted polymers/magnetic nanoparticles prepared by simple physical blending was adopted, which enabled magnetic solid-phase extraction. Thus, the resultant material can be separated from the solvent rapidly and conveniently by a magnet. Two kinds of molecularly imprinted polymer/magnetic nanoparticles materials were fabricated, and the characteristics of materials such as the ratio, pH, amount, desorption, and regeneration were investigated. The analytes were quantified by ultra high performance liquid chromatography coupled to an electrospray ionization tandem mass spectrometer operating in multiple reaction monitoring modes. The detection limit of the method was 0.003-0.3 μg/L, and the detection capability was 0.01-0.3 μg/L. The recoveries of these compounds were 65.7-114% at three spiked levels. Reproducibility represented by relative standard deviation was 11.2% or less. The method was successfully applied to the screening of real samples obtained from local markets and confirmation of the suspected target analytes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

β-Hydroxybutyrate modulates N-type calcium channels in rat sympathetic neurons by acting as an agonist for the G-protein-coupled receptor FFA3.

PubMed

Won, Yu-Jin; Lu, Van B; Puhl, Henry L; Ikeda, Stephen R

2013-12-04

Free fatty acids receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43), for which the short-chain fatty acids (SCFAs) acetate and propionate are agonist, have emerged as important G-protein-coupled receptors influenced by diet and gut flora composition. A recent study (Kimura et al., 2011) demonstrated functional expression of FFA3 in the rodent sympathetic nervous system (SNS) providing a potential link between nutritional status and autonomic function. However, little is known of the source of endogenous ligands, signaling pathways, or effectors in sympathetic neurons. In this study, we found that FFA3 and FFA2 are unevenly expressed in the rat SNS with higher transcript levels in prevertebral (e.g., celiac-superior mesenteric and major pelvic) versus paravertebral (e.g., superior cervical and stellate) ganglia. FFA3, whether heterologously or natively expressed, coupled via PTX-sensitive G-proteins to produce voltage-dependent inhibition of N-type Ca(2+) channels (Cav2.2) in sympathetic neurons. In addition to acetate and propionate, we show that β-hydroxybutyrate (BHB), a metabolite produced during ketogenic conditions, is also an FFA3 agonist. This contrasts with previous interpretations of BHB as an antagonist at FFA3. Together, these results indicate that endogenous BHB levels, especially when elevated under certain conditions, such as starvation, diabetic ketoacidosis, and ketogenic diets, play a potentially important role in regulating the activity of the SNS through FFA3.

Activation of Cyclic AMP Synthesis by Full and Partial Beta-Adrenergic Receptor Agonists in Chicken Skeletal Muscle Cells

NASA Technical Reports Server (NTRS)

Young, R. B.; Bridge, K. Y.

2003-01-01

Several beta-adrenergic receptor (bAR) agonists are known to cause hypertrophy of skeletal muscle tissue. Accordingly, five bAR agonists encompassing a range in activity from strong to weak were evaluated for their ability to stimulate CAMP accumulation in embryonic chicken skeletal muscle cells in culture. Two strong agonists (epinephrine and isoproterenol), one moderate agonist (albuterol), and two weak agonists known to cause hypertrophy in animals (clenbuterol and cimaterol) were studied. Dose response curves were determined over six orders of magnitude in concentration for each agonist, and values were determined for their maximum stimulation of CAMP synthesis rate (Bmax) and the agonist concentration at which 50% stimulation of CAMP synthesis (EC50) occurred. Bmax values decreased in the following order: isoproterenol, epinephrine, albuterol, cimaterol, clenbuterol. Cimaterol and clenbuterol at their Bmax concentrations were approximately 15-fold weaker than isoproterenol in stimulating the rate of CAMP synthesis. When cimaterol and clenbuterol were added to culture media at concentrations known to cause significant muscle hypertrophy in animals, there was no detectable effect on stimulation of CAMP synthesis. Finally, these same levels of cimaterol and clenbuterol did not antagonize the stimulation of CAMP by either epinephrine or isoproterenol.

Identification of novel selective V2 receptor non-peptide agonists.

PubMed

Del Tredici, Andria L; Vanover, Kim E; Knapp, Anne E; Bertozzi, Sine M; Nash, Norman R; Burstein, Ethan S; Lameh, Jelveh; Currier, Erika A; Davis, Robert E; Brann, Mark R; Mohell, Nina; Olsson, Roger; Piu, Fabrice

2008-10-30

Peptides with agonist activity at the vasopressin V(2) receptor are used clinically to treat fluid homeostasis disorders such as polyuria and central diabetes insipidus. Of these peptides, the most commonly used is desmopressin, which displays poor bioavailability as well as potent activity at the V(1b) receptor, with possible stress-related adverse effects. Thus, there is a strong need for the development of small molecule chemistries with selective V(2) receptor agonist activity. Using the functional cell-based assay Receptor Selection and Amplification Technology (R-SAT((R))), a screening effort identified three small molecule chemotypes (AC-94544, AC-88324, and AC-110484) with selective agonist activity at the V(2) receptor. One of these compounds, AC-94544, displayed over 180-fold selectivity at the V(2) receptor compared to related vasopressin and oxytocin receptors and no activity at 28 other G protein-coupled receptors (GPCRs). All three compounds also showed partial agonist activity at the V(2) receptor in a cAMP accumulation assay. In addition, in a rat model of central diabetes insipidus, AC-94544 was able to significantly reduce urine output in a dose-dependent manner. Thus, AC-94544, AC-88324, and AC-110484 represent novel opportunities for the treatment of disorders associated with V(2) receptor agonist deficiency.

Development of a Efficient and Sensitive Dispersive Liquid–Liquid Microextraction Technique for Extraction and Preconcentration of 10 β2-Agonists in Animal Urine

PubMed Central

2015-01-01

Dispersive liquid–liquid microextraction (DLLME) coupled with ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) was developed for the extraction and determination of 10 β2-agonists in animal urine. Some experimental parameters, such as the type and volume of the extraction solvent, the concentration of the dispersant, the salt concentration, the pH value of the sample solution, the extraction time and the speed of centrifugation, were investigated and optimized. Under the optimized conditions, a good enrichment factors (4.8 to 32.3) were obtained for the extraction. The enrichment factor show that the concentration rate of DLLME is significantly higher than other pretreatment methods, and the detection sensitivity has been greatly improved. The calibration curves were linear, the correlation coefficient ranged from 0.9928 to 0.9999 for the concentration range of 0.05 to 50 ngmL-1 and 0.1 to 50 ngmL-1, and the relative standard deviations (RSDs, n = 15, intra and inter-day precision) at a concentration of 5 ngmL-1 were in the range of 1.8 to 14.6%. The limits of detection (LODs) for the 10 β2-agonists, based on a signal-to-noise ratio (S/N) of 3, were in the range of 0.01 to 0.03 ngmL-1. The proposed method was used to identify β2-agonists in three types of animal urine (swine, cattle, sheep), and the relative recoveries from each matrix were in the range of 89.2 to 106.8%, 90.0 to 109.8% and 89.2 to 107.2%, respectively. PMID:26348922

Determination of beta-agonists in swine hair by μFIA and chemiluminescence.

PubMed

Chen, Xu; Luo, Yong; Shi, Bo; Gao, Zhigang; Du, Yuguang; Liu, Xianming; Zhao, Weijie; Lin, Bingcheng

2015-04-01

β-Agonists are a group of illegal feed additives. In this paper, it was found that the light emission produced by the oxidation of luminol by potassium ferricyanide was enhanced by the β-agonists (ractopamine, salbutamol, and terbutaline). Based on chemiluminescence phenomenon, a novel, rapid, and sensitive microflow injection analysis system on a microfluidic glass chip was established for determination of the β-agonists. The chip was fabricated from two glass plates (64 mm × 32 mm) with microchannels of 200 μm width and 100 μm depth. The detection limits were achieved at 2.0 × 10(-8) mol/L of ractopamine, 1.0 × 10(-8) mol/L of terbutaline and 5.0 × 10(-7) mol/L of salbutamol. In this report, our method was applied for determination of the β-agonists in swine hair from three different sources with satisfactory results. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural basis for regulation of human calcium-sensing receptor by magnesium ions and an unexpected tryptophan derivative co-agonist.

PubMed

Zhang, Chen; Zhang, Tuo; Zou, Juan; Miller, Cassandra Lynn; Gorkhali, Rakshya; Yang, Jeong-Yeh; Schilmiller, Anthony; Wang, Shuo; Huang, Kenneth; Brown, Edward M; Moremen, Kelley W; Hu, Jian; Yang, Jenny J

2016-05-01

Ca(2+)-sensing receptors (CaSRs) modulate calcium and magnesium homeostasis and many (patho)physiological processes by responding to extracellular stimuli, including divalent cations and amino acids. We report the first crystal structure of the extracellular domain (ECD) of human CaSR bound with Mg(2+) and a tryptophan derivative ligand at 2.1 Å. The structure reveals key determinants for cooperative activation by metal ions and aromatic amino acids. The unexpected tryptophan derivative was bound in the hinge region between two globular ECD subdomains, and represents a novel high-affinity co-agonist of CaSR. The dissection of structure-function relations by mutagenesis, biochemical, and functional studies provides insights into the molecular basis of human diseases arising from CaSR mutations. The data also provide a novel paradigm for understanding the mechanism of CaSR-mediated signaling that is likely shared by the other family C GPCR [G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor] members and can facilitate the development of novel CaSR-based therapeutics.

Analysis of L-serine-O-phosphate in cerebrospinal spinal fluid by derivatization-liquid chromatography/mass spectrometry.

PubMed

McNaney, Colleen A; Benitex, Yulia; Luchetti, David; Labasi, Jeffrey M; Olah, Timothy V; Morgan, Daniel G; Drexler, Dieter M

2014-05-01

L-serine-O-phosphate (L-SOP), the precursor of L-serine, is a potent agonist against the group III metabotropic glutamate receptors (mGluRs) and, thus, is of interest as a potential biomarker for monitoring modulation of neurotransmitter release. So far, no reports are available on the analysis of L-SOP in cerebrospinal fluid (CSF). Here a novel method is presented to determine L-SOP levels in CSF employing precolumn derivatization with (5-N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) coupled to liquid chromatography/mass spectrometry (derivatization-LC/MS, d-LC/MS). Copyright © 2014 Elsevier Inc. All rights reserved.

Co-Expression of Regulator of G Protein Signaling 4 (RGS4) and the MU Opioid Receptor in Regions of Rat Brain: Evidence That RGS4 Attenuates MU Opioid Receptor Signaling

DTIC Science & Technology

2003-01-01

coupled receptor signal transduction proposes that agonist-induced conformational changes in the receptor result in an enhanced release of GDP...Regulators of G protein Signalling (RGS) proteins influence G protein-coupled receptor signal transduction by enhancing the intrinsic GTPase activity...of G proteins. The RGS- enhanced GTPase activity of G proteins may be responsible for the desensitization of certain G protein-coupled receptors

Differential requirements of arrestin-3 and clathrin for ligand-dependent and -independent internalization of human G protein-coupled receptor 40.

PubMed

Qian, Jing; Wu, Chun; Chen, Xiaopan; Li, Xiangmei; Ying, Guoyuan; Jin, Lili; Ma, Qiang; Li, Guo; Shi, Ying; Zhang, Guozheng; Zhou, Naiming

2014-11-01

G protein-coupled receptor 40 (GPR40) is believed to be an attractive target to enhance insulin secretion in patients with type 2 diabetes. GPR40 has been found to couple to Gq protein, leading to the activation of phospholipase C and subsequent increases in the intracellular Ca(2+) level. However, the underlying mechanisms that regulate the internalization and desensitization of GPR40 remain to be elucidated. In the present study, a construct of GPR40 fused with enhanced green fluorescent protein (EGFP) at its C-terminus was constructed for direct imaging of the localization and internalization of GPR40 by confocal microscopy. In stably transfected HEK-293 cells, GPR40 receptors underwent rapid agonist-induced internalization and constitutive ligand-independent internalization. Our data demonstrated that the agonist-mediated internalization of GPR40 was significantly blocked by hypertonic sucrose treatment and by siRNA mediated depletion of the heavy chain of clathrin. In contrast, constitutive GPR40 internalization was not affected by hypertonic sucrose or by knock-down of clathrin expression, but it was affected by treatment with methyl-β-cyclodextrin (MβCD) and nystatin. Furthermore, our results using an arrestin-3-EGFP redistribution assay and siRNA-mediated knock-down of arrestin-3 and GRK2 expression revealed that arrestin-3 and GRK2 play an essential role in the regulation of agonist-mediated GPR40 internalization, but are not involved in the regulation of constitutive GPR40 internalization. Additionally, our observation showed that upon activation by agonist, the internalized GPR40 receptors were rapidly recycled back to the plasma membrane via Rab4/Rab5 positive endosomes, whereas the constitutively internalized GPR40 receptors were recycled back to the cell surface through Rab5 positive endosomes. Because FFA receptors exhibit a high level of homology, our observations could be applicable to other members of this family. Copyright © 2014 Elsevier Inc. All rights reserved.

Functional efficacy of adenosine A2A receptor agonists is positively correlated to their receptor residence time

PubMed Central

Guo, Dong; Mulder-Krieger, Thea; IJzerman, Adriaan P; Heitman, Laura H

2012-01-01

BACKGROUND AND PURPOSE The adenosine A2A receptor belongs to the superfamily of GPCRs and is a promising therapeutic target. Traditionally, the discovery of novel agents for the A2A receptor has been guided by their affinity for the receptor. This parameter is determined under equilibrium conditions, largely ignoring the kinetic aspects of the ligand-receptor interaction. The aim of this study was to assess the binding kinetics of A2A receptor agonists and explore a possible relationship with their functional efficacy. EXPERIMENTAL APPROACH We set up, validated and optimized a kinetic radioligand binding assay (a so-called competition association assay) at the A2A receptor from which the binding kinetics of unlabelled ligands were determined. Subsequently, functional efficacies of A2A receptor agonists were determined in two different assays: a novel label-free impedance-based assay and a more traditional cAMP determination. KEY RESULTS A simplified competition association assay yielded an accurate determination of the association and dissociation rates of unlabelled A2A receptor ligands at their receptor. A correlation was observed between the receptor residence time of A2A receptor agonists and their intrinsic efficacies in both functional assays. The affinity of A2A receptor agonists was not correlated to their functional efficacy. CONCLUSIONS AND IMPLICATIONS This study indicates that the molecular basis of different agonist efficacies at the A2A receptor lies within their different residence times at this receptor. PMID:22324512

Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target.

PubMed

Brown, Dean G; Brown, Giles A; Centrella, Paolo; Certel, Kaan; Cooke, Robert M; Cuozzo, John W; Dekker, Niek; Dumelin, Christoph E; Ferguson, Andrew; Fiez-Vandal, Cédric; Geschwindner, Stefan; Guié, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Schlenker, Oliver; Sigel, Eric A; Snijder, Arjan; Soutter, Holly T; Sundström, Linda; Troast, Dawn M; Wiggin, Giselle; Zhang, Jing; Zhang, Ying; Clark, Matthew A

2018-06-01

The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.

Synthesis and pharmacological characterization of novel inverse agonists acting on the viral-encoded chemokine receptor US28.

PubMed

Hulshof, Janneke W; Vischer, Henry F; Verheij, Mark H P; Fratantoni, Silvina A; Smit, Martine J; de Esch, Iwan J P; Leurs, Rob

2006-11-01

G-protein coupled receptors encoded by viruses represent an unexplored class of potential drug targets. In this study, we describe the synthesis and pharmacological characterization of the first class of inverse agonists acting on the HCMV-encoded receptor US28. It is shown that replacement of the 4-hydroxy group of lead compound 1 with a methylamine group results in a significant 6-fold increase in affinity. Interestingly, increasing the rigidity of the spacer by the introduction of a double bond also leads to a significant increase in binding affinity compared to 1. These novel inverse agonists serve as valuable tools to elucidate the role of constitutive signaling in the pathogenesis of viral infection and may have therapeutic potential as leads for new antiviral drugs.

GnRH antagonist versus long agonist protocols in IVF: a systematic review and meta-analysis accounting for patient type.

PubMed

Lambalk, C B; Banga, F R; Huirne, J A; Toftager, M; Pinborg, A; Homburg, R; van der Veen, F; van Wely, M

2017-09-01

Most reviews of IVF ovarian stimulation protocols have insufficiently accounted for various patient populations, such as ovulatory women, women with polycystic ovary syndrome (PCOS) or women with poor ovarian response, and have included studies in which the agonist or antagonist was not the only variable between the compared study arms. The aim of the current study was to compare GnRH antagonist protocols versus standard long agonist protocols in couples undergoing IVF or ICSI, while accounting for various patient populations and treatment schedules. The Cochrane Menstrual Disorders and Subfertility Review Group specialized register of controlled trials and Pubmed and Embase databases were searched from inception until June 2016. Eligible trials were those that compared GnRH antagonist protocols and standard long GnRH agonist protocols in couples undergoing IVF or ICSI. The primary outcome was ongoing pregnancy rate. Secondary outcomes were: live birth rate, clinical pregnancy rate, number of oocytes retrieved and safety with regard to ovarian hyperstimulation syndrome (OHSS). Separate comparisons were performed for the general IVF population, women with PCOS and women with poor ovarian response. Pre-planned subgroup analyses were performed for various antagonist treatment schedules. We included 50 studies. Of these, 34 studies reported on general IVF patients, 10 studies reported on PCOS patients and 6 studies reported on poor responders. In general IVF patients, ongoing pregnancy rate was significantly lower in the antagonist group compared with the agonist group (RR 0.89, 95% CI 0.82-0.96). In women with PCOS and in women with poor ovarian response, there was no evidence of a difference in ongoing pregnancy between the antagonist and agonist groups (RR 0.97, 95% CI 0.84-1.11 and RR 0.87, 95% CI 0.65-1.17, respectively). Subgroup analyses for various antagonist treatment schedules compared to the long protocol GnRH agonist showed a significantly lower ongoing pregnancy rate when the oral hormonal programming pill (OHP) pretreatment was combined with a flexible protocol (RR 0.74, 95% CI 0.59-0.91) while without OHP, the RR was 0.84, 95% CI 0.71-1.0. Subgroup analysis for the fixed antagonist schedule demonstrated no evidence of a significant difference with or without OHP (RR 0.94, 95% CI 0.79-1.12 and RR 0.94, 95% CI 0.83-1.05, respectively). Antagonists resulted in significantly lower OHSS rates both in the general IVF patients and in women with PCOS (RR 0.63, 95% CI 0.50-0.81 and RR 0.53, 95% CI 0.30-0.95, respectively). No data on OHSS was available from trials in poor responders. In a general IVF population, GnRH antagonists are associated with lower ongoing pregnancy rates when compared to long protocol agonists, but also with lower OHSS rates. Within this population, antagonist treatment prevents one case of OHSS in 40 patients but results in one less ongoing pregnancy out of every 28 women treated. Thus standard use of the long GnRH agonist treatment is perhaps still the approach of choice for prevention of premature luteinization. In couples with PCOS and poor responders, GnRH antagonists do not seem to compromise ongoing pregnancy rates and are associated with less OHSS and therefore could be considered as standard treatment. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Potent and Orally Bioavailable GPR142 Agonists as Novel Insulin Secretagogues for the Treatment of Type 2 Diabetes

PubMed Central

2013-01-01

GPR142 is a G protein-coupled receptor that is predominantly expressed in pancreatic β-cells. GPR142 agonists stimulate insulin secretion in the presence of high glucose concentration, so that they could be novel insulin secretagogues with reduced or no risk of hypoglycemia. We report here the optimization of HTS hit compound 1 toward a proof of concept compound 33, which showed potent glucose lowering effects during an oral glucose tolerance test in mice and monkeys. PMID:24900747

Discovery of novel brain permeable and G protein-biased beta-1 adrenergic receptor partial agonists for the treatment of neurocognitive disorders

PubMed Central

Yi, Bitna; Jahangir, Alam; Evans, Andrew K.; Briggs, Denise; Ravina, Kristine; Ernest, Jacqueline; Farimani, Amir B.; Sun, Wenchao; Rajadas, Jayakumar; Green, Michael; Feinberg, Evan N.; Pande, Vijay S.

2017-01-01

The beta-1 adrenergic receptor (ADRB1) is a promising therapeutic target intrinsically involved in the cognitive deficits and pathological features associated with Alzheimer’s disease (AD). Evidence indicates that ADRB1 plays an important role in regulating neuroinflammatory processes, and activation of ADRB1 may produce neuroprotective effects in neuroinflammatory diseases. Novel small molecule modulators of ADRB1, engineered to be highly brain permeable and functionally selective for the G protein with partial agonistic activity, could have tremendous value both as pharmacological tools and potential lead molecules for further preclinical development. The present study describes our ongoing efforts toward the discovery of functionally selective partial agonists of ADRB1 that have potential therapeutic value for AD and neuroinflammatory disorders, which has led to the identification of the molecule STD-101-D1. As a functionally selective agonist of ADRB1, STD-101-D1 produces partial agonistic activity on G protein signaling with an EC50 value in the low nanomolar range, but engages very little beta-arrestin recruitment compared to the unbiased agonist isoproterenol. STD-101-D1 also inhibits the tumor necrosis factor α (TNFα) response induced by lipopolysaccharide (LPS) both in vitro and in vivo, and shows high brain penetration. Other than the therapeutic role, this newly identified, functionally selective, partial agonist of ADRB1 is an invaluable research tool to study mechanisms of G protein-coupled receptor signal transduction. PMID:28746336

Discovery of novel brain permeable and G protein-biased beta-1 adrenergic receptor partial agonists for the treatment of neurocognitive disorders.

PubMed

Yi, Bitna; Jahangir, Alam; Evans, Andrew K; Briggs, Denise; Ravina, Kristine; Ernest, Jacqueline; Farimani, Amir B; Sun, Wenchao; Rajadas, Jayakumar; Green, Michael; Feinberg, Evan N; Pande, Vijay S; Shamloo, Mehrdad

2017-01-01

The beta-1 adrenergic receptor (ADRB1) is a promising therapeutic target intrinsically involved in the cognitive deficits and pathological features associated with Alzheimer's disease (AD). Evidence indicates that ADRB1 plays an important role in regulating neuroinflammatory processes, and activation of ADRB1 may produce neuroprotective effects in neuroinflammatory diseases. Novel small molecule modulators of ADRB1, engineered to be highly brain permeable and functionally selective for the G protein with partial agonistic activity, could have tremendous value both as pharmacological tools and potential lead molecules for further preclinical development. The present study describes our ongoing efforts toward the discovery of functionally selective partial agonists of ADRB1 that have potential therapeutic value for AD and neuroinflammatory disorders, which has led to the identification of the molecule STD-101-D1. As a functionally selective agonist of ADRB1, STD-101-D1 produces partial agonistic activity on G protein signaling with an EC50 value in the low nanomolar range, but engages very little beta-arrestin recruitment compared to the unbiased agonist isoproterenol. STD-101-D1 also inhibits the tumor necrosis factor α (TNFα) response induced by lipopolysaccharide (LPS) both in vitro and in vivo, and shows high brain penetration. Other than the therapeutic role, this newly identified, functionally selective, partial agonist of ADRB1 is an invaluable research tool to study mechanisms of G protein-coupled receptor signal transduction.

Metabolomic analysis of swine urine treated with β2-agonists by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

PubMed

Wu, Yuping; Bi, Yanfeng; Bingga, Gali; Li, Xiaowei; Zhang, Suxia; Li, Jiancheng; Li, Hui; Ding, Shuangyang; Xia, Xi

2015-06-26

The illegal use of β2-agonists in livestock production was previously detected by efficient methods based on mass spectrometry to control the residues of these drugs. Nevertheless, such methods still remain a challenging task for authorities who monitor these residues because the use of "cocktails" composed of mixtures of low amounts of several substances as well as the synthesis of new compounds of unknown structure prevent efficient prevention of illegal use of growth-promoting agents. Here, we outlined a metabolomics-based strategy for detecting the use of "cocktails" composed of mixtures of low amounts of three β2-agonists via urine profiling. Urine profiles of controls and swine treated with mixture of low amounts of three substances (clenbuterol, salbutamol, and ractopamine) were analyzed with ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The metabolic differences between controls and β2-agonists-treated groups were compared using multivariate data analysis. Fourteen metabolites were identified related with the β2-agonists treatment, while two co-biomarkers, 2-indolecarboxylic acid and fluorometholone acetate, either in single or "cocktails" of low-dose mixture of clenbuterol, salbutamol, and ractopamine, could be considered as diagnostic markers for the detection of illegal use of β2-agonists. The results of depletion study demonstrated that it is practical to use the markers for monitoring of β2-agonists. Copyright © 2015 Elsevier B.V. All rights reserved.

Functioning of the dimeric GABAB receptor extracellular domain revealed by glycan wedge scanning

PubMed Central

Rondard, Philippe; Huang, Siluo; Monnier, Carine; Tu, Haijun; Blanchard, Bertrand; Oueslati, Nadia; Malhaire, Fanny; Li, Ying; Trinquet, Eric; Labesse, Gilles; Pin, Jean-Philippe; Liu, Jianfeng

2008-01-01

The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABAB1 and GABAB2. GABAB1 binds agonists, whereas GABAB2 is required for trafficking GABAB1 to the cell surface, increasing agonist affinity to GABAB1, and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABAB1 VFT leads to GABAB2 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABAB VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABAB2, including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation. PMID:18388862

Activation of Cyclic AMP Synthesis by Full and Partial Beta-Adrenergic Receptor Agonists in Chicken Skeletal Muscle Cells

NASA Technical Reports Server (NTRS)

Young, R. B.; Bridge, K. Y.; Cureri, Peter A. (Technical Monitor)

2002-01-01

Several beta-adrenergic receptor (bAR) agonists are known to cause hypertrophy of skeletal muscle tissue. Accordingly, five bAR agonists encompassing a range in activity from strong to weak were evaluated for their ability to stimulate cAMP accumulation in embryonic chicken skeletal muscle cells in culture. Two strong agonists (epinephrine and isoproterenol), one moderate agonist (albuterol), and two weak agonists known to cause hypertrophy in animals (clenbuterol and cimaterol) were studied. Dose response curves were determined over six orders of magnitude in concentration for each agonist, and values were determined for their maximum stimulation of cAMP synthesis rate (Bmax) and the agonist concentration at which 50% stimulation of cAMP synthesis (EC50) occurred. Bmax values decreased in the following order: isoproterenol, epinephrine, albuterol, cimaterol, clenbuterol. Cimaterol and clenbuterol at their Bmax concentrations were approximately 15-fold weaker than isoproterenol in stimulating the rate of cAMP synthesis. When cimaterol and clenbuterol were added to culture media at concentrations known to cause significant muscle hypertrophy in animals, there was no detectable effect on stimulation of cAMP synthesis. Finally, these same levels of cimaterol and clenbuterol did not antagonize the stimulation of cAMP by either epinephrine or isoproterenol.

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the nativemore » ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.« less

Emergent biological properties of arrestin pathway-selective biased agonism.

PubMed

Appleton, Kathryn M; Luttrell, Louis M

2013-06-01

Our growing appreciation of the pluridimensionality of G protein-coupled receptor (GPCR) signaling, combined with the phenomenon of orthosteric ligand "bias", has created the possibility of drugs that selectively modulate different aspects of GPCR function for therapeutic benefit. When viewed from the short-term perspective, e.g. changes in receptor conformation, effector coupling or second messenger generation, biased ligands appear to activate a subset of the response profile produced by a conventional agonist. Yet when examined in vivo, the limited data available suggest that biased ligand effects can diverge from their conventional counterparts in ways that cannot be predicted from their in vitro efficacy profile. What is currently missing, at least with respect to G protein and arrestin pathway-selective ligands, is a rational framework for relating the in vitro efficacy of a "biased" agonist to its in vivo actions that will enable drug screening programs to identify ligands with the desired biological effects.

Altered M(1) muscarinic acetylcholine receptor (CHRM1)-Galpha(q/11) coupling in a schizophrenia endophenotype.

PubMed

Salah-Uddin, Hasib; Scarr, Elizabeth; Pavey, Geoffrey; Harris, Kriss; Hagan, Jim J; Dean, Brian; Challiss, R A John; Watson, Jeannette M

2009-08-01

Alterations in muscarinic acetylcholine receptor (CHRM) populations have been implicated in the pathology of schizophrenia. Here we have assessed whether the receptor function of the M(1) subtype (CHRM1) is altered in a sub-population of patients with schizophrenia, defined by marked (60-80%) reductions in cortical [3H]-pirenzepine (PZP) binding, and termed 'muscarinic receptor-deficit schizophrenia' (MRDS). Using a [35S]-GTPgammaS-Galpha(q/11) immunocapture method we have assessed whether CHRM1 signalling in human cortex (Brodmann area 9 (BA9)) is altered in post mortem tissue from a MRDS group compared with a subgroup of patients with schizophrenia displaying normal PZP binding, and controls with no known history of psychiatric or neurological disorders. The CHRM agonist (oxotremorine-M) and a CHRM1-selective agonist (AC-42) increased Galpha(q/11)-[35S]-GTPgammaS binding, with AC-42 producing responses that were approximately 50% of those maximally evoked by the full agonist, oxotremorine-M, in control and subgroups of patients with schizophrenia. However, the potency of oxotremorine-M to stimulate Galpha(q/11)-[35S]-GTPgammaS binding was significantly decreased in the MRDS group (pEC(50) (M)=5.69+/-0.16) compared with the control group (6.17+/-0.10) and the non-MRDS group (6.05+/-0.07). The levels of Galpha(q/11) protein present in BA9 did not vary with diagnosis. Maximal oxotremorine-M-stimulated Galpha(q/11)-[35S]-GTPgammaS binding in BA9 membranes was significantly increased in the MRDS group compared with the control group. Similar, though non-statistically significant, trends were observed for AC-42. These data provide evidence that both orthosterically and allosterically acting CHRM agonists can stimulate a receptor-driven functional response ([35S]-GTPgammaS binding to Galpha(q/11)) in membranes prepared from post mortem human dorsolateral prefrontal cortex of patients with schizophrenia and controls . Furthermore, in a subgroup of patients with schizophrenia displaying markedly decreased PZP binding (MRDS) we have shown that although agonist potency may decrease, the efficacy of CHRM1-Galpha(q/11) coupling increases, suggesting an adaptative change in receptor-G protein coupling efficiency in this endophenotype of patients with schizophrenia.

Structure/function relationships of calcitonin analogues as agonists, antagonists, or inverse agonists in a constitutively activated receptor cell system.

PubMed

Pozvek, G; Hilton, J M; Quiza, M; Houssami, S; Sexton, P M

1997-04-01

The structure/function relationship of salmon calcitonin (sCT) analogues was investigated in heterologous calcitonin receptor (CTR) expression systems. sCT analogues with progressive amino-terminal truncations intermediate of sCT-(1-32) to sCT-(8-32) were examined for their ability to act as agonists, antagonists, or inverse agonists. Two CTR cell clones, B8-H10 and G12-E12, which express approximately 5 million and 25,000 C1b receptors/cell, respectively, were used for this study. The B8-H10 clone has an approximately 80-fold increase in basal levels of intracellular cAMP due to constitutive activation of the overexpressed receptor. In whole-cell competition binding studies, sCT-(1-32) was more potent than any of its amino-terminally truncated analogues in competition for 125I-sCT binding. In cAMP accumulation studies, sCT-(1-32) and modified analogues sCT-(2-32) and sCT-(3-32) had agonist activities. SDZ-216-710, with an amino-terminal truncation of four amino acids, behaved as a partial agonist/antagonist, whereas amino-terminal truncations of six or seven amino acid residues produced a 16-fold reduction in basal cAMP levels and attenuated the response to the agonist sCT-(1-32) in the constitutively active CTR system. This inverse agonist effect was insensitive to pertussis toxin inhibition. In contrast, the inverse agonist activity of these peptides was not observed in the nonconstitutively active CTR system, in which sCT analogues with amino-terminal truncations of four or more amino acids behaved as neutral competitive antagonists. These results suggest that the inverse agonist activity is mediated by stabilization of the inactive state of the receptor, which does not couple to G protein, and attenuates basal signaling initiated by ligand-independent activation of the effector adenylyl cyclase.

NMR spectroscopy of the ligand binding core of ionotropic glutamate receptor 2 bound to 5-substituted willardiine partial agonists

PubMed Central

Fenwick, Michael K.; Oswald, Robert E.

2008-01-01

Glutamate receptors mediate neuronal intercommunication in the central nervous system by coupling extracellular neurotransmitter-receptor interactions to ion channel conductivity. To gain insight into structural and dynamical factors that underlie this coupling, solution NMR experiments were performed on the bi-lobed ligand-binding core of glutamate receptor 2 in complexes with a set of willardiine partial agonists. These agonists are valuable for studying structure-function relationships because their 5-position substituent size is correlated with ligand efficacy and extent of receptor desensitization whereas the substituent electronegativity is correlated with ligand potency. NMR results show that the protein backbone amide chemical shift deviations correlate mainly with efficacy and extent of desensitization. Pronounced deviations occur at specific residues in the ligand-binding site and in the two helical segments that join the lobes by a disulfide bond. Experiments detecting conformational exchange show that micro- to millisecond timescale motions also occur near the disulfide bond and vary largely with efficacy and extent of desensitization. These results thus identify regions displaying structural and dynamical dissimilarity arising from differences in ligand-protein interactions and lobe closure which may play a critical role in receptor response. Furthermore, measures of line broadening and conformational exchange for a portion of the ligand-binding site correlate with ligand EC50 data. These results do not have any correlate in the currently available crystal structures and thus provide a novel view of ligand-binding events that may be associated with agonist potency differences. PMID:18387631

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors.

PubMed

Koshimizu, Taka-Aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-05-03

Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations.

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors

PubMed Central

Koshimizu, Taka-aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-01-01

Reducing Na+ in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na+-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na+ sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na+ increased cell surface [3H]AVP binding and decreased receptor internalization. Substitution of Na+ by Cs+ or NH4+ inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na+ over Cs+. Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations. PMID:27138239

PAR-2 triggers placenta-derived protease-induced altered VE-cadherin reorganization at endothelial junctions in preeclampsia.

PubMed

Gu, Y; Groome, L J; Alexander, J S; Wang, Y

2012-10-01

PAR-2 is a G-protein coupled protease receptor whose activation in endothelial cells (ECs) is associated with increased solute permeability. VE-cadherin is an endothelial-specific junction protein, which exhibits a disorganized distribution at cell junction during inflammation and is a useful indicator of endothelial barrier dysfunction. In the present study, we tested the hypothesis that PAR-2 activation mediates placenta-derived chymotrypsin-like protease (CLP)-induced endothelial junction disturbance and permeability in preeclampsia (PE). PAR-2 and VE-cadherin were examined by immunofluorescent staining. Specific CLP induced PAR-2 activation and altered VE-cadherin distribution was assessed following depletion of protease chymotrypsin in the placental conditioned medium and after PAR-2 siRNA. VE-cadherin assembly was determined by treating cells with protease chymotrypsin and/or the specific PAR-2 agonist SLIGKV-NH2. Our results showed: 1) placental conditioned medium not only disturbed VE-cadherin distribution at cell junctions but also activated PAR-2 in ECs; 2) PAR-2 siRNA blocked the placental conditioned medium induced PAR-2 upregulation and disorganization of VE-cadherin at cell junctions; 3) PAR-2 agonist induced PAR-2 activation and VE-cadherin reorganization were dose-dependent; and 4) PAR-2 agonist could stimulate ERK1/2 activation. These results strongly suggest that proteases produced by the placenta elicit endothelial barrier dysfunction via a PAR-2 signaling regulatory mechanism in PE. Copyright © 2012 Elsevier Ltd. All rights reserved.

PAR-2 triggers placenta-derived protease-induced altered VE-cadherin reorganization at endothelial junctions in preeclampsia

PubMed Central

Gu, Yang; Groome, Lynn J.; Alexander, J. Steven; Wang, Yuping

2014-01-01

PAR-2 is a G-protein coupled protease receptor whose activation in endothelial cells (ECs) is associated with increased solute permeability. VE-cadherin is an endothelial specific junction protein, which exhibits a disorganized distribution at cell junction during inflammation and is a useful indicator of endothelial barrier dysfunction. In the present study, we tested the hypothesis that PAR-2 activation mediates placenta-derived chymotrypsin-like protease (CLP)-induced endothelial junction disturbance and permeability in preeclampsia (PE). PAR-2 and VE-cadherin were examined by immunofluorescent staining. Specific CLP-induced PAR-2 activation and altered VE-cadherin distribution was assessed following depletion of protease chymotrypsin in the placental conditioned medium and after PAR-2 siRNA. VE-cadherin assembly was determined by treating cells with protease chymotrypsin and/or the specific PAR-2 agonist SLIGKV-NH2. Our results showed: 1) placental conditioned medium not only disturbed VE-cadherin distribution at cell junctions but also activated PAR-2 in ECs; 2) PAR-2 siRNA blocked the placental conditioned medium induced PAR-2 upregulation and disorganization of VE-cadherin at cell junctions; 3) PAR-2 agonist induced PAR-2 activation and VE-cadherin reorganization were dose-dependent; and 4) PAR-2 agonist could stimulate ERK1/2 activation. These results strongly suggest that proteases produced by the placenta elicit endothelial barrier dysfunction via a PAR-2 signaling regulatory mechanism in PE. PMID:22840244

Nicotinic Receptor Transduction Zone: Invariant Arginine Couples to Multiple Electron-Rich Residues

PubMed Central

Mukhtasimova, Nuriya; Sine, Steven M.

2013-01-01

Summary Gating of the muscle-type acetylcholine receptor (AChR) channel depends on communication between the ACh-binding site and the remote ion channel. A key region for this communication is located within the structural transition zone between the ligand-binding and pore domains. Here, stemming from β-strand 10 of the binding domain, the invariant αArg209 lodges within the hydrophobic interior of the subunit and is essential for rapid and efficient channel gating. Previous charge-reversal experiments showed that the contribution of αArg209 to channel gating depends strongly on αGlu45, also within this region. Here we determine whether the contribution of αArg209 to channel gating depends on additional anionic or electron-rich residues in this region. Also, to reconcile diverging findings in the literature, we compare the dependence of αArg209 on αGlu45 in AChRs from different species, and compare the full agonist ACh with the weak agonist choline. Our findings reveal that the contribution of αArg209 to channel gating depends on additional nearby electron-rich residues, consistent with both electrostatic and steric contributions. Furthermore, αArg209 and αGlu45 show a strong interdependence in both human and mouse AChRs, whereas the functional consequences of the mutation αE45R depend on the agonist. The emerging picture shows a multifaceted network of interdependent residues that are required for communication between the ligand-binding and pore domains. PMID:23442857

Impaired activation of adenylyl cyclase in lung of the Basenji-greyhound model of airway hyperresponsiveness: decreased numbers of high affinity beta-adrenoceptors.

PubMed Central

Emala, C. W.; Aryana, A.; Hirshman, C. A.

1996-01-01

1. To evaluate mechanisms involved in the impaired beta-adrenoceptor stimulation of adenylyl cyclase in tissues from the Basenji-greyhound (BG) dog model of airway hyperresponsiveness, we compared agonist and antagonist binding affinity of beta-adrenoceptors, beta-adrenoceptor subtypes, percentage of beta-adrenoceptors sequestered, and coupling of the beta-adrenoceptor to Gs alpha in lung membranes from BG and control mongrel dogs. We found that lung membranes from the BG dog had higher total numbers of beta-adrenoceptors with a greater percentage of receptors of the beta 2 subtype as compared to mongrel lung membranes. 2. Agonist and antagonist binding affinity and the percentage of beta-adrenoceptors sequestered were not different in BG and mongrel dog lung membranes. However, the percentage of beta-adrenoceptors in the high affinity state for agonist was decreased in BG lung membranes suggesting an uncoupling of the receptor from Gs alpha. 3. Impaired coupling between the beta-adrenoceptor and G protein documented by the decreased numbers of beta-adrenoceptors in the high affinity state in BG lung membranes, is a plausible explanation for the reduced stimulation of adenylyl cyclase and the resultant reduction in airway smooth muscle relaxation in this model. PMID:8864536

A pH-sensitive fluor, CypHer 5, used to monitor agonist-induced G protein-coupled receptor internalization in live cells.

PubMed

Adie, E J; Kalinka, S; Smith, L; Francis, M J; Marenghi, A; Cooper, M E; Briggs, M; Michael, N P; Milligan, G; Game, S

2002-11-01

G protein-coupled receptors (GPCRs) are the largest family of proteins involved in transmembrane signal transduction and are actively studied because of their suitability as therapeutic small-molecule drug targets. Agonist activation of GPCRs almost invariably results in the receptor being desensitized. One of the key events in receptor desensitization is the sequestration of the receptor from the cell surface into acidic intracellular endosomes. Therefore, a convenient, generic, and noninvasive monitor of this process is desirable. A novel, pH-sensitive, red-excited fluorescent dye, CypHer 5, was synthesized. This dye is non-fluorescent at neutral pH and is fluorescent at acidic pH. Anti-epitope antibodies labeled with this dye were internalized in an agonist concentration- and time-dependent manner, following binding on live cells to a range of GPCRs that had been modified to incorporate the epitope tags in their extracellular N-terminal domain. This resulted in a large signal increase over background. When protonated, the red fluorescence of CypHer 5 provides a generic reagent suitable for monitoring the internalization of GPCRs into acidic vesicles. This approach should be amenable to the study of many other classes of cell surface receptors that also internalize following stimulation.

Differential manipulation of arrestin-3 binding to basal and agonist-activated G protein-coupled receptors.

PubMed

Prokop, Susanne; Perry, Nicole A; Vishnivetskiy, Sergey A; Toth, Andras D; Inoue, Asuka; Milligan, Graeme; Iverson, Tina M; Hunyady, Laszlo; Gurevich, Vsevolod V

2017-08-01

Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M 2 muscarinic receptor, so that agonist activation of the M 2 did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M 2 , whereas its interactions with other receptors, including the β 2 -adrenergic receptor and the D 1 and D 2 dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-induced arrestin-3 binding to the β 2 -adrenergic and D 2 dopamine receptors, while reducing its interaction with the D 1 dopamine receptor. These mutations represent the first example of altering arrestin specificity via enhancement of the arrestin-receptor interactions rather than selective reduction of the binding to certain subtypes. Copyright © 2017. Published by Elsevier Inc.

Purinergic signaling pathways in endocrine system.

PubMed

Bjelobaba, Ivana; Janjic, Marija M; Stojilkovic, Stanko S

2015-09-01

Adenosine-5'-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5'-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5'-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5'-triphosphate hydrolysis to adenosine-5'-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling. Published by Elsevier B.V.

Purinergic Signaling Pathways in Endocrine System

PubMed Central

Bjelobaba, Ivana; Janjic, Marija M.; Stojilkovic, Stanko S.

2015-01-01

Adenosine-5′-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5′-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5′-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5′-triphosphate hydrolysis to adenosine-5′-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling. PMID:25960051

Adaptability and selectivity of human peroxisome proliferator-activated receptor (PPAR) pan agonists revealed from crystal structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oyama, Takuji; Toyota, Kenji; Waku, Tsuyoshi

2009-08-01

The structures of the ligand-binding domains (LBDs) of human peroxisome proliferator-activated receptors (PPARα, PPARγ and PPARδ) in complexes with a pan agonist, an α/δ dual agonist and a PPARδ-specific agonist were determined. The results explain how each ligand is recognized by the PPAR LBDs at an atomic level. Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor family, which is defined as transcriptional factors that are activated by the binding of ligands to their ligand-binding domains (LBDs). Although the three PPAR subtypes display different tissue distribution patterns and distinct pharmacological profiles, they all are essentially related to fatty-acid andmore » glucose metabolism. Since the PPARs share similar three-dimensional structures within the LBDs, synthetic ligands which simultaneously activate two or all of the PPARs could be potent candidates in terms of drugs for the treatment of abnormal metabolic homeostasis. The structures of several PPAR LBDs were determined in complex with synthetic ligands, derivatives of 3-(4-alkoxyphenyl)propanoic acid, which exhibit unique agonistic activities. The PPARα and PPARγ LBDs were complexed with the same pan agonist, TIPP-703, which activates all three PPARs and their crystal structures were determined. The two LBD–ligand complex structures revealed how the pan agonist is adapted to the similar, but significantly different, ligand-binding pockets of the PPARs. The structures of the PPARδ LBD in complex with an α/δ-selective ligand, TIPP-401, and with a related δ-specific ligand, TIPP-204, were also determined. The comparison between the two PPARδ complexes revealed how each ligand exhibits either a ‘dual selective’ or ‘single specific’ binding mode.« less

Functional assessment of recombinant human alpha(2)-adrenoceptor subtypes with cytosensor microphysiometry.

PubMed

Pihlavisto, M; Scheinin, M

1999-12-03

We applied the Cytosensor Microphysiometry system to study the three human alpha(2)-adrenoceptor subtypes, alpha(2A), alpha(2B) and alpha(2C), expressed in Chinese hamster ovary (CHO) cells, and assessed its potential in the quantitative monitoring of agonist activity. The natural full agonist, (-)-noradrenaline, was used to define agonist efficacy. The imidazole derivative dexmedetomidine was a potent full agonist of all three receptor subtypes. The imidazolines clonidine and UK 14,304 (5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) appeared to be partial agonists at alpha(2B)-adrenoceptors (E(max) approximately 60% of (-)-noradrenaline) but full agonists at alpha(2A)- and alpha(2C)-adrenoceptors. The responses mediated by all three alpha(2)-adrenoceptor subtypes were partly inhibited by the sodium-hydrogen (Na(+)/H(+)) exchange inhibitor, MIA (5-(N-methyl-N-isobutyl)-amiloride). The agonist responses were totally abolished by pretreatment with pertussis toxin in cells with alpha(2A)- and alpha(2C)-adrenoceptors, and partly abolished in cells with alpha(2B)-adrenoceptors. The residual signal in alpha(2B)-cells was sensitive to the intracellular Ca(2+)chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester). Cholera toxin (which acts on G(s)-proteins) had no effect on the agonist responses. The results suggest that the extracellular acidification responses mediated by all three human alpha(2)-adrenoceptor subtypes are dependent on Na(+)/H(+)exchange and G(i/o) pathways, and that alpha(2B)-adrenoceptors are capable of coupling to another, G(i/o)-independent and Ca(2+)-dependent signaling pathway.

Functional Selectivity of Allosteric Interactions within G Protein–Coupled Receptor Oligomers: The Dopamine D1-D3 Receptor Heterotetramer

PubMed Central

Guitart, Xavier; Navarro, Gemma; Moreno, Estefania; Yano, Hideaki; Cai, Ning-Sheng; Sánchez-Soto, Marta; Kumar-Barodia, Sandeep; Naidu, Yamini T.; Mallol, Josefa; Cortés, Antoni; Lluís, Carme; Canela, Enric I.; Casadó, Vicent; McCormick, Peter J.

2014-01-01

The dopamine D1 receptor–D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa–induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques, and cell-signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R–D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and Gi proteins, respectively. Coactivation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of β-arrestin-1, and to a positive cross talk of D1R and D3R agonists at the level of mitogen-activated protein kinase (MAPK) signaling. Furthermore, D1R or D3R antagonists counteracted β-arrestin-1 recruitment and MAPK activation induced by D3R and D1R agonists, respectively (cross-antagonism). Positive cross talk and cross-antagonism at the MAPK level were counteracted by specific synthetic peptides with amino acid sequences corresponding to D1R transmembrane (TM) domains TM5 and TM6, which also selectively modified the quaternary structure of the D1R-D3R heteromer, as demonstrated by complementation of hemiproteins of yellow fluorescence protein fused to D1R and D3R. These results demonstrate functional selectivity of allosteric modulations within the D1R-D3R heteromer, which can be involved with the reported behavioral synergism of D1R and D3R agonists. PMID:25097189

Effect of beta-ADrenergic Agonist on Cyclic AMP Synthesis in Chicken Skeletal Muscle Cells in Culture

NASA Technical Reports Server (NTRS)

Young, R. B.; Bridge, K. Y.; Rose, M. Franklin (Technical Monitor)

2000-01-01

Several beta-adrenergic receptor (bAR) agonists are known to cause hypertrophy of skeletal muscle tissue. Because it seems logical that these agonists exert their action on muscle through stimulation of cAMP synthesis, five bAR agonists encompassing a range in activity from strong to weak were evaluated for their ability to stimulate cAMP accumulation in embryonic chicken skeletal muscle cells in culture. Two strong agonists (epinephrine and isoproterenol), one moderate agonist (albuterol), and two weak agonists known to cause hypertrophy in animals (clenbuterol and cimaterol) were studied. Dose response curves were determined over six orders of magnitude in concentration for each agonist, and values were determined for their maximum stimulation of cAMP synthesis rate (Bmax) and the agonist concentration at which 50% stimulation of cAMP synthesis (EC50) occurred. Bmax values decreased in the following order: isoproterenol, epinephrine, albuterol, cimaterol, clenbuterol. Cimaterol and clenbuterol at their Bmax levels were approximately 15-fold weaker than isoproterenol in stimulating the rate of cAMP synthesis. In addition, the EC50 values for isoproterenol, cimaterol, clenbuterol, epinephrine, and albuterol were 360 nM, 630 nM, 900 nM, 2,470 nM, and 3,650 nM, respectively. Finally, dose response curves show that the concentrations of cimaterol and clenbuterol in culture media at concentrations known to cause significant muscle hypertrophy in animals had no detectable effect on stimulation of CAMP accumulation in chicken skeletal muscle cells.

Nucleotide-binding oligomerization domain 2 (NOD2) activation induces apoptosis of human oral squamous cell carcinoma cells.

PubMed

Yoon, Hyo-Eun; Ahn, Mee-Young; Kwon, Seong-Min; Kim, Dong-Jae; Lee, Jun; Yoon, Jung-Hoon

2016-04-01

Microbial Pattern-recognition receptors (PRRs), such as nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. This study was designed to determine the effect of NOD1 and NOD2 agonist on innate immune responses and antitumor activity in oral squamous cell carcinoma (OSCC) cells. NODs expression was examined by RT-PCR, and IL-8 production by NODs agonist was examined by ELISA. Western blot analysis was performed to determine the MAPK activation in response to their agonist. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the MDP-induced cell death. The levels of NODs were apparently expressed in OSCC cells. NODs agonist, Tri-DAP and MDP, led to the production of IL-8 and MAPK activation. NOD2 agonist, MDP, inhibited the proliferation of YD-10B cells in a dose-dependent manner. Also, the ratio of Annexin V-positive cells and cleaved PARP was increased by MDP treatment in YD-10B cells, suggesting that MDP-induced cell death in YD-10B cells may be owing to apoptosis. Our results indicate that NODs are functionally expressed in OSCC cells and can trigger innate immune responses. In addition, NOD2 agonist inhibited cell proliferation and induced apoptosis. These findings provide the potential value of MDP as novel candidates for antitumor agents of OSCC. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Agonist-Directed Desensitization of the β2-Adrenergic Receptor

PubMed Central

Goral, Vasiliy; Jin, Yan; Sun, Haiyan; Ferrie, Ann M.; Wu, Qi; Fang, Ye

2011-01-01

The β2-adrenergic receptor (β2AR) agonists with reduced tachyphylaxis may offer new therapeutic agents with improved tolerance profile. However, receptor desensitization assays are often inferred at the single signaling molecule level, thus ligand-directed desensitization is poorly understood. Here we report a label-free biosensor whole cell assay with microfluidics to determine ligand-directed desensitization of the β2AR. Together with mechanistic deconvolution using small molecule inhibitors, the receptor desensitization and resensitization patterns under the short-term agonist exposure manifested the long-acting agonism of salmeterol, and differentiated the mechanisms of agonist-directed desensitization between a full agonist epinephrine and a partial agonist pindolol. This study reveals the cellular mechanisms of agonist-selective β2AR desensitization at the whole cell level. PMID:21541288

Discrimination between olfactory receptor agonists and non-agonists.

PubMed

Topin, Jérémie; de March, Claire A; Charlier, Landry; Ronin, Catherine; Antonczak, Serge; Golebiowski, Jérôme

2014-08-11

A joint approach combining free-energy calculations and calcium-imaging assays on the broadly tuned human 1G1 olfactory receptor is reported. The free energy of binding of ten odorants was computed by means of molecular-dynamics simulations. This state function allows separating the experimentally determined eight agonists from the two non-agonists. This study constitutes a proof-of-principle for the computational deorphanization of olfactory receptors. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novel 2-aminotetralin and 3-aminochroman derivatives as selective serotonin 5-HT7 receptor agonists and antagonists.

PubMed

Holmberg, Pär; Sohn, Daniel; Leideborg, Robert; Caldirola, Patrizia; Zlatoidsky, Pavel; Hanson, Sverker; Mohell, Nina; Rosqvist, Susanne; Nordvall, Gunnar; Johansson, Anette M; Johansson, Rolf

2004-07-29

The understanding of the physiological role of the G-protein coupled serotonin 5-HT(7) receptor is largely rudimentary. Therefore, selective and potent pharmacological tools will add to the understanding of serotonergic effects mediated through this receptor. In this report, we describe two compound classes, chromans and tetralins, encompassing compounds with nanomolar affinity for the 5-HT(7) receptor and with good selectivity. Within theses classes, we have discovered both agonists and antagonists that can be used for further understanding of the pharmacology of the 5-HT(7) receptor.

Ligand-based receptor tyrosine kinase partial agonists: New paradigm for cancer drug discovery?

PubMed

Riese, David J

2011-02-01

INTRODUCTION: Receptor tyrosine kinases (RTKs) are validated targets for oncology drug discovery and several RTK antagonists have been approved for the treatment of human malignancies. Nonetheless, the discovery and development of RTK antagonists has lagged behind the discovery and development of agents that target G-protein coupled receptors. In part, this is because it has been difficult to discover analogs of naturally-occurring RTK agonists that function as antagonists. AREAS COVERED: Here we describe ligands of ErbB receptors that function as partial agonists for these receptors, thereby enabling these ligands to antagonize the activity of full agonists for these receptors. We provide insights into the mechanisms by which these ligands function as antagonists. We discuss how information concerning these mechanisms can be translated into screens for novel small molecule- and antibody-based antagonists of ErbB receptors and how such antagonists hold great potential as targeted cancer chemotherapeutics. EXPERT OPINION: While there have been a number of important key findings into this field, the identification of the structural basis of ligand functional specificity is still of the greatest importance. While it is true that, with some notable exceptions, peptide hormones and growth factors have not proven to be good platforms for oncology drug discovery; addressing the fundamental issues of antagonistic partial agonists for receptor tyrosine kinases has the potential to steer oncology drug discovery in new directions. Mechanism based approaches are now emerging to enable the discovery of RTK partial agonists that may antagonize both agonist-dependent and -independent RTK signaling and may hold tremendous promise as targeted cancer chemotherapeutics.

Activation and inhibition of adenylyl cyclase isoforms by forskolin analogs.

PubMed

Pinto, Cibele; Papa, Dan; Hübner, Melanie; Mou, Tung-Chung; Lushington, Gerald H; Seifert, Roland

2008-04-01

Adenylyl cyclase (AC) isoforms 1 to 9 are differentially expressed in tissues and constitute an interesting drug target. ACs 1 to 8 are activated by the diterpene, forskolin (FS). It is unfortunate that there is a paucity of AC isoform-selective activators. To develop such compounds, an understanding of the structure/activity relationships of diterpenes is necessary. Therefore, we examined the effects of FS and nine FS analogs on ACs 1, 2, and 5 expressed in Spodoptera frugiperda insect cells. Diterpenes showed the highest potencies at AC1 and the lowest potencies at AC2. We identified full agonists, partial agonists, antagonists, and inverse agonists, i.e., diterpenes that reduced basal AC activity. Each AC isoform exhibited a distinct pharmacological profile. AC2 showed the highest basal activity of all AC isoforms and highest sensitivity to inverse agonistic effects of 1-deoxy-forskolin, 7-deacetyl-1,9-dideoxy-forskolin, and, particularly, BODIPY-forskolin. In contrast, BODIPY-forskolin acted as partial agonist at the other ACs. 1-Deoxy-forskolin analogs were devoid of agonistic activity at ACs but antagonized the effects of FS in a mixed competitive/noncompetitive manner. At purified catalytic AC subunits, BODIPY-forskolin acted as weak partial agonist/strong partial antagonist. Molecular modeling revealed that the BODIPY group rotates promiscuously outside of the FS-binding site. Collectively, ACs are not uniformly activated and inhibited by FS and FS analogs, demonstrating the feasibility to design isoform-selective FS analogs. The two- and multiple-state models, originally developed to conceptualize ligand effects at G-protein-coupled receptors, can be applied to ACs to explain certain experimental data.

The costo-uterine muscle of the rat contains a homogeneous population of beta-adrenoceptors.

PubMed Central

Hartley, M. L.; Pennefather, J. N.

1985-01-01

The effects of two selective beta-adrenoceptor antagonists on the inhibitory responses to some sympathomimetic amines of electrically-stimulated preparations of costo-uterine muscle, taken from virgin rats, have been examined quantitatively. pA2 values for the antagonist, atenolol (beta 1-selective) and ICI 118,551 (beta 2-selective) were obtained using as agonists, fenoterol (beta 2-selective agonist) and noradrenaline (alpha- and beta-adrenoceptor agonist, beta 1-selective); and in addition, with ICI 118,551 only, isoprenaline (beta-agonist, non-selective) and adrenaline (alpha- and beta-adrenoceptor agonist, beta 2-selective). Catecholamine uptake mechanisms and alpha-adrenoceptors were not blocked in any of these experiments. Atenolol competitively antagonized the effects of fenoterol and noradrenaline to a similar extent, the pA2 values being 5.4 and 5.7, respectively. ICI 118,551 competitively antagonized the effects of fenoterol, isoprenaline, adrenaline and noradrenaline to a similar extent; pA2 values ranged from 8.7 with noradrenaline to 9.1 with isoprenaline. These results extend our previous observations which indicated that the adrenoceptors mediating inhibition of electrically-evoked contractions of costo-uterine muscle of the virgin rat are homogeneous and of the beta 2-subtype. The potency of the beta 1-selective agonist RO 363 in producing inhibition of electrically-evoked contractions of this tissue was also examined. RO 363 was 200 times less potent than isoprenaline but was a full agonist. This indicates that there is efficient coupling between beta 2-adrenoceptor activation and tissue response in this non-innervated preparation. PMID:2858239

Adenosine-A1 Receptor Agonist Induced Hyperalgesic Priming Type II

PubMed Central

Araldi, Dioneia; Ferrari, Luiz F.; Levine, Jon D.

2016-01-01

We have recently shown that repeated exposure of the peripheral terminal of the primary afferent nociceptor to the mu-opioid receptor (MOR) agonist DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt) induces a model of the transition to chronic pain that we have termed Type II hyperalgesic priming. Similar to Type I hyperalgesic priming, there is a markedly prolonged response to subsequent administration of proalgesic cytokines, prototypically prostaglandin E2 (PGE2). However, Type II hyperalgesic priming differs from Type I in being rapidly induced, protein kinase A (PKA), rather than PKCε dependent, not reversed by a protein translation inhibitor, occurring in female as well as in male rats, and isolectin B4-negative neuron dependent. We report that as with the repeated injection of a MOR agonist, the repeated administration of an agonist at the A1-adenosine receptor, also a Gi-protein coupled receptor, N6-Cyclopentyladenosine (CPA), also produces priming similar to DAMGO-induced Type II hyperalgesic priming. In this study we demonstrate that priming induced by repeated exposure to this A1-adenosine receptor agonist shares the same mechanisms as MOR-agonist induced priming. However, the prolongation of PGE2 hyperalgesia induced by repeated administration of CPA depends on G-protein αi subunit activation, differently from DAMGO-induced Type II priming, in which it depends on the β/γ subunit. These data implicate a novel form of Gi-protein signaling pathway in the Type II hyperalgesic priming induced by repeated administration of an agonist at A1-adenosine receptor to the peripheral terminal of the nociceptor. PMID:26588695

Structure and dynamics of a constitutively active neurotensin receptor

DOE PAGES

Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo; ...

2016-12-07

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecularmore » dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. In conclusion, the loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.« less

Structure and dynamics of a constitutively active neurotensin receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecularmore » dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. In conclusion, the loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.« less

Targeting GLP-1 receptor trafficking to improve agonist efficacy.

PubMed

Jones, Ben; Buenaventura, Teresa; Kanda, Nisha; Chabosseau, Pauline; Owen, Bryn M; Scott, Rebecca; Goldin, Robert; Angkathunyakul, Napat; Corrêa, Ivan R; Bosco, Domenico; Johnson, Paul R; Piemonti, Lorenzo; Marchetti, Piero; Shapiro, A M James; Cochran, Blake J; Hanyaloglu, Aylin C; Inoue, Asuka; Tan, Tricia; Rutter, Guy A; Tomas, Alejandra; Bloom, Stephen R

2018-04-23

Glucagon-like peptide-1 receptor (GLP-1R) activation promotes insulin secretion from pancreatic beta cells, causes weight loss, and is an important pharmacological target in type 2 diabetes (T2D). Like other G protein-coupled receptors, the GLP-1R undergoes agonist-mediated endocytosis, but the functional and therapeutic consequences of modulating GLP-1R endocytic trafficking have not been clearly defined. Here, we investigate a series of biased GLP-1R agonists with variable propensities for GLP-1R internalization and recycling. Compared to a panel of FDA-approved GLP-1 mimetics, compounds that retain GLP-1R at the plasma membrane produce greater long-term insulin release, which is dependent on a reduction in β-arrestin recruitment and faster agonist dissociation rates. Such molecules elicit glycemic benefits in mice without concomitant increases in signs of nausea, a common side effect of GLP-1 therapies. Our study identifies a set of agents with specific GLP-1R trafficking profiles and the potential for greater efficacy and tolerability as T2D treatments.

Neuroreceptor Activation by Vibration-Assisted Tunneling

PubMed Central

Hoehn, Ross D.; Nichols, David; Neven, Hartmut; Kais, Sabre

2015-01-01

G protein-coupled receptors (GPCRs) constitute a large family of receptor proteins that sense molecular signals on the exterior of a cell and activate signal transduction pathways within the cell. Modeling how an agonist activates such a receptor is fundamental for an understanding of a wide variety of physiological processes and it is of tremendous value for pharmacology and drug design. Inelastic electron tunneling spectroscopy (IETS) has been proposed as a model for the mechanism by which olfactory GPCRs are activated by a bound agonist. We apply this hyothesis to GPCRs within the mammalian nervous system using quantum chemical modeling. We found that non-endogenous agonists of the serotonin receptor share a particular IET spectral aspect both amongst each other and with the serotonin molecule: a peak whose intensity scales with the known agonist potencies. We propose an experiential validation of this model by utilizing lysergic acid dimethylamide (DAM-57), an ergot derivative, and its deuterated isotopologues; we also provide theoretical predictions for comparison to experiment. If validated our theory may provide new avenues for guided drug design and elevate methods of in silico potency/activity prediction. PMID:25909758

Structure and dynamics of a constitutively active neurotensin receptor

PubMed Central

Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo; Botos, Istvan; White, Courtney F.; Du, Haijuan; Vaidehi, Nagarajan; Grisshammer, Reinhard

2016-01-01

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist. PMID:27924846

N-Arachidonyl Glycine Does Not Activate G Protein–Coupled Receptor 18 Signaling via Canonical Pathways

PubMed Central

Lu, Van B.; Puhl, Henry L.

2013-01-01

Recent studies propose that N-arachidonyl glycine (NAGly), a carboxylic analogue of anandamide, is an endogenous ligand of the Gαi/o protein–coupled receptor 18 (GPR18). However, a high-throughput β-arrestin–based screen failed to detect activation of GPR18 by NAGly (Yin et al., 2009; JBC, 18:12328). To address this inconsistency, this study investigated GPR18 coupling in a native neuronal system with endogenous signaling pathways and effectors. GPR18 was heterologously expressed in rat sympathetic neurons, and the modulation of N-type (Cav2.2) calcium channels was examined. Proper expression and trafficking of receptor were confirmed by the “rim-like” fluorescence of fluorescently tagged receptor and the positive staining of external hemagglutinin-tagged GPR18-expressing cells. Application of NAGly on GPR18-expressing neurons did not inhibit calcium currents but instead potentiated currents in a voltage-dependent manner, similar to what has previously been reported (Guo et al., 2008; J Neurophysiol, 100:1147). Other proposed agonists of GPR18, including anandamide and abnormal cannabidiol, also failed to induce inhibition of calcium currents. Mutants of GPR18, designed to constitutively activate receptors, did not tonically inhibit calcium currents, indicating a lack of GPR18 activation or coupling to endogenous G proteins. Other downstream effectors of Gαi/o-coupled receptors, G protein–coupled inwardly rectifying potassium channels and adenylate cyclase, were not modulated by GPR18 signaling. Furthermore, GPR18 did not couple to other G proteins tested: Gαs, Gαz, and Gα15. These results suggest NAGly is not an agonist for GPR18 or that GPR18 signaling involves noncanonical pathways not examined in these studies. PMID:23104136

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs).

PubMed

Li, Lingyong; Homan, Kristoff T; Vishnivetskiy, Sergey A; Manglik, Aashish; Tesmer, John J G; Gurevich, Vsevolod V; Gurevich, Eugenia V

2015-04-24

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential regulation of serotonin-1A receptor-stimulated [35S]GTP gamma S binding in the dorsal raphe nucleus by citalopram and escitalopram.

PubMed

Rossi, Dania V; Burke, Teresa F; Hensler, Julie G

2008-03-31

The effect of chronic citalopram or escitalopram administration on 5-HT1A receptor function in the dorsal raphe nucleus was determined by measuring [35S]GTP gamma S binding stimulated by the 5-HT1A receptor agonist (R)-(+)-8-OH-DPAT (1nM-10 microM). Although chronic administration of citalopram or escitalopram has been shown to desensitize somatodendritic 5-HT1A autoreceptors, we found that escitalopram treatment decreased the efficacy of 5-HT1A receptors to activate G proteins, whereas citalopram treatment did not. The binding of [3H]8-OH-DPAT to the coupled, high affinity agonist state of the receptor was not altered by either treatment. Interestingly, escitalopram administration resulted in greater occupancy of serotonin transporter sites as measured by the inhibition of [3H]cyanoimipramine binding. As the binding and action of escitalopram is limited by the inactive enantiomer R-citalopram present in racemic citalopram, we propose that the regulation of 5-HT1A receptor function in the dorsal raphe nucleus at the level of receptor-G protein interaction may be a result of greater inhibition of the serotonin transporter by escitalopram.

Differential regulation of serotonin-1A receptor stimulated [35S]GTPγS binding in the dorsal raphe nucleus by citalopram and escitalopram

PubMed Central

Rossi, Dania V.; Burke, Teresa F.; Hensler, Julie G.

2008-01-01

The effect of chronic citalopram or escitalopram administration on 5-HT1A receptor function in the dorsal raphe nucleus was determined by measuring [35S]GTPγS binding stimulated by the 5-HT1A receptor agonist (R)-(+)-8-OH-DPAT (1nM-10μM). Although chronic administration of citalopram or escitalopram has been shown to desensitize somatodendritic 5-HT1A autoreceptors, we found that escitalopram treatment decreased the efficacy of 5-HT1A receptors to activate G-proteins, whereas citalopram treatment did not. The binding of [3H]8-OH-DPAT to the coupled, high affinity agonist state of the receptor was not altered by either treatment. Interestingly, escitalopram administration resulted in greater occupancy of serotonin transporter sites as measured by the inhibition of [3H]cyanoimipramine binding. As the binding and action of escitalopram is limited by the inactive enantiomer R-citalopram present in racemic citalopram, we propose that the regulation of 5-HT1A receptor function in the dorsal raphe nucleus at the level of receptor-G protein interaction may be a result of greater inhibition of the serotonin transporter by escitalopram. PMID:18289523

A unified view of "how allostery works".

PubMed

Tsai, Chung-Jung; Nussinov, Ruth

2014-02-01

The question of how allostery works was posed almost 50 years ago. Since then it has been the focus of much effort. This is for two reasons: first, the intellectual curiosity of basic science and the desire to understand fundamental phenomena, and second, its vast practical importance. Allostery is at play in all processes in the living cell, and increasingly in drug discovery. Many models have been successfully formulated, and are able to describe allostery even in the absence of a detailed structural mechanism. However, conceptual schemes designed to qualitatively explain allosteric mechanisms usually lack a quantitative mathematical model, and are unable to link its thermodynamic and structural foundations. This hampers insight into oncogenic mutations in cancer progression and biased agonists' actions. Here, we describe how allostery works from three different standpoints: thermodynamics, free energy landscape of population shift, and structure; all with exactly the same allosteric descriptors. This results in a unified view which not only clarifies the elusive allosteric mechanism but also provides structural grasp of agonist-mediated signaling pathways, and guides allosteric drug discovery. Of note, the unified view reasons that allosteric coupling (or communication) does not determine the allosteric efficacy; however, a communication channel is what makes potential binding sites allosteric.

IMAGING ASSESSMENT OF G-PROTEIN-COUPLED ESTROGEN RECEPTOR ACTIVATION

EPA Science Inventory

We expect to provide a targeted imaging assay that links chemical mechanism of toxicity to target organ effects and heart physiology within individual 96-hpf zebrafish. Importantly, with the utilization of selective, non-toxic agonists or antagonists for additional xenobiot...

Regulation of mGlu4 metabotropic glutamate receptor signaling by type-2 G-protein coupled receptor kinase (GRK2).

PubMed

Iacovelli, L; Capobianco, L; Iula, M; Di Giorgi Gerevini, V; Picascia, A; Blahos, J; Melchiorri, D; Nicoletti, F; De Blasi, A

2004-05-01

We examined the role of G-protein coupled receptor kinase-2 (GRK2) in the homologous desensitization of mGlu4 metabotropic glutamate receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Receptor activation with the agonist l-2-amino-4-phosphonobutanoate (l-AP4) stimulated at least two distinct signaling pathways: inhibition of cAMP formation and activation of the mitogen-activated protein kinase (MAPK) pathway [assessed by Western blot analysis of phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2]. Activation of both pathways was attenuated by pertussis toxin. Overexpression of GRK2 (but not GRK4) largely attenuated the stimulation of the MAPK pathway by l-AP4, whereas it slightly potentiated the inhibition of FSK-stimulated cAMP formation. Transfection with a kinase-dead mutant of GRK2 (GRK2-K220R) or with the C-terminal fragment of GRK2 also reduced the mGlu4-mediated stimulation of MAPK, suggesting that GRK2 binds to the Gbetagamma subunits to inhibit signal propagation toward the MAPK pathway. This was confirmed by the evidence that GRK2 coimmunoprecipitated with Gbetagamma subunits in an agonist-dependent manner. Finally, neither GRK2 nor its kinase-dead mutant had any effect on agonist-induced mGlu4 receptor internalization in HEK293 cells transiently transfected with GFP-tagged receptors. Agonist-dependent internalization was instead abolished by a negative-dominant mutant of dynamin, which also reduced the stimulation of MAPK pathway by l-AP4. We speculate that GRK2 acts as a "switch molecule" by inhibiting the mGlu4 receptor-mediated stimulation of MAPK and therefore directing the signal propagation toward the inhibition of adenylyl cyclase.

Phosphatase-Resistant Analogues of Lysophosphatidic Acid

PubMed Central

Prestwich, Glenn D.; Gajewiak, Joanna; Zhang, Honglu; Xu, Xiaoyu; Yang, Guanghui; Serban, Monica

2008-01-01

Isoform-selective agonists and antagonists of the lysophosphatidic acid (LPA) G protein coupled receptors (GPCRs) have important potential applications in cell biology and therapy. LPA GPCRs regulate cancer cell proliferation, invasion, angiogenesis, and also biochemical resistance to chemotherapy- and radiotherapy-induced apoptosis. LPA and its analogues also are feedback inhibitors of the enzyme lysophospholipase D (lysoPLD, a.k.a., autotaxin, ATX), a central regulator of invasion and metastasis. For cancer therapy, the optimal therapeutic profile would be a metabolically stabilized, pan-LPA receptor antagonist that also inhibited lysoPLD. For protection of gastrointestinal mucosa and lymphocytes, LPA agonists would be desirable to minimize or reverse radiation or chemical-induced injury. Analogues of lysophosphatidic acid (LPA) that are chemically modified to be less susceptible to phospholipases and phosphatases show activity as long-lived receptor-specific agonists and antagonists for LPA receptors, as well as inhibitors for the lysoPLD activity of ATX. PMID:18454946

Protection of Primary Dopaminergic Midbrain Neurons by GPR139 Agonists Supports Different Mechanisms of MPP+ and Rotenone Toxicity

PubMed Central

Bayer Andersen, Kirsten; Leander Johansen, Jens; Hentzer, Morten; Smith, Garrick Paul; Dietz, Gunnar P. H.

2016-01-01

The G-protein coupled receptor 139 (GPR139) is expressed specifically in the brain in areas of relevance for motor control. GPR139 function and signal transduction pathways are elusive, and results in the literature are even contradictory. Here, we examined the potential neuroprotective effect of GPR139 agonism in primary culture models of dopaminergic (DA) neuronal degeneration. We find that in vitro GPR139 agonists protected primary mesencephalic DA neurons against 1-methyl-4-phenylpyridinium (MPP+)-mediated degeneration. Protection was concentration-dependent and could be blocked by a GPR139 antagonist. However, the protection of DA neurons was not found against rotenone or 6-hydroxydopamine (6-OHDA) mediated degeneration. Our results support differential mechanisms of toxicity for those substances commonly used in Parkinson’s disease (PD) models and potential for GPR139 agonists in neuroprotection. PMID:27445691

Simulations of Biased Agonists in the β2 Adrenergic Receptor with Accelerated Molecular Dynamics

PubMed Central

2013-01-01

The biased agonism of the G protein-coupled receptors (GPCRs), where in addition to a traditional G protein-signaling pathway a GPCR promotes intracellular signals though β-arrestin, is a novel paradigm in pharmacology. Biochemical and biophysical studies have suggested that a GPCR forms a distinct ensemble of conformations signaling through the G protein and β-arrestin. Here we report on the dynamics of the β2 adrenergic receptor bound to the β-arrestin and G protein-biased agonists and the empty receptor to further characterize the receptor conformational changes caused by biased agonists. We use conventional and accelerated molecular dynamics (aMD) simulations to explore the conformational transitions of the GPCR from the active state to the inactive state. We found that aMD simulations enable monitoring of the transition within the nanosecond time scale while capturing the known microscopic characteristics of the inactive states, such as the ionic lock, the inward position of F6.44, and water clusters. Distinct conformational states are shown to be stabilized by each biased agonist. In particular, in simulations of the receptor with the β-arrestin-biased agonist N-cyclopentylbutanepherine, we observe a different pattern of motions in helix 7 when compared to simulations with the G protein-biased agonist salbutamol that involves perturbations of the network of interactions within the NPxxY motif. Understanding the network of interactions induced by biased ligands and the subsequent receptor conformational shifts will lead to development of more efficient drugs. PMID:23879802

Preclinical evaluation of SMM-189, a cannabinoid receptor 2-specific inverse agonist

PubMed Central

Presley, Chaela; Abidi, Ammaar; Suryawanshi, Satyendra; Mustafa, Suni; Meibohm, Bernd; Moore, Bob M

2015-01-01

Cannabinoid receptor 2 agonists and inverse agonists are emerging as new therapeutic options for a spectrum of autoimmune-related disease. Of particular interest, is the ability of CB2 ligands to regulate microglia function in neurodegenerative diseases and traumatic brain injury. We have previously reported the receptor affinity of 3′,5′-dichloro-2,6-dihydroxy-biphenyl-4-yl)-phenyl-methanone (SMM-189) and the characterization of the beneficial effects of SMM-189 in the mouse model of mild traumatic brain injury. Herein, we report the further characterization of SMM-189 as a potent and selective CB2 inverse agonist, which acts as a noncompetitive inhibitor of CP 55,940. The ability of SMM-189 to regulate microglial activation, in terms of chemokine expression and cell morphology, has been determined. Finally, we have determined that SMM-189 possesses acceptable biopharmaceutical properties indicating that the triaryl class of CB2 inverse agonists are viable compounds for continued preclinical development for the treatment of neurodegenerative disorders and traumatic brain injury. PMID:26196013

Preclinical evaluation of SMM-189, a cannabinoid receptor 2-specific inverse agonist.

PubMed

Presley, Chaela; Abidi, Ammaar; Suryawanshi, Satyendra; Mustafa, Suni; Meibohm, Bernd; Moore, Bob M

2015-08-01

Cannabinoid receptor 2 agonists and inverse agonists are emerging as new therapeutic options for a spectrum of autoimmune-related disease. Of particular interest, is the ability of CB2 ligands to regulate microglia function in neurodegenerative diseases and traumatic brain injury. We have previously reported the receptor affinity of 3',5'-dichloro-2,6-dihydroxy-biphenyl-4-yl)-phenyl-methanone (SMM-189) and the characterization of the beneficial effects of SMM-189 in the mouse model of mild traumatic brain injury. Herein, we report the further characterization of SMM-189 as a potent and selective CB2 inverse agonist, which acts as a noncompetitive inhibitor of CP 55,940. The ability of SMM-189 to regulate microglial activation, in terms of chemokine expression and cell morphology, has been determined. Finally, we have determined that SMM-189 possesses acceptable biopharmaceutical properties indicating that the triaryl class of CB2 inverse agonists are viable compounds for continued preclinical development for the treatment of neurodegenerative disorders and traumatic brain injury.

Crystal Structure of Oligomeric β1-Adrenergic G Protein- Coupled Receptors in Ligand-Free Basal State

PubMed Central

Huang, Jianyun; Chen, Shuai; Zhang, J. Jillian; Huang, Xin-Yun

2013-01-01

G protein-coupled receptors (GPCRs) mediate transmembrane signaling. Before ligand binding, GPCRs exist in a basal state. Crystal structures of several GPCRs bound with antagonists or agonists have been solved. However, the crystal structure of the ligand-free basal state of a GPCR, the starting point of GPCR activation and function, has not been determined. Here we report the X-ray crystal structure of the first ligand-free basal state of a GPCR in a lipid membrane-like environment. Oligomeric turkey β1-adrenergic receptors display two alternating dimer interfaces. One interface involves the transmembrane domain (TM) 1, TM2, the C-terminal H8, and the extracellular loop 1. The other interface engages residues from TM4, TM5, the intracellular loop 2 and the extracellular loop 2. Structural comparisons show that this ligand-free state is in an inactive conformation. This provides the structural information regarding GPCR dimerization and oligomerization. PMID:23435379

The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts*

PubMed Central

Jensen, Dane D.; Godfrey, Cody B.; Niklas, Christian; Canals, Meritxell; Kocan, Martina; Poole, Daniel P.; Murphy, Jane E.; Alemi, Farzad; Cottrell, Graeme S.; Korbmacher, Christoph; Lambert, Nevin A.; Bunnett, Nigel W.; Corvera, Carlos U.

2013-01-01

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists. PMID:23818521

Cannabinoid 1 (CB1) receptors coupled to cholinergic motorneurones inhibit neurogenic circular muscle contractility in the human colon

PubMed Central

Hinds, Nicholas M; Ullrich, Katja; Smid, Scott D

2006-01-01

The effects of cannabinoid subtype 1 (CB1) receptor activation were determined on smooth muscle, inhibitory and excitatory motorneuronal function in strips of human colonic longitudinal muscle (LM) and circular muscle (CM) in vitro. Electrical field stimulation (EFS; 0.5–20 Hz, 50 V) evoked a relaxation in LM and CM precontracted with a neurokinin-2 (NK-2) selective receptor agonist (β-ala8-neurokinin A; 10−6 M) in the presence of atropine (10−6 M); this was unaltered following pretreatment with the CB1-receptor selective agonist arachidonyl-2-chloroethylamide (ACEA; 10−6 M). In the presence of nitric oxide synthase blockade with N-nitro-L-arginine (10−4 M), EFS evoked a frequency-dependent ‘on-contraction' during stimulation and an ‘off-contraction' following stimulus cessation. On-contractions were significantly inhibited in CM strips by pretreatment with ACEA (10−6 M). These inhibitory effects were reversed in the presence of the CB1 receptor-selective antagonist N-(piperidine-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (10−7 M). ACEA did not alter LM or CM contractile responses to acetylcholine or NK-2 receptor-evoked contraction. Immunohistochemical studies revealed a colocalisation of CB1 receptors to cholinergic neurones in the human colon based on colabelling with choline acetyltransferase, in addition to CB1 receptor labelling in unidentified structures in the CM. In conclusion, activation of CB1 receptors coupled to cholinergic motorneurones selectively and reversibly inhibits excitatory nerve transmission in colonic human colonic CM. These results provide evidence of a direct role for cannabinoids in the modulation of motor activity in the human colon by coupling to cholinergic motorneurones. PMID:16520743

Evaluation of commercial ß-agonists, dietary protein, and shade on fecal shedding of Escherichia coli O157:H7 from feedlot cattle

USDA-ARS?s Scientific Manuscript database

Escherichia coli O157:H7 is a foodborne pathogen commonly associated with cattle feces. Diet, including dietary supplements such as b-agonists, may impact fecal shedding of this pathogen. A series of three experiments were conducted to determine if the b- agonists ractopamine hydrochloride (RAC) or ...

Toward an understanding of agonist binding to human Orexin-1 and Orexin-2 receptors with G-protein-coupled receptor modeling and site-directed mutagenesis.

PubMed

Heifetz, Alexander; Barker, Oliver; Morris, G Benjamin; Law, Richard J; Slack, Mark; Biggin, Philip C

2013-11-19

The class A G-protein-coupled receptors (GPCRs) Orexin-1 (OX1) and Orexin-2 (OX2) are located predominantly in the brain and are linked to a range of different physiological functions, including the control of feeding, energy metabolism, modulation of neuro-endocrine function, and regulation of the sleep-wake cycle. The natural agonists for OX1 and OX2 are two neuropeptides, Orexin-A and Orexin-B, which have activity at both receptors. Site-directed mutagenesis (SDM) has been reported on both the receptors and the peptides and has provided important insight into key features responsible for agonist activity. However, the structural interpretation of how these data are linked together is still lacking. In this work, we produced and used SDM data, homology modeling followed by MD simulation, and ensemble-flexible docking to generate binding poses of the Orexin peptides in the OX receptors to rationalize the SDM data. We also developed a protein pairwise similarity comparing method (ProS) and a GPCR-likeness assessment score (GLAS) to explore the structural data generated within a molecular dynamics simulation and to help distinguish between different GPCR substates. The results demonstrate how these newly developed methods of structural assessment for GPCRs can be used to provide a working model of neuropeptide-Orexin receptor interaction.

Ca(2+)-sensitive tyrosine kinase Pyk2/CAK beta-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy.

PubMed

Hirotani, Shinichi; Higuchi, Yoshiharu; Nishida, Kazuhiko; Nakayama, Hiroyuki; Yamaguchi, Osamu; Hikoso, Shungo; Takeda, Toshihiro; Kashiwase, Kazunori; Watanabe, Tetsuya; Asahi, Michio; Taniike, Masayuki; Tsujimoto, Ikuko; Matsumura, Yasushi; Sasaki, Terukatsu; Hori, Masatsugu; Otsu, Kinya

2004-06-01

G-protein-coupled receptor agonists including endothelin-1 (ET-1) and phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Others and we previously reported that Rac1 signaling pathway plays an important role in this agonist-induced cardiomyocyte hypertrophy. In this study reported here, we found that a Ca(2+)-sensitive non-receptor tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2)/cell adhesion kinase beta (CAKbeta), is involved in ET-1- and PE-induced cardiomyocyte hypertrophy medicated through Rac1 activation. ET-1, PE or the Ca(2+) inophore, ionomycin, stimulated a rapid increase in tyrosine phosphorylation of Pyk2. The tyrosine phosphorylation of Pyk2 was suppressed by the Ca(2+) chelator, BAPTA. ET-1- or PE-induced increases in [(3)H]-leucine incorporation and expression of atrial natriuretic factor and the enhancement of sarcomere organization. Infection of cardiomyocytes with an adenovirus expressing a mutant Pyk2 which lacked its kinase domain or its ability to bind to c-Src, eliminated ET-1- and PE-induced hypertrophic responses. Inhibition of Pyk2 activation also suppressed Rac1 activation and reactive oxygen species (ROS) production. These findings suggest that the signal transduction pathway leading to hypertrophy involves Ca(2+)-induced Pyk2 activation followed by Rac1-dependent ROS production.

The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

PubMed Central

Yao, Xiao Jie; Vélez Ruiz, Gisselle; Whorton, Matthew R.; Rasmussen, Søren G. F.; DeVree, Brian T.; Deupi, Xavier; Sunahara, Roger K.; Kobilka, Brian

2009-01-01

G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the β2 adrenergic receptor (β2AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The β2AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist- and Gs-induced conformational changes. In the absence of an agonist, the β2AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the β2AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the β2AR and the structural basis for ligand efficacy. PMID:19470481

Coexpression of human somatostatin receptor-2 (SSTR2) and SSTR3 modulates antiproliferative signaling and apoptosis

PubMed Central

2012-01-01

Background Somatostatin (SST) via five Gi coupled receptors namely SSTR1-5 is known to inhibit cell proliferation by cytostatic and cytotoxic mechanisms. Heterodimerization plays a crucial role in modulating the signal transduction pathways of SSTR subtypes. In the present study, we investigated human SSTR2/SSTR3 heterodimerization, internalization, MAPK signaling, cell proliferation and apoptosis in HEK-293 cells in response to SST and specific agonists for SSTR2 and SSTR3. Results Although in basal conditions, SSTR2 and SSTR3 colocalize at the plasma membrane and exhibit heterodimerization, the cell surface distribution of both receptors decreased upon agonist activation and was accompanied by a parallel increase in intracellular colocalization. Receptors activation by SST and specific agonists significantly decreased cAMP levels in cotransfected cells in comparison to control. Agonist-mediated modulation of pERK1/2 was time and concentration-dependent, and pronounced in serum-deprived conditions. pERK1/2 was inhibited in response to SST; conversely receptor-specific agonist treatment caused inhibition at lower concentration and activation at higher concentration. Strikingly, ERK1/2 phosphorylation was sustained upon prolonged treatment with SST but not with receptor-specific agonists. On the other hand, SST and receptor-specific agonists modulated p38 phosphorylation time-dependently. The receptor activation in cotransfected cells exhibits Gi-dependent inhibition of cell proliferation attributed to increased PARP-1 expression and TUNEL staining, whereas induction of p21 and p27Kip1 suggests a cytostatic effect. Conclusion Our study provides new insights in SSTR2/SSTR3 mediated signaling which might help in better understanding of the molecular interactions involving SSTRs in tumor biology. PMID:22651821

Downregulation of kinin B1 receptor function by B2 receptor heterodimerization and signaling.

PubMed

Zhang, Xianming; Brovkovych, Viktor; Zhang, Yongkang; Tan, Fulong; Skidgel, Randal A

2015-01-01

Signaling through the G protein-coupled kinin receptors B1 (kB1R) and B2 (kB2R) plays a critical role in inflammatory responses mediated by activation of the kallikrein-kinin system. The kB2R is constitutively expressed and rapidly desensitized in response to agonist whereas kB1R expression is upregulated by inflammatory stimuli and it is resistant to internalization and desensitization. Here we show that the kB1R heterodimerizes with kB2Rs in co-transfected HEK293 cells and natively expressing endothelial cells, resulting in significant internalization and desensitization of the kB1R response in cells pre-treated with kB2R agonist. However, pre-treatment of cells with kB1R agonist did not affect subsequent kB2R responses. Agonists of other G protein-coupled receptors (thrombin, lysophosphatidic acid) had no effect on a subsequent kB1R response. The loss of kB1R response after pretreatment with kB2R agonist was partially reversed with kB2R mutant Y129S, which blocks kB2R signaling without affecting endocytosis, or T342A, which signals like wild type but is not endocytosed. Co-endocytosis of the kB1R with kB2R was dependent on β-arrestin and clathrin-coated pits but not caveolae. The sorting pathway of kB1R and kB2R after endocytosis differed as recycling of kB1R to the cell surface was much slower than that of kB2R. In cytokine-treated human lung microvascular endothelial cells, pre-treatment with kB2R agonist inhibited kB1R-mediated increase in transendothelial electrical resistance (TER) caused by kB1R stimulation (to generate nitric oxide) and blocked the profound drop in TER caused by kB1R activation in the presence of pyrogallol (a superoxide generator). Thus, kB1R function can be downregulated by kB2R co-endocytosis and signaling, suggesting new approaches to control kB1R signaling in pathological conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

Downregulation of kinin B1 receptor function by B2 receptor heterodimerization and signaling

PubMed Central

Zhang, Xianming; Brovkovych, Viktor; Zhang, Yongkang; Tan, Fulong; Skidgel, Randal A.

2014-01-01

Signaling through the G protein-coupled kinin receptors B1 (kB1R) and B2 (kB2R) plays a critical role in inflammatory responses mediated by activation of the kallikrein-kinin system. The kB2R is constitutively expressed and rapidly desensitized in response to agonist whereas kB1R expression is upregulated by inflammatory stimuli and it is resistant to internalization and desensitization. Here we show that the kB1R heterodimerizes with kB2Rs in co-transfected HEK293 cells and natively expressing endothelial cells, resulting in significant internalization and desensitization of the kB1R response in cells pre-treated with kB2R agonist. However, pre-treatment of cells with kB1R agonist did not affect subsequent kB2R responses. Agonists of other G protein-coupled receptors (thrombin, lysophosphatidic acid) had no effect on a subsequent kB1R response. The loss of kB1R response after pretreatment with kB2R agonist was partially reversed with kB2R mutant Y129S, which blocks kB2R signaling without affecting endocytosis, or T342A, which signals like wild type but is not endocytosed. Co-endocytosis of the kB1R with kB2R was dependent on β-arrestin and clathrin-coated pits but not caveolae. The sorting pathway of kB1R and kB2R after endocytosis differed as recycling of kB1R to the cell surface was much slower than that of kB2R. In cytokine-treated human lung microvascular endothelial cells, pre-treatment with kB2R agonist inhibited kB1R-mediated increase in transendothelial electrical resistance (TER) caused by kB1R stimulation (to generate nitric oxide) and blocked the profound drop in TER caused by kB1R activation in the presence of pyrogallol (a superoxide generator). Thus, kB1R function can be downregulated by kB2R co-endocytosis and signaling, suggesting new approaches to control kB1R signaling in pathological conditions. PMID:25289859

Molecular interactions between fenoterol stereoisomers and derivatives and the β₂-adrenergic receptor binding site studied by docking and molecular dynamics simulations.

PubMed

Plazinska, Anita; Kolinski, Michal; Wainer, Irving W; Jozwiak, Krzysztof

2013-11-01

The β2 adrenergic receptor (β2-AR) has become a model system for studying the ligand recognition process and mechanism of the G protein coupled receptors activation. In the present study stereoisomers of fenoterol and some of its derivatives (N = 94 molecules) were used as molecular probes to identify differences in stereo-recognition interactions between β2-AR and structurally similar agonists. The present study aimed at determining the 3D molecular models of the fenoterol derivative-β2-AR complexes. Molecular models of β2-AR have been developed by using the crystal structure of the human β2-AR T4 lysozyme fusion protein with bound (S)-carazolol (PDB ID: 2RH1) and more recently reported structure of a nanobody-stabilized active state of the β2-AR with the bound full agonist BI-167107 (PDB ID: 3P0G). The docking procedure allowed us to study the similarities and differences in the recognition binding site(s) for tested ligands. The agonist molecules occupied the same binding region, between TM III, TM V, TM VI and TM VII. The residues identified by us during docking procedure (Ser203, Ser207, Asp113, Lys305, Asn312, Tyr308, Asp192) were experimentally indicated in functional and biophysical studies as being very important for the agonist-receptor interactions. Moreover, the additional space, an extension of the orthosteric pocket, was identified and described. Furthermore, the molecular dynamics simulations were used to study the molecular mechanism of interaction between ligands ((R,R')- and (S,S')-fenoterol) and β2-AR. Our research offers new insights into the ligand stereoselective interaction with one of the most important GPCR member. This study may also facilitate the design of improved selective medications, which can be used to treat, prevent and control heart failure symptoms.

Central Agonism of GPR120 Acutely Inhibits Food Intake and Food Reward and Chronically Suppresses Anxiety-Like Behavior in Mice

PubMed Central

Fisette, Alexandre; Fernandes, Maria F.; Hryhorczuk, Cécile; Poitout, Vincent; Alquier, Thierry; Fulton, Stephanie

2016-01-01

Background: GPR120 (FFAR4) is a G-protein coupled receptor implicated in the development of obesity and the antiinflammatory and insulin-sensitizing effects of omega-3 (ω-3) polyunsaturated fatty acids. Increasing central ω-3 polyunsaturated fatty acid levels has been shown to have both anorectic and anxiolytic actions. Despite the strong clinical interest in GPR120, its role in the brain is largely unknown, and thus we sought to determine the impact of central GPR120 pharmacological activation on energy balance, food reward, and anxiety-like behavior. Methods: Male C57Bl/6 mice with intracerebroventricular cannulae received a single injection (0.1 or 1 µM) or continuous 2-week infusion (1 µM/d; mini-pump) of a GPR120 agonist or vehicle. Free-feeding intake, operant lever-pressing for palatable food, energy expenditure (indirect calorimetry), and body weight were measured. GPR120 mRNA expression was measured in pertinent brain areas. Anxiety-like behavior was assessed in the elevated-plus maze and open field test. Results: GPR120 agonist injections substantially reduced chow intake during 4 hours postinjection, suppressed the rewarding effects of high-fat/-sugar food, and blunted approach-avoidance behavior in the open field. Conversely, prolonged central GPR120 agonist infusions reduced anxiety-like behavior in the elevated-plus maze and open field, yet failed to affect free-feeding intake, energy expenditure, and body weight on a high-fat diet. Conclusion: Acute reductions in food intake and food reward suggest that GPR120 could mediate the effects of central ω-3 polyunsaturated fatty acids to inhibit appetite. The anxiolytic effect elicited by GPR120 agonist infusions favors the testing of compounds that can enter the brain to activate GPR120 for the mitigation of anxiety. PMID:26888796

Nicotinic receptor transduction zone: invariant arginine couples to multiple electron-rich residues.

PubMed

Mukhtasimova, Nuriya; Sine, Steven M

2013-01-22

Gating of the muscle-type acetylcholine receptor (AChR) channel depends on communication between the ACh-binding site and the remote ion channel. A key region for this communication is located within the structural transition zone between the ligand-binding and pore domains. Here, stemming from β-strand 10 of the binding domain, the invariant αArg209 lodges within the hydrophobic interior of the subunit and is essential for rapid and efficient channel gating. Previous charge-reversal experiments showed that the contribution of αArg209 to channel gating depends strongly on αGlu45, also within this region. Here we determine whether the contribution of αArg209 to channel gating depends on additional anionic or electron-rich residues in this region. Also, to reconcile diverging findings in the literature, we compare the dependence of αArg209 on αGlu45 in AChRs from different species, and compare the full agonist ACh with the weak agonist choline. Our findings reveal that the contribution of αArg209 to channel gating depends on additional nearby electron-rich residues, consistent with both electrostatic and steric contributions. Furthermore, αArg209 and αGlu45 show a strong interdependence in both human and mouse AChRs, whereas the functional consequences of the mutation αE45R depend on the agonist. The emerging picture shows a multifaceted network of interdependent residues that are required for communication between the ligand-binding and pore domains. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding.

PubMed

Jung, Seung-Ryoung; Kushmerick, Christopher; Seo, Jong Bae; Koh, Duk-Su; Hille, Bertil

2017-07-11

Binding of agonists to G-protein-coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M 1 muscarinic acetylcholine receptors (M 1 Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M 1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M 1 R, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane.

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding

PubMed Central

Jung, Seung-Ryoung; Kushmerick, Christopher; Seo, Jong Bae; Koh, Duk-Su

2017-01-01

Binding of agonists to G-protein–coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M1 muscarinic acetylcholine receptors (M1Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M1R, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane. PMID:28652372

Induction of selective blood-tumor barrier permeability and macromolecular transport by a biostable kinin B1 receptor agonist in a glioma rat model.

PubMed

Côté, Jérôme; Bovenzi, Veronica; Savard, Martin; Dubuc, Céléna; Fortier, Audrey; Neugebauer, Witold; Tremblay, Luc; Müller-Esterl, Werner; Tsanaclis, Ana-Maria; Lepage, Martin; Fortin, David; Gobeil, Fernand

2012-01-01

Treatment of malignant glioma with chemotherapy is limited mostly because of delivery impediment related to the blood-brain tumor barrier (BTB). B1 receptors (B1R), inducible prototypical G-protein coupled receptors (GPCR) can regulate permeability of vessels including possibly that of brain tumors. Here, we determine the extent of BTB permeability induced by the natural and synthetic peptide B1R agonists, LysdesArg(9)BK (LDBK) and SarLys[dPhe(8)]desArg(9)BK (NG29), in syngeneic F98 glioma-implanted Fischer rats. Ten days after tumor inoculation, we detected the presence of B1R on tumor cells and associated vasculature. NG29 infusion increased brain distribution volume and uptake profiles of paramagnetic probes (Magnevist and Gadomer) at tumoral sites (T(1)-weighted imaging). These effects were blocked by B1R antagonist and non-selective cyclooxygenase inhibitors, but not by B2R antagonist and non-selective nitric oxide synthase inhibitors. Consistent with MRI data, systemic co-administration of NG29 improved brain tumor delivery of Carboplatin chemotherapy (ICP-Mass spectrometry). We also detected elevated B1R expression in clinical samples of high-grade glioma. Our results documented a novel GPCR-signaling mechanism for promoting transient BTB disruption, involving activation of B1R and ensuing production of COX metabolites. They also underlined the potential value of synthetic biostable B1R agonists as selective BTB modulators for local delivery of different sized-therapeutics at (peri)tumoral sites.

Induction of Selective Blood-Tumor Barrier Permeability and Macromolecular Transport by a Biostable Kinin B1 Receptor Agonist in a Glioma Rat Model

PubMed Central

Côté, Jérôme; Bovenzi, Veronica; Savard, Martin; Dubuc, Céléna; Fortier, Audrey; Neugebauer, Witold; Tremblay, Luc; Müller-Esterl, Werner; Tsanaclis, Ana-Maria; Lepage, Martin; Fortin, David; Gobeil, Fernand

2012-01-01

Treatment of malignant glioma with chemotherapy is limited mostly because of delivery impediment related to the blood-brain tumor barrier (BTB). B1 receptors (B1R), inducible prototypical G-protein coupled receptors (GPCR) can regulate permeability of vessels including possibly that of brain tumors. Here, we determine the extent of BTB permeability induced by the natural and synthetic peptide B1R agonists, LysdesArg9BK (LDBK) and SarLys[dPhe8]desArg9BK (NG29), in syngeneic F98 glioma-implanted Fischer rats. Ten days after tumor inoculation, we detected the presence of B1R on tumor cells and associated vasculature. NG29 infusion increased brain distribution volume and uptake profiles of paramagnetic probes (Magnevist and Gadomer) at tumoral sites (T 1-weighted imaging). These effects were blocked by B1R antagonist and non-selective cyclooxygenase inhibitors, but not by B2R antagonist and non-selective nitric oxide synthase inhibitors. Consistent with MRI data, systemic co-administration of NG29 improved brain tumor delivery of Carboplatin chemotherapy (ICP-Mass spectrometry). We also detected elevated B1R expression in clinical samples of high-grade glioma. Our results documented a novel GPCR-signaling mechanism for promoting transient BTB disruption, involving activation of B1R and ensuing production of COX metabolites. They also underlined the potential value of synthetic biostable B1R agonists as selective BTB modulators for local delivery of different sized-therapeutics at (peri)tumoral sites. PMID:22629405

Characterization of muscarinic receptors on intact human neuroblastoma cells: coupling to phosphoinositide hydrolysis and phosphorylation by phorbol esters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serra, M.; Watson, M.; Roeske, W.R.

Cloned human neuroblastoma cells (SH-SY5Y) were grown. High affinity binding of (/sup 3/H)(-)quinuclidinyl benzilate ((/sup 3/H)(-)QNB) and its quaternary derivative (/sup 3/H)(-)methyl QNB to muscarinic receptors (MR) on intact SH-SY5Y cells was studied. A 30 min rinse time gave a ratio of specific/total binding of 90% for both ligands. Association rates of (/sup 3/H)(-)QNB and (/sup 3/H)(-)methyl QNB were determined. Both ligands reached steady state by 60 min at 37/sup 0/C. Rates of dissociation for both radioligands were biphasic, although (/sup 3/H)(-)methyl QNB was faster. Saturation studies yielded K/sub d/ (dissociation constant) values of 16 and 260 pM and B/submore » max/ (maximal MR density) values of 172 and 134 fmoles/mg prot for (/sup 3/H)(-)QNB and (/sup 3/H)(-)methyl QNB, respectively. Activation of protein kinase C by phorbol esters produced increased phosphorylation of cellular proteins. Pretreatment with 100 nM of 4..beta..-phorbol 12..beta..-myristate 13..cap alpha..-acetate (PMA) induced a decrease in agonist affinity for MR, suggesting a PMA-promoted phosphorylation of the MR protein. Phosphoinositide (PhI) turnover was measured by MR agonist-induced accumulation of inositol-1-phosphate in the presence of Li/sup + +/ (10 mM). Only carbachol and acetylcholine elicited potent responses with oxotremorine (16%) pilocarpine (17%) and McN-A-343 (8%) appearing to be weak partial agonist of low efficacy.« less

Opioid agonist efficacy predicts the magnitude of tolerance and the regulation of mu-opioid receptors and dynamin-2.

PubMed

Pawar, Mohit; Kumar, Priyank; Sunkaraneni, Soujanya; Sirohi, Sunil; Walker, Ellen A; Yoburn, Byron C

2007-06-01

It has been proposed that opioid agonist efficacy may play a role in tolerance and the regulation of opioid receptor density. To address this issue, the present studies estimated the in vivo efficacy of three opioid agonists and then examined changes in spinal mu-opioid receptor density following chronic treatment in the mouse. In addition, tolerance and regulation of the trafficking protein dynamin-2 were determined. To evaluate efficacy, the method of irreversible receptor alkylation was employed and the efficacy parameter tau estimated. Mice were injected with the irreversible mu-opioid receptor antagonist clocinnamox (0.32-25.6 mg/kg, i.p), and 24 h later, the analgesic potency of s.c. morphine, oxycodone and etorphine were determined. Clocinnamox dose-dependently antagonized the analgesic effects of morphine, etorphine and oxycodone. The shift to the right of the dose-response curves was greater for morphine and oxycodone compared to etorphine and the highest dose of clocinnamox reduced the maximal effect of morphine and oxycodone, but not etorphine. The order of efficacy calculated from these results was etorphine>morphine>oxycodone. Other mice were infused for 7 days with oxycodone (10-150 mg/kg/day, s.c.) or etorphine (50-250 microg/kg/day, s.c.) and the analgesic potency of s.c. morphine determined. The low efficacy agonist (oxycodone) produced more tolerance than the high efficacy agonist (etorphine) at equi-effective infusion doses. In saturation binding experiments, the low efficacy opioid agonists (morphine, oxycodone) did not regulate the density of spinal mu-opioid receptors, while etorphine produced approximately 40% reduction in mu-opioid receptor density. Furthermore, etorphine increased spinal dynamin-2 abundance, while oxycodone did not produce any significant change in dynamin-2 abundance. Overall, these data indicate that high efficacy agonists produce less tolerance at equi-effective doses. Furthermore, increased efficacy was associated with mu-opioid receptor downregulation and dynamin-2 upregulation. Conversely, lower efficacy agonists produced more tolerance at equi-effective doses, but did not regulate mu-opioid receptor density or dynamin-2 abundance. Taken together, these studies indicate that agonist efficacy plays an important role in tolerance and regulation of receptors and trafficking proteins.

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity.

PubMed

Joseph, Christine G; Wilczynski, Andrzej; Holder, Jerry R; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Haskell-Luevano, Carrie

2003-12-01

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111-113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7-9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.

Exploring the binding energy profiles of full agonists, partial agonists, and antagonists of the α7 nicotinic acetylcholine receptor.

PubMed

Tabassum, Nargis; Ma, Qianyun; Wu, Guanzhao; Jiang, Tao; Yu, Rilei

2017-09-01

Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop receptor family and are important drug targets for the treatment of neurological diseases. However, the precise determinants of the binding efficacies of ligands for these receptors are unclear. Therefore, in this study, the binding energy profiles of various ligands (full agonists, partial agonists, and antagonists) were quantified by docking those ligands with structural ensembles of the α7 nAChR exhibiting different degrees of C-loop closure. This approximate treatment of interactions suggested that full agonists, partial agonists, and antagonists of the α7 nAChR possess distinctive binding energy profiles. Results from docking revealed that ligand binding efficacy may be related to the capacity of the ligand to stabilize conformational states with a closed C loop.

Methods to Quantify Cell Signaling and GPCR Receptor Ligand Bias: Characterization of Drugs that Target the Endocannabinoid Receptors in Huntington's Disease.

PubMed

Bagher, Amina M; Laprairie, Robert B; Kelly, Melanie E M; Denovan-Wright, Eileen M

2018-01-01

G protein-coupled receptors (GPCRs) interact with multiple intracellular effector proteins such that different ligands may preferentially activate one signal pathway over others, a phenomenon known as signaling bias. Signaling bias can be quantified to optimize drug selection for preclinical research. Here, we describe moderate-throughput methods to quantify signaling bias of known and novel compounds. In the example provided, we describe a method to define cannabinoid-signaling bias in a cell culture model of Huntington's disease (HD). Decreasing type 1 cannabinoid receptor (CB 1 ) levels is correlated with chorea and cognitive deficits in HD. There is evidence that elevating CB 1 levels and/or signaling may be beneficial for HD patients while decreasing CB 1 levels and/or signaling may be detrimental. Recent studies have found that Gα i/o -biased CB 1 agonists activate extracellular signal-regulated kinase (ERK), increase CB 1 protein levels, and improve viability of cells expressing mutant huntingtin. In contrast, CB 1 agonists that are β-arrestin1-biased were found to reduce CB 1 protein levels and cell viability. Measuring agonist bias of known and novel CB 1 agonists will provide important data that predict CB 1 -specific agonists that might be beneficial in animal models of HD and, following animal testing, in HD patients. This method can also be applied to study signaling bias for other GPCRs.

Intermediate closed state for glycine receptor function revealed by cysteine cross-linking.

PubMed

Prevost, Marie S; Moraga-Cid, Gustavo; Van Renterghem, Catherine; Edelstein, Stuart J; Changeux, Jean-Pierre; Corringer, Pierre-Jean

2013-10-15

Pentameric ligand-gated ion channels (pLGICs) mediate signal transmission by coupling the binding of extracellular ligands to the opening of their ion channel. Agonist binding elicits activation and desensitization of pLGICs, through several conformational states, that are, thus far, incompletely characterized at the structural level. We previously reported for GLIC, a prokaryotic pLGIC, that cross-linking of a pair of cysteines at both sides of the extracellular and transmembrane domain interface stabilizes a locally closed (LC) X-ray structure. Here, we introduced the homologous pair of cysteines on the human α1 glycine receptor. We show by electrophysiology that cysteine cross-linking produces a gain-of-function phenotype characterized by concomitant constitutive openings, increased agonist potency, and equalization of efficacies of full and partial agonists. However, it also produces a reduction of maximal currents at saturating agonist concentrations without change of the unitary channel conductance, an effect reversed by the positive allosteric modulator propofol. The cross-linking thus favors a unique closed state distinct from the resting and longest-lived desensitized states. Fitting the data according to a three-state allosteric model suggests that it could correspond to a LC conformation. Its plausible assignment to a gating intermediate or a fast-desensitized state is discussed. Overall, our data show that relative movement of two loops at the extracellular-transmembrane interface accompanies orthosteric agonist-mediated gating.

Determination of ragaglitazar, a novel dual acting peroxisome proliferator-activated receptor (PPAR) alpha and -gamma agonist, in human plasma by high-performance liquid chromatography coupled with tandem mass spectrometry.

PubMed

Andersen, Michael P; Nielsen, Karin K

2003-05-05

A sensitive and specific LC/MS/MS method has been developed and validated for determination of ragaglitazar (NNC 61-0029 or DRF 2725) in human plasma. After solid-phase extraction (SPEC((R)) PLUS C(8)) of plasma, separation was performed on a Symmetry Shield RP8 column (mobile phase: acetonitrile: 10 mM ammonium acetate, pH 5.6 (40:60 v/v)). Two ranges were validated having LLOQs of either 0.500 or 100 ng/ml and linearity up to either 500 or 50000 ng/ml. The intra-assay precision and accuracy were 1.1% to 15.7% and 85.8% to 118.2% (range 0.500-500 ng/ml) and 2.0% to 8.8% and 92.9% to 104.8% (range 100-50000 ng/ml). The method was applied for determination of ragaglitazar in plasma from phase 1 and 2 clinical studies.

Activation of single heteromeric GABAA receptor ion channels by full and partial agonists

PubMed Central

Mortensen, Martin; Kristiansen, Uffe; Ebert, Bjarke; Frølund, Bente; Krogsgaard-Larsen, Povl; Smart, Trevor G

2004-01-01

The linkage between agonist binding and the activation of a GABAA receptor ion channel is yet to be resolved. This aspect was examined on human recombinant α1β2γ2S GABAA receptors expressed in human embryonic kidney cells using the following series of receptor agonists: GABA, isoguvacine, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), isonipecotic acid, piperidine-4-sulphonic acid (P4S), imidazole-4-acetic acid (IAA), 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL) and 5-(4-piperidyl)-3-isoxazolol (4-PIOL). Whole-cell concentration–response curves enabled the agonists to be categorized into four classes based upon their maximum responses. Single channel analyses revealed that the channel conductance of 25–27 pS was unaffected by the agonists. However, two open states were resolved from the open period distributions with mean open times reduced 5-fold by the weakest partial agonists. Using saturating agonist concentrations, estimates of the channel shutting rate, α, ranged from 200 to 600 s−1. The shut period distributions were described by three or four components and for the weakest partial agonists, the interburst shut periods increased whilst the mean burst durations and longest burst lengths were reduced relative to the full agonists. From the burst analyses, the opening rates for channel activation, β, and the total dissociation rates, k−1, for the agonists leaving the receptor were estimated. The agonist efficacies were larger for the full agonists (E ∼7−9) compared to the weak partial agonists (∼0.4–0.6). Overall, changes in agonist efficacy largely determined the different agonist profiles with contributions from the agonist affinities and the degree of receptor desensitization. From this we conclude that GABAA receptor activation does not occur in a switch-like manner since the agonist recognition sites are flexible, accommodating diverse agonist structures which differentially influence the opening and shutting rates of the ion channel. PMID:14990676

Discovery of a Selective S1P1 Receptor Agonist Efficacious at Low Oral Dose and Devoid of Effects on Heart Rate.

PubMed

Demont, Emmanuel H; Andrews, Benjamin I; Bit, Rino A; Campbell, Colin A; Cooke, Jason W B; Deeks, Nigel; Desai, Sapna; Dowell, Simon J; Gaskin, Pam; Gray, James R J; Haynes, Andrea; Holmes, Duncan S; Kumar, Umesh; Morse, Mary A; Osborne, Greg J; Panchal, Terry; Patel, Bela; Perboni, Alcide; Taylor, Simon; Watson, Robert; Witherington, Jason; Willis, Robert

2011-06-09

Gilenya (fingolimod, FTY720) was recently approved by the U.S. FDA for the treatment of patients with remitting relapsing multiple sclerosis (RRMS). It is a potent agonist of four of the five sphingosine 1-phosphate (S1P) G-protein-coupled receptors (S1P1 and S1P3-5). It has been postulated that fingolimod's efficacy is due to S1P1 agonism, while its cardiovascular side effects (transient bradycardia and hypertension) are due to S1P3 agonism. We have discovered a series of selective S1P1 agonists, which includes 3-[6-(5-{3-cyano-4-[(1-methylethyl)oxy]phenyl}-1,2,4-oxadiazol-3-yl)-5-methyl-3,4-dihydro-2(1H)-isoquinolinyl]propanoate, 20, a potent, S1P3-sparing, orally active S1P1 agonist. Compound 20 is as efficacious as fingolimod in a collagen-induced arthritis model and shows excellent pharmacokinetic properties preclinically. Importantly, the selectivity of 20 against S1P3 is responsible for an absence of cardiovascular signal in telemetered rats, even at high dose levels.

Discovery of a Selective S1P1 Receptor Agonist Efficacious at Low Oral Dose and Devoid of Effects on Heart Rate

PubMed Central

2011-01-01

Gilenya (fingolimod, FTY720) was recently approved by the U.S. FDA for the treatment of patients with remitting relapsing multiple sclerosis (RRMS). It is a potent agonist of four of the five sphingosine 1-phosphate (S1P) G-protein-coupled receptors (S1P1 and S1P3−5). It has been postulated that fingolimod's efficacy is due to S1P1 agonism, while its cardiovascular side effects (transient bradycardia and hypertension) are due to S1P3 agonism. We have discovered a series of selective S1P1 agonists, which includes 3-[6-(5-{3-cyano-4-[(1-methylethyl)oxy]phenyl}-1,2,4-oxadiazol-3-yl)-5-methyl-3,4-dihydro-2(1H)-isoquinolinyl]propanoate, 20, a potent, S1P3-sparing, orally active S1P1 agonist. Compound 20 is as efficacious as fingolimod in a collagen-induced arthritis model and shows excellent pharmacokinetic properties preclinically. Importantly, the selectivity of 20 against S1P3 is responsible for an absence of cardiovascular signal in telemetered rats, even at high dose levels. PMID:24900328

The GIT–PIX complexes regulate the chemotactic response of rat basophilic leukaemia cells

PubMed Central

Gavina, Manuela; Za, Lorena; Molteni, Raffaella; Pardi, Ruggero; Curtis, Ivan de

2009-01-01

Background information. Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT–PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G-protein-coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl-Met-Leu-Phe) peptide, in a rat basophilic leukaemia RBL-2H3 cell line stably expressing an HA (haemagglutinin)-tagged receptor for the fMLP peptide. Results. Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1–PIX and GIT2–PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist-induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist-enhanced cell spreading was inhibited. Analysis of agonist-stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans-well migration. The internalization of the formyl-peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody-enhanced agonist-induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors. Conclusions. Our results show that endogenous GIT complexes are involved in the regulation of chemoattractant-induced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G-protein-coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist-induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins. PMID:19912111

Exploring pharmacological activities and signaling of morphinans substituted in position 6 as potent agonists interacting with the μ opioid receptor

PubMed Central

2014-01-01

Background Opioid analgesics are the most effective drugs for the treatment of moderate to severe pain. However, they also produce several adverse effects that can complicate pain management. The μ opioid (MOP) receptor, a G protein-coupled receptor, is recognized as the opioid receptor type which primarily mediates the pharmacological actions of clinically used opioid agonists. The morphinan class of analgesics including morphine and oxycodone are of main importance as therapeutically valuable drugs. Though the natural alkaloid morphine contains a C-6-hydroxyl group and the semisynthetic derivative oxycodone has a 6-carbonyl function, chemical approaches have uncovered that functionalizing position 6 gives rise to a range of diverse activities. Hence, position 6 of N-methylmorphinans is one of the most manipulated sites, and is established to play a key role in ligand binding at the MOP receptor, efficacy, signaling, and analgesic potency. We have earlier reported on a chemically innovative modification in oxycodone resulting in novel morphinans with 6-acrylonitrile incorporated substructures. Results This study describes in vitro and in vivo pharmacological activities and signaling of new morphinans substituted in position 6 with acrylonitrile and amido functions as potent agonists and antinociceptive agents interacting with MOP receptors. We show that the presence of a 6-cyano group in N-methylmorphinans has a strong influence on the binding to the opioid receptors and post-receptor signaling. One 6-cyano-N-methylmorphinan of the series was identified as the highest affinity and most selective MOP agonist, and very potent in stimulating G protein coupling and intracellular calcium release through the MOP receptor. In vivo, this MOP agonist showed to be greatly effective against thermal and chemical nociception in mice with marked increased antinociceptive potency than the lead molecule oxycodone. Conclusion Development of such novel chemotypes by targeting position 6 provides valuable insights on ligand-receptor interaction and molecular mode of action, and may aid in identification of opioid therapeutics with enhanced analgesic properties and fewer undesirable effects. PMID:25059282

Functional selectivity of allosteric interactions within G protein-coupled receptor oligomers: the dopamine D1-D3 receptor heterotetramer.

PubMed

Guitart, Xavier; Navarro, Gemma; Moreno, Estefania; Yano, Hideaki; Cai, Ning-Sheng; Sánchez-Soto, Marta; Kumar-Barodia, Sandeep; Naidu, Yamini T; Mallol, Josefa; Cortés, Antoni; Lluís, Carme; Canela, Enric I; Casadó, Vicent; McCormick, Peter J; Ferré, Sergi

2014-10-01

The dopamine D1 receptor-D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa-induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques, and cell-signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R-D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and Gi proteins, respectively. Coactivation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of β-arrestin-1, and to a positive cross talk of D1R and D3R agonists at the level of mitogen-activated protein kinase (MAPK) signaling. Furthermore, D1R or D3R antagonists counteracted β-arrestin-1 recruitment and MAPK activation induced by D3R and D1R agonists, respectively (cross-antagonism). Positive cross talk and cross-antagonism at the MAPK level were counteracted by specific synthetic peptides with amino acid sequences corresponding to D1R transmembrane (TM) domains TM5 and TM6, which also selectively modified the quaternary structure of the D1R-D3R heteromer, as demonstrated by complementation of hemiproteins of yellow fluorescence protein fused to D1R and D3R. These results demonstrate functional selectivity of allosteric modulations within the D1R-D3R heteromer, which can be involved with the reported behavioral synergism of D1R and D3R agonists. U.S. Government work not protected by U.S. copyright.

Vasoconstriction induced by G1, a G-protein-coupled oestrogen receptor1 (GPER-1) agonist, in the isolated perfused rat kidney.

PubMed

Kurt, Akif Hakan; Buyukafsar, Kansu

2013-02-28

Vascular effects of the G protein-coupled oestrogen receptor1 (GPER-1) agonist, G1 (10(-7)-5×10(-6) M), the main oestrogenic hormone, 17β-estradiol (10(-9)-10(-4) M), the NR3A1 agonist, PPT (10(-8)-10(-5) M), the NR3A2 agonist DPN (10(-8)-10(-5) M), and the classical oestrogen receptor blocker but also a GPER agonist, ICI-182780 (10(-8)-3×10(-6) M), were investigated on the perfusion pressure in the isolated rat kidney. To seek cellular mechanisms involved in GPER-1-induced signalling we tested several compounds including the inhibitors of Rho-kinase (ROCK) (Y-27632), tyrosine kinase (genistein), p38MAPK (SB203580), p44/42MAPK (PD98059), protein kinase C (PKC) (GF109203X), Jun-kinase (JNK) (SP600125), phosphatidylinositol-3-kinase (PI3K) (LY294002), Ca(2+) channels (nifedipine), GPER-1 (G15) and epidermal growth factor (EGF) receptor kinase (AG-1478). Moreover, the effect of saponin (50mg/ml) that was used for endothelium removal was explored on G1-elicited vascular action. G1, 17β-estradiol and ICI-182780 but not PPT and DPN induced vasoconstrictions in basal renal perfusion pressure. In contrast, G1 promoted vasodilatation when the perfusion pressure was elevated in advance by phenylephrine. G1-elicited vasoconstriction was not modified by endothelial removal; however, it was markedly inhibited by GPER-1 antagonist, G15. The vasoconstrictor response to G1 was also significantly attenuated by Y-27632, PD98059, SB203580, GF109203X, genistein, AG-1478, and nifedipine, but not LY294002 and SP600125. Western blotting indicated the expression of GPER-1 in renal artery, medulla and cortex of rat kidney. In conclusion, GPER-1 could substantially modulate vascular responses through a variety of signalling pathways including ROCK, PKC, p38 MAPK, p42/44 MAPK, tyrosine kinase, EGF receptor kinase and VOCC but not JNK or PI3K in isolated perfused rat kidney. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, B.T.

1989-06-01

Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand (3H)-8-cyclopentyl-1,3-diproylxanthine ((3H)CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or themore » maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that (3H) CPX is an antagonist radioligand. Competition curves for (3H) CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific (3H)CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid).« less

An EP2 Agonist Facilitates NMDA-Induced Outward Currents and Inhibits Dendritic Beading through Activation of BK Channels in Mouse Cortical Neurons

PubMed Central

Hayashi, Yoshinori; Morinaga, Saori; Liu, Xia; Zhang, Jing; Wu, Zhou; Yokoyama, Takeshi; Nakanishi, Hiroshi

2016-01-01

Prostaglandin E2 (PGE2), a major metabolite of arachidonic acid produced by cyclooxygenase pathways, exerts its bioactive responses by activating four E-prostanoid receptor subtypes, EP1, EP2, EP3, and EP4. PGE2 enables modulating N-methyl-D-aspartate (NMDA) receptor-mediated responses. However, the effect of E-prostanoid receptor agonists on large-conductance Ca2+-activated K+ (BK) channels, which are functionally coupled with NMDA receptors, remains unclear. Here, we showed that EP2 receptor-mediated signaling pathways increased NMDA-induced outward currents (I NMDA-OUT), which are associated with the BK channel activation. Patch-clamp recordings from the acutely dissociated mouse cortical neurons revealed that an EP2 receptor agonist activated I NMDA-OUT, whereas an EP3 receptor agonist reduced it. Agonists of EP1 or EP4 receptors showed no significant effects on I NMDA-OUT. A direct perfusion of 3,5′-cyclic adenosine monophosphate (cAMP) through the patch pipette facilitated I NMDA-OUT, which was abolished by the presence of protein kinase A (PKA) inhibitor. Furthermore, facilitation of I NMDA-OUT caused by an EP2 receptor agonist was significantly suppressed by PKA inhibitor. Finally, the activation of BK channels through EP2 receptors facilitated the recovery phase of NMDA-induced dendritic beading in the primary cultured cortical neurons. These results suggest that a direct activation of BK channels by EP2 receptor-mediated signaling pathways plays neuroprotective roles in cortical neurons. PMID:27298516

Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, M.H.; Neubig, R.R.

1986-03-05

High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonistmore » (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.« less

Caged Naloxone Reveals Opioid Signaling Deactivation Kinetics

PubMed Central

Banghart, Matthew R.; Shah, Ruchir C.; Lavis, Luke D.

2013-01-01

The spatiotemporal dynamics of opioid signaling in the brain remain poorly defined. Photoactivatable opioid ligands provide a means to quantitatively measure these dynamics and their underlying mechanisms in brain tissue. Although activation kinetics can be assessed using caged agonists, deactivation kinetics are obscured by slow clearance of agonist in tissue. To reveal deactivation kinetics of opioid signaling we developed a caged competitive antagonist that can be quickly photoreleased in sufficient concentrations to render agonist dissociation effectively irreversible. Carboxynitroveratryl-naloxone (CNV-NLX), a caged analog of the competitive opioid antagonist NLX, was readily synthesized from commercially available NLX in good yield and found to be devoid of antagonist activity at heterologously expressed opioid receptors. Photolysis in slices of rat locus coeruleus produced a rapid inhibition of the ionic currents evoked by multiple agonists of the μ-opioid receptor (MOR), but not of α-adrenergic receptors, which activate the same pool of ion channels. Using the high-affinity peptide agonist dermorphin, we established conditions under which light-driven deactivation rates are independent of agonist concentration and thus intrinsic to the agonist-receptor complex. Under these conditions, some MOR agonists yielded deactivation rates that are limited by G protein signaling, whereas others appeared limited by agonist dissociation. Therefore, the choice of agonist determines which feature of receptor signaling is unmasked by CNV-NLX photolysis. PMID:23960100

Fight tactics in wood ants: individuals in smaller groups fight harder but die faster

PubMed Central

Batchelor, Tim P.; Briffa, Mark

2011-01-01

When social animals engage in inter-group contests, the outcome is determined by group sizes and individual masses, which together determine group resource-holding potential (‘group RHP’). Individuals that perceive themselves as being in a group with high RHP may receive a motivational increase and increase their aggression levels. Alternatively, individuals in lower RHP groups may increase their aggression levels in an attempt to overcome the RHP deficit. We investigate how ‘group RHP’ influences agonistic tactics in red wood ants Formica rufa. Larger groups had higher total agonistic indices, but per capita agonistic indices were highest in the smallest groups, indicating that individuals in smaller groups fought harder. Agonistic indices were influenced by relative mean mass, focal group size, opponent group size and opponent group agonistic index. Focal group attrition rates decreased as focal group relative agonistic indices increased and there was a strong negative influence of relative mean mass. The highest focal attrition rates were received when opponent groups were numerically large and composed of large individuals. Thus, fight tactics in F. rufa seem to vary with both aspects of group RHP, group size and the individual attributes of group members, indicating that information on these are available to fighting ants. PMID:21389029

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raffa, R.B.; Baldy, W.J. Jr.; Shank, R.P.

Tritiated (D-Ala2,NMePhe4,Gly-ol5)-enkephalin ((3H)DAGO) was used to examine mu-opioid receptor number and mu-ligand binding in brain synaptic membranes (P2 fraction) from C57BL/6J-bgJ/bgJ (beige-J) mice, a strain with combined deficiencies in immunological function (resembling Chediak-Higashi syndrome) and analgesic response to mu-opioid agonists such as morphine and DAGO. As controls, white mice, beige-J littermates (normally responsive to mu-opioid agonists), and a known mu-deficient strain (CXBK) were also examined. Neither the KD (0.47 to 0.49 nM) nor the Bmax (153 to 168 fmol/mg protein) determined for beige-J mice was significantly different from values determined for littermates or white mice. In contrast, the Bmax ofmore » CXBK mice (66 fmol/mg protein) was clearly less than that of the other strains. The analgesic defect of beige-J mice, therefore, is not likely due to an insufficient number of mu-opioid receptors, as it presumably is in CXBK mice. Carbachol (200 micrograms/ml), which partly corrects the analgesic defect of beige-J mice, had no effect on (3H)DAGO binding either acutely in vitro or chronically ex vivo after administration to beige-J mice for three weeks. Hence, the analgesic defect of beige-J mice appears to be due to some defect in the mu-opioid receptor-effector coupling mechanism or to some endogenous substance that inhibits binding of mu-opioid ligands to otherwise functional receptors.« less

Estrogen receptor β selective agonist ameliorates liver cirrhosis in rats by inhibiting the activation and proliferation of hepatic stellate cells.

PubMed

Zhang, Bin; Zhang, Cheng-Gang; Ji, Lin-Hua; Zhao, Gang; Wu, Zhi-Yong

2018-03-01

The aim of this study is to explore the roles of estrogen receptor (ER) subtypes and corresponding agonists/antagonists on the development of cirrhosis and activation and proliferation of hepatic stellate cells (HSCs). Carbon tetrachloride (CCl 4 )-induced cirrhotic ovariectomized rats were administered non-selective ER agonist (β-estradiol, E2), ER selective agonists (ERα agonist, propylpyrazoletriol; ERβ agonist, diarylpropionitrile [DPN]; and G-protein-coupled ER [GPER] agonist, G1), or E2 + ER selective antagonists (ERα antagonist, MPP; ERβ antagonist, PHTPP; and GPER antagonist, G15) for 12 weeks. The expression of the three ER subtypes in livers and HSCs and the effects of the drugs on hepatic fibrosis, isolated HSCs, and uteri were evaluated. Selective ER agonists/antagonists had various effects on CCl 4 -induced cirrhosis. The cirrhotic rats in the CCl 4  + E2, CCl 4  + DPN, CCl 4  + E2 + MPP, and CCl 4  + E2 + G15 groups presented reduced fibrosis scores, compared with those in the CCl 4 group. The cirrhotic rats in the E2 + PHTPP group presented increased fibrosis scores that similar to those in the CCl 4 group. The ovariectomized rats had enlarged uteri with increased uterus indexes after E2 administration; however, the proliferative effects of E2 were partially blocked by MPP or G15, but not PHTPP. In the in vitro study, DPN attenuated the transformation of quiescent HSCs to activated phenotype, suppressed collagen I, and α-smooth muscle actin expression. DPN also suppressed platelet-derived growth factor-induced proliferation in cultured HSCs, which was reversed by PHTPP. The antifibrogenic effects of estrogen were mediated by ERβ but not ERα or GPER. The ERβ selective agonist exerted a fibrosuppressive effect by inhibiting the activation and proliferation of HSCs, but did not induce uterine hyperplasia. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Metformin sensitizes triple-negative breast cancer to proapoptotic TRAIL receptor agonists by suppressing XIAP expression.

PubMed

Strekalova, Elena; Malin, Dmitry; Rajanala, Harisha; Cryns, Vincent L

2017-06-01

Despite robust antitumor activity in diverse preclinical models, TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists have not demonstrated efficacy in clinical trials, underscoring the need to identify agents that enhance their activity. We postulated that the metabolic stress induced by the diabetes drug metformin would sensitize breast cancer cells to TRAIL receptor agonists. Human triple (estrogen receptor, progesterone receptor, and HER2)-negative breast cancer (TNBC) cell lines were treated with TRAIL receptor agonists (monoclonal antibodies or TRAIL peptide), metformin, or the combination. The effects on cell survival, caspase activation, and expression of TRAIL receptors and the antiapoptotic protein XIAP were determined. In addition, XIAP was silenced by RNAi in TNBC cells and the effects on sensitivity to TRAIL were determined. The antitumor effects of metformin, TRAIL, or the combination were evaluated in an orthotopic model of metastatic TNBC. Metformin sensitized diverse TNBC cells to TRAIL receptor agonists. Metformin selectively enhanced the sensitivity of transformed breast epithelial cells to TRAIL receptor agonist-induced caspase activation and apoptosis with little effect on untransformed breast epithelial cells. These effects of metformin were accompanied by robust reductions in the protein levels of XIAP, a negative regulator of TRAIL-induced apoptosis. Silencing XIAP in TNBC cells mimicked the TRAIL-sensitizing effects of metformin. Metformin also enhanced the antitumor effects of TRAIL in a metastatic murine TNBC model. Our findings indicate that metformin enhances the activity of TRAIL receptor agonists, thereby supporting the rationale for additional translational studies combining these agents.

Novel 4-Substituted-N,N-dimethyltetrahydronaphthalen-2-amines: Synthesis, Affinity, and In Silico Docking Studies at Serotonin 5-HT2-type and Histamine H1 G Protein-Coupled Receptors

PubMed Central

Sakhuja, Rajeev; Kondabolu, Krishnakanth; Córdova-Sintjago, Tania; Travers, Sean; Vincek, Adam S.; Kim, Myong Sang; Abboud, Khalil A.; Fang, Lijuan; Sun, Zhuming; Canal, Clinton E.; Booth, Raymond G.

2015-01-01

Syntheses were undertaken of derivatives of (2S, 4R)-(−)-trans-4-phenyl-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (4-phenyl-2-dimethylaminotetralin, PAT), a stereospecific agonist at the serotonin 5-HT2C G protein-coupled receptor (GPCR), with inverse agonist activity at 5-HT2A and 5-HT2B GPCRs. Molecular changes were made at the PAT C(4)-position, while preserving N, N-dimethyl substitution at the 2-position as well as trans-stereochemistry, structural features previously shown to be optimal for 5-HT2 binding. Affinities of analogs were determined at recombinant human 5-HT2 GPCRs in comparison to the phylogenetically closely-related histamine H1 GPCR, and in silico ligand docking studies were conducted at receptor molecular models to help interpret pharmacological results and guide future ligand design. In most cases, C(4)-substituted PAT analogs exhibited the same stereoselectivity ([−]-trans > [+]-trans) as the parent PAT across 5-HT2 and H1 GPCRs, albeit, with variable receptor selectivity. 4-(4′-substituted)-PAT analogs, however, demonstrated reversed stereoselectivity ([2S, 4R]-[+]-trans > [2S, 4R]-[−]-trans), with absolute configuration confirmed by single X-ray crystallographic data for the 4-(4′-Cl)-PAT analog. Pharmacological affinity results and computational results herein support further PAT drug development studies and provide a basis for predicting and interpreting translational results, including, for (+)-trans-4-(4′-Cl)-PAT and (−)-trans-4-(3′-Br)-PAT that were previously shown to be more potent and efficacious than their corresponding enantiomers in rodent models of psychoses, psychostimulant-induced behaviors, and compulsive feeding (‘binge-eating’). PMID:25703249

The metabotropic glutamate receptors: structure, activation mechanism and pharmacology.

PubMed

Pin, Jean-Philippe; Acher, Francine

2002-06-01

The metabotropic glutamate receptors are G-protein coupled receptors (GPCR) involved in the regulation of many synapses, including most glutamatergic fast excitatory synapses. Eight subtypes have been identified that can be classified into three groups. The molecular characterization of these receptors revealed proteins much more complex than any other GPCRs. They are composed of a Venus Flytrap (VFT) module where glutamate binds, connected to a heptahelical domain responsible for G-protein coupling. Recent data including the structure of the VFT module determined with and without glutamate, indicate that these receptors function as dimers. Moreover a number of intracellular proteins can regulate their targeting and transduction mechanism. Such structural features of mGlu receptors offer multiple possibilities for synthetic compounds to modulate their activity. In addition to agonists and competitive antagonists acting at the glutamate binding site, a number of non-competitive antagonists with inverse agonist activity, and positive allosteric modulators have been discovered. These later compounds share specific properties that make them good candidates for therapeutic applications. First, their non-amino acid structure makes them pass more easily the blood brain barrier. Second, they are much more selective than any other compound identified so far, being the first subtype selective molecules. Third, for the negative modulators, their non competitive mechanism of action makes them relatively unaffected by high concentrations of glutamate that may be present in disease states (e.g. stroke, epilepsy, neuropathic pain, etc.). Fourth, like the benzodiazepines acting at the GABA(A) receptors, the positive modulators offer a new way to increase the activity of these receptors in vivo, with a low risk of inducing their desensitization. The present review article focuses on the specific structural features of these receptors and highlights the various possibilities these offer for drug development.

Apparent loss-of-function mutant GPCRs revealed as constitutively desensitized receptors.

PubMed

Wilbanks, Alyson M; Laporte, Stéphane A; Bohn, Laura M; Barak, Larry S; Caron, Marc G

2002-10-08

The DRY motif is a triplet amino acid sequence (aspartic acid, arginine, and tyrosine) that is highly conserved in G protein-coupled receptors (GPCRs). Recently, we have shown that a molecular determinant for nephrogenic diabetes insipidus, the vasopressin receptor with a substitution at the DRY motif arginine (V2R R137H), is a constitutively desensitized receptor that is unable to couple to G proteins due to its constitutive association with beta-arrestin [Barak, L. S. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 93-98]. Additionally, the mutant receptors are localized in endocytic vesicles, identical to wild-type receptors stimulated with agonist. In this study, we asked whether the constitutively desensitized phenotype observed in the V2R R137H represents a general paradigm that may be extended to other GPCRs. We show that arginine substitutions in the DRY motifs of the alpha(1B) adrenergic receptor (alpha(1B)-AR) and angiotensin II type 1A receptor (AT(1A)R) result in receptors that are uncoupled from G proteins, associated with beta-arrestins, and found localized in endocytic vesicles rather than at the plasma membrane in the absence of agonists. The localization of the alpha(1B)-ARs and AT(1A)Rs with arginine substitutions can be restored to the plasma membrane by either using selective antagonists or preventing the endocytosis of the beta-arrestin-receptor complexes. These results indicate that the arginine residue of the DRY motif is essential for preserving the localization of the inactive receptor complex. Furthermore, constitutive desensitization may underlie some loss-of-function receptor phenotypes and represent an unappreciated mechanism of hormonal resistance.

Divergent agonist selectivity in activating β1- and β2-adrenoceptors for G-protein and arrestin coupling.

PubMed

Casella, Ida; Ambrosio, Caterina; Grò, Maria Cristina; Molinari, Paola; Costa, Tommaso

2011-08-15

The functional selectivity of adrenergic ligands for activation of β1- and β2-AR (adrenoceptor) subtypes has been extensively studied in cAMP signalling. Much less is known about ligand selectivity for arrestin-mediated signalling pathways. In the present study we used resonance energy transfer methods to compare the ability of β1- and β2-ARs to form a complex with the G-protein β-subunit or β-arrestin-2 in response to a variety of agonists with various degrees of efficacy. The profiles of β1-/β2-AR selectivity of the ligands for the two receptor-transducer interactions were sharply different. For G-protein coupling, the majority of ligands were more effective in activating the β2-AR, whereas for arrestin coupling the relationship was reversed. These data indicate that the β1-AR interacts more efficiently than β2-AR with arrestin, but less efficiently than β2-AR with G-protein. A group of ligands exhibited β1-AR-selective efficacy in driving the coupling to arrestin. Dobutamine, a member of this group, had 70% of the adrenaline (epinephrine) effect on arrestin via β1-AR, but acted as a competitive antagonist of adrenaline via β2-AR. Thus the structure of such ligands appears to induce an arrestin-interacting form of the receptor only when bound to the β1-AR subtype. © The Authors Journal compilation © 2011 Biochemical Society

Synthesis of two potent glucocorticoid receptor agonists labeled with carbon-14 and stable isotopes.

PubMed

Latli, Bachir; Reeves, Jonathan T; Tan, Zhulin; Hrapchak, Matt; Song, Jinhua J; Busacca, Carl B; Senanayake, Chris H

2015-01-01

Two potent glucocorticoid receptor agonists were prepared labeled with carbon-14 and with stable isotopes to perform drug metabolism, pharmacokinetics, and bioanalytical studies. Carbon-14 labeled (1) was obtained from an enantiopure alkyne (5) via a Sonogashira coupling to a previously reported 5-amino-4-iodo-[2-(14)C]pyrimidine [(14)C]-(6), followed by a base-mediated cyclization (1) in 72% overall radiochemical yield. Carbon-14 labeled (2) was prepared in five steps employing a key benzoic acid intermediate [(14)C]-(13), which was synthesized in one pot from enolization of trifluoromethylketone (12), followed by bromine-magnesium exchange and then electrophile trapping reaction with [(14)C]-carbon dioxide. A chiral auxiliary (S)-1-(4-methoxyphenyl)ethylamine was then coupled to this acid to give [(14)C]-(15). Propargylation and separation of diastereoisomers by crystallizations gave the desired diastereomer [(14)C]-(17) in 34% yield. Sonogashira coupling to iodopyridine (10) followed by cyclization to the azaindole [(14)C]-(18) and finally removal of the chiral auxiliary gave [(14)C]-(2) in 7% overall yield. For stable isotope syntheses, [(13)C6]-(1) was obtained in three steps using [(13)C4]-(6) and trimethylsilylacetylene-[(13)C2] in 26% yield, while [(2)H5]-(2) was obtained by first preparing the iodopyridine [(2)H5]-(10) in five steps. Then, Sonogashira coupling to chiral alkyne (24) and cyclization gave [(2)H5]-(2) in 42% overall yield. Copyright © 2015 John Wiley & Sons, Ltd.

Rotigotine is a potent agonist at dopamine D1 receptors as well as at dopamine D2 and D3 receptors.

PubMed

Wood, Martyn; Dubois, Vanessa; Scheller, Dieter; Gillard, Michel

2015-02-01

Rotigotine acts as a dopamine receptor agonist with high affinity for the dopamine D2, D3, D4 and D5 receptors but with a low affinity for the dopamine D1 receptor. We have investigated this further in radioligand binding and functional studies and compared the profile of rotigotine with that of other drugs used in the treatment of Parkinson's disease (PD). The binding of rotigotine to human dopamine D1, D2, D3, D4 and D5 receptors was determined in radioligand binding studies using [(3)H]rotigotine and compared with that of standard antagonist radioligands. Functional interactions of rotigotine with human dopamine receptors was also determined. [(3)H]rotigotine can be used as an agonist radioligand to label all dopamine receptor subtypes and this can be important to derive agonist affinity estimates. Rotigotine maintains this high affinity in functional studies at all dopamine receptors especially D1, D2 and D3 receptors and, to a lesser extent, D4 and D5 receptors. Rotigotine, like apomorphine but unlike ropinirole and pramipexole, was a potent agonist at all dopamine receptors. Rotigotine is a high-potency agonist at human dopamine D1, D2 and D3 receptors with a lower potency at D4 and D5 receptors. These studies differentiate rotigotine from conventional dopamine D2 agonists, used in the treatment of PD, such as ropinirole and pramipexole which lack activity at the D1 and D5 receptors, but resembles that of apomorphine which has greater efficacy in PD than other dopamine agonists but has suboptimal pharmacokinetic properties. © 2014 The British Pharmacological Society.

Rotigotine is a potent agonist at dopamine D1 receptors as well as at dopamine D2 and D3 receptors

PubMed Central

Wood, Martyn; Dubois, Vanessa; Scheller, Dieter; Gillard, Michel

2015-01-01

Background and Purpose Rotigotine acts as a dopamine receptor agonist with high affinity for the dopamine D2, D3, D4 and D5 receptors but with a low affinity for the dopamine D1 receptor. We have investigated this further in radioligand binding and functional studies and compared the profile of rotigotine with that of other drugs used in the treatment of Parkinson's disease (PD). Experimental Approach The binding of rotigotine to human dopamine D1, D2, D3, D4 and D5 receptors was determined in radioligand binding studies using [3H]rotigotine and compared with that of standard antagonist radioligands. Functional interactions of rotigotine with human dopamine receptors was also determined. Key Results [3H]rotigotine can be used as an agonist radioligand to label all dopamine receptor subtypes and this can be important to derive agonist affinity estimates. Rotigotine maintains this high affinity in functional studies at all dopamine receptors especially D1, D2 and D3 receptors and, to a lesser extent, D4 and D5 receptors. Rotigotine, like apomorphine but unlike ropinirole and pramipexole, was a potent agonist at all dopamine receptors. Conclusions and Implications Rotigotine is a high-potency agonist at human dopamine D1, D2 and D3 receptors with a lower potency at D4 and D5 receptors. These studies differentiate rotigotine from conventional dopamine D2 agonists, used in the treatment of PD, such as ropinirole and pramipexole which lack activity at the D1 and D5 receptors, but resembles that of apomorphine which has greater efficacy in PD than other dopamine agonists but has suboptimal pharmacokinetic properties. PMID:25339241

Interaction of fenoterol stereoisomers with β2-adrenoceptor-G sα fusion proteins: antagonist and agonist competition binding.

PubMed

Reinartz, Michael T; Kälble, Solveig; Wainer, Irving W; Seifert, Roland

2015-05-01

The specific interaction between G-protein-coupled receptors and ligand is the starting point for downstream signaling. Fenoterol stereoisomers were successfully used to probe ligand-specific activation (functional selectivity) of the β2-adrenoceptor (β2AR) (Reinartz et al. 2015). In the present study, we extended the pharmacological profile of fenoterol stereoisomers using β2AR-Gsα fusion proteins in agonist and antagonist competition binding assays. Dissociations between binding affinities and effector potencies were found for (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol. Our data corroborate former studies on the importance of the aminoalkyl moiety of fenoterol derivatives for functional selectivity.

Allosteric Modulation of Chemoattractant Receptors

PubMed Central

Allegretti, Marcello; Cesta, Maria Candida; Locati, Massimo

2016-01-01

Chemoattractants control selective leukocyte homing via interactions with a dedicated family of related G protein-coupled receptor (GPCR). Emerging evidence indicates that the signaling activity of these receptors, as for other GPCR, is influenced by allosteric modulators, which interact with the receptor in a binding site distinct from the binding site of the agonist and modulate the receptor signaling activity in response to the orthosteric ligand. Allosteric modulators have a number of potential advantages over orthosteric agonists/antagonists as therapeutic agents and offer unprecedented opportunities to identify extremely selective drug leads. Here, we resume evidence of allosterism in the context of chemoattractant receptors, discussing in particular its functional impact on functional selectivity and probe/concentration dependence of orthosteric ligands activities. PMID:27199992

Theranostic Prospects of Gastrin-Releasing Peptide Receptor-Radioantagonists in Oncology.

PubMed

Maina, Theodosia; Nock, Berthold A; Kulkarni, Harshad; Singh, Aviral; Baum, Richard P

2017-07-01

Gastrin-releasing peptide receptors (GRPRs) represent attractive targets for cancer diagnosis and therapy owing to their overexpression in widespread human tumors. Bombesin (BBN) analogues coupled to suitable chelators for stable radiometal binding have been proposed for diagnostic imaging and radionuclide therapy (theranostics) of GRPR-positive tumors. Recently, interest has shifted from BBN-like receptor agonists to GRPR-radioantagonists, because radioantagonists do not induce adverse effects after injection to patients and display superior pharmacokinetic in vivo profiles. Thus, they seem more advantageous for clinical use compared to agonists. Newer developments highlighting the theranostic potential of GRPR-radioantagonists in cancer patient management are presented herein. Copyright © 2017 Elsevier Inc. All rights reserved.

Electrical Stimulation Decreases Coupling Efficiency Between Beta-Adrenergic Receptors and Cyclic AMP Production in Cultured Muscle Cells

NASA Technical Reports Server (NTRS)

Young, R. B.; Bridge, K. Y.

1999-01-01

Electrical stimulation of skeletal muscle cells in culture is an effective way to simulate the effects of muscle contraction and its effects on gene expression in muscle cells. Expression of the beta-adrenergic receptor and its coupling to cyclic AMP synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this project was to determine if electrical stimulation altered the beta-adrenergic response in muscle cells. Chicken skeletal muscle cells that had been grown for seven days in culture were subjected to electrical stimulation for an additional two days at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. At the end of this two-day stimulation period, beta-adrenergic receptor population was measured by the binding of tritium-labeled CGP-12177 to muscle cells, and coupling to cAMP synthesis was measured by Radioimmunoassay (RIA) after treating the cells for 10 min with the potent (beta)AR agonist, isoproterenol. The number of beta adrenergic receptors and the basal levels of intracellular cyclic AMP were not affected by electrical stimulation. However, the ability of these cells to synthesize cyclic AMP was reduced by approximately 50%. Thus, an enhanced level of contraction reduces the coupling efficiency of beta-adrenergic receptors for cyclic AMP production.

Structure of a nanobody-stabilized active state of the β(2) adrenoceptor.

PubMed

Rasmussen, Søren G F; Choi, Hee-Jung; Fung, Juan Jose; Pardon, Els; Casarosa, Paola; Chae, Pil Seok; Devree, Brian T; Rosenbaum, Daniel M; Thian, Foon Sun; Kobilka, Tong Sun; Schnapp, Andreas; Konetzki, Ingo; Sunahara, Roger K; Gellman, Samuel H; Pautsch, Alexander; Steyaert, Jan; Weis, William I; Kobilka, Brian K

2011-01-13

G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β(2) adrenergic receptor (β(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β(2)AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11 Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.

Rapid effects of the G-protein coupled oestrogen receptor (GPER) on learning and dorsal hippocampus dendritic spines in female mice.

PubMed

Gabor, Christopher; Lymer, Jennifer; Phan, Anna; Choleris, Elena

2015-10-01

Recently, oestrogen receptors (ERs) have been implicated in rapid learning processes. We have previously shown that 17β-estradiol, ERα and ERβ agonists can improve learning within 40 min of drug administration in mice. However, oestrogen action at the classical receptors may only in part explain these rapid learning effects. Chronic treatment of a G-protein coupled oestrogen receptor (GPER) agonist has been shown to affect learning and memory in ovariectomized rats, yet little is known about its rapid learning effects. Therefore we investigated whether the GPER agonist G-1 at 1 μg/kg, 6 μg/kg, 10 μg/kg, and 30 μg/kg could affect social recognition, object recognition, and object placement learning in ovariectomized CD1 mice within 40 min of drug administration. We also examined rapid effects of G-1 on CA1 hippocampal dendritic spine density and length within 40 min of drug administration, but in the absence of any learning tests. Results suggest a rapid enhancing effect of GPER activation on social recognition, object recognition and object placement learning. G-1 treatment also resulted in increased dendritic spine density in the stratum radiatum of the CA1 hippocampus. Hence GPER, along with the classical ERs, may mediate the rapid effects of oestrogen on learning and neuronal plasticity. To our knowledge, this is the first report of GPER effects occurring within a 40 min time frame. Copyright © 2015 Elsevier Inc. All rights reserved.

Stimulation of the p38 Mitogen-activated Protein Kinase Pathway in Neonatal Rat Ventricular Myocytes by the G Protein–coupled Receptor Agonists, Endothelin-1 and Phenylephrine: A Role in Cardiac Myocyte Hypertrophy?

PubMed Central

Clerk, Angela; Michael, Ashour; Sugden, Peter H.

1998-01-01

We examined the activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by the G protein–coupled receptor agonists, endothelin-1 and phenylephrine in primary cultures of cardiac myocytes from neonatal rat hearts. Both agonists increased the phosphorylation (activation) of p38-MAPK by ∼12-fold. A p38-MAPK substrate, MAPK-activated protein kinase 2 (MAPKAPK2), was activated approximately fourfold and 10 μM SB203580, a p38-MAPK inhibitor, abolished this activation. Phosphorylation of the MAPKAPK2 substrate, heat shock protein 25/27, was also increased. Using selective inhibitors, activation of the p38-MAPK pathway by endothelin-1 was shown to involve protein kinase C but not Gi/Go nor the extracellularly responsive kinase (ERK) pathway. SB203580 failed to inhibit the morphological changes associated with cardiac myocyte hypertrophy induced by endothelin-1 or phenylephrine between 4 and 24 h. However, it decreased the myofibrillar organization and cell profile at 48 h. In contrast, inhibition of the ERK cascade with PD98059 prevented the increase in myofibrillar organization but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response. PMID:9679149

Morphine-like Opiates Selectively Antagonize Receptor-Arrestin Interactions*

PubMed Central

Molinari, Paola; Vezzi, Vanessa; Sbraccia, Maria; Grò, Cristina; Riitano, Daniela; Ambrosio, Caterina; Casella, Ida; Costa, Tommaso

2010-01-01

The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of μ and δ receptors with G protein or β-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent Gβ1. In this system, the agonist-induced enhancement of BRET (indicating shortening of distance between the two proteins) was Gα-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at δ and μ receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (δ) or partial agonists (μ) for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation. PMID:20189994

Comparison of hippocampal G protein activation by 5-HT(1A) receptor agonists and the atypical antipsychotics clozapine and S16924.

PubMed

Newman-Tancredi, A; Rivet, J-M; Cussac, D; Touzard, M; Chaput, C; Marini, L; Millan, M J

2003-09-01

This study employed [(35)S]guanosine 5'- O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding to compare the actions of antipsychotic agents known to stimulate cloned, human 5-HT(1A) receptors with those of reference agonists at postsynaptic 5-HT(1A) receptors. In rat hippocampal membranes, the following order of efficacy was observed (maximum efficacy, E(max), values relative to 5-HT=100): (+)8-OH-DPAT (85), flesinoxan (62), eltoprazine (60), S14506 (59), S16924 (48), buspirone (41), S15535 (22), clozapine (22), ziprasidone (21), pindolol (7), p-MPPI (0), WAY100,635 (0), spiperone (0). Despite differences in species and tissue source, the efficacy and potency (pEC(50)) of agonists (with the exception of clozapine) correlated well with those determined previously at human 5-HT(1A) receptors expressed in Chinese hamster ovary (CHO) cells. In contrast, clozapine was more potent at hippocampal membranes. The selective antagonists p-MPPI and WAY100,635 abolished stimulation of binding by (+)8-OH-DPAT, clozapine and S16924 (p-MPPI), indicating that these actions were mediated specifically by 5-HT(1A) receptors. Clozapine and S16924 also attenuated 5-HT- and (+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding, consistent with partial agonist properties. In [(35)S]GTPgammaS autoradiographic studies, 5-HT-induced stimulation, mediated through 5-HT(1A) receptors, was more potent in the septum (pEC(50) approximately 6.5) than in the dentate gyrus of the hippocampus (pEC(50) approximately 5) suggesting potential differences in coupling efficiency or G protein expression. Though clozapine (30 and 100 microM) did not enhance [(35)S]GTPgammaS labelling in any structure, S16924 (10 micro M) modestly increased [(35)S]GTPgammaS labelling in the dentate gyrus. On the other hand, both these antipsychotic agents attenuated 5-HT (10 microM)-stimulated [(35)S]GTPgammaS binding in the dentate gyrus and septum. In conclusion, clozapine, S16924 and ziprasidone act as partial agonists for G protein activation at postsynaptic 5-HT(1A) receptors in the hippocampus. These data support a role of postsynaptic 5-HT(1A) receptors in the functional profiles of certain antipsychotic agents.

Regulation of Glucose Homeostasis by GLP-1

PubMed Central

Nadkarni, Prashant; Chepurny, Oleg G.; Holz, George G.

2014-01-01

Glucagon-like peptide-1(7–36)amide (GLP-1) is a secreted peptide that acts as a key determinant of blood glucose homeostasis by virtue of its abilities to slow gastric emptying, to enhance pancreatic insulin secretion, and to suppress pancreatic glucagon secretion. GLP-1 is secreted from L cells of the gastrointestinal mucosa in response to a meal, and the blood glucose-lowering action of GLP-1 is terminated due to its enzymatic degradation by dipeptidyl-peptidase-IV (DPP-IV). Released GLP-1 activates enteric and autonomic reflexes while also circulating as an incretin hormone to control endocrine pancreas function. The GLP-1 receptor (GLP-1R) is a G protein-coupled receptor that is activated directly or indirectly by blood glucose-lowering agents currently in use for the treatment of type 2 diabetes mellitus (T2DM). These therapeutic agents include GLP-1R agonists (exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, and langlenatide) and DPP-IV inhibitors (sitagliptin, vildagliptin, saxagliptin, linagliptin, and alogliptin). Investigational agents for use in the treatment of T2DM include GPR119 and GPR40 receptor agonists that stimulate the release of GLP-1 from L cells. Summarized here is the role of GLP-1 to control blood glucose homeo-stasis, with special emphasis on the advantages and limitations of GLP-1-based therapeutics. PMID:24373234

Pharmacological analysis of zebrafish lphn3.1 morphant larvae suggests that saturated dopaminergic signaling could underlie the ADHD-like locomotor hyperactivity.

PubMed

Lange, Merlin; Froc, Cynthia; Grunwald, Hannah; Norton, William H J; Bally-Cuif, Laure

2018-06-08

Polymorphisms in the gene coding for the adhesion G-protein coupled receptor LPHN3 are a risk factor for attention-deficit/hyperactivity disorder (ADHD). Transient down-regulation of latrophilin3.1 (lphn3.1), the zebrafish LPHN3 homologue, causes hyperactivity. Zebrafish injected with a lphn3.1-specific morpholino are hyperactive and display an impairment in dopaminergic neuron development. In the present study we used lphn3.1 morphants to further characterize the changes to dopaminergic signaling that trigger hyperactivity. We applied dopamine agonists (Apomorphine, Quinpirole, SKF-38393) and antagonists (Haloperidol, Eticlopride, SCH-23390) to Lphn3.1 morpholino-injected or control-injected animals. The percentage of change in locomotor activity was then determined at three different time periods (10-20 min, 30-40 min and 60-70 min). Our results show that drugs targeting dopamine receptors appear to elicit similar effects on locomotion in zebrafish larvae and mammals. In addition, we observed that lphn3.1 morphants have an overall hyposensitivity to dopamine agonists and antagonists compared to control fish. These results are compatible with a model whereby dopaminergic neurotransmission is saturated in lphn3.1 morphants. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Differential Modulation of Beta-Adrenergic Receptor Signaling by Trace Amine-Associated Receptor 1 Agonists

PubMed Central

Nürnberg, Daniela; Grüters, Annette; Führer-Sakel, Dagmar; Krude, Heiko; Köhrle, Josef; Schöneberg, Torsten; Biebermann, Heike

2011-01-01

Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes. PMID:22073124

A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perez-Aguilar, Jose Manuel; Shan, Jufang; LeVine, Michael V.

With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT 2AR) in the absence of ligand and bound to four distinct serotonergic agonists. Themore » 5-HT 2AR is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT 2AR agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT 2AR interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. Lastly, the findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT 2AR activation.« less

A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2

DOE PAGES

Perez-Aguilar, Jose Manuel; Shan, Jufang; LeVine, Michael V.; ...

2014-10-14

With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT 2AR) in the absence of ligand and bound to four distinct serotonergic agonists. Themore » 5-HT 2AR is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT 2AR agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT 2AR interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. Lastly, the findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT 2AR activation.« less

Agonist-dependent consequences of proline to alanine substitution in the transmembrane helices of the calcitonin receptor

PubMed Central

Bailey, R J; Hay, D L

2007-01-01

Background and purpose: Transmembrane proline (P) residues in family A G protein-coupled receptors (GPCRs) form functionally important kinks in their helices. These residues are little studied in family B GPCRs but experiments with the VPAC1 receptor and calcitonin receptor-like receptor (CL) show parallels with family A receptors. We sought to determine the function of these residues in the insert negative form of the human calcitonin receptor, a close relative of CL. Experimental approach: Proline residues within the transmembrane domains of the calcitonin receptor (P246, P249, P280, P326, P336) were individually mutated to alanine (A) using site-directed mutagenesis. Receptors were transiently transfected into Cos-7 cells using polyethylenimine and salmon and human calcitonin-induced cAMP responses measured. Salmon and human calcitonin competition binding experiments were also performed and receptor cell-surface expression assessed by whole cell ELISA. Key results: P246A, P249A and P280A were wild-type in terms of human calcitonin-induced cAMP activation. P326A and P336A had reduced function (165 and 12-fold, respectively). In membranes, human calcitonin binding was not detectable for any mutant receptor but in whole cells, binding was detected for all mutants apart from P326A. Salmon calcitonin activated mutant and wild-type receptors equally, although Bmax values were reduced for all mutants apart from P326A. Conclusions and Implications: P326 and P336 are important for the function of human calcitonin receptors and are likely to be involved in generating receptor conformations appropriate for agonist binding and receptor activation. However, agonist-specific effects were observed , implying distinct conformations of the human calcitonin receptor. PMID:17486143

Development of 7TM receptor-ligand complex models using ligand-biased, semi-empirical helix-bundle repacking in torsion space: application to the agonist interaction of the human dopamine D2 receptor.

PubMed

Malo, Marcus; Persson, Ronnie; Svensson, Peder; Luthman, Kristina; Brive, Lars

2013-03-01

Prediction of 3D structures of membrane proteins, and of G-protein coupled receptors (GPCRs) in particular, is motivated by their importance in biological systems and the difficulties associated with experimental structure determination. In the present study, a novel method for the prediction of 3D structures of the membrane-embedded region of helical membrane proteins is presented. A large pool of candidate models are produced by repacking of the helices of a homology model using Monte Carlo sampling in torsion space, followed by ranking based on their geometric and ligand-binding properties. The trajectory is directed by weak initial restraints to orient helices towards the original model to improve computation efficiency, and by a ligand to guide the receptor towards a chosen conformational state. The method was validated by construction of the β1 adrenergic receptor model in complex with (S)-cyanopindolol using bovine rhodopsin as template. In addition, models of the dopamine D2 receptor were produced with the selective and rigid agonist (R)-N-propylapomorphine ((R)-NPA) present. A second quality assessment was implemented by evaluating the results from docking of a library of 29 ligands with known activity, which further discriminated between receptor models. Agonist binding and recognition by the dopamine D2 receptor is interpreted using the 3D structure model resulting from the approach. This method has a potential for modeling of all types of helical transmembrane proteins for which a structural template with sequence homology sufficient for homology modeling is not available or is in an incorrect conformational state, but for which sufficient empirical information is accessible.

Estrogen receptor α activation enhances its cell surface localization and improves myocardial redox status in ovariectomized rats.

PubMed

Steagall, Rebecca J; Yao, Fanrong; Shaikh, Saame Raza; Abdel-Rahman, Abdel A

2017-08-01

Little is known about the role of subcellular trafficking of estrogen receptor (ER) subtypes in the acute estrogen (E 2 )-mediated alleviation of oxidative stress. We tested the hypothesis that ERα migration to the cardiac myocyte membrane mediates the acute E 2 -dependent improvement of cellular redox status. Myocardial distribution of subcellular ERα, ERβ and G-protein coupled estrogen receptor (GPER) was determined in proestrus sham-operated (SO) and in ovariectomized (OVX) rats, acutely treated with E 2 (1μg/kg) or a selective ERα (PPT), ERβ (DPN) or GPER (G1) agonist (10μg/kg), by immunofluorescence and Western blot. We measured ROS and malondialdehyde (MDA) levels, and catalase and superoxide dismutase (SOD) activities to evaluate myocardial antioxidant/redox status. Compared with SO, OVX rats exhibited higher myocardial ROS and MDA levels, reduced catalase and SOD activities, along with diminished ERα, and enhanced ERβ and GPER, localization at cardiomyocyte membrane. Acute E 2 or an ERα (PPT), but not ERβ (DPN) or GPER (G1), agonist reversed these responses in OVX rats and resulted in higher ERα/ERβ and ERα/GPER ratios at the cardiomyocytes membrane. PPT or DPN enhanced myocardial Akt phosphorylation. We present the first evidence that preferential aggregation of ERα at the cardiomyocytes plasma membrane is ERα-dependent, and underlies E 2 -mediated reduction in oxidative stress, at least partly, via the enhancements of myocardial catalase and SOD activities in OVX rats. The findings highlight ERα agonists as potential therapeutics for restoring the myocardial redox status following E 2 depletion in postmenopausal women. Copyright © 2017 Elsevier Inc. All rights reserved.

GPER agonist dilates mesenteric arteries via PI3K-Akt-eNOS and potassium channels in both sexes.

PubMed

Peixoto, Pollyana; Aires, Rosária Dias; Lemos, Virgínia Soares; Bissoli, Nazaré Souza; Santos, Roger Lyrio Dos

2017-08-15

The action of oestrogen has traditionally been attributed to the activation of nuclear receptors (ERα and ERβ). A third receptor, the G protein-coupled oestrogen receptor (GPER), has been described as mediator of the rapid action of oestrogen. Based on the possible protective role of oestrogen in the cardiovascular system, the present study was designed to determine whether selective GPER activation induces relaxation of mesenteric resistance arteries in both sexes and which signalling pathways are involved. Third-order mesenteric arteries were isolated, and concentration-response curves were plotted following the cumulative addition of the selective GPER agonist G-1 (1nM-10μM) following induction of contraction with phenylephrine (3μM). The vasodilatory effects of G-1 were assessed before and after removal of the endothelium or incubation for 30min with nitric oxide synthase (N ω -nitro-L-arginine methyl ester - L-NAME, 300μM) and cyclooxygenase (indomethacin - INDO, 10μM) inhibitors alone or combined, PI3K-Akt pathway inhibitor (LY-294,002, 2.5μM) or a potassium channel blocker (tetraethylammonium - TEA, 5mM). GPER immunolocalisation was also performed on the investigated arteries. The tested GPER agonist induced concentration-dependent relaxation of the mesenteric resistance arteries without differences related to sex that were partially endothelium dependent, mainly mediated by the PI3K-Akt-eNOS pathway and attenuated by nonspecific potassium channel blockade. In addition, the endothelial GPER immunolocalisation was stronger among females. This evidence provides a new perspective for understanding the mechanisms involved in the vascular responses triggered by oestrogen via GPER in both sexes. Copyright © 2017 Elsevier Inc. All rights reserved.

Cryo-EM structure of the serotonin 5-HT1B receptor coupled to heterotrimeric Go.

PubMed

García-Nafría, Javier; Nehmé, Rony; Edwards, Patricia C; Tate, Christopher G

2018-06-20

G-protein-coupled receptors (GPCRs) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different α-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of G s to four different GPCRs have been elucidated 1-4 , but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT 1B receptor (5-HT 1B R) bound to the agonist donitriptan and coupled to an engineered G o heterotrimer. In this complex, 5-HT 1B R is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the β 2 -adrenoceptor (β 2 AR) 3 or the adenosine A 2A receptor (A 2A R) 1 in complex with G s . In contrast to the complexes with G s , the gap between the receptor and the Gβ-subunit in the G o -5-HT 1B R complex precludes molecular contacts, and the interface between the Gα-subunit of G o and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the G o α-subunit. The molecular variations between the interfaces of G o and G s in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling.

G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na/sup +/ channel interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

1988-01-12

The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na/sup +/ channels is a coupled event mediated by guanine nucleotide binding protein(s) (G-protein(s)). These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of (/sup 3/H) acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of (/sup 3/H)batrachotoxin to Na/sup +/ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinicallymore » induced /sup 22/Na/sup +/ uptake in the presence and absence of tetrodotoxin, which blocks Na/sup +/ channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na/sup +/channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na/sup +/ channel-is such that at resting potential the muscarinic receptor induces opening of Na/sup +/ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.« less

Reciprocal regulation of platelet responses to P2Y and thromboxane receptor activation.

PubMed

Barton, J F; Hardy, A R; Poole, A W; Mundell, S J

2008-03-01

Thromboxane A(2) and ADP are two major platelet agonists that stimulate two sets of G protein-coupled receptors to activate platelets. Although aggregation responses to ADP and thromboxane desensitize, there are no reports currently addressing whether activation by one agonist may heterologously desensitize responses to the other. To demonstrate whether responses to ADP or U46619 may be modulated by prior treatment of platelets with the alternate agonist, revealing a level of cross-desensitization between receptor systems. Here we show that pretreatment of platelets with either agonist substantially desensitizes aggregation responses to the other agonist. Calcium responses to thromboxane receptor activation are desensitized by preactivation of P2Y(1) but not P2Y(12) receptors. This heterologous desensitization is mediated by a protein kinase C (PKC)-independent mechanism. Reciprocally, calcium responses to ADP are desensitized by pretreatment of platelets with the thromboxane analogue, U46619, and P2Y(12)-mediated inhibition of adenylate cyclase is also desensitized by pretreatment with U46619. In this direction, desensitization is comprised of two components, a true heterologous component that is PKC-independent, and a homologous component that is mediated through stimulated release of dense granule ADP. This study reveals cross-desensitization between ADP and thromboxane receptor signaling in human platelets. Cross-desensitization is mediated by protein kinases, involving PKC-dependent and independent pathways, and indicates that alterations in the activation state of one receptor may have effects upon the sensitivity of the other receptor system.

Guanine nucleotide regulatory protein co-purifies with the D/sub 2/-dopamine receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Senogles, S.E.; Caron, M.G.

1986-05-01

The D/sub 2/-dopamine receptor from bovine anterior pituitary was purified approx.1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with /sup 3/H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D/sub 2/ receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 ..mu..M NPA. /sup 35/S-GTP..gamma..Smore » binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D/sub 2/-dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D/sub 2/-dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes.« less

Agonist-induced CXCR4 and CB2 Heterodimerization Inhibits Gα13/RhoA-mediated Migration.

PubMed

Scarlett, Kisha A; White, El-Shaddai Z; Coke, Christopher J; Carter, Jada R; Bryant, Latoya K; Hinton, Cimona V

2018-04-01

G-protein-coupled receptor (GPCR) heterodimerization has emerged as a means by which alternative signaling entities can be created; yet, how receptor heterodimers affect receptor pharmacology remains unknown. Previous observations suggested a biochemical antagonism between GPCRs, CXCR4 and CB2 (CNR2), where agonist-bound CXCR4 and agonist-bound CB2 formed a physiologically nonfunctional heterodimer on the membrane of cancer cells, inhibiting their metastatic potential in vitro However, the reduced signaling entities responsible for the observed functional outputs remain elusive. This study now delineates the signaling mechanism whereby heterodimeric association between CXCR4 and CB2, induced by simultaneous agonist treatment, results in decreased CXCR4-mediated cell migration, invasion, and adhesion through inhibition of the Gα13/RhoA signaling axis. Activation of CXCR4 by its cognate ligand, CXCL12, stimulates Gα13 (GNA13), and subsequently, the small GTPase RhoA, which is required for directional cell migration and the metastatic potential of cancer cells. These studies in prostate cancer cells demonstrate decreased protein expression levels of Gα13 and RhoA upon simultaneous CXCR4/CB2 agonist stimulation. Furthermore, the agonist-induced heterodimer abrogated RhoA-mediated cytoskeletal rearrangement resulting in the attenuation of cell migration and invasion of an endothelial cell barrier. Finally, a reduction was observed in the expression of integrin α5 (ITGA5) upon heterodimerization, supported by decreased cell adhesion to extracellular matrices in vitro Taken together, the data identify a novel pharmacologic mechanism for the modulation of tumor cell migration and invasion in the context of metastatic disease. Implications: This study investigates a signaling mechanism by which GPCR heterodimerization inhibits cancer cell migration. Mol Cancer Res; 16(4); 728-39. ©2018 AACR . ©2018 American Association for Cancer Research.

Putative antipsychotics with pronounced agonism at serotonin 5-HT1A and partial agonist activity at dopamine D2 receptors disrupt basal PPI of the startle reflex in rats.

PubMed

Auclair, Agnès L; Galinier, Alexandra; Besnard, Joël; Newman-Tancredi, Adrian; Depoortère, Ronan

2007-07-01

Prepulse inhibition (PPI) of the startle reflex has been extensively studied because it is disrupted in several psychiatric diseases, most notably schizophrenia. In rats, and to a lesser extent, in humans, PPI can be diminished by dopamine (DA) D(2)/D(3) and serotonin 5-HT(1A) receptor agonists. A novel class of potential antipsychotics (SSR181507, bifeprunox, and SLV313) possess partial agonist/antagonist properties at D(2) receptors and various levels of 5-HT(1A) activation. It thus appeared warranted to assess, in Sprague-Dawley rats, the effects of these antipsychotics on basal PPI. SSR181507, sarizotan, and bifeprunox decreased PPI, with a near-complete abolition at 2.5-10 mg/kg; SLV313 had a significant effect at 0.16 mg/kg only. Co-treatment with the 5-HT(1A) receptor antagonist WAY100,635 (0.63 mg/kg) showed that the 5-HT(1A) agonist activity of SSR181507 was responsible for its effect. By contrast, antipsychotics with low affinity and/or efficacy at 5-HT(1A) receptors, such as aripiprazole (another DA D(2)/D(3) and 5-HT(1A) ligand), and established typical and atypical antipsychotics (haloperidol, clozapine, risperidone, olanzapine, quetiapine, and ziprasidone) had no effect on basal PPI (0.01-2.5 to 2.5-40 mg/kg). The present data demonstrate that some putative antipsychotics with pronounced 5-HT(1A) agonist activity, coupled with partial agonist activity at DA D(2) receptors, markedly diminish PPI of the startle reflex in rats. These data raise the issue of the influence of such compounds on sensorimotor gating in humans.

Arrestin binds to different phosphorylated regions of the thyrotropin-releasing hormone receptor with distinct functional consequences.

PubMed

Jones, Brian W; Hinkle, Patricia M

2008-07-01

Arrestin binding to agonist-occupied phosphorylated G protein-coupled receptors typically increases the affinity of agonist binding, increases resistance of receptor-bound agonist to removal with high acid/salt buffer, and leads to receptor desensitization and internalization. We tested whether thyrotropin-releasing hormone (TRH) receptors lacking phosphosites in the C-terminal tail could form stable and functional complexes with arrestin. Fibroblasts from mice lacking arrestins 2 and 3 were used to distinguish between arrestin-dependent and -independent effects. Arrestin did not promote internalization or desensitization of a receptor that had key Ser/Thr phosphosites mutated to Ala (4Ala receptor). Nevertheless, arrestin greatly increased acid/salt resistance and the affinity of 4Ala receptor for TRH. Truncation of 4Ala receptor just distal to the key phosphosites (4AlaStop receptor) abolished arrestin-dependent acid/salt resistance but not the effect of arrestin on agonist affinity. Arrestin formed stable complexes with activated wild-type and 4Ala receptors but not with 4AlaStop receptor, as measured by translocation of arrestin-green fluorescent protein to the plasma membrane or chemical cross-linking. An arrestin mutant that does not interact with clathrin and AP2 did not internalize receptor but still promoted high affinity TRH binding, acid/salt resistance, and desensitization. A sterically restricted arrestin mutant did not cause receptor internalization or desensitization but did promote acid/salt resistance and high agonist affinity. The results demonstrate that arrestin binds to proximal or distal phosphosites in the receptor tail. Arrestin binding at either site causes increased agonist affinity and acid/salt resistance, but only the proximal phosphosites evoke the necessary conformational changes in arrestin for receptor desensitization and internalization.

Identification of specific sites in the third intracellular loop and carboxyl terminus of the Bombyx mori PBAN receptor crucial for ligand-induced internalization

USDA-ARS?s Scientific Manuscript database

Sex pheromone production in most moths is mediated by the pheromone biosynthesis activating neuropeptide receptor (PBANR). Similar to other rhodopsin-like G protein-coupled receptors, the silkmoth Bombyx mori PBANR (BmPBANR) undergoes agonist-induced internalization. Despite interest in developing...

Calcium-dependent mitochondrial cAMP production enhances aldosterone secretion.

PubMed

Katona, Dávid; Rajki, Anikó; Di Benedetto, Giulietta; Pozzan, Tullio; Spät, András

2015-09-05

Glomerulosa cells secrete aldosterone in response to agonists coupled to Ca(2+) increases such as angiotensin II and corticotrophin, coupled to a cAMP dependent pathway. A recently recognized interaction between Ca(2+) and cAMP is the Ca(2+)-induced cAMP formation in the mitochondrial matrix. Here we describe that soluble adenylyl cyclase (sAC) is expressed in H295R adrenocortical cells. Mitochondrial cAMP formation, monitored with a mitochondria-targeted fluorescent sensor (4mtH30), is enhanced by HCO3(-) and the Ca(2+) mobilizing agonist angiotensin II. The effect of angiotensin II is inhibited by 2-OHE, an inhibitor of sAC, and by RNA interference of sAC, but enhanced by an inhibitor of phosphodiesterase PDE2A. Heterologous expression of the Ca(2+) binding protein S100G within the mitochondrial matrix attenuates angiotensin II-induced mitochondrial cAMP formation. Inhibition and knockdown of sAC significantly reduce angiotensin II-induced aldosterone production. These data provide the first evidence for a cell-specific functional role of mitochondrial cAMP. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The putative G-protein coupled estrogen receptor agonist G-1 suppresses proliferation of ovarian and breast cancer cells in a GPER-independent manner

PubMed Central

Wang, Cheng; Lv, Xiangmin; Jiang, Chao; Davis, John S

2012-01-01

G-protein coupled estrogen receptor 1 (GPER) plays an important role in mediating estrogen action in many different tissues under both physiological and pathological conditions. G-1 (1-[4-(6-bromobenzo[1,3]dioxol-5yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta [c]quinolin-8-yl]-ethanone) has been developed as a selective GPER agonist to distinguish estrogen actions mediated by GPER from those mediated by classic estrogen receptors. In the present study, we surprisingly found that G-1 suppressed proliferation and induced apoptosis of KGN cells (a human ovarian granulosa cell tumor cell line), actions that were not blocked by a selective GPER antagonist G15 or siRNA knockdown of GPER. G-1 also suppressed proliferation and induced cell apoptosis in GPER-negative HEK-293 cells and MDA-MB 231 breast cancer cells. Our results demonstrate that G-1 suppresses proliferation of ovarian and breast cancer cells in a GPER-independent manner. G-1 may be a candidate for the development of drugs against ovarian and breast cancer. PMID:23145207

Structure and functional interaction of the extracellular domain of human GABA[subscript B] receptor GBR2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geng, Yong; Xiong, Dazhi; Mosyak, Lidia

2012-10-24

Inhibitory neurotransmission is mediated primarily by GABA. The metabotropic GABA{sub B} receptor is a G protein-coupled receptor central to mammalian brain function. Malfunction of GABA{sub B} receptor has been implicated in several neurological disorders. GABA{sub B} receptor functions as a heterodimeric assembly of GBR1 and GBR2 subunits, where GBR1 is responsible for ligand-binding and GBR2 is responsible for G protein coupling. Here we demonstrate that the GBR2 ectodomain directly interacts with the GBR1 ectodomain to increase agonist affinity by selectively stabilizing the agonist-bound conformation of GBR1. We present the crystal structure of the GBR2 ectodomain, which reveals a polar heterodimericmore » interface. We also identify specific heterodimer contacts from both subunits, and GBR1 residues involved in ligand recognition. Lastly, our structural and functional data indicate that the GBR2 ectodomain adopts a constitutively open conformation, suggesting a structural asymmetry in the active state of GABA{sub B} receptor that is unique to the GABAergic system.« less

CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release*

PubMed Central

Malenczyk, Katarzyna; Jazurek, Magdalena; Keimpema, Erik; Silvestri, Cristoforo; Janikiewicz, Justyna; Mackie, Ken; Di Marzo, Vincenzo; Redowicz, Maria J.; Harkany, Tibor; Dobrzyn, Agnieszka

2013-01-01

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB1 cannabinoid receptors (CB1Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB1R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB1Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB1Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes. PMID:24089517

G protein-coupled estrogen receptor is required for the neuritogenic mechanism of 17β-estradiol in developing hippocampal neurons.

PubMed

Ruiz-Palmero, Isabel; Hernando, Maria; Garcia-Segura, Luis M; Arevalo, Maria-Angeles

2013-06-15

Estradiol promotes neuritogenesis in developing hippocampal neurons by a mechanism involving the upregulation of neurogenin 3, a Notch-regulated transcription factor. In this study we have explored whether G-protein coupled estrogen receptor 1 (GPER) participates in this hormonal action. GPER agonists (17β-estradiol, G1, ICI 182,780) increased neurogenin 3 expression and neuritogenesis in mouse primary hippocampal neurons and this effect was blocked by the GPER antagonist G15 and by a siRNA for GPER. In addition, GPER agonists increased Akt phosphorylation in ser473, which is indicative of the activation of phosphoinositide-3-kinase (PI3K). G15 or GPER silencing prevented the estrogenic induction of Akt phosphorylation. Furthermore, the PI3K inhibitor wortmannin prevented the effect of G1 and estradiol on neurogenin 3 expression and the effect of estradiol on neuritogenesis. These findings suggest that GPER participates in the control of hippocampal neuritogenesis by a mechanism involving the activation of PI3K signaling. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weber, Thomas J.; Markillie, Lye MENG.

The thromboxane A{sub 2} (TXA{sub 2}) receptor (TP) is represented by two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP isoforms may be regulated by novel ligands termed the isoprostanes, which paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes (8-iso-PG{sub 2{sub {alpha}}} and 8-iso-PGE{sub 2}) regulate the activity of TP isoforms expressed in Chinese Hamster Ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist (U46619) in CHO cellsmore » transfected with the human TP-P and TP-E receptors and this response was fully inhibited by TP antagonists (ISAP, SQ29,548). AP-1-luciferase activity was potently (nM) increased by 8-iso-PGE2 in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, while 8-iso-PGF{sub 2{sub {alpha}}} was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE{sub 2} mediated AP-1-luciferase activity, indicating that this response is not dependent on de novo TXA2 biosynthesis. Interestingly, 8-iso-PGE{sub 2}-mediated AP-1-luciferase activity was near maximal in naive cells between 1-10 nM concentrations, and this response was not inhibited by TP antagonist or reproduced by agonists for TP or EP1/EP3 receptors. These observations (1) support a role for novel ligands in the regulation of TP-dependent signaling, (2) indicate that TP-P and TP-E couple to AP-1, (3) provide further evidence that isoprostanes function as TP agonists in a cell-type specific fashion, and (4) indicate that additional targets regulated by 8-iso-PGE{sub 2} couple to AP-1.« less

Action of novel antipsychotics at human dopamine D3 receptors coupled to G protein and ERK1/2 activation.

PubMed

Bruins Slot, Liesbeth A; Palmier, Christiane; Tardif, Stéphanie; Cussac, Didier

2007-08-01

The effects of new generation antipsychotic drugs (APDs) targeting dopamine D(2) and serotonin 5-HT(1A) receptors were compared with typical and atypical APDs on phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and measures of G protein activation in CHO cell lines stably expressing the human dopamine D(3) receptor. The preferential dopamine D(3) agonists (+)-7-OH-DPAT and PD128907, like dopamine and quinelorane, efficaciously stimulated ERK 1/2 phosphorylation at dopamine D(3) receptors. In contrast, in [(35)S]GTPgammaS binding experiments, (+)-7-OH-DPAT exhibited partial agonist properties, while PD128907 and quinelorane maintained full agonist properties. The preferential dopamine D(3) ligand BP 897 and the antidyskinetic sarizotan partially activated ERK 1/2 phosphorylation while exerting no agonist activity on GTPgammaS binding, suggesting signal amplification at the MAP kinase level. Antipsychotics differed in their ability to inhibit both agonist-stimulated GTPgammaS binding and ERK 1/2 phosphorylation, but all typical and atypical compounds tested acted as dopamine D(3) receptor antagonists with the exception of n-desmethylclozapine, the active metabolite of clozapine, which partially activated dopamine D(3) receptor-mediated ERK 1/2 phosphorylation. Among the new generation dopamine D(2)/serotonin 5-HT(1A) antipsychotics, only F 15063 and SLV313 acted as pure dopamine D(3) receptor antagonists, bifeprunox was highly efficacious whereas SSR181507 and aripiprazole showed marked partial agonist properties for ERK 1/2 phosphorylation. In contrast, in the GTPgammaS binding study, aripiprazole was devoid of agonist properties and bifeprunox, and to an even lesser extent SSR181507, only weakly stimulated GTPgammaS binding. In summary, these findings underline the differences of dopamine D(3) properties of new generation antipsychotics which may need to be considered in understanding their diverse therapeutic actions.

Structure-activity relationships of the unique and potent agouti-related protein (AGRP)-melanocortin chimeric Tyr-c[beta-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template.

PubMed

Wilczynski, Andrzej; Wilson, Krista R; Scott, Joseph W; Edison, Arthur S; Haskell-Luevano, Carrie

2005-04-21

The melanocortin receptor system consists of endogenous agonists, antagonists, G-protein coupled receptors, and auxiliary proteins that are involved in the regulation of complex physiological functions such as energy and weight homeostasis, feeding behavior, inflammation, sexual function, pigmentation, and exocrine gland function. Herein, we report the structure-activity relationship (SAR) of a new chimeric hAGRP-melanocortin agonist peptide template Tyr-c[beta-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) that was characterized using amino acids previously reported in other melanocortin agonist templates. Twenty peptides were examined in this study, and six peptides were selected for (1)H NMR and computer-assisted molecular modeling structural analysis. The most notable results include the identification that modification of the chimeric template at the His position with Pro and Phe resulted in ligands that were nM mouse melanocortin-3 receptor (mMC3R) antagonists and nM mouse melanocortin-4 receptor (mMC4R) agonists. The peptides Tyr-c[beta-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) and Tyr-c[beta-Asp-His-DNal(1')-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) resulted in 730- and 560-fold, respectively, mMC4R versus mMC3R selective agonists that also possessed nM agonist potency at the mMC1R and mMC5R. Structural studies identified a reverse turn occurring in the His-DPhe-Arg-Trp domain, with subtle differences observed that may account for the differences in melanocortin receptor pharmacology. Specifically, a gamma-turn secondary structure involving the DPhe(4) in the central position of the Tyr-c[beta-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) peptide may differentiate the mixed mMC3R antagonist and mMC4R agonist pharmacology.

Heterotrimeric G Protein-coupled Receptor Signaling in Yeast Mating Pheromone Response.

PubMed

Alvaro, Christopher G; Thorner, Jeremy

2016-04-08

The DNAs encoding the receptors that respond to the peptide mating pheromones of the budding yeastSaccharomyces cerevisiaewere isolated in 1985, and were the very first genes for agonist-binding heterotrimeric G protein-coupled receptors (GPCRs) to be cloned in any organism. Now, over 30 years later, this yeast and its receptors continue to provide a pathfinding experimental paradigm for investigating GPCR-initiated signaling and its regulation, as described in this retrospective overview. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Control of yeast mating signal transduction by a mammalian. beta. sub 2 -adrenergic receptor and G sub s. alpha. subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

King, K.; Caron, M.G.; Lefkowitz, R.J.

1990-10-05

To facilitate functional and mechanistic studies of receptor-G protein interactions by expression of the human {beta}{sub 2}-adrenergic receptor (h{beta}-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h{beta}-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h{beta}-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h{beta}-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by {beta}-adrenergic receptor agonists was achieved in cells coexpressing h{beta}-AR andmore » a mammalian G protein (G{sub s}) {alpha} subunit - demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.« less

Assignment of the {beta}-arrestin 1 gene (ARRB1) to human chromosome 11q13

DOE Office of Scientific and Technical Information (OSTI.GOV)

Calabrese, G.; Morizio, E.; Palka, G.

1994-11-01

Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor, and its functional cofactor, {beta}-arrestin. {beta}ARK is a member of a multigene family, consisting of six known subtypes, which have also been named G-protein-coupled receptor kinases (GRK 1 to 6) due to the apparently unique functional association of such kinases with this receptor family. The gene for {beta}ARK1 has been localized to human chromosome 11q13. The four members of the arrestin/{beta}-arrestin gene family identified so far are arrestin, X-arrestin, {beta}-arrestin 1, and {beta}-arrestin 2. Here themore » authors report the chromosome mapping of the human gene for {beta}-arrestin 1 (ARRB1) to chromosome 11q13 by fluorescence in situ hybridization (FISH). Two-color FISH confirmed that the two genes coding for the functionally related proteins {beta}ARK1 and {beta}arrestin 1 both map to 11q13. 16 refs., 1 fig., 1 tab.« less

Anti-Brownian ELectrokinetic (ABEL) trapping of single β2-adrenergic receptors in the absence and presence of agonist

NASA Astrophysics Data System (ADS)

Bockenhauer, Samuel; Fuerstenberg, Alexandre; Yao, Xiao Jie; Kobilka, Brian K.; Moerner, W. E.

2012-02-01

The ABEL trap allows trapping of single biomolecules in solution for extended observation without immobilization. The essential idea combines fluorescence-based position estimation with fast electrokinetic feedback in a microfluidic geometry to counter the Brownian motion of a single nanoscale object, hence maintaining its position in the field of view for hundreds of milliseconds to seconds. Such prolonged observation of single proteins allows access to slow dynamics, as probed by any available photophysical observables. We have used the ABEL trap to study conformational dynamics of the β2-adrenergic receptor, a key G-protein coupled receptor and drug target, in the absence and presence of agonist. A single environment-sensitive dye reports on the receptor microenvironment, providing a real-time readout of conformational change for each trapped receptor. The focus of this paper will be a quantitative comparison of the ligandfree and agonist-bound receptor data from our ABEL trap experiments. We observe a small but clearly detectable shift in conformational equilibria and a lengthening of fluctuation timescales upon binding of agonist. In order to quantify the shift in state distributions and timescales, we apply nonparametric statistical tests to place error bounds on the resulting single-molecule distributions.

Ocular Purine Receptors as Drug Targets in the Eye

PubMed Central

Civan, Mortimer M.

2016-01-01

Abstract Agonists and antagonists of various subtypes of G protein coupled adenosine receptors (ARs), P2Y receptors (P2YRs), and ATP-gated P2X receptor ion channels (P2XRs) are under consideration as agents for the treatment of ocular diseases, including glaucoma and dry eye. Numerous nucleoside and nonnucleoside modulators of the receptors are available as research tools and potential therapeutic molecules. Three of the 4 subtypes of ARs have been exploited with clinical candidate molecules for treatment of the eye: A1, A2A, and A3. An A1AR agonist is in clinical trials for glaucoma, A2AAR reduces neuroinflammation, A3AR protects retinal ganglion cells from apoptosis, and both A3AR agonists and antagonists had been reported to lower intraocular pressure (IOP). Extracellular concentrations of endogenous nucleotides, including dinucleoside polyphosphates, are increased in pathological states, activating P2Y and P2XRs throughout the eye. P2YR agonists, including P2Y2 and P2Y6, lower IOP. Antagonists of the P2X7R prevent the ATP-induced neuronal apoptosis in the retina. Thus, modulators of the purinome in the eye might be a source of new therapies for ocular diseases. PMID:27574786

Ocular Purine Receptors as Drug Targets in the Eye.

PubMed

Jacobson, Kenneth A; Civan, Mortimer M

2016-10-01

Agonists and antagonists of various subtypes of G protein coupled adenosine receptors (ARs), P2Y receptors (P2YRs), and ATP-gated P2X receptor ion channels (P2XRs) are under consideration as agents for the treatment of ocular diseases, including glaucoma and dry eye. Numerous nucleoside and nonnucleoside modulators of the receptors are available as research tools and potential therapeutic molecules. Three of the 4 subtypes of ARs have been exploited with clinical candidate molecules for treatment of the eye: A 1 , A 2A , and A 3 . An A 1 AR agonist is in clinical trials for glaucoma, A 2A AR reduces neuroinflammation, A 3 AR protects retinal ganglion cells from apoptosis, and both A 3 AR agonists and antagonists had been reported to lower intraocular pressure (IOP). Extracellular concentrations of endogenous nucleotides, including dinucleoside polyphosphates, are increased in pathological states, activating P2Y and P2XRs throughout the eye. P2YR agonists, including P2Y 2 and P2Y 6 , lower IOP. Antagonists of the P2X7R prevent the ATP-induced neuronal apoptosis in the retina. Thus, modulators of the purinome in the eye might be a source of new therapies for ocular diseases.

Evaluation of partial beta-adrenoceptor agonist activity.

PubMed

Lipworth, B J; Grove, A

1997-01-01

A partial beta-adrenoceptor (beta-AR) agonist will exhibit opposite agonist and antagonist activity depending on the prevailing degree of adrenergic tone or the presence of a beta-AR agonist with higher intrinsic activity. In vivo partial beta-AR agonist activity will be evident at rest with low endogenous adrenergic tone, as for example with chronotropicity (beta 1/beta 2), inotropicity (beta 1) or peripheral vasodilatation and finger tremor (beta 2). beta-AR blocking drugs which have partial agonist activity may exhibit a better therapeutic profile when used for hypertension because of maintained cardiac output without increased systemic vascular resistance, along with an improved lipid profile. In the presence of raised endogenous adrenergic tone such as exercise or an exogenous full agonist, beta-AR subtype antagonist activity will become evident in terms of effects on exercise induced heart rate (beta 1) and potassium (beta 2) responses. Reduction of exercise heart rate will occur to a lesser degree in the case of a beta-adrenoceptor blocker with partial beta 1-AR agonist activity compared with a beta-adrenoceptor blocker devoid of partial agonist activity. This may result in reduced therapeutic efficacy in the treatment of angina on effort when using beta-AR blocking drugs with partial beta 1-AR agonist activity. Effects on exercise hyperkalaemia are determined by the balance between beta 2-AR partial agonist activity and endogenous adrenergic activity. For predominantly beta 2-AR agonist such as salmeterol and salbutamol, potentiation of exercise hyperkalaemia occurs. For predominantly beta 2-AR antagonists such as carteolol, either potentiation or attenuation of exercise hyperkalaemia occurs at low and high doses respectively. beta 2-AR partial agonist activity may also be expressed as antagonism in the presence of an exogenous full agonist, as for example attenuation of fenoterol induced responses by salmeterol. Studies are required to investigate whether this phenomenon is relevant in the setting of acute severe asthma.

Sensitivity of GBM cells to cAMP agonist-mediated apoptosis correlates with CD44 expression and agonist resistance with MAPK signaling.

PubMed

Daniel, Paul M; Filiz, Gulay; Mantamadiotis, Theo

2016-12-01

In some cell types, activation of the second messenger cAMP leads to increased expression of proapoptotic Bim and subsequent cell death. We demonstrate that suppression of the cAMP pathway is a common event across many cancers and that pharmacological activation of cAMP in glioblastoma (GBM) cells leads to enhanced BIM expression and apoptosis in specific GBM cell types. We identified the MAPK signaling axis as the determinant of cAMP agonist sensitivity in GBM cells, with high MAPK activity corresponding to cAMP resistance and low activity corresponding to sensitization to cAMP-induced apoptosis. Sensitive cells were efficiently killed by cAMP agonists alone, while targeting both the cAMP and MAPK pathways in resistant GBM cells resulted in efficient apoptosis. We also show that CD44 is differentially expressed in cAMP agonist-sensitive and -resistant cells. We thus propose that CD44 may be a useful biomarker for distinguishing tumors that may be sensitive to cAMP agonists alone or cAMP agonists in combination with other pathway inhibitors. This suggests that using existing chemotherapeutic compounds in combination with existing FDA-approved cAMP agonists may fast track trials toward improved therapies for difficult-to-treat cancers, such as GBM.

Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor*

PubMed Central

Clayton, Cecilea C.; Donthamsetti, Prashant; Lambert, Nevin A.; Javitch, Jonathan A.; Neve, Kim A.

2014-01-01

Arrestins mediate desensitization and internalization of G protein-coupled receptors and also direct receptor signaling toward heterotrimeric G protein-independent signaling pathways. We previously identified a four-residue segment (residues 212–215) of the dopamine D2 receptor that is necessary for arrestin binding in an in vitro heterologous expression system but that also impairs receptor expression. We now describe the characterization of additional mutations at that arrestin binding site in the third intracellular loop. Mutating two (residues 214 and 215) or three (residues 213–215) of the four residues to alanine partially decreased agonist-induced recruitment of arrestin3 without altering activation of a G protein. Arrestin-dependent receptor internalization, which requires arrestin binding to β2-adaptin (the β2 subunit of the clathrin-associated adaptor protein AP2) and clathrin, was disproportionately affected by the three-residue mutation, with no agonist-induced internalization observed even in the presence of overexpressed arrestin or G protein-coupled receptor kinase 2. The disjunction between arrestin recruitment and internalization could not be explained by alterations in the time course of the receptor-arrestin interaction, the recruitment of G protein-coupled receptor kinase 2, or the receptor-induced interaction between arrestin and β2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified. PMID:25336643

Structurally related odorant ligands of the olfactory receptor OR51E2 differentially promote metastasis emergence and tumor growth

PubMed Central

Sanz, Guenhaël; Leray, Isabelle; Grébert, Denise; Antoine, Sharmilee; Acquistapace, Adrien; Muscat, Adeline; Boukadiri, Abdelhak; Mir, Lluis M.

2017-01-01

Olfactory receptors are G protein-coupled receptors. Some of them are expressed in tumor cells, such as the OR51E2 receptor overexpressed in LNCaP prostate cancer cells. It is considered a prostate tumor marker. We previously demonstrated that this receptor is able to promote LNCaP cell invasiveness in vitro upon stimulation with its odorant agonist β-ionone, leading to increased generation of metastases in vivo. In the present study, we show that even a relatively short exposure to β-ionone is sufficient to promote metastasis emergence. Moreover, α-ionone, considered an OR51E2 antagonist, in fact promotes prostate tumor growth in vivo. The combination of α-ionone with β-ionone triggers a higher increase in the total tumor burden than each molecule alone. To support the in vivo results, we demonstrate in vitro that α-ionone is a real agonist of OR51E2, mainly sustaining LNCaP cell growth, while β-ionone mainly promotes cell invasiveness. So, while structurally close, α-ionone and β-ionone appear to induce different cellular effects, both leading to increased tumor aggressiveness. This behaviour could be explained by a different coupling to downstream effectors, as it has been reported for the so-called biased ligands of other G protein-coupled receptors. PMID:28032594

DEVELOPMENT AND CHARACTERIZATION OF A CELL LINE THAT STABLY EXPRESSES AN ESTROGEN-RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ESTROGEN RECEPTOR AGONIST AND ANTAGONISTS

EPA Science Inventory

Screening for endocrine disrupting chemicals (EDCs) that act as estrogens or antiestrogens relies on the use of in vitro binding and gene expression assays coupled with short-term diagnostic in vivo assays. Although binding assays are useful to identify chemicals that are competi...

Molecular recognition at adenine nucleotide (P2) receptors in platelets.

PubMed

Jacobson, Kenneth A; Mamedova, Liaman; Joshi, Bhalchandra V; Besada, Pedro; Costanzi, Stefano

2005-04-01

Transmembrane signaling through P2Y receptors for extracellular nucleotides controls a diverse array of cellular processes, including thrombosis. Selective agonists and antagonists of the two P2Y receptors present on the platelet surface-the G (q)-coupled P2Y (1) subtype and the G (i)-coupled P2Y (12) subtype-are now known. High-affinity antagonists of each have been developed from nucleotide structures. The (N)-methanocarba bisphosphate derivatives MRS2279 and MRS2500 are potent and selective P2Y (1) receptor antagonists. The carbocyclic nucleoside AZD6140 is an uncharged, orally active P2Y (12) receptor antagonist of nM affinity. Another nucleotide receptor on the platelet surface, the P2X (1) receptor, the activation of which may also be proaggregatory, especially under conditions of high shear stress, has high-affinity ligands, although high selectivity has not yet been achieved. Although alpha,beta-methylene-adenosine triphosphate (ATP) is the classic agonist for the P2X (1) receptor, where it causes rapid desensitization, the agonist BzATP is among the most potent in activating this subtype. The aromatic sulfonates NF279 and NF449 are potent antagonists of the P2X (1) receptor. The structures of the two platelet P2Y receptors have been modeled, based on a rhodopsin template, to explain the basis for nucleotide recognition within the putative transmembrane binding sites. The P2Y (1) receptor model, especially, has been exploited in the design and optimization of antagonists targeted to interact selectively with that subtype.

Recruitment of β-arrestin2 to the dopamine D2 receptor: Insights into anti-psychotic and anti-parkinsonian drug receptor signaling

PubMed Central

Klewe, Ib V.; Nielsen, Søren M.; Tarpø, Louise; Urizar, Eneko; Dipace, Concetta; Javitch, Jonathan A.; Gether, Ulrik; Egebjerg, Jan; Christensen, Kenneth V.

2013-01-01

Drugs acting at dopamine D2-like receptors play a pivotal role in the treatment of both schizophrenia and Parkinson’s disease. Recent studies have demonstrated a role for G-protein independent D2 receptor signaling pathways acting through β-arrestin. In this study we describe the establishment of a Bioluminescence Resonance Energy Transfer (BRET) assay for measuring dopamine induced recruitment of human β-arrestin2 to the human dopamine D2 receptor. Dopamine, as well as the dopamine receptor agonists pramipexole and quinpirole, acted as full agonists in the assay as reflected by their ability to elicit marked concentration dependent increases in the BRET signal signifying β-arrestin2 recruitment to the D2 receptor. As expected from their effect on G-protein coupling and cAMP levels mediated through the D2 receptor RNPA, pergolide, apomorphine, ropinirole, bromocriptine, 3PPP, terguride, aripiprazole, SNPA all acted as partial agonists with decreasing efficacy in the BRET assay. In contrast, a wide selection of typical and atypical anti-psychotics was incapable of stimulating β-arrestin2 recruitment to the D2 receptor. Moreover, we observed that haloperidol, sertindole, olanzapine, clozapine and ziprasidone all fully inhibited the dopamine induced β-arrestin2 recruitment to D2 receptor (short variant) in a concentration dependent manner. We conclude that most anti-psychotics are incapable of stimulating β-arrestin2 recruitment to the dopamine D2 receptor, in accordance with their antagonistic properties at the level of G-protein coupling. PMID:18455202

Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1

PubMed Central

Fay, Jonathan F.; Farrens, David L.

2015-01-01

G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1—it simultaneously increases agonist binding, decreases G-protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling. PMID:26100912

Gi-coupled γ-aminobutyric acid-B receptors cross-regulate phospholipase C and calcium in airway smooth muscle.

PubMed

Mizuta, Kentaro; Mizuta, Fumiko; Xu, Dingbang; Masaki, Eiji; Panettieri, Reynold A; Emala, Charles W

2011-12-01

γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system, and exerts its actions via both ionotropic (GABA(A)) and metabotropic (GABA(B)) receptors. Although the functional expression of GABA(B) receptors coupled to the G(i) protein was reported for airway smooth muscle, the role of GABA(B) receptors in airway responsiveness remains unclear. We investigated whether G(i)-coupled GABA(B) receptors cross-regulate phospholipase C (PLC), an enzyme classically regulated by G(q)-coupled receptors in human airway smooth muscle cells. Both the GABA(B)-selective agonist baclofen and the endogenous ligand GABA significantly increased the synthesis of inositol phosphate, whereas GABA(A) receptor agonists, muscimol, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol exerted no effect. The baclofen-induced synthesis of inositol phosphate and transient increases in [Ca(2+)](i) were blocked by CGP35348 and CGP55845 (selective GABA(B) antagonists), pertussis toxin (PTX, which inactivates the G(i) protein), gallein (a G(βγ) signaling inhibitor), U73122 (an inhibitor of PLC-β), and xestospongin C, an inositol 1,4,5-triphosphate receptor blocker. Baclofen also potentiated the bradykinin-induced synthesis of inositol phosphate and transient increases in [Ca(2+)](i), which were blocked by CGP35348 or PTX. Moreover, baclofen potentiated the substance P-induced contraction of airway smooth muscle in isolated guinea pig tracheal rings. In conclusion, the stimulation of GABA(B) receptors in human airway smooth muscle cells rapidly mobilizes intracellular Ca(2+) stores by the synthesis of inositol phosphate via the activation of PLC-β, which is stimulated by G(βγ) protein liberated from G(i) proteins coupled to GABA(B) receptors. Furthermore, crosstalk between GABA(B) receptors and G(q)-coupled receptors potentiates the synthesis of inositol phosphate, transient increases in [Ca(2+)](i), and smooth muscle contraction through G(i) proteins.

Gi-Coupled γ-Aminobutyric Acid–B Receptors Cross-Regulate Phospholipase C and Calcium in Airway Smooth Muscle

PubMed Central

Mizuta, Kentaro; Mizuta, Fumiko; Xu, Dingbang; Masaki, Eiji; Panettieri, Reynold A.

2011-01-01

γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system, and exerts its actions via both ionotropic (GABAA) and metabotropic (GABAB) receptors. Although the functional expression of GABAB receptors coupled to the Gi protein was reported for airway smooth muscle, the role of GABAB receptors in airway responsiveness remains unclear. We investigated whether Gi-coupled GABAB receptors cross-regulate phospholipase C (PLC), an enzyme classically regulated by Gq-coupled receptors in human airway smooth muscle cells. Both the GABAB-selective agonist baclofen and the endogenous ligand GABA significantly increased the synthesis of inositol phosphate, whereas GABAA receptor agonists, muscimol, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol exerted no effect. The baclofen-induced synthesis of inositol phosphate and transient increases in [Ca2+]i were blocked by CGP35348 and CGP55845 (selective GABAB antagonists), pertussis toxin (PTX, which inactivates the Gi protein), gallein (a Gβγ signaling inhibitor), U73122 (an inhibitor of PLC-β), and xestospongin C, an inositol 1,4,5-triphosphate receptor blocker. Baclofen also potentiated the bradykinin-induced synthesis of inositol phosphate and transient increases in [Ca2+]i, which were blocked by CGP35348 or PTX. Moreover, baclofen potentiated the substance P–induced contraction of airway smooth muscle in isolated guinea pig tracheal rings. In conclusion, the stimulation of GABAB receptors in human airway smooth muscle cells rapidly mobilizes intracellular Ca2+ stores by the synthesis of inositol phosphate via the activation of PLC-β, which is stimulated by Gβγ protein liberated from Gi proteins coupled to GABAB receptors. Furthermore, crosstalk between GABAB receptors and Gq-coupled receptors potentiates the synthesis of inositol phosphate, transient increases in [Ca2+]i, and smooth muscle contraction through Gi proteins. PMID:21719794

An Elevation in Physical Coupling of Type 1 IP3 Receptors to TRPC3 Channels Constricts Mesenteric Arteries in Genetic Hypertension

PubMed Central

Adebiyi, Adebowale; Thomas-Gatewood, Candice M.; Leo, M. Dennis; Kidd, Michael W.; Neeb, Zachary P.; Jaggar, Jonathan H.

2013-01-01

Hypertension is associated with an elevation in agonist-induced vasoconstriction, but mechanisms involved require further investigation. Many vasoconstrictors bind to phospholipase C-coupled receptors, leading to an elevation in inositol 1,4,5-trisphosphate (IP3) that activates sarcoplasmic reticulum (SR) IP3 receptors (IP3Rs). In cerebral artery myocytes, IP3Rs release SR Ca2+ and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current (ICat) activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP3Rs control vascular contractility in systemic arteries and IP3R involvement in elevated agonist-induced vasoconstriction during hypertension. Total and plasma membrane-localized TRPC3 protein was ~2.7- and 2-fold higher in mesenteric arteries of hypertensive spontaneously hypertensive rats (SHR) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP3R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-hypertensive SHR and WKY rats. Control, IP3- and endothelin-1 (ET-1)-induced FRET between IP3R1 and TRPC3 was higher in hypertensive SHR than WKY myocytes. IP3-induced ICat was ~3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and CIRBP-TAT, an IP3R-TRP physical coupling inhibitor, reduced IP3-induced ICat and ET-1-induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a SR Ca2+-ATPase blocker, did not alter ET-1-stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP3R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP3R1 to TRPC3 channel molecular coupling augments ET-1-induced vasoconstriction during hypertension. PMID:23045459

Opioid-receptor subtype agonist-induced enhancements of sucrose intake are dependent upon sucrose concentration.

PubMed

Ruegg, H; Yu, W Z; Bodnar, R J

1997-07-01

Selective mu ([D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO)), delta1 ([D-Pen2, D-Pen5]-enkephalin (DPDPE)), delta2 ([D-Ala2, Glu4]-Deltorphin (Delt II)), kappa1 (U50488H) and kappa3 (naloxone benzoylhydrazone (NalBzOH)) opioid agonists each stimulate food intake in rats. Whereas studies with selective opioid antagonists implicate mu and kappa1 receptors in the mediation of sucrose intake, studies with selective opioid agonists implicate mu and delta receptors in the mediation of saccharin intake. The present study determined if specific delta1, delta2, kappa1, kappa3 and mu opioid-receptor subtype agonists produced similar alterations in sucrose intake as a function of sucrose concentration (0.5%, 2.5%, 10%) across a 1-h time-course. Each of these agonists significantly increased sucrose intake with variations in pattern, magnitude, and consistency as a function of sucrose concentration. Whereas the mu opioid agonist, DAMGO, and the delta1 opioid agonist, DPDPE, each enhanced sucrose intake at higher (2.5%, 10%), but not lower (0.5%), concentrations, the delta2 opioid agonist, Delt II, increased sucrose intake at lower (0.5%, 2.5%), but not higher (10%), concentrations. Kappa opioid agonists produced less consistent effects. The kappa1 opioid agonist, U50488H, increased sucrose intake at high (10%) concentrations and decreased sucrose intake at low (0.5%) concentrations, and the kappa3 opioid agonist, NalBzOH, inconsistently increased sucrose intake at the 0.5% (20 microg) and 10% (1 microg) concentrations. Thus, these data further implicate mu, delta1, and delta2 opioid mediation of palatable intake, particularly of its orosensory characteristics.

Agonism and Antagonism at the Insulin Receptor

PubMed Central

Knudsen, Louise; Hansen, Bo Falck; Jensen, Pia; Pedersen, Thomas Åskov; Vestergaard, Kirsten; Schäffer, Lauge; Blagoev, Blagoy; Oleksiewicz, Martin B.; Kiselyov, Vladislav V.; De Meyts, Pierre

2012-01-01

Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B’29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1–10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints (3H-thymidine incorporation), and not on metabolic endpoints (14C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity (3H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B’29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed. PMID:23300584

Muscarinic and nicotinic acetylcholine receptor agonists: current scenario in Alzheimer's disease therapy.

PubMed

Verma, Stuti; Kumar, Ashwini; Tripathi, Timir; Kumar, Awanish

2018-04-16

Alzheimer's disease (AD) has become the primary cause of dementia. It shows a progressive cognitive dysfunction with degenerating neurons. Acetylcholine receptors (AChRs) propagate the cognitive ability and it consists of two primary members namely muscarinic (mAChRs) and nicotinic receptors (nAChRs). Where mAChRs is G-protein coupled receptor, (nAChRs) are ligand-gated ion channels. The conventional therapeutic regimen for AD consists of three acetylcholinestearse inhibitors while a single NMDA receptor antagonist. Researchers around the globe are developing new and modifying the existing AChRs agonists to develop lead candidates with lower risk to benefit ratio where benefits clearly outweigh the adverse events. We have searched PubMed, MEDLINE, Google scholar, Science Direct and, Web of Science with keywords "Muscarinic/Nicotinic acetylcholine receptor, agonists and, AD". The literature search included articles written in English. Scientific relevance for clinical studies, basic science studies is eligibility criteria for articles referred in this paper. M1 is the primary muscarinic subtype while α7 is the primary nAChR subtype that is responsible for cognition and memory and these two have been the major recent experimental targets for mAChR agonist strategy. The last cholinergic receptor agonist to enter phase 3 trial was EVP-6124 (Enceniclin) but was withdrawn due to severe gastrointestinal adverse effects. We aim to present an overview of the efforts and achievements in targeting Muscarinic and Nicotinic acetylcholine receptor in the current review for development of better AD therapeutics. © 2018 Royal Pharmaceutical Society.

Extent of Vascular Remodeling Is Dependent on the Balance Between Estrogen Receptor α and G-Protein-Coupled Estrogen Receptor.

PubMed

Gros, Robert; Hussain, Yasin; Chorazyczewski, Jozef; Pickering, J Geoffrey; Ding, Qingming; Feldman, Ross D

2016-11-01

Estrogens are important regulators of cardiovascular function. Some of estrogen's cardiovascular effects are mediated by a G-protein-coupled receptor mechanism, namely, G-protein-coupled estrogen receptor (GPER). Estradiol-mediated regulation of vascular cell programmed cell death reflects the balance of the opposing actions of GPER versus estrogen receptor α (ERα). However, the significance of these opposing actions on the regulation of vascular smooth muscle cell proliferation or migration in vitro is unclear, and the significance in vivo is unknown. To determine the effects of GPER activation in vitro, we studied rat aortic vascular smooth muscle cells maintained in primary culture. GPER was reintroduced using adenoviral gene transfer. Both estradiol and G1, a GPER agonist, inhibited both proliferation and cell migration effects that were blocked by the GPER antagonist, G15. To determine the importance of the GPER-ERα balance in regulating vascular remodeling in a rat model of carotid ligation, we studied the effects of upregulation of GPER expression versus downregulation of ERα. Reintroduction of GPER significantly attenuated the extent of medial hypertrophy and attenuated the extent of CD45 labeling. Downregulation of ERα expression comparably attenuated the extent of medial hypertrophy and inflammation after carotid ligation. These studies demonstrate that the balance between GPER and ERα regulates vascular remodeling. Receptor-specific modulation of estrogen's effects may be an important new approach in modifying vascular remodeling in both acute settings like vascular injury and perhaps in longer term regulation like in hypertension. © 2016 American Heart Association, Inc.

Imaging Agonist-Induced D2/D3 Receptor Desensitization and Internalization In Vivo with PET/fMRI.

PubMed

Sander, Christin Y; Hooker, Jacob M; Catana, Ciprian; Rosen, Bruce R; Mandeville, Joseph B

2016-04-01

This study investigated the dynamics of dopamine receptor desensitization and internalization, thereby proposing a new technique for non-invasive, in vivo measurements of receptor adaptations. The D2/D3 agonist quinpirole, which induces receptor internalization in vitro, was administered at graded doses in non-human primates while imaging with simultaneous positron emission tomography (PET) and functional magnetic resonance imaging (fMRI). A pronounced temporal divergence between receptor occupancy and fMRI signal was observed: occupancy remained elevated while fMRI responded transiently. Analogous experiments with an antagonist (prochlorperazine) and a lower-affinity agonist (ropinirole) exhibited reduced temporal dissociation between occupancy and function, consistent with a mechanism of desensitization and internalization that depends upon drug efficacy and affinity. We postulated a model that incorporates internalization into a neurovascular-coupling relationship. This model yielded in vivo desensitization/internalization rates (0.2/min for quinpirole) consistent with published in vitro measurements. Overall, these results suggest that simultaneous PET/fMRI enables characterization of dynamic neuroreceptor adaptations in vivo, and may offer a first non-invasive method for assessing receptor desensitization and internalization.

Imaging Agonist-Induced D2/D3 Receptor Desensitization and Internalization In Vivo with PET/fMRI

PubMed Central

Sander, Christin Y; Hooker, Jacob M; Catana, Ciprian; Rosen, Bruce R; Mandeville, Joseph B

2016-01-01

This study investigated the dynamics of dopamine receptor desensitization and internalization, thereby proposing a new technique for non-invasive, in vivo measurements of receptor adaptations. The D2/D3 agonist quinpirole, which induces receptor internalization in vitro, was administered at graded doses in non-human primates while imaging with simultaneous positron emission tomography (PET) and functional magnetic resonance imaging (fMRI). A pronounced temporal divergence between receptor occupancy and fMRI signal was observed: occupancy remained elevated while fMRI responded transiently. Analogous experiments with an antagonist (prochlorperazine) and a lower-affinity agonist (ropinirole) exhibited reduced temporal dissociation between occupancy and function, consistent with a mechanism of desensitization and internalization that depends upon drug efficacy and affinity. We postulated a model that incorporates internalization into a neurovascular-coupling relationship. This model yielded in vivo desensitization/internalization rates (0.2/min for quinpirole) consistent with published in vitro measurements. Overall, these results suggest that simultaneous PET/fMRI enables characterization of dynamic neuroreceptor adaptations in vivo, and may offer a first non-invasive method for assessing receptor desensitization and internalization. PMID:26388148

Synthesis and structure-activity relationship of the first nonpeptidergic inverse agonists for the human cytomegalovirus encoded chemokine receptor US28.

PubMed

Hulshof, Janneke W; Casarosa, Paola; Menge, Wiro M P B; Kuusisto, Leena M S; van der Goot, Henk; Smit, Martine J; de Esch, Iwan J P; Leurs, Rob

2005-10-06

US28 is a human cytomegalovirus (HCMV) encoded G-protein-coupled receptor that signals in a constitutively active manner. Recently, we identified 1 [5-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenylpentanenitrile] as the first reported nonpeptidergic inverse agonist for a viral-encoded chemokine receptor. Interestingly, this compound is able to partially inhibit the viral entry of HIV-1. In this study we describe the synthesis of 1 and several of its analogues and unique structure-activity relationships for this first class of small-molecule ligands for the chemokine receptor US28. Moreover, the compounds have been pharmacologically characterized as inverse agonists on US28. By modification of lead structure 1, it is shown that a 4-phenylpiperidine moiety is essential for affinity and activity. Other structural features of 1 are shown to be of less importance. These compounds define the first SAR of ligands on a viral GPCR (US28) and may therefore serve as important tools to investigate the significance of US28-mediated constitutive activity during viral infection.

Palmitoylation and membrane cholesterol stabilize μ-opioid receptor homodimerization and G protein coupling

PubMed Central

2012-01-01

Background A cholesterol-palmitoyl interaction has been reported to occur in the dimeric interface of the β2-adrenergic receptor crystal structure. We sought to investigate whether a similar phenomenon could be observed with μ-opioid receptor (OPRM1), and if so, to assess the role of cholesterol in this class of G protein-coupled receptor (GPCR) signaling. Results C3.55(170) was determined to be the palmitoylation site of OPRM1. Mutation of this Cys to Ala did not affect the binding of agonists, but attenuated receptor signaling and decreased cholesterol associated with the receptor signaling complex. In addition, both attenuation of receptor palmitoylation (by mutation of C3.55[170] to Ala) and inhibition of cholesterol synthesis (by treating the cells with simvastatin, a HMG-CoA reductase inhibitor) impaired receptor signaling, possibly by decreasing receptor homodimerization and Gαi2 coupling; this was demonstrated by co-immunoprecipitation, immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) analyses. A computational model of the OPRM1 homodimer structure indicated that a specific cholesterol-palmitoyl interaction can facilitate OPRM1 homodimerization at the TMH4-TMH4 interface. Conclusions We demonstrate that C3.55(170) is the palmitoylation site of OPRM1 and identify a cholesterol-palmitoyl interaction in the OPRM1 complex. Our findings suggest that this interaction contributes to OPRM1 signaling by facilitating receptor homodimerization and G protein coupling. This conclusion is supported by computational modeling of the OPRM1 homodimer. PMID:22429589

Pramipexole in the treatment of Parkinson's disease: new developments.

PubMed

Möller, J Carsten; Oertel, Wolfgang H

2005-09-01

The nonergot dopamine agonist pramipexole is an efficient and safe drug for the treatment of Parkinson's disease. Clinicians may favor pramipexole over other dopamine agonists because of its suggested higher tolerability with respect to peripheral dopaminergic side effects. Importantly, nonergot dopamine agonists such as pramipexole may not cause restrictive valvular heart disease and may therefore represent the first choice in patients with valvular lesions under treatment with ergot dopamine agonists. However, particular caution has to be exercised in younger Parkinson's disease patients with a shorter disease duration regarding the occurrence of sudden onset of sleep. In light of cost-effectiveness and quality-of-life issues, its final significance for the initial treatment of patients with early Parkinson's disease remains to be determined.

Peroxisome proliferator-activated receptor-{gamma} agonists inhibit the replication of respiratory syncytial virus (RSV) in human lung epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arnold, Ralf; Koenig, Wolfgang

2006-07-05

We have previously shown that peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) agonists inhibited the inflammatory response of RSV-infected human lung epithelial cells. In this study, we supply evidence that specific PPAR{gamma} agonists (15d-PGJ{sub 2}, ciglitazone, troglitazone, Fmoc-Leu) efficiently blocked the RSV-induced cytotoxicity and development of syncytia in tissue culture (A549, HEp-2). All PPAR{gamma} agonists under study markedly inhibited the cell surface expression of the viral G and F protein on RSV-infected A549 cells. This was paralleled by a reduced cellular amount of N protein-encoding mRNA determined by real-time RT-PCR. Concomitantly, a reduced release of infectious progeny virus into the cell supernatants ofmore » human lung epithelial cells (A549, normal human bronchial epithelial cells (NHBE)) was observed. Similar results were obtained regardless whether PPAR{gamma} agonists were added prior to RSV infection or thereafter, suggesting that the agonists inhibited viral gene expression and not the primary adhesion or fusion process.« less

Comparison of the ultrashort gonadotropin-releasing hormone agonist-antagonist protocol with microdose flare -up protocol in poor responders: a preliminary study.

PubMed

Berker, Bülent; Duvan, Candan İltemir; Kaya, Cemil; Aytaç, Ruşen; Satıroğlu, Hakan

2010-01-01

To determine the potential effect of the ultrashort gonadotropin-releasing hormone (GnRH) agonist/GnRH antagonist protocol versus the microdose GnRH agonist protocol in poor responders undergoing intracytoplasmic sperm injection (ICSI). The patients in the Agonist-Antagonist Group (n=41) were administered the ultrashort GnRH-agonist/ antagonist protocol, while the patients in the Microdose Group (n=41) were stimulated according to the microdose flare-up protocol. The mean number of mature oocytes retrieved was the primary outcome measure. Fertilization rate, implantation rate per embryo and clinical pregnancy rates were secondary outcome measures. There was no differenc between the mean number of mature oocytes retrieved in the two groups. There were also no statistical differences between the two groups in terms of peak serum E2 level, canceled cycles, endometrial thickness on hCG day, number of 2 pronucleus and number of embryos transferred. However, the total gonadotropin consumption and duration of stimulation were significantly higher with the Agonist-Antagonist Group compared with the Microdose Group. The implantation and clinical pregnancy rates were similar between the two groups. Despite the high dose of gonadotropin consumption and longer duration of stimulation with the ultrashort GnRH agonist/ antagonist protocol, it seems that the Agonist-Antagonist Protocol is not inferior to the microdose protocol in poor responders undergoing ICSI.

Dopamine D2-like receptor signaling suppresses human osteoclastogenesis.

PubMed

Hanami, Kentaro; Nakano, Kazuhisa; Saito, Kazuyoshi; Okada, Yosuke; Yamaoka, Kunihiro; Kubo, Satoshi; Kondo, Masahiro; Tanaka, Yoshiya

2013-09-01

Dopamine, a major neurotransmitter, transmits signals via five different seven-transmembrane G protein-coupled receptors termed D1 to D5. Although the relevance of neuroendocrine system to bone metabolism has been emerging, the precise effects of dopaminergic signaling upon osteoclastogenesis remain unknown. Here, we demonstrate that human monocyte-derived osteoclast precursor cells express all dopamine-receptor subtypes. Dopamine and dopamine D2-like receptor agonists such as pramipexole and quinpirole reduced the formation of TRAP-positive multi-nucleated cells, cathepsin K mRNA expression, and pit formation area in vitro. These inhibitory effects were reversed by pre-treatment with a D2-like receptor antagonist haloperidol or a Gαi inhibitor pertussis toxin, but not with the D1-like receptor antagonist SCH-23390. Dopamine and dopamine D2-like receptor agonists, but not a D1-like receptor agonist, suppressed intracellular cAMP concentration as well as RANKL-meditated induction of c-Fos and NFATc1 mRNA expression in human osteoclast precursor cells. Finally, the dopamine D2-like receptor agonist suppressed LPS-induced osteoclast formation in murine bone marrow culture ex vivo. These findings indicate that dopaminergic signaling plays an important role in bone homeostasis via direct effects upon osteoclast differentiation and further suggest that the clinical use of neuroleptics is likely to affect bone mass. Copyright © 2013 Elsevier Inc. All rights reserved.

Ligand binding modes from low resolution GPCR models and mutagenesis: chicken bitter taste receptor as a test-case.

PubMed

Di Pizio, Antonella; Kruetzfeldt, Louisa-Marie; Cheled-Shoval, Shira; Meyerhof, Wolfgang; Behrens, Maik; Niv, Masha Y

2017-08-15

Bitter taste is one of the basic taste modalities, warning against consuming potential poisons. Bitter compounds activate members of the bitter taste receptor (Tas2r) subfamily of G protein-coupled receptors (GPCRs). The number of functional Tas2rs is species-dependent. Chickens represent an intriguing minimalistic model, because they detect the bitter taste of structurally different molecules with merely three bitter taste receptor subtypes. We investigated the binding modes of several known agonists of a representative chicken bitter taste receptor, ggTas2r1. Because of low sequence similarity between ggTas2r1 and crystallized GPCRs (~10% identity, ~30% similarity at most), the combination of computational approaches with site-directed mutagenesis was used to characterize the agonist-bound conformation of ggTas2r1 binding site between TMs 3, 5, 6 and 7. We found that the ligand interactions with N93 in TM3 and/or N247 in TM5, combined with hydrophobic contacts, are typically involved in agonist recognition. Next, the ggTas2r1 structural model was successfully used to identify three quinine analogues (epiquinidine, ethylhydrocupreine, quinidine) as new ggTas2r1 agonists. The integrated approach validated here may be applicable to additional cases where the sequence identity of the GPCR of interest and the existing experimental structures is low.

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells.

PubMed

Ehre, Camille; Rossi, Andrea H; Abdullah, Lubna H; De Pestel, Kathleen; Hill, Sandra; Olsen, John C; Davis, C William

2005-01-01

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G(q)-coupled P2Y(2) receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of beta- and gamma-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of beta- or gamma-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca(2+)-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

Augmentation of cutaneous immune responses by ATP gamma S: purinergic agonists define a novel class of immunologic adjuvants.

PubMed

Granstein, Richard D; Ding, Wanhong; Huang, Jing; Holzer, Aton; Gallo, Richard L; Di Nardo, Anna; Wagner, John A

2005-06-15

Extracellular nucleotides activate ligand-gated P2XR ion channels and G protein-coupled P2YRs. In this study we report that intradermal administration of ATPgammaS, a hydrolysis-resistant P2 agonist, results in an enhanced contact hypersensitivity response in mice. Furthermore, ATPgammaS enhanced the induction of delayed-type hypersensitivity to a model tumor vaccine in mice and enhanced the Ag-presenting function of Langerhans cells (LCs) in vitro. Exposure of a LC-like cell line to ATPgammaS in the presence of LPS and GM-CSF augmented the induction of I-A, CD80, CD86, IL-1beta, and IL-12 p40 while inhibiting the expression of IL-10, suggesting that the immunostimulatory activities of purinergic agonists in the skin are mediated at least in part by P2Rs on APCs. In this regard, an LC-like cell line was found to express mRNA for P2X(1), P2X(7), P2Y(1), P2Y(2), P2Y(4), P2Y(9), and P2Y(11) receptors. We suggest that ATP, when released after trauma or infection, may act as an endogenous adjuvant to enhance the immune response, and that P2 agonists may augment the efficacy of vaccines.

First administration of cytidine diphosphocholine and galantamine in schizophrenia: a sustained alpha7 nicotinic agonist strategy.

PubMed

Deutsch, Stephen I; Schwartz, Barbara L; Schooler, Nina R; Rosse, Richard B; Mastropaolo, John; Gaskins, Brooke

2008-01-01

Converging lines of evidence suggest pathophysiology of alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) in schizophrenia. This pilot study was designed to test the tolerability, safety, and preliminary efficacy of chronic administration of an alpha7 nAChR agonist strategy involving combination treatment of cytidine diphosphocholine (CDP-choline; 2 g/d), a dietary source of the alpha7 nAChR agonist choline, and galantamine (24 mg/d), a positive allosteric modulator of nAChRs that was prescribed to prevent choline from becoming a functional antagonist and improve the efficiency of coupling the binding of choline to channel opening. The combination of CDP-choline and galantamine was administered to 6 schizophrenic patients with residual symptoms in a 12-week, open-label trial. Patients were maintained on stable dose regimens of antipsychotic medications for 4 weeks before study entry and for the trial duration. All reached target doses of both agents and completed the trial. Transient side effects resolved without slowing of dose titration. Gastrointestinal adverse effects were most common. Of the 6 patients, 5 showed reduction in Clinical Global Impressions severity scores and Positive and Negative Syndrome Scale total scores. Three patients requested continuation of the adjunctive combination at the end of the trial. These results suggest further investigation of the combination of CDP-choline and galantamine as an alpha7 nAChR agonist intervention.

Modeling ligand recognition at the P2Y12 receptor in light of X-ray structural information

NASA Astrophysics Data System (ADS)

Paoletta, Silvia; Sabbadin, Davide; von Kügelgen, Ivar; Hinz, Sonja; Katritch, Vsevolod; Hoffmann, Kristina; Abdelrahman, Aliaa; Straßburger, Jens; Baqi, Younis; Zhao, Qiang; Stevens, Raymond C.; Moro, Stefano; Müller, Christa E.; Jacobson, Kenneth A.

2015-08-01

The G protein-coupled P2Y12 receptor (P2Y12R) is an important antithrombotic target and of great interest for pharmaceutical discovery. Its recently solved, highly divergent crystallographic structures in complex either with nucleotides (full or partial agonist) or with a nonnucleotide antagonist raise the question of which structure is more useful to understand ligand recognition. Therefore, we performed extensive molecular modeling studies based on these structures and mutagenesis, to predict the binding modes of major classes of P2Y12R ligands previously reported. Various nucleotide derivatives docked readily to the agonist-bound P2Y12R, but uncharged nucleotide-like antagonist ticagrelor required a hybrid receptor resembling the agonist-bound P2Y12R except for the top portion of TM6. Supervised molecular dynamics (SuMD) of ticagrelor binding indicated interactions with the extracellular regions of P2Y12R, defining possible meta-binding sites. Ureas, sulfonylureas, sulfonamides, anthraquinones and glutamic acid piperazines docked readily to the antagonist-bound P2Y12R. Docking dinucleotides at both agonist- and antagonist-bound structures suggested interactions with two P2Y12R pockets. Thus, our structure-based approach consistently rationalized the main structure-activity relationships within each ligand class, giving useful information for designing improved ligands.

Electromembrane extraction of gonadotropin-releasing hormone agonists from plasma and wastewater samples.

PubMed

Nojavan, Saeed; Bidarmanesh, Tina; Mohammadi, Ali; Yaripour, Saeid

2016-03-01

In the present study, for the first time electromembrane extraction followed by high performance liquid chromatography coupled with ultraviolet detection was optimized and validated for quantification of four gonadotropin-releasing hormone agonist anticancer peptides (alarelin, leuprolide, buserelin and triptorelin) in biological and aqueous samples. The parameters influencing electromigration were investigated and optimized. The membrane consists 95% of 1-octanol and 5% di-(2-ethylhexyl)-phosphate immobilized in the pores of a hollow fiber. A 20 V electrical field was applied to make the analytes migrate from sample solution with pH 7.0, through the supported liquid membrane into an acidic acceptor solution with pH 1.0 which was located inside the lumen of hollow fiber. Extraction recoveries in the range of 49 and 71% within 15 min extraction time were obtained in different biological matrices which resulted in preconcentration factors in the range of 82-118 and satisfactory repeatability (7.1 < RSD% < 19.8). The method offers good linearity (2.0-1000 ng/mL) with estimation of regression coefficient higher than 0.998. The procedure allows very low detection and quantitation limits of 0.2 and 0.6 ng/mL, respectively. Finally, it was applied to determination and quantification of peptides in human plasma and wastewater samples and satisfactory results were yielded. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy.

PubMed

Rahmeh, Rita; Damian, Marjorie; Cottet, Martin; Orcel, Hélène; Mendre, Christiane; Durroux, Thierry; Sharma, K Shivaji; Durand, Grégory; Pucci, Bernard; Trinquet, Eric; Zwier, Jurriaan M; Deupi, Xavier; Bron, Patrick; Banères, Jean-Louis; Mouillac, Bernard; Granier, Sébastien

2012-04-24

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

G Protein-Coupled Estrogen Receptor (GPER) Expression in Normal and Abnormal Endometrium

PubMed Central

Lessey, Bruce A.; Taylor, Robert N.; Wang, Wei; Bagchi, Milan K.; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A.; Young, Steven L.

2012-01-01

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen’s importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis. PMID:22378861

G protein-coupled estrogen receptor (GPER) expression in normal and abnormal endometrium.

PubMed

Plante, Beth J; Lessey, Bruce A; Taylor, Robert N; Wang, Wei; Bagchi, Milan K; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A; Young, Steven L

2012-07-01

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen's importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis.

An Efficient Metadynamics-Based Protocol To Model the Binding Affinity and the Transition State Ensemble of G-Protein-Coupled Receptor Ligands.

PubMed

Saleh, Noureldin; Ibrahim, Passainte; Saladino, Giorgio; Gervasio, Francesco Luigi; Clark, Timothy

2017-05-22

A generally applicable metadynamics scheme for predicting the free energy profile of ligand binding to G-protein-coupled receptors (GPCRs) is described. A common and effective collective variable (CV) has been defined using the ideally placed and highly conserved Trp6.48 as a reference point for ligand-GPCR distance measurement and the common orientation of GPCRs in the cell membrane. Using this single CV together with well-tempered multiple-walker metadynamics with a funnel-like boundary allows an efficient exploration of the entire ligand binding path from the extracellular medium to the orthosteric binding site, including vestibule and intermediate sites. The protocol can be used with X-ray structures or high-quality homology models (based on a high-quality template and after thorough refinement) for the receptor and is universally applicable to agonists, antagonists, and partial and reverse agonists. The root-mean-square error (RMSE) in predicted binding free energies for 12 diverse ligands in five receptors (a total of 23 data points) is surprisingly small (less than 1 kcal mol -1 ). The RMSEs for simulations that use receptor X-ray structures and homology models are very similar.

Accelerated molecular dynamics simulations of ligand binding to a muscarinic G-protein-coupled receptor.

PubMed

Kappel, Kalli; Miao, Yinglong; McCammon, J Andrew

2015-11-01

Elucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein-coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc) and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein-ligand binding, especially the drug recognition of GPCRs.

The G protein-coupled receptor GPR30 mediates the proliferative and invasive effects induced by hydroxytamoxifen in endometrial cancer cells.

PubMed

Du, Gui-Qiang; Zhou, Long; Chen, Xiao-Yue; Wan, Xiao-Ping; He, Yin-Yan

2012-04-06

The selective ER modulator tamoxifen (TAM(1)) is the most widely used ER antagonist for treatment of women with hormone-dependent breast tumor. However, long-term treatment is associated with an increased risk of endometrial cancer. The aim of the present study was to demonstrate new insight into the role of G-protein coupled receptor 30 (GPR30) in the activity of TAM, which promoted endometrial cancer. In endometrial cancer cell lines ISHIKAWA and KLE, the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, 17β-estradiol (E2) and G1, a non-steroidal GPR30-specific agonist to promote cell proliferation and invasion was evaluated. All agents above induced high proliferative and invasive effects, while the down-regulation of GPR30 or the interruption of MAPK signal pathway partly or completely prevented the action of the regent. Moreover, the RNA and protein expression of GPR30 was up-regulated by G1, E2 or OHT in both cell lines. The present study provided a new insight into the mechanism involved in the agonistic activity exerted by TAM in the uterus. Copyright © 2012 Elsevier Inc. All rights reserved.

Modification on ursodeoxycholic acid (UDCA) scaffold. discovery of bile acid derivatives as selective agonists of cell-surface G-protein coupled bile acid receptor 1 (GP-BAR1).

PubMed

Sepe, Valentina; Renga, Barbara; Festa, Carmen; D'Amore, Claudio; Masullo, Dario; Cipriani, Sabrina; Di Leva, Francesco Saverio; Monti, Maria Chiara; Novellino, Ettore; Limongelli, Vittorio; Zampella, Angela; Fiorucci, Stefano

2014-09-25

Bile acids are signaling molecules interacting with the nuclear receptor FXR and the G-protein coupled receptor 1 (GP-BAR1/TGR5). GP-BAR1 is a promising pharmacological target for the treatment of steatohepatitis, type 2 diabetes, and obesity. Endogenous bile acids and currently available semisynthetic bile acids are poorly selective toward GP-BAR1 and FXR. Thus, in the present study we have investigated around the structure of UDCA, a clinically used bile acid devoid of FXR agonist activity, to develop a large family of side chain modified 3α,7β-dihydroxyl cholanoids that selectively activate GP-BAR1. In vivo and in vitro pharmacological evaluation demonstrated that administration of compound 16 selectively increases the expression of pro-glucagon 1, a GP-BAR1 target, in the small intestine, while it had no effect on FXR target genes in the liver. Further, compound 16 results in a significant reshaping of bile acid pool in a rodent model of cholestasis. These data demonstrate that UDCA is a useful scaffold to generate novel and selective steroidal ligands for GP-BAR1.

Scale-Up Synthesis and Identification of GLYX-13, a NMDAR Glycine-Site Partial Agonist for the Treatment of Major Depressive Disorder.

PubMed

Li, Wenchao; Liu, Jingjian; Fan, Minghua; Li, Zhongtang; Chen, Yin; Zhang, Guisen; Huang, Zhuo; Zhang, Liangren

2018-04-24

GLYX-13, a NMDAR glycine-site partial agonist, was discovered as a promising antidepressant with rapidly acting effects but no ketamine-like side effects. However, the reported synthetic process route had deficiencies of low yield and the use of unfriendly reagents. Here, we report a scaled-up synthesis of GLYX-13 with an overall yield of 30% on the hectogram scale with a column chromatography-free strategy, where the coupling and deprotection reaction conditions were systematically optimized. Meanwhile, the absolute configuration of precursor compound of GLYX-13 was identified by X-ray single crystal diffraction. Finally, the activity of GLYX-13 was verified in the cortical neurons of mice through whole-cell voltage-clamp technique.

Effects of a combined treatment with GPR30 agonist G-1 and herceptin on growth and gene expression of human breast cancer cell lines.

PubMed

Lubig, Julia; Lattrich, Claus; Springwald, Anette; Häring, Julia; Schüler, Susanne; Ortmann, Olaf; Treeck, Oliver

2012-06-01

Expression of G-protein-coupled receptor 30 (GPR30) is present in HER2-overexpressing breast cancer. In this study, we examined to what extent GPR30-agonist G-1 would affect the antitumoral action of trastuzumab (Herceptin). Combined treatment with both drugs exerted an additive growth-inhibitory effect on breast cancer cells which was accompanied by a significant decline of cyclin A2 expression both on the protein and the mRNA level. Combined treatment also resulted in expression changes of c-fos, cyclin D1, or p21/WAF-1. The results of our study encourage further attempts to test the relevance of these in vitro data in the clinical setting.

Hypoxia increases pulmonary arterial thromboxane receptor internalization independent of receptor sensitization.

PubMed

Fediuk, J; Sikarwar, A S; Lizotte, P P; Hinton, M; Nolette, N; Dakshinamurti, S

2015-02-01

Persistent Pulmonary Hypertension of the Newborn (PPHN) is characterized by sustained vasospasm and an increased thromboxane:prostacyclin ratio. Thromboxane (TP) receptors signal via Gαq to mobilize IP3 and Ca(2+), causing pulmonary arterial constriction. We have previously reported increased TP internalization in hypoxic pulmonary arterial (PA) myocytes. Serum-deprived PA myocytes were grown in normoxia (NM) or hypoxia (HM) for 72 h. TP localization was visualized in agonist-naïve and -challenged NM and HM by immunocytochemistry. Pathways for agonist-induced TP receptor internalization were determined by inhibiting caveolin- or clathrin-mediated endocytosis, and caveolar fractionation. Roles of actin and tubulin in TP receptor internalization were assessed using inhibitors of tubulin, actin-stabilizing or -destabilizing agents. PKA, PKC or GRK activation and inhibition were used to determine the kinase responsible for post-agonist receptor internalization. Agonist-naïve HM had decreased cell surface TP, and greater TP internalization after agonist challenge. TP protein did not sort with caveolin-rich fractions. Inhibition of clathrin prevented TP internalization. Both actin-stabilizing and -destabilizing agents prevented TP endocytosis in NM, while normalizing TP internalization in HM. Velocity of TP internalization was unaffected by PKA activity, but PKC activation normalized TP receptor internalization in HM. GRK inhibition had no effect. We conclude that in hypoxic myocytes, TP is internalized faster and to a greater extent than in normoxic controls. Internalization of the agonist-challenged TP requires clathrin, dynamic actin and is sensitive to PKC activity. TP receptor trafficking and signaling in hypoxia are pivotal to understanding increased vasoconstrictor sensitivity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

PubMed

Law, Wenjing; Wuescher, Leah M; Ortega, Amanda; Hapiak, Vera M; Komuniecki, Patricia R; Komuniecki, Richard

2015-04-01

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets.

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening

PubMed Central

Law, Wenjing; Wuescher, Leah M.; Ortega, Amanda; Hapiak, Vera M.; Komuniecki, Patricia R.; Komuniecki, Richard

2015-01-01

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets. PMID:25928899

A Role for Sigma Receptors in Stimulant Self Administration and Addiction

PubMed Central

Katz, Jonathan L.; Su, Tsung-Ping; Hiranita, Takato; Hayashi, Teruo; Tanda, Gianluigi; Kopajtic, Theresa; Tsai, Shang-Yi

2011-01-01

Sigma1 receptors (σ1Rs) represent a structurally unique class of intracellular proteins that function as chaperones. σ1Rs translocate from the mitochondria-associated membrane to the cell nucleus or cell membrane, and through protein-protein interactions influence several targets, including ion channels, G-protein-coupled receptors, lipids, and other signaling proteins. Several studies have demonstrated that σR antagonists block stimulant-induced behavioral effects, including ambulatory activity, sensitization, and acute toxicities. Curiously, the effects of stimulants have been blocked by σR antagonists tested under place-conditioning but not self-administration procedures, indicating fundamental differences in the mechanisms underlying these two effects. The self administration of σR agonists has been found in subjects previously trained to self administer cocaine. The reinforcing effects of the σR agonists were blocked by σR antagonists. Additionally, σR agonists were found to increase dopamine concentrations in the nucleus accumbens shell, a brain region considered important for the reinforcing effects of abused drugs. Although the effects of the σR agonist, DTG, on dopamine were obtained at doses that approximated those that maintained self administration behavior those of another agonist, PRE-084 required higher doses. The effects of DTG were antagonized by non-selective or a preferential σ2R antagonist but not by a preferential σ1R antagonist. The effects of PRE-084 on dopamine were insensitive to σR antagonists. The data suggest that the self administration of σR agonists is independent of dopamine and the findings are discussed in light of a hypothesis that cocaine has both intracellular actions mediated by σRs, as well as extracellular actions mediated through conventionally studied mechanisms. The co-activation and potential interactions among these mechanisms, in particular those involving the intracellular chaperone σRs, may lead to the pernicious addictive effects of stimulant drugs. PMID:21904468

A Role for Sigma Receptors in Stimulant Self Administration and Addiction.

PubMed

Katz, Jonathan L; Su, Tsung-Ping; Hiranita, Takato; Hayashi, Teruo; Tanda, Gianluigi; Kopajtic, Theresa; Tsai, Shang-Yi

2011-01-01

Sigma(1) receptors (σ(1)Rs) represent a structurally unique class of intracellular proteins that function as chaperones. σ(1)Rs translocate from the mitochondria-associated membrane to the cell nucleus or cell membrane, and through protein-protein interactions influence several targets, including ion channels, G-protein-coupled receptors, lipids, and other signaling proteins. Several studies have demonstrated that σR antagonists block stimulant-induced behavioral effects, including ambulatory activity, sensitization, and acute toxicities. Curiously, the effects of stimulants have been blocked by σR antagonists tested under place-conditioning but not self-administration procedures, indicating fundamental differences in the mechanisms underlying these two effects. The self administration of σR agonists has been found in subjects previously trained to self administer cocaine. The reinforcing effects of the σR agonists were blocked by σR antagonists. Additionally, σR agonists were found to increase dopamine concentrations in the nucleus accumbens shell, a brain region considered important for the reinforcing effects of abused drugs. Although the effects of the σR agonist, DTG, on dopamine were obtained at doses that approximated those that maintained self administration behavior those of another agonist, PRE-084 required higher doses. The effects of DTG were antagonized by non-selective or a preferential σ(2)R antagonist but not by a preferential σ(1)R antagonist. The effects of PRE-084 on dopamine were insensitive to σR antagonists. The data suggest that the self administration of σR agonists is independent of dopamine and the findings are discussed in light of a hypothesis that cocaine has both intracellular actions mediated by σRs, as well as extracellular actions mediated through conventionally studied mechanisms. The co-activation and potential interactions among these mechanisms, in particular those involving the intracellular chaperone σRs, may lead to the pernicious addictive effects of stimulant drugs.

Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5-HT2C Receptor Underlying the Pharmacology of Distinct Ligands.

PubMed

Liu, Yue; Canal, Clinton E; Cordova-Sintjago, Tania C; Zhu, Wanying; Booth, Raymond G

2017-01-18

While exploring the structure-activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT 2C receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT 2C receptors. In HEK293 cells expressing human 5-HT 2C-INI receptors, for example, (-)-trans-3'-Br-PAT and (-)-trans-3'-Cl-PAT are agonists regarding Gα q -inositol phosphate signaling, whereas (-)-trans-3'-CF 3 -PAT is an inverse agonist. To investigate the ligand-receptor interactions that govern this change in function, we performed site-directed mutagenesis of 14 amino acids of the 5-HT 2C receptor based on molecular modeling and reported G protein-coupled receptor crystal structures, followed by molecular pharmacology studies. We found that S3.36, T3.37, and F5.47 in the orthosteric binding pocket are critical for affinity (K i ) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC 50 functional potencies of PATs. We discovered that when residue S5.43, N6.55, or both are mutated to alanine, (-)-trans-3'-CF 3 -PAT switches from inverse agonist to agonist function, and when N6.55 is mutated to leucine, (-)-trans-3'-Br-PAT switches from agonist to inverse agonist function. Notably, most point-mutations that affected PAT pharmacology did not significantly alter affinity (K D ) of the antagonist radioligand [ 3 H]mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT 2C ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT 2C receptor residues important for pharmacology of one ligand are not necessarily important for another ligand.

The four As associated with pathological Parkinson disease gamblers: anxiety, anger, age, and agonists.

PubMed

Shapiro, Michael A; Chang, Yu Ling; Munson, Sarah K; Jacobson, Charles E; Rodriguez, Ramon L; Skidmore, Frank M; Okun, Michael S; Fernandez, Hubert H

2007-02-01

Several studies have related pathological gambling in PD to dopamine agonist therapy. A mail-in survey was sent to PD patients seen at the University of Florida Movement Disorders Center to determine gambling frequency and behavior, and any lifestyle or environmental factors associated with compulsive gambling in PD. 462 surveys were sent and 127 completed surveys were returned, of which ten were from patients who met criteria for compulsive gambling. All ten were taking dopamine agonists coincident with the compulsive gambling. Compulsive gamblers were younger, and psychological distress measures revealed that compulsive gamblers exhibited higher levels of anxiety, anger, and confusion. Thus in this cohort, we have uncovered the several characteristics of the most likely PD compulsive gambler, namely: (young) age, "angry", "anxious", and using a (dopamine) agonist.

Oral vitamin-A-coupled valsartan nanomedicine: High hepatic stellate cell receptors accessibility and prolonged enterohepatic residence.

PubMed

El-Mezayen, Nesrine S; El-Hadidy, Wessam F; El-Refaie, Wessam M; Shalaby, Thanaa I; Khattab, Mahmoud M; El-Khatib, Aiman S

2018-05-22

So far, liver fibrosis still has no clinically-approved treatment. The loss of stored vitamin-A (V A ) in hepatic stellate cells (HSCs), the main regulators to hepatic fibrosis, can be applied as a mechanism for their targeting. Valsartan is a good candidate for this approach; it is a marketed oral-therapy with inverse- and partial-agonistic activity to the over-expressed angiotensin-II type1 receptor (AT1R) and depleted nuclear peroxisome proliferator-activated receptor-gamma (PPAR-γ), respectively, in activated HSCs. However, efficacy on AT1R and PPAR-γ necessitates high drug permeability which is lacking in valsartan. In the current study, liposomes were used as nanocarriers for valsartan to improve its permeability and hence efficacy. They were coupled to V A and characterized for HSCs-targeting. Tracing of orally-administered fluorescently-labeled V A -coupled liposomes in normal rats and their fluorescence intensity quantification in different organs convincingly demonstrated their intestinal entrapment. On the other hands, their administration to rats with induced fibrosis revealed preferential hepatic, and less intestinal, accumulation which lasted up to six days. This indicated their uptake by intestinal stellate cells that acted as a depot for their release over time. Confocal microscopical examination of immunofluorescently-stained HSCs in liver sections, with considerable formula accumulation, confirmed HSCs-targeting and nuclear uptake. Consequently, V A -coupled valsartan-loaded liposomes (VLC)-therapy resulted in profound re-expression of hepatic Mas-receptor and PPAR-γ, potent reduction of fibrogenic mediators' level and nearly normal liver function tests. Therefore, VLC epitomizes a promising antifibrotic therapy with exceptional extended action and additional PPAR-γ agonistic activity. Copyright © 2018 Elsevier B.V. All rights reserved.

Pharmacological lineage analysis revealed the binding affinity of broad-spectrum substance P antagonists to receptors for gonadotropin-releasing peptide.

PubMed

Arai, Kazune; Kashiwazaki, Aki; Fujiwara, Yoko; Tsuchiya, Hiroyoshi; Sakai, Nobuya; Shibata, Katsushi; Koshimizu, Taka-aki

2015-02-15

A group of synthetic substance P (SP) antagonists, such as [Arg(6),D-Trp(7,9),N(Me)Phe(8)]-substance P(6-11) and [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]-substance P, bind to a range of distinct G-protein-coupled receptor (GPCR) family members, including V1a vasopressin receptors, and they competitively inhibit agonist binding. This extended accessibility enabled us to identify a GPCR subset with a partially conserved binding site structure. By combining pharmacological data and amino acid sequence homology matrices, a pharmacological lineage of GPCRs that are sensitive to these two SP antagonists was constructed. We found that sensitivity to the SP antagonists was not limited to the Gq-protein-coupled V1a and V1b receptors; Gs-coupled V2 receptors and oxytocin receptors, which couple with both Gq and Gi, also demonstrated sensitivity. Unexpectedly, a dendrogram based on the amino acid sequences of 222 known GPCRs showed that a group of receptors sensitive to the SP antagonists are located in close proximity to vasopressin/oxytocin receptors. Gonadotropin-releasing peptide receptors, located near the vasopressin receptors in the dendrogram, were also sensitive to the SP analogs, whereas α1B adrenergic receptors, located more distantly from the vasopressin receptors, were not sensitive. Our finding suggests that pharmacological lineage analysis is useful in selecting subsets of candidate receptors that contain a conserved binding site for a ligand with broad-spectrum binding abilities. The knowledge that the binding site of the two broad-spectrum SP analogs partially overlaps with that of distinct peptide agonists is valuable for understanding the specificity/broadness of peptide ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel Analogues of (R)-5-(Methylamino)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (Sumanirole) Provide Clues to Dopamine D2/D3 Receptor Agonist Selectivity

PubMed Central

2016-01-01

Novel 1-, 5-, and 8-substituted analogues of sumanirole (1), a dopamine D2/D3 receptor (D2R/D3R) agonist, were synthesized. Binding affinities at both D2R and D3R were higher when determined in competition with the agonist radioligand [3H]7-hydroxy-N,N-dipropyl-2-aminotetralin (7-OH-DPAT) than with the antagonist radioligand [3H]N-methylspiperone. Although 1 was confirmed as a D2R-preferential agonist, its selectivity in binding and functional studies was lower than previously reported. All analogues were determined to be D2R/D3R agonists in both GoBRET and mitogenesis functional assays. Loss of efficacy was detected for the N-1-substituted analogues at D3R. In contrast, the N-5-alkyl-substituted analogues, and notably the n-butyl-arylamides (22b and 22c), all showed improved affinity at D2R over 1 with neither a loss of efficacy nor an increase in selectivity. Computational modeling provided a structural basis for the D2R selectivity of 1, illustrating how subtle differences in the highly homologous orthosteric binding site (OBS) differentially affect D2R/D3R affinity and functional efficacy. PMID:27035329

Adenosine A2A receptor agonists with potent antiplatelet activity.

PubMed

Fuentes, Eduardo; Fuentes, Manuel; Caballero, Julio; Palomo, Iván; Hinz, Sonja; El-Tayeb, Ali; Müller, Christa E

2018-05-01

Selected adenosine A 2A receptor agonists (PSB-15826, PSB-12404, and PSB-16301) have been evaluated as new antiplatelet agents. In addition, radioligand-binding studies and receptor-docking experiments were performed in order to explain their differential biological effects on a molecular level. Among the tested adenosine derivatives, PSB-15826 was the most potent compound to inhibit platelet aggregation (EC 50 0.32 ± 0.05 µmol/L) and platelet P-selectin cell-surface localization (EC 50 0.062 ± 0.2 µmol/L), and to increase intraplatelets cAMP levels (EC 50 0.24 ± 0.01 µmol/L). The compound was more active than CGS21680 (EC 50 0.97±0.07 µmol/L) and equipotent to NECA (EC 50 0.31 ± 0.05 µmol/L) in platelet aggregation induced by ADP. In contrast to the results from cAMP assays, K i values determined in radioligand-binding studies were not predictive of the A 2A agonists' antiplatelet activity. Docking studies revealed the key molecular determinants of this new family of adenosine A 2A receptor agonists: differences in activities are related to π-stacking interactions between the ligands and the residue His264 in the extracellular loop of the adenosine A 2A receptor which may result in increased residence times. In conclusion, these results provide an improved understanding of the requirements of antiplatelet adenosine A 2A receptor agonists.

The effect of fenoterol stereochemistry on the β2 adrenergic receptor system: ligand directed chiral recognition

PubMed Central

Jozwiak, Krzysztof; Plazinska, Anita; Toll, Lawrence; Jimenez, Lucita; Woo, Anthony Yiu-Ho; Xiao, Rui-Ping; Wainer, Irving W.

2011-01-01

The β2 adrenergic receptor (β2-AR) is a model system for studying the ligand recognition process in G-protein coupled receptors. Fenoterol (FEN) is a β2-AR selective agonist that has two centers of chirality and exists as four stereoisomers. Radioligand binding studies determined that stereochemistry greatly influences the binding affinity. Subsequent Van’t Hoff analysis shows very different thermodynamics of binding depending on the stereoconfiguration of the molecule. The binding of (S,x’)-isomers is almost entirely enthalpy controlled whereas binding of (R,x’)-isomers is purely entropy driven. Stereochemistry of FEN molecule also affects the coupling of the receptor to different G proteins. In a rat cardiomyocyte contractility model, (R,R’)-FEN was shown to selectively activate Gs protein signaling while the (S,R’)- isomer activated both Gi and Gs protein. The overall data demonstrate that the chirality at the two chiral centers of the FEN molecule influences the magnitude of binding affinity, thermodynamics of local interactions within the binding site and the global mechanism of β2-AR activation. Differences in thermodynamic parameters and non-uniform G-protein coupling suggest a mechanism of chiral recognition in which observed enantioselectivities arise from the interaction of the (R,x’)-FEN stereoisomers with a different receptor conformation than the one with which the (S,x’)-isomer interacts. PMID:21618615

Effect of fenoterol stereochemistry on the β2 adrenergic receptor system: ligand-directed chiral recognition.

PubMed

Jozwiak, Krzysztof; Plazinska, Anita; Toll, Lawrence; Jimenez, Lucita; Woo, Anthony Yiu-Ho; Xiao, Rui-Ping; Wainer, Irving W

2011-01-01

The β(2) adrenergic receptor (β(2)-AR) is a model system for studying the ligand recognition process in G protein-coupled receptors. Fenoterol (FEN) is a β(2)-AR selective agonist that has two centers of chirality and exists as four stereoisomers. Radioligand binding studies determined that stereochemistry greatly influences the binding affinity. Subsequent Van't Hoff analysis shows very different thermodynamics of binding depending on the stereoconfiguration of the molecule. The binding of (S,x')-isomers is almost entirely enthalpy controlled whereas binding of (R,x')-isomers is purely entropy driven. Stereochemistry of FEN molecule also affects the coupling of the receptor to different G proteins. In a rat cardiomyocyte contractility model, (R,R')-FEN was shown to selectively activate G(s) protein signaling while the (S,R')-isomer activated both G(i) and G(s) protein. The overall data demonstrate that the chirality at the two chiral centers of the FEN molecule influences the magnitude of binding affinity, thermodynamics of local interactions within the binding site, and the global mechanism of β(2)-AR activation. Differences in thermodynamic parameters and nonuniform G-protein coupling suggest a mechanism of chiral recognition in which observed enantioselectivities arise from the interaction of the (R,x')-FEN stereoisomers with a different receptor conformation than the one with which the (S,x')-isomer interacts. Copyright © 2011 Wiley-Liss, Inc.

Mixed nicotinic-muscarinic properties of the alpha9 nicotinic cholinergic receptor.

PubMed

Verbitsky, M; Rothlin, C V; Katz, E; Elgoyhen, A B

2000-10-01

The rat alpha9 nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus laevis oocytes and tested for its sensitivity to a wide variety of cholinergic compounds. Acetylcholine (ACh), carbachol, choline and methylcarbachol elicited agonist-evoked currents, giving maximal or near maximal responses. Both the nicotinic agonist suberyldicholine as well as the muscarinic agonists McN-A-343 and methylfurtrethonium behaved as weak partial agonists of the receptor. Most classical cholinergic compounds tested, being either nicotinic (nicotine, epibatidine, cytisine, methyllycaconitine, mecamylamine, dihydro-beta-erythroidine), or muscarinic (muscarine, atropine, gallamine, pilocarpine, bethanechol) agonists and antagonists, blocked the recombinant alpha9 receptor. Block by nicotine, epibatidine, cytisine, methyllycaconitine and atropine was overcome at high ACh concentrations, suggesting a competitive type of block. The present results indicate that alpha9 displays mixed nicotinic-muscarinic features that resemble the ones described for the cholinergic receptor of cochlear outer hair cells (OHCs). We suggest that alpha9 contains the structural determinants responsible for the pharmacological properties of the native receptor.

Microdose gonadotropin-releasing hormone agonist in the absence of exogenous gonadotropins is not sufficient to induce multiple follicle development.

PubMed

Chung, Karine; Fogle, Robin; Bendikson, Kristin; Christenson, Kamilee; Paulson, Richard

2011-01-01

Because the effectiveness of the "microdose flare" stimulation protocol often is attributed to the dramatic endogenous gonadotropin release induced by the GnRH agonist, the aim of this study was to determine whether use of microdose GnRH agonist alone could induce multiple ovarian follicle development in normal responders. Based on these data, the duration of gonadotropin rise is approximately 24 to 48 hours and is too brief to sustain continued multiple follicle growth. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Regulation of ERRα Gene Expression by Estrogen Receptor Agonists and Antagonists in SKBR3 Breast Cancer Cells: Differential Molecular Mechanisms Mediated by G Protein-Coupled Receptor GPR30/GPER-1

PubMed Central

Li, Yin; Birnbaumer, Lutz; Teng, Christina T.

2010-01-01

In selected tissues and cell lines, 17β-estradiol (E2) regulates the expression of estrogen-related receptor α (ERRα), a member of the orphan nuclear receptor family. This effect is thought to be mediated by the estrogen receptor α (ERα). However in the ERα- and ERβ-negative SKBR3 breast cancer cell line, physiological levels of E2 also stimulate ERRα expression. Here, we explored the molecular mechanism that mediates estrogen action in ER-negative breast cancer cells. We observed that E2, the ERα agonist, as well as the ERα antagonists ICI 182,780 and tamoxifen (TAM), a selective ER modulator, stimulate the transcriptional activity of the ERRα gene and increase the production of ERRα protein in SKBR3 cells. Moreover, the ERRα downstream target genes expression and cellular proliferation are also increased. We show further that the G protein-coupled receptor GPR30/GPER-1 (GPER-1) mediates these effects. The GPER-1 specific ligand G-1 mimics the actions of E2, ICI 182,780, and TAM on ERRα expression, and changing the levels of GPER-1 mRNA by overexpression or small interfering RNA knockdown affected the expression of ERRα accordingly. Utilizing inhibitors, we delineate a different downstream pathway for ER agonist and ER antagonist-triggered signaling through GPER-1. We also find differential histone acetylation and transcription factor recruitment at distinct nucleosomes of the ERRα promoter, depending on whether the cells are activated with E2 or with ER antagonists. These findings provide insight into the molecular mechanisms of GPER-1/ERRα-mediated signaling and may be relevant to what happens in breast cancer cells escaping inhibitory control by TAM. PMID:20211987

Kinase cascades and ligand-directed signaling at the kappa opioid receptor.

PubMed

Bruchas, Michael R; Chavkin, Charles

2010-06-01

The dynorphin/kappa opioid receptor (KOR) system has been implicated as a critical component of the stress response. Stress-induced activation of dynorphin-KOR is well known to produce analgesia, and more recently, it has been implicated as a mediator of stress-induced responses including anxiety, depression, and reinstatement of drug seeking. Drugs selectively targeting specific KOR signaling pathways may prove potentially useful as therapeutic treatments for mood and addiction disorders. KOR is a member of the seven transmembrane spanning (7TM) G-protein coupled receptor (GPCR) superfamily. KOR activation of pertussis toxin-sensitive G proteins leads to Galphai/o inhibition of adenylyl cyclase production of cAMP and releases Gbetagamma, which modulates the conductances of Ca(+2) and K(+) channels. In addition, KOR agonists activate kinase cascades including G-protein coupled Receptor Kinases (GRK) and members of the mitogen-activated protein kinase (MAPK) family: ERK1/2, p38 and JNK. Recent pharmacological data suggests that GPCRs exist as dynamic, multi-conformational protein complexes that can be directed by specific ligands towards distinct signaling pathways. Ligand-induced conformations of KOR that evoke beta-arrestin-dependent p38 MAPK activation result in aversion; whereas ligand-induced conformations that activate JNK without activating arrestin produce long-lasting inactivation of KOR signaling. In this review, we discuss the current status of KOR signal transduction research and the data that support two novel hypotheses: (1) KOR selective partial agonists that do not efficiently activate p38 MAPK may be useful analgesics without producing the dysphoric or hallucinogenic effects of selective, highly efficacious KOR agonists and (2) KOR antagonists that do not activate JNK may be effective short-acting drugs that may promote stress-resilience.

Identification of GPCR-Interacting Cytosolic Proteins Using HDL Particles and Mass Spectrometry-Based Proteomic Approach

PubMed Central

Chung, Ka Young; Day, Peter W.; Vélez-Ruiz, Gisselle; Sunahara, Roger K.; Kobilka, Brian K.

2013-01-01

G protein-coupled receptors (GPCRs) have critical roles in various physiological and pathophysiological processes, and more than 40% of marketed drugs target GPCRs. Although the canonical downstream target of an agonist-activated GPCR is a G protein heterotrimer; there is a growing body of evidence suggesting that other signaling molecules interact, directly or indirectly, with GPCRs. However, due to the low abundance in the intact cell system and poor solubility of GPCRs, identification of these GPCR-interacting molecules remains challenging. Here, we establish a strategy to overcome these difficulties by using high-density lipoprotein (HDL) particles. We used the β2-adrenergic receptor (β2AR), a GPCR involved in regulating cardiovascular physiology, as a model system. We reconstituted purified β2AR in HDL particles, to mimic the plasma membrane environment, and used the reconstituted receptor as bait to pull-down binding partners from rat heart cytosol. A total of 293 proteins were identified in the full agonist-activated β2AR pull-down, 242 proteins in the inverse agonist-activated β2AR pull-down, and 210 proteins were commonly identified in both pull-downs. A small subset of the β2AR-interacting proteins isolated was confirmed by Western blot; three known β2AR-interacting proteins (Gsα, NHERF-2, and Grb2) and 3 newly identified known β2AR-interacting proteins (AMPKα, acetyl-CoA carboxylase, and UBC-13). Profiling of the identified proteins showed a clear bias toward intracellular signal transduction pathways, which is consistent with the role of β2AR as a cell signaling molecule. This study suggests that HDL particle-reconstituted GPCRs can provide an effective platform method for the identification of GPCR binding partners coupled with a mass spectrometry-based proteomic analysis. PMID:23372797

Nigrostriatal lesion induces D2-modulated phase-locked activity in the basal ganglia of rats.

PubMed

Zold, Camila L; Ballion, Bérangère; Riquelme, Luis A; Gonon, François; Murer, M Gustavo

2007-04-01

There is a debate as to what modifications of neuronal activity underlie the clinical manifestations of Parkinson's disease and the efficacy of antiparkinsonian pharmacotherapy. Previous studies suggest that release of GABAergic striatopallidal neurons from D2 receptor-mediated inhibition allows spreading of cortical rhythms to the globus pallidus (GP) in rats with 6-hydroxydopamine-induced nigrostriatal lesions. Here this abnormal spreading was thoroughly investigated. In control urethane-anaesthetized rats most GP neurons were excited during the active part of cortical slow waves ('direct-phase' neurons). Two neuronal populations having opposite phase relationships with cortical and striatal activity coexisted in the GP of 6-hydroxydopamine-lesioned rats. 'Inverse-phase' GP units exhibited reduced firing coupled to striatal activation during slow waves, suggesting that this GP oscillation was driven by striatopallidal hyperactivity. Half of the pallidonigral neurons identified by antidromic stimulation exhibited inverse-phase activity. Therefore, spreading of inverse-phase oscillations through pallidonigral axons might contribute to the abnormal direct-phase cortical entrainment of basal ganglia output described previously. Systemic administration of the D2 agonist quinpirole to 6-hydroxydopamine-lesioned rats reduced GP inverse-phase coupling with slow waves, and this effect was reversed by the D2 antagonist eticlopride. Because striatopallidal hyperactivity was only slightly reduced by quinpirole, other mechanisms might have contributed to the effect of quinpirole on GP oscillations. These results suggest that antiparkinsonian efficacy may rely on other actions of D2 agonists on basal ganglia activity. However, abnormal slow rhythms may promote enduring changes in functional connectivity along the striatopallidal axis, contributing to D2 agonist-resistant clinical signs of parkinsonism.

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

PubMed

Pydi, Sai P; Jaggupilli, Appalaraju; Nelson, Ken M; Abrams, Suzanne R; Bhullar, Rajinder P; Loewen, Michele C; Chelikani, Prashen

2015-04-28

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 Gamma-Aminobutyric Acid B Receptor in Depression and Reward.

PubMed

Jacobson, Laura H; Vlachou, Styliani; Slattery, David A; Li, Xia; Cryan, John F

2018-06-01

The metabotropic gamma-aminobutyric acid B (GABA B ) receptor was the first described obligate G protein-coupled receptor heterodimer and continues to set the stage for discoveries in G protein-coupled receptor signaling complexity. In this review, dedicated to the life and work of Athina Markou, we explore the role of GABA B receptors in depression, reward, and the convergence of these domains in anhedonia, a shared symptom of major depressive disorder and withdrawal from drugs of abuse. GABA B receptor expression and function are enhanced by antidepressants and reduced in animal models of depression. Generally, GABA B receptor antagonists are antidepressant-like and agonists are pro-depressive. Exceptions to this rule likely reflect the differential influence of GABA B1 isoforms in depression-related behavior and neurobiology, including the anhedonic effects of social stress. A wealth of data implicate GABA B receptors in the rewarding effects of drugs of abuse. We focus on nicotine as an example. GABA B receptor activation attenuates, and deactivation enhances, nicotine reward and associated neurobiological changes. In nicotine withdrawal, however, GABA B receptor agonists, antagonists, and positive allosteric modulators enhance anhedonia, perhaps owing to differential effects of GABA B1 isoforms on the dopaminergic system. Nicotine cue-induced reinstatement is more reliably attenuated by GABA B receptor activation. Separation of desirable and undesirable side effects of agonists is achievable with positive allosteric modulators, which are poised to enter clinical studies for drug abuse. GABA B1 isoforms are key to understanding the neurobiology of anhedonia, whereas allosteric modulators may offer a mechanism for targeting specific brain regions and processes associated with reward and depression. Copyright © 2018 Society of Biological Psychiatry. All rights reserved.

Activation of the G protein-coupled estrogen receptor, but not estrogen receptor α or β, rapidly enhances social learning.

PubMed

Ervin, Kelsy Sharice Jean; Mulvale, Erin; Gallagher, Nicola; Roussel, Véronique; Choleris, Elena

2015-08-01

Social learning is a highly adaptive process by which an animal acquires information from a conspecific. While estrogens are known to modulate learning and memory, much of this research focuses on individual learning. Estrogens have been shown to enhance social learning on a long-term time scale, likely via genomic mechanisms. Estrogens have also been shown to affect individual learning on a rapid time scale through cell-signaling cascades, rather than via genomic effects, suggesting they may also rapidly influence social learning. We therefore investigated the effects of 17β-estradiol and involvement of the estrogen receptors (ERs) using the ERα agonist propyl pyrazole triol, the ERβ agonist diarylpropionitrile, and the G protein-coupled ER 1 (GPER1) agonist G1 on the social transmission of food preferences (STFP) task, within a time scale that focused on the rapid effects of estrogens. General ER activation with 17β-estradiol resulted in a modest facilitation of social learning, with mice showing a preference up to 30min of testing. Specific activation of the GPER1 also rapidly enhanced social learning, with mice showing a socially learned preference up to 2h of testing. ERα activation instead shortened the expression of a socially learned food preference, while ERβ activation had little to no effects. Thus, rapid estrogenic modulation of social learning in the STFP may be the outcome of competing action at the three main receptors. Hence, estrogens' rapid effects on social learning likely depend on the specific ERs present in brain regions recruited during social learning. Copyright © 2015 Elsevier Ltd. All rights reserved.

Lesioning of TRPV1 Expressing Primary Afferent Neurons Prevents PAR-2 Induced Motility, but Not Mechanical Hypersensitivity in the Rat Colon

PubMed Central

Suckow, Shelby K.; Anderson, Ethan M.; Caudle, Robert M.

2011-01-01

Background Proteinase activated receptor 2 (PAR-2) is expressed by many neurons in the colon, including primary afferent neurons that co-express transient receptor potential vanilloid 1 (TRPV1). Activation of PAR-2 receptors was previously found to enhance colonic motility, increase secretion and produce hypersensitivity to mechanical stimuli. This study examined the functional role of TRPV1/PAR-2 expressing neurons that innervate the colon by lesioning TRPV1 bearing neurons with the highly selective and potent TRPV1 agonist resiniferatoxin. Methods Colonic motility in response to PAR-2 activation was evaluated in vitro using isolated segments of descending colon and in vivo using manometry. Colonic mechanical nociceptive thresholds were measured using colorectal distension. TRPV1 expressing neurons were selectively lesioned with resiniferatoxin. Key Results In vitro the PAR-2 agonists trypsin and SLIGRL did not alter contractions of colon segments when applied alone, however, the agents enhanced acetylcholine stimulated contraction. In vivo, PAR-2 agonists administered intraluminally induced contractions of the colon and produced hypersensitivity to colorectal distention. The PAR-2 agonist enhancement of colonic contraction was eliminated when TRPV1 expressing neurons were lesioned with resiniferatoxin, but the PAR-2 agonist induced hypersensitivity remained in the lesioned animals. Conclusions and Inferences Our findings indicate that TRPV1/PAR-2 expressing primary afferent neurons mediate an extrinsic motor reflex pathway in the colon. These data, coupled with our previous studies, also indicate that the recently described colospinal afferent neurons are nociceptive, suggesting that these neurons may be useful targets for the pharmacological control of pain in diseases such as irritable bowel syndrome. PMID:22168801

Lesioning of TRPV1 expressing primary afferent neurons prevents PAR-2 induced motility, but not mechanical hypersensitivity in the rat colon.

PubMed

Suckow, S K; Anderson, E M; Caudle, R M

2012-03-01

Proteinase activated receptor 2 (PAR-2) is expressed by many neurons in the colon, including primary afferent neurons that co-express transient receptor potential vanilloid 1 (TRPV1). Activation of PAR-2 receptors was previously found to enhance colonic motility, increase secretion and produce hypersensitivity to mechanical stimuli. This study examined the functional role of TRPV1/PAR-2 expressing neurons that innervate the colon by lesioning TRPV1 bearing neurons with the highly selective and potent TRPV1 agonist resiniferatoxin. Colonic motility in response to PAR-2 activation was evaluated in vitro using isolated segments of descending colon and in vivo using manometry. Colonic mechanical nociceptive thresholds were measured using colorectal distension. Transient receptor potential vanilloid 1 expressing neurons were selectively lesioned with resiniferatoxin. In vitro, the PAR-2 agonists, trypsin and SLIGRL did not alter contractions of colon segments when applied alone, however, the agents enhanced acetylcholine stimulated contraction. In vivo, PAR-2 agonists administered intraluminally induced contractions of the colon and produced hypersensitivity to colorectal distention. The PAR-2 agonist enhancement of colonic contraction was eliminated when TRPV1 expressing neurons were lesioned with resiniferatoxin, but the PAR-2 agonist induced hypersensitivity remained in the lesioned animals. Our findings indicate that TRPV1/PAR-2 expressing primary afferent neurons mediate an extrinsic motor reflex pathway in the colon. These data, coupled with our previous studies, also indicate that the recently described colospinal afferent neurons are nociceptive, suggesting that these neurons may be useful targets for the pharmacological control of pain in diseases such as irritable bowel syndrome. © 2011 Blackwell Publishing Ltd.

Structure-activity relationships of the melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 at the mouse melanocortin receptors. Part 3: modifications at the Arg position.

PubMed

Holder, Jerry Ryan; Xiang, Zhimin; Bauzo, Rayna M; Haskell-Luevano, Carrie

2003-01-01

The melanocortin pathway is involved in the regulation of several physiological functions including skin pigmentation, steroidogenesis, obesity, energy homeostasis, and exocrine gland function. This melanocortin pathway consists of five known G-protein coupled receptors, endogenous agonists derived from the proopiomelanocortin (POMC) gene transcript, the endogenous antagonists Agouti and the Agouti-related protein (AGRP) and signals through the intracellular cAMP signal transduction pathway. The melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) located in the brain are implicated as participating in the metabolic and food intake aspects of energy homeostasis and are stimulated by melanocortin agonists such as alpha-melanocyte stimulation hormone (alpha-MSH). All the endogenous (POMC-derived) melanocortin agonists contain the putative message sequence "His-Phe-Arg-Trp." Herein, we report 12 tetrapeptides, based upon the template Ac-His(6)-DPhe(7)-Arg(8)-Trp(9)-NH(2) (alpha-MSH numbering) that have been modified at the Arg(8) position by neutral, basic, or acidic amino acid side chains. These peptides have been pharmacologically characterized for agonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. The most notable results of this study include the observation that removal of the guanidinyl side chain moiety results in decreased melanocortin receptor potency, but that this Arg(8) side chain is not critical for melanocortin receptor agonist activity. Additionally, incorporation of the homoArg(8) residue results in 56-fold MC4R versus MC3R selectivity, and the Orn(8) residue results in 123-fold MC4R versus MC5R and 63-fold MC5R versus MC3R selectivity. Copyright 2002 Elsevier Science Inc.

The A3 adenosine receptor agonist CF502 inhibits the PI3K, PKB/Akt and NF-kappaB signaling pathway in synoviocytes from rheumatoid arthritis patients and in adjuvant-induced arthritis rats.

PubMed

Ochaion, A; Bar-Yehuda, S; Cohen, S; Amital, H; Jacobson, K A; Joshi, B V; Gao, Z G; Barer, F; Patoka, R; Del Valle, L; Perez-Liz, G; Fishman, P

2008-08-15

The A(3) adenosine receptor (A(3)AR) is over-expressed in inflammatory cells and was defined as a target to combat inflammation. Synthetic agonists to this receptor, such as IB-MECA and Cl-IB-MECA, exert an anti-inflammatory effect in experimental animal models of adjuvant- and collagen-induced arthritis. In this study we present a novel A(3)AR agonist, CF502, with high affinity and selectivity at the human A(3)AR. CF502 induced a dose dependent inhibitory effect on the proliferation of fibroblast-like synoviocytes (FLS) via de-regulation of the nuclear factor-kappa B (NF-kappaB) signaling pathway. Furthermore, CF502 markedly suppressed the clinical and pathological manifestations of adjuvant-induced arthritis (AIA) in a rat experimental model when given orally at a low dose (100 microg/kg). As is typical of other G-protein coupled receptors, the A(3)AR expression level was down-regulated shortly after treatment with agonist CF502 in paw and in peripheral blood mononuclear cells (PBMCs) derived from treated AIA animals. Subsequently, a decrease in the expression levels of protein kinase B/Akt (PKB/Akt), IkappaB kinase (IKK), I kappa B (IkappaB), NF-kappaB and tumor necrosis factor-alpha (TNF-alpha) took place. In addition, the expression levels of glycogen synthase kinase-3 beta (GSK-3beta), beta-catenin, and poly(ADP-ribose)polymerase (PARP), known to control the level and activity of NF-kappaB, were down-regulated upon treatment with CF502. Taken together, CF502 inhibits FLS growth and the inflammatory manifestations of arthritis, supporting the development of A(3)AR agonists for the treatment of rheumatoid arthritis.

Identification of Novel Transplantable GPCR Recycling Motif for Drug Discovery

PubMed Central

Nooh, Mohammed M.; Mancarella, Salvatore; Bahouth, Suleiman W.

2016-01-01

β1-adrenergic receptor (β1-AR) agonists and antagonists are widely used in the treatment of major cardiovascular diseases such as heart failure and hypertension. The β1-AR like other G protein-couple receptors (GPCR) is endocytosed in response to intense agonist activation. Recycling of the agonist-internalized β1-AR is dependent on its carboxy-terminal type-1 PSD-95/DLG/ZO1 (PDZ) and on phospho-serine312 in the third intracellular loop of the β1-AR. Progressive elongation of the β1-AR at its C-tail inactivated the PDZ-biding domain and inhibited the recycling of the β1-AR. However, fusing a twenty amino acid peptide derived from the multiple cloning region of the mammalian expression vector pCDNA3 to the C-tail of the β1-AR (β1-AR[+20]) produced a chimeric β1-AR that recycled rapidly and efficiently. The β1-AR[+20] recycled in a type-1 PDZ and phospho-Ser312-independent manner, indicating that this peptide provided a general GPCR recycling signal. Fusing the enhanced yellow fluorescent protein (EYFP) down-stream of β1-AR[+20] generated a β1-AR-EYFP chimera that was expressed on the membrane and recycled efficiently after agonist-induced internalization. This construct trafficked in a PDZ-SNX27/retromer-independent manner. We also fused EYFP to the N-terminus of the β1-AR to created EYFP-WT β1-AR. This construct recycled in PDZ and SNX27/retromer dependent manner. These β1-AR-EYFP constructs would be useful for high throughput screening (HTS) programs to identify new entities that would interfere with the recycling of agonist internalized GPCR that traffic in PDZ-dependent vs. PDZ-independent roadmaps. PMID:27645110

A dual agonist of farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, INT-767, reverses age-related kidney disease in mice.

PubMed

Wang, Xiaoxin X; Luo, Yuhuan; Wang, Dong; Adorini, Luciano; Pruzanski, Mark; Dobrinskikh, Evgenia; Levi, Moshe

2017-07-21

Even in healthy individuals, renal function gradually declines during aging. However, an observed variation in the rate of this decline has raised the possibility of slowing or delaying age-related kidney disease. One of the most successful interventional measures that slows down and delays age-related kidney disease is caloric restriction. We undertook the present studies to search for potential factors that are regulated by caloric restriction and act as caloric restriction mimetics. Based on our prior studies with the bile acid-activated nuclear hormone receptor farnesoid X receptor (FXR) and G protein-coupled membrane receptor TGR5 that demonstrated beneficial effects of FXR and TGR5 activation in the kidney, we reasoned that FXR and TGR5 could be excellent candidates. We therefore determined the effects of aging and caloric restriction on the expression of FXR and TGR5 in the kidney. We found that FXR and TGR5 expression levels are decreased in the aging kidney and that caloric restriction prevents these age-related decreases. Interestingly, in long-lived Ames dwarf mice, renal FXR and TGR5 expression levels were also increased. A 2-month treatment of 22-month-old C57BL/6J mice with the FXR-TGR5 dual agonist INT-767 induced caloric restriction-like effects and reversed age-related increases in proteinuria, podocyte injury, fibronectin accumulation, TGF-β expression, and, most notably, age-related impairments in mitochondrial biogenesis and mitochondrial function. Furthermore, in podocytes cultured in serum obtained from old mice, INT-767 prevented the increases in the proinflammatory markers TNF-α, toll-like receptor 2 (TLR2), and TLR4. In summary, our results indicate that FXR and TGR5 may play an important role in modulation of age-related kidney disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

RNA sequencing to determine the contribution of kinase receptor transactivation to G protein coupled receptor signalling in vascular smooth muscle cells.

PubMed

Kamato, Danielle; Bhaskarala, Venkata Vijayanand; Mantri, Nitin; Oh, Tae Gyu; Ling, Dora; Janke, Reearna; Zheng, Wenhua; Little, Peter J; Osman, Narin

2017-01-01

G protein coupled receptor (GPCR) signalling covers three major mechanisms. GPCR agonist engagement allows for the G proteins to bind to the receptor leading to a classical downstream signalling cascade. The second mechanism is via the utilization of the β-arrestin signalling molecule and thirdly via transactivation dependent signalling. GPCRs can transactivate protein tyrosine kinase receptors (PTKR) to activate respective downstream signalling intermediates. In the past decade GPCR transactivation dependent signalling was expanded to show transactivation of serine/threonine kinase receptors (S/TKR). Kinase receptor transactivation enormously broadens the GPCR signalling paradigm. This work utilizes next generation RNA-sequencing to study the contribution of transactivation dependent signalling to total protease activated receptor (PAR)-1 signalling. Transactivation, assessed as gene expression, accounted for 50 percent of the total genes regulated by thrombin acting through PAR-1 in human coronary artery smooth muscle cells. GPCR transactivation of PTKRs is approximately equally important as the transactivation of the S/TKR with 209 and 177 genes regulated respectively, via either signalling pathway. This work shows that genome wide studies can provide powerful insights into GPCR mediated signalling pathways.

Circadian clock component REV-ERBα controls homeostatic regulation of pulmonary inflammation.

PubMed

Pariollaud, Marie; Gibbs, Julie E; Hopwood, Thomas W; Brown, Sheila; Begley, Nicola; Vonslow, Ryan; Poolman, Toryn; Guo, Baoqiang; Saer, Ben; Jones, D Heulyn; Tellam, James P; Bresciani, Stefano; Tomkinson, Nicholas Co; Wojno-Picon, Justyna; Cooper, Anthony Wj; Daniels, Dion A; Trump, Ryan P; Grant, Daniel; Zuercher, William; Willson, Timothy M; MacDonald, Andrew S; Bolognese, Brian; Podolin, Patricia L; Sanchez, Yolanda; Loudon, Andrew Si; Ray, David W

2018-06-01

Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERBα as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBβ in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous proinflammatory mechanism in unchallenged cells. However, REV-ERBα plays the dominant role, as deletion of REV-ERBβ alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation, and used this to reveal how proinflammatory cytokines trigger rapid degradation of REV-ERBα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERBα protein couple the core clock to innate immunity.

Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Calabrese, G.; Sallese, M.; Stornaiuolo, A.

1994-09-01

Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment ofmore » the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.« less

Muscarinic Control of MIN6 Pancreatic β Cells Is Enhanced by Impaired Amino Acid Signaling*

PubMed Central

Guerra, Marcy L.; Wauson, Eric M.; McGlynn, Kathleen; Cobb, Melanie H.

2014-01-01

We have shown recently that the class C G protein-coupled receptor T1R1/T1R3 taste receptor complex is an early amino acid sensor in MIN6 pancreatic β cells. Amino acids are unable to activate ERK1/2 in β cells in which T1R3 has been depleted. The muscarinic receptor agonist carbachol activated ERK1/2 better in T1R3-depleted cells than in control cells. Ligands that activate certain G protein-coupled receptors in pancreatic β cells potentiate glucose-stimulated insulin secretion. Among these is the M3 muscarinic acetylcholine receptor, the major muscarinic receptor in β cells. We found that expression of M3 receptors increased in T1R3-depleted MIN6 cells and that calcium responses were altered. To determine whether these changes were related to impaired amino acid signaling, we compared responses in cells exposed to reduced amino acid concentrations. M3 receptor expression was increased, and some, but not all, changes in calcium signaling were mimicked. These findings suggest that M3 acetylcholine receptors are increased in β cells as a mechanism to compensate for amino acid deficiency. PMID:24695728

Comparison of the ultrashort gonadotropin-releasing hormone agonist-antagonist protocol with microdose flare -up protocol in poor responders: a preliminary study

PubMed Central

Berker, Bülent; Duvan, Candan İltemir; Kaya, Cemil; Aytaç, Ruşen; Şatıroğlu, Hakan

2010-01-01

Objective To determine the potential effect of the ultrashort gonadotropin-releasing hormone (GnRH) agonist/GnRH antagonist protocol versus the microdose GnRH agonist protocol in poor responders undergoing intracytoplasmic sperm injection (ICSI). Material and Methods The patients in the Agonist-Antagonist Group (n=41) were administered the ultrashort GnRH-agonist/ antagonist protocol, while the patients in the Microdose Group (n=41) were stimulated according to the microdose flare-up protocol. The mean number of mature oocytes retrieved was the primary outcome measure. Fertilization rate, implantation rate per embryo and clinical pregnancy rates were secondary outcome measures. Results There was no differenc between the mean number of mature oocytes retrieved in the two groups. There were also no statistical differences between the two groups in terms of peak serum E2 level, canceled cycles, endometrial thickness on hCG day, number of 2 pronucleus and number of embryos transferred. However, the total gonadotropin consumption and duration of stimulation were significantly higher with the Agonist-Antagonist Group compared with the Microdose Group. The implantation and clinical pregnancy rates were similar between the two groups. Conclusion Despite the high dose of gonadotropin consumption and longer duration of stimulation with the ultrashort GnRH agonist/ antagonist protocol, it seems that the Agonist-Antagonist Protocol is not inferior to the microdose protocol in poor responders undergoing ICSI. PMID:24591934

Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists

PubMed Central

Charfi, Iness; Nagi, Karim; Mnie-Filali, Ouissame; Thibault, Dominic; Balboni, Gianfranco; Schiller, Peter W.; Trudeau, Louis-Eric

2014-01-01

Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of Emax values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase Emax values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells. PMID:24022593

Structural Determinants of the Transient Receptor Potential 1 (TRPV1) Channel Activation by Phospholipid Analogs*

PubMed Central

Morales-Lázaro, Sara L.; Serrano-Flores, Barbara; Llorente, Itzel; Hernández-García, Enrique; González-Ramírez, Ricardo; Banerjee, Souvik; Miller, Duane; Gududuru, Veeresh; Fells, James; Norman, Derek; Tigyi, Gabor; Escalante-Alcalde, Diana; Rosenbaum, Tamara

2014-01-01

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1. PMID:25035428

Endothelin-converting enzyme 1 degrades neuropeptides in endosomes to control receptor recycling.

PubMed

Roosterman, Dirk; Cottrell, Graeme S; Padilla, Benjamin E; Muller, Laurent; Eckman, Christopher B; Bunnett, Nigel W; Steinhoff, Martin

2007-07-10

Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with beta-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide-receptor-arrestin complex is sequestered into endosomes. Although dissociation of beta-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase endothelin-converting enzyme 1 (ECE-1), determines stability of the peptide-receptor-arrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK1R) induced membrane translocation of beta-arrestins followed by trafficking of the SP-NK1R-beta-arrestin complex to early endosomes containing ECE-1a-d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; beta-arrestins returned to the cytosol, and the NK1R, freed from beta-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK1R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK3R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptide-receptor-arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control beta-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.

Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor.

PubMed

Yang, Zhenlin; Han, Shuo; Keller, Max; Kaiser, Anette; Bender, Brian J; Bosse, Mathias; Burkert, Kerstin; Kögler, Lisa M; Wifling, David; Bernhardt, Guenther; Plank, Nicole; Littmann, Timo; Schmidt, Peter; Yi, Cuiying; Li, Beibei; Ye, Sheng; Zhang, Rongguang; Xu, Bo; Larhammar, Dan; Stevens, Raymond C; Huster, Daniel; Meiler, Jens; Zhao, Qiang; Beck-Sickinger, Annette G; Buschauer, Armin; Wu, Beili

2018-04-01

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y 1 , Y 2 , Y 4 and Y 5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y 1 receptor (Y 1 R) 4 . A number of peptides and small-molecule compounds have been characterized as Y 1 R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y 1 R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y 1 R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y 1 R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y 1 R can enable structure-based drug discovery that targets NPY receptors.

Structural determinants of the transient receptor potential 1 (TRPV1) channel activation by phospholipid analogs.

PubMed

Morales-Lázaro, Sara L; Serrano-Flores, Barbara; Llorente, Itzel; Hernández-García, Enrique; González-Ramírez, Ricardo; Banerjee, Souvik; Miller, Duane; Gududuru, Veeresh; Fells, James; Norman, Derek; Tigyi, Gabor; Escalante-Alcalde, Diana; Rosenbaum, Tamara

2014-08-29

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110gamma.

PubMed

Guillermet-Guibert, Julie; Bjorklof, Katja; Salpekar, Ashreena; Gonella, Cristiano; Ramadani, Faruk; Bilancio, Antonio; Meek, Stephen; Smith, Andrew J H; Okkenhaug, Klaus; Vanhaesebroeck, Bart

2008-06-17

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.

The p110β isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110γ

PubMed Central

Guillermet-Guibert, Julie; Bjorklof, Katja; Salpekar, Ashreena; Gonella, Cristiano; Ramadani, Faruk; Bilancio, Antonio; Meek, Stephen; Smith, Andrew J. H.; Okkenhaug, Klaus; Vanhaesebroeck, Bart

2008-01-01

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110α, p110β, and p110δ) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110α and p110δ to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110γ class IB PI3K lack SH2 domains and instead couple p110γ to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110β and cells derived from a p110β-deficient mouse line, that p110β is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110β and p110γ contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110β but not p110γ, p110β mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110γ in these cells reduced the contribution of p110β to GPCR signaling. Taken together, these data show that p110β and p110γ can couple redundantly to the same GPCR agonists. p110β, which shows a much broader tissue distribution than the leukocyte-restricted p110γ, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110γ expression is low or absent. PMID:18544649

Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1.

PubMed

Bhattacharya, Sayak; Mahavadi, Sunila; Al-Shboul, Othman; Rajagopal, Senthilkumar; Grider, John R; Murthy, Karnam S

2013-08-01

Caveolae act as scaffolding proteins for several G protein-coupled receptor signaling molecules to regulate their activity. Caveolin-1, the predominant isoform in smooth muscle, drives the formation of caveolae. The precise role of caveolin-1 and caveolae as scaffolds for G protein-coupled receptor signaling and contraction in gastrointestinal muscle is unclear. Thus the aim of this study was to examine the role of caveolin-1 in the regulation of Gq- and Gi-coupled receptor signaling. RT-PCR, Western blot, and radioligand-binding studies demonstrated the selective expression of M2 and M3 receptors in gastric smooth muscle cells. Carbachol (CCh) stimulated phosphatidylinositol (PI) hydrolysis, Rho kinase and zipper-interacting protein (ZIP) kinase activity, induced myosin phosphatase 1 (MYPT1) phosphorylation (at Thr(696)) and 20-kDa myosin light chain (MLC20) phosphorylation (at Ser(19)) and muscle contraction, and inhibited cAMP formation. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activity, phosphorylation of MYPT1 and MLC20, and muscle contraction in response to CCh were attenuated by methyl β-cyclodextrin (MβCD) or caveolin-1 small interfering RNA (siRNA). Similar inhibition of PI hydrolysis, Rho kinase, and ZIP kinase activity and muscle contraction in response to CCh and gastric emptying in vivo was obtained in caveolin-1-knockout mice compared with wild-type mice. Agonist-induced internalization of M2, but not M3, receptors was blocked by MβCD or caveolin-1 siRNA. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activities in response to other Gq-coupled receptor agonists such as histamine and substance P was also attenuated by MβCD or caveolin-1 siRNA. Taken together, these results suggest that caveolin-1 facilitates signaling by Gq-coupled receptors and contributes to enhanced smooth muscle function.

Structure-based drug design for G protein-coupled receptors.

PubMed

Congreve, Miles; Dias, João M; Marshall, Fiona H

2014-01-01

Our understanding of the structural biology of G protein-coupled receptors has undergone a transformation over the past 5 years. New protein-ligand complexes are described almost monthly in high profile journals. Appreciation of how small molecules and natural ligands bind to their receptors has the potential to impact enormously how medicinal chemists approach this major class of receptor targets. An outline of the key topics in this field and some recent examples of structure- and fragment-based drug design are described. A table is presented with example views of each G protein-coupled receptor for which there is a published X-ray structure, including interactions with small molecule antagonists, partial and full agonists. The possible implications of these new data for drug design are discussed. © 2014 Elsevier B.V. All rights reserved.

Drug Discovery Targeting Serotonin G Protein-Coupled Receptors in the Treatment of Neuropsychiatric Disorders

NASA Astrophysics Data System (ADS)

Felsing, Daniel E.

Clinical data show that activation of 5-HT2C G protein-coupled receptors (GPCRs) can treat obesity (lorcaserin/BelviqRTM) and psychotic disorders (aripiprazole/Abilify.), including schizophrenia. 5-HT2C GPCRs are members of the 5-HT2 sub-family of 5-HT GPCRs, which include 5-HT2A, 5-HT2B, and 5-HT 2C GPCRs. 5-HT2C is structurally similar to 5-HT2A and 5-HT2B GPCRs, but activation of 5-HT2A and/or 5-HT 2B causes deleterious effects, including hallucinations and cardiac valvulopathy. Thus, there is a challenge to develop drugs that selectively activate only 5-HT2C. Prolonged activation of GPCRs by agonists reduces their function via a regulatory process called desensitization. This has clinical relevance, as 45% of drugs approved by the FDA target GPCRs, and agonist drugs (e.g., morphine) typically lose efficacy over time due to desensitization, which invites tolerance. Agonists that cause less desensitization may show extended clinical efficacy as well as a more acceptable clinical dose range. We hypothesized that structurally distinct agonists of the 5-HT2C receptor may cause varying degrees of desensitization by stabilizing unique 5-HT2C receptor conformations. Discovery of 5-HT2C agonists that exhibit minimal desensitization is therapeutically relevant for the pharmacotherapeutic treatment of chronic diseases such as obesity and psychotic disorders. The 5-HT7 receptor has recently been discovered as a druggable target, and selective activation of the 5-HT7 receptor has been shown to alleviate locomotor deficits in mouse models of Rett Syndrome. Additionally, buspirone has been shown to display therapeutically relevant affinity at 5-HT 1A and is currently in phase II clinical trials to treat stereotypy in children with autism. The 5-PAT chemical scaffold shows high affinity towards the 5-HT7 and 5-HT1A receptors. Modulations around the 5-phenyl moiety were able to improve selectivity in binding towards the 5-HT 7 receptor, whereas modulations of the alkyl chains bonded to the vital basic nitrogen modulated 5-HT1A selectivity. The lead candidate, (+)-o-F-5-PAT, was shown effective in attenuating three separate murine models of stereotypy and two models of drug-induced hyperlocomotion. Therefore, the 5-PAT chemical scaffold is a unique chemical scaffold enabling discrimination of therapeutic function of the 5-HT7 and 5-HT 1A receptors in vivo..

Determination of Zilpaterol in Horse Urine from Withdrawal Day 0 to Day 21 by UPLC-MS/MS and ELISA

USDA-ARS?s Scientific Manuscript database

Zilpaterol is a beta-adrenergic agonist that is approved in the United States, Mexico and South Africa as a feed additive to increase weight gain and improve feed efficiency of cattle. Beta-adrenergic agonists have a long history of illicit and off-label use in livestock, competitive sport animals,...

The four As associated with pathological Parkinson disease gamblers: anxiety, anger, age, and agonists

PubMed Central

Shapiro, Michael A; Chang, Yu Ling; Munson, Sarah K; Jacobson, Charles E; Rodriguez, Ramon L; Skidmore, Frank M; Okun, Michael S; Fernandez, Hubert H

2007-01-01

Several studies have related pathological gambling in PD to dopamine agonist therapy. A mail-in survey was sent to PD patients seen at the University of Florida Movement Disorders Center to determine gambling frequency and behavior, and any lifestyle or environmental factors associated with compulsive gambling in PD. 462 surveys were sent and 127 completed surveys were returned, of which ten were from patients who met criteria for compulsive gambling. All ten were taking dopamine agonists coincident with the compulsive gambling. Compulsive gamblers were younger, and psychological distress measures revealed that compulsive gamblers exhibited higher levels of anxiety, anger, and confusion. Thus in this cohort, we have uncovered the several characteristics of the most likely PD compulsive gambler, namely: (young) age, “angry”, “anxious”, and using a (dopamine) agonist. PMID:19300546

[Rapid determination of illicit beta2-agonist additives in health foods and traditional Chinese patent medicines with DCBI-MS/MS method].

PubMed

Hou, Yu-Lan; Wu, Shuang; Wang, Hua; Zhao, Yong; Liao, Peng; Tian, Qing-Qing; Sun, Wen-Jian; Chen, Bo

2013-01-01

A novel rapid method for detection of the illicit beta2-agonist additives in health foods and traditional Chinese patent medicines was developed with the desorption corona beam ionization mass spectrometry (DCBI-MS) technique. The DCBI conditions including temperature and sample volume were optimized according to the resulting mass spectra intensity. Matrix effect on 9 beta2-agonists additives was not significant in the proposed rapid determination procedure. All of the 9 target molecules were detected within 1 min. Quantification was achieved based on the typical fragment ion in MS2 spectra of each analyte. The method showed good linear coefficients in the range of 1-100 mg x L(-1) for all analytes. The relative deviation values were between 14.29% and 25.13%. Ten claimed antitussive and antiasthmatic health foods and traditional Chinese patent medicines from local pharmacies were analyzed. All of them were negative with the proposed DCBI-MS method. Without tedious sample pretreatments, the developed DCBI-MS is simple, rapid and sensitive for rapid qualification and semi-quantification of the illicit beta2-agonist additives in health foods and traditional Chinese patent medicines.

Luteinizing hormone-releasing hormone (LHRH) and its analogs for contraception in women: a review.

PubMed

Thau, R B

1984-02-01

In animals, LHRH agonists have multiple sites of action including the pituitary, the gonads, and the reproductive tract. In humans, the major antifertility action of this class of peptides is believed to be mediated via the pituitary. Studies in women have indicated that potent LHRH agonists can block ovulation when administered once daily. In the volunteers who have used these agents no serious side effects were observed, although some women experienced irregular bleeding or amenorrhea. It is anticipated that formal clinical trials could be conducted in the near future to determine the efficacy of continuous LHRH agonist administration. Early attempts to use an LHRH agonist to produce luteal insufficiency, luteolysis, or interruption of pregnancy have either been unsuccessful or the results are still too preliminary to ascertain whether these approaches warrant further trials. LHRH antagonists are believed to act by inhibiting the action of LHRH on the pituitary. Although some of these peptides are known to be active in women, very large doses have been required. Recently several investigators have produced LHRH antagonists with increased potency. In the near future, it should be possible to determine whether these peptides should be considered as potential contraceptives in men or in women.

Docosahexaenoic acid, G protein-coupled receptors, and melanoma: is G protein-coupled receptor 40 a potential therapeutic target?

PubMed

Nehra, Deepika; Pan, Amy H; Le, Hau D; Fallon, Erica M; Carlson, Sarah J; Kalish, Brian T; Puder, Mark

2014-05-15

To determine the effect of docosahexaenoic acid (DHA) on the growth of human melanoma in vitro and in vivo and to better understand the potential role of the G protein-coupled receptors (GPRs) in mediating this effect. For in vitro studies, human melanoma and control fibroblast cells were treated with DHA and TAK-875 (selective GPR40 agonist) and a cell viability assay was performed to determine cell counts. A murine subcutaneous xenograft model of human melanoma was used to test the effect of dietary treatment with an omega-3 fatty acid (FA) rich diet compared with an omega-6 FA rich diet on the growth of human melanoma in vivo. A similar animal model was used to test the effect of oral TAK-875 on the growth of established melanoma tumors in vivo. DHA has an inhibitory effect on the growth of human melanoma both in vitro and in vivo. Tumors from animals on the omega-3 FA rich diet were 69% smaller in weight (P = 0.005) and 76% smaller in volume compared with tumors from animals on the omega-6 FA rich diet. TAK-875 has an inhibitory effect on the growth of human melanoma both in vitro and in vivo. Tumors from animals treated with TAK-875 were 46% smaller in weight (P = 0.07), 62% smaller in volume (P = 0.03), and grew 77% slower (P = 0.04) compared with the placebo group. DHA and TAK-875 have a profound and selective inhibitory effect on the growth of human melanoma both in vitro and in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

Pharmacological characterization of extracellular acidification rate responses in human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells

PubMed Central

Coldwell, M C; Boyfield, I; Brown, A M; Stemp, G; Middlemiss, D N

1999-01-01

This study characterized pharmacologically the functional responses to agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) zreceptors separately expressed in cloned cells using the cytosensor microphysiometer. Dopaminergic receptor agonists caused increases in extracellular acidification rate in adherent Chinese hamster ovary (CHO) clones expressing hD2, hD3 or hD4 receptors. Acidification rate responses to agonists in other cell lines expressing these receptors were smaller than those in adherent CHO cells. The time courses and maximum increases in acidification rate of the agonist responses in adherent CHO cells were different between the three dopamine receptor clones. Responses were blocked by pretreatment of cells with pertussis toxin or amiloride analogues. Most agonists had full intrinsic activity at each of the dopamine receptor subtypes, as compared to quinpirole, however both enantiomers of UH-232 and (−)3-PPP were partial agonists in this assay system. The functional potency of full agonists at each of the three receptors expressed in CHO cells was either higher than, or similar to, the apparent inhibition constants (Ki) determined in [125I]-iodosulpride competition binding studies. Functional selectivities of the agonists were less than radioligand binding selectivities. The rank orders of agonist potencies and selectivities were similar, but not identical, to the rank orders of radioligand binding affinities and selectivities. The dopamine receptor antagonists, iodosulpride and clozapine, had no effect on basal acidification rates but inhibited acidification responses in CHO cells to quinpirole in an apparently competitive manner. Antagonist potencies closely matched their radioligand binding affinities in these cells. PMID:10455259

Characterization of the intrinsic activity for a novel class of cannabinoid receptor ligands: Indole Quinuclidine analogues

PubMed Central

Franks, Lirit N.; Ford, Benjamin M.; Madadi, Nikhil R.; Penthala, Narsimha R.; Crooks, Peter A.; Prather, Paul L.

2014-01-01

Our laboratory recently reported that a group of novel indole quinuclidine analogues bind with nanomolar affinity to cannabinoid type-1 and type-2 receptors. This study characterized the intrinsic activity of these compounds by determining whether they exhibit agonist, antagonist, or inverse agonist activity at cannabinoid type-1 and/or type-2 receptors. Cannabinoid receptors activate Gi/Go-proteins that then proceed to inhibit activity of the downstream intracellular effector adenylyl cyclase. Therefore, intrinsic activity was quantified by measuring the ability of compounds to modulate levels of intracellular cAMP in intact cells. Concerning cannabinoid type-1 receptors endogenously expressed in Neuro2A cells, a single analogue exhibited agonist activity, while eight acted as neutral antagonists and two possessed inverse agonist activity. For cannabinoid type-2 receptors stably expressed in CHO cells, all but two analogues acted as agonists; these two exceptions exhibited inverse agonist activity. Confirming specificity at cannabinoid type-1 receptors, modulation of adenylyl cyclase activity by all proposed agonists and inverse agonists was blocked by co-incubation with the neutral cannabinoid type-1 antagonist O-2050. All proposed cannabinoid type-1 receptor antagonists attenuated adenylyl cyclase modulation by cannabinoid agonist CP-55,940. Specificity at cannabinoid type-2 receptors was confirmed by failure of all compounds to modulate adenylyl cyclase activity in CHO cells devoid of cannabinoid type-2 receptors. Further characterization of select analogues demonstrated concentration-dependent modulation of adenylyl cyclase activity with potencies similar to their respective affinities for cannabinoid receptors. Therefore, indole quinuclidines are a novel structural class of compounds exhibiting high affinity and a range of intrinsic activity at cannabinoid type-1 and type-2 receptors. PMID:24858620

Differentiation of δ, μ, and κ opioid receptor agonists based on pharmacophore development and computed physicochemical properties

NASA Astrophysics Data System (ADS)

Filizola, Marta; Villar, Hugo O.; Loew, Gilda H.

2001-04-01

Compounds that bind with significant affinity to the opioid receptor types, δ, μ, and κ, with different combinations of activation and inhibition at these three receptors could be promising behaviorally selective agents. Working on this hypothesis, the chemical moieties common to three different sets of opioid receptor agonists with significant affinity for each of the three receptor types δ, μ, or κ were identified. Using a distance analysis approach, common geometric arrangements of these chemical moieties were found for selected δ, μ, or κ opioid agonists. The chemical and geometric commonalities among agonists at each opioid receptor type were then compared with a non-specific opioid recognition pharmacophore recently developed. The comparison provided identification of the additional requirements for activation of δ, μ, and κ opioid receptors. The distance analysis approach was able to clearly discriminate κ-agonists, while global molecular properties for all compounds were calculated to identify additional requirements for activation of δ and μ receptors. Comparisons of the combined geometric and physicochemical properties calculated for each of the three sets of agonists allowed the determination of unique requirements for activation of each of the three opioid receptors. These results can be used to improve the activation selectivity of known opioid agonists and as a guide for the identification of novel selective opioid ligands with potential therapeutic usefulness.

Involvement of the Calcium-sensing Receptor in Human Taste Perception

PubMed Central

Ohsu, Takeaki; Amino, Yusuke; Nagasaki, Hiroaki; Yamanaka, Tomohiko; Takeshita, Sen; Hatanaka, Toshihiro; Maruyama, Yutaka; Miyamura, Naohiro; Eto, Yuzuru

2010-01-01

By human sensory analyses, we found that various extracellular calcium-sensing receptor (CaSR) agonists enhance sweet, salty, and umami tastes, although they have no taste themselves. These characteristics are known as “kokumi taste” and often appear in traditional Japanese cuisine. Although GSH is a typical kokumi taste substance (taste enhancer), its mode of action is poorly understood. Here, we demonstrate how the kokumi taste is enhanced by the CaSR, a close relative of the class C G-protein-coupled receptors T1R1, T1R2, and T1R3 (sweet and umami receptors). We identified a large number of CaSR agonist γ-glutamyl peptides, including GSH (γ-Glu-Cys-Gly) and γ-Glu-Val-Gly, and showed that these peptides elicit the kokumi taste. Further analyses revealed that some known CaSR agonists such as Ca2+, protamine, polylysine, l-histidine, and cinacalcet (a calcium-mimetic drug) also elicit the kokumi taste and that the CaSR-specific antagonist, NPS-2143, significantly suppresses the kokumi taste. This is the first report indicating a distinct function of the CaSR in human taste perception. PMID:19892707

Melanocortin Antagonist Tetrapeptides with Minimal Agonist Activity at the Mouse Melanocortin-3 Receptor

PubMed Central

2014-01-01

The melanocortin system regulates many important functions in the body. There are five melanocortin G protein-coupled receptor subtypes known to date. Herein, we report a structure–activity relationship (SAR) study of a tetrapeptide lead discovered through a double substitution strategy at the melanocortin core His-Phe-Arg-Trp sequence. Several compounds were identified with micromolar agonist activity at the mouse melanocortin-1 (mMC1R) and mouse melanocortin-5 receptor (mMC5R) subtypes, weak antagonist activity at the mouse melanocortin-3 receptor (mMC3R), and potent antagonist activity at the mouse melanocortin-4 receptor (mMC4R). Two compounds (2 and 3) were nanomolar mMC4R antagonists with no mMC3R antagonist activity observed. Additionally, we identified three tetrapeptide MC3R antagonists (1, 6, and 7) that possess minimal mMC3R agonist activity only at 100 μM, not commonly observed for mMC3R/mMC4R antagonists. These novel molecular templates have the potential as molecular probes to better differentiate the roles of the centrally expressed MC3 and MC4 receptors. PMID:25699138

Medium-chain fatty acid-sensing receptor, GPR84, is a proinflammatory receptor.

PubMed

Suzuki, Masakatsu; Takaishi, Sachiko; Nagasaki, Miyuki; Onozawa, Yoshiko; Iino, Ikue; Maeda, Hiroaki; Komai, Tomoaki; Oda, Tomiichiro

2013-04-12

G protein-coupled receptor 84 (GPR84) is a putative receptor for medium-chain fatty acids (MCFAs), whose pathophysiological roles have not yet been clarified. Here, we show that GPR84 was activated by MCFAs with the hydroxyl group at the 2- or 3-position more effectively than nonhydroxylated MCFAs. We also identified a surrogate agonist, 6-n-octylaminouracil (6-OAU), for GPR84. These potential ligands and the surrogate agonist, 6-OAU, stimulated [(35)S]GTP binding and accumulated phosphoinositides in a GPR84-dependent manner. The surrogate agonist, 6-OAU, internalized GPR84-EGFP from the cell surface. Both the potential ligands and 6-OAU elicited chemotaxis of human polymorphonuclear leukocytes (PMNs) and macrophages and amplified LPS-stimulated production of the proinflammatory cytokine IL-8 from PMNs and TNFα from macrophages. Furthermore, the intravenous injection of 6-OAU raised the blood CXCL1 level in rats, and the inoculation of 6-OAU into the rat air pouch accumulated PMNs and macrophages in the site. Our results indicate a proinflammatory role of GPR84, suggesting that the receptor may be a novel target to treat chronic low grade inflammation associated-disease.

Medium-chain Fatty Acid-sensing Receptor, GPR84, Is a Proinflammatory Receptor

PubMed Central

Suzuki, Masakatsu; Takaishi, Sachiko; Nagasaki, Miyuki; Onozawa, Yoshiko; Iino, Ikue; Maeda, Hiroaki; Komai, Tomoaki; Oda, Tomiichiro

2013-01-01

G protein-coupled receptor 84 (GPR84) is a putative receptor for medium-chain fatty acids (MCFAs), whose pathophysiological roles have not yet been clarified. Here, we show that GPR84 was activated by MCFAs with the hydroxyl group at the 2- or 3-position more effectively than nonhydroxylated MCFAs. We also identified a surrogate agonist, 6-n-octylaminouracil (6-OAU), for GPR84. These potential ligands and the surrogate agonist, 6-OAU, stimulated [35S]GTP binding and accumulated phosphoinositides in a GPR84-dependent manner. The surrogate agonist, 6-OAU, internalized GPR84-EGFP from the cell surface. Both the potential ligands and 6-OAU elicited chemotaxis of human polymorphonuclear leukocytes (PMNs) and macrophages and amplified LPS-stimulated production of the proinflammatory cytokine IL-8 from PMNs and TNFα from macrophages. Furthermore, the intravenous injection of 6-OAU raised the blood CXCL1 level in rats, and the inoculation of 6-OAU into the rat air pouch accumulated PMNs and macrophages in the site. Our results indicate a proinflammatory role of GPR84, suggesting that the receptor may be a novel target to treat chronic low grade inflammation associated-disease. PMID:23449982

GPER activation is effective in protecting against inflammation-induced nigral dopaminergic loss and motor function impairment.

PubMed

Mendes-Oliveira, Julieta; Lopes Campos, Filipa; Videira, Rita Alexandra; Baltazar, Graça

2017-08-01

Increasing evidence suggest that excessive inflammatory responses from overactivated microglia play a critical role in Parkinson's disease (PD), contributing to, or exacerbating, nigral dopaminergic (DA) degeneration. Recent results from our group and others demonstrated that selective activation of G protein-coupled estrogen receptor (GPER) with the agonist G1 can protect DA neurons from 1-methyl-4-phenylpyridinium (MPP + ) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxins. However, it is not known whether modulation of microglial responses is one of the mechanisms by which G1 exerts its DA neuroprotective effects. We analyzed, in the N9 microglial cell line, the effect of G1 on microglial activation induced by lipopolysaccharide (LPS) exposure. The results revealed that G1 significantly decrease phagocytic activity, expression of inducible nitric oxide synthase (iNOS) and release of nitric oxide (NO) induced by LPS. To determine the relevance of this anti-inflammatory effect to the protection of nigral DA cells, the effect of G1 was analyzed in male mice injected unilaterally in the substantia nigra (SN) with LPS. Although G1 treatment did not decrease LPS-induced increase of ionized calcium binding adaptor molecule 1 (iba-1) positive cells it significantly reduced interleukin-1beta (IL-1β), cluster of differentiation 68 (CD68) and iNOS mRNA levels, and totally inhibited nigral DA cell loss and, as a consequence, protected the motor function. In summary, our findings demonstrated that the G1 agonist is able to modulate microglial responses and to protect DA neurons and motor functions against a lesion induced by an inflammatory insult. Since G1 lacks the feminizing effects associated with agonists of the classical estrogen receptors (ERs), the use of G1 to selectively activate the GPER may be a promising strategy for the development of new therapeutics for the treatment of PD and other neuroinflammatory diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Conformational changes in the M2 muscarinic receptor induced by membrane voltage and agonist binding

PubMed Central

Navarro-Polanco, Ricardo A; Galindo, Eloy G Moreno; Ferrer-Villada, Tania; Arias, Marcelo; Rigby, J Ryan; Sánchez-Chapula, José A; Tristani-Firouzi, Martin

2011-01-01

Abstract The ability to sense transmembrane voltage is a central feature of many membrane proteins, most notably voltage-gated ion channels. Gating current measurements provide valuable information on protein conformational changes induced by voltage. The recent observation that muscarinic G-protein-coupled receptors (GPCRs) generate gating currents confirms their intrinsic capacity to sense the membrane electrical field. Here, we studied the effect of voltage on agonist activation of M2 muscarinic receptors (M2R) in atrial myocytes and how agonist binding alters M2R gating currents. Membrane depolarization decreased the potency of acetylcholine (ACh), but increased the potency and efficacy of pilocarpine (Pilo), as measured by ACh-activated K+ current, IKACh. Voltage-induced conformational changes in M2R were modified in a ligand-selective manner: ACh reduced gating charge displacement while Pilo increased the amount of charge displaced. Thus, these ligands manifest opposite voltage-dependent IKACh modulation and exert opposite effects on M2R gating charge displacement. Finally, mutations in the putative ligand binding site perturbed the movement of the M2R voltage sensor. Our data suggest that changes in voltage induce conformational changes in the ligand binding site that alter the agonist–receptor interaction in a ligand-dependent manner. Voltage-dependent GPCR modulation has important implications for cellular signalling in excitable tissues. Gating current measurement allows for the tracking of subtle conformational changes in the receptor that accompany agonist binding and changes in membrane voltage. PMID:21282291

Modifying Expression Modes of Human Neurotensin Receptor Type 1 Alters Sensing Capabilities for Agonists in Yeast Signaling Biosensor.

PubMed

Hashi, Hiroki; Nakamura, Yasuyuki; Ishii, Jun; Kondo, Akihiko

2018-04-01

Neurotensin receptor type 1 (NTSR1), a member of the G-protein-coupled receptor (GPCR) family, is naturally activated by binding of a neurotensin peptide, leading to a variety of physiological effects. The budding yeast Saccharomyces cerevisiae is a proven host organism for assaying the agonistic activation of human GPCRs. Previous studies showed that yeast cells can functionally express human NTSR1 receptor, permitting the detection of neurotensin-promoted signaling using a ZsGreen fluorescent reporter gene. However, the fluorescence intensity (sensitivity) of NTSR1-expressing yeast cells is low compared to that of yeast cells expressing other human GPCRs (e.g., human somatostatin receptors). The present study sought to increase the sensitivity of the NTSR1-expressing yeast for use as a fluorescent biosensor, including modification of the expression of human NTSR1 in yeast. Changes in the transcription, translation, and transport of the receptor are attempted by altering the promoter, consensus Kozak-like sequence, and secretion signal sequences of the NTSR1-encoding gene. The resulting yeast cells exhibited increased sensitivity to exogenously added peptide. The cells are further engineered by using cell-surface display technology to ensure that the agonistic peptides are secreted and tethered to the yeast cell wall, yielding cells with enhanced NTSR1 activation. This yeast biosensor holds promise for the identification of agonists to treat NTSR1-related diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The two-state dimer receptor model: a general model for receptor dimers.

PubMed

Franco, Rafael; Casadó, Vicent; Mallol, Josefa; Ferrada, Carla; Ferré, Sergi; Fuxe, Kjell; Cortés, Antoni; Ciruela, Francisco; Lluis, Carmen; Canela, Enric I

2006-06-01

Nonlinear Scatchard plots are often found for agonist binding to G-protein-coupled receptors. Because there is clear evidence of receptor dimerization, these nonlinear Scatchard plots can reflect cooperativity on agonist binding to the two binding sites in the dimer. According to this, the "two-state dimer receptor model" has been recently derived. In this article, the performance of the model has been analyzed in fitting data of agonist binding to A(1) adenosine receptors, which are an example of receptor displaying concave downward Scatchard plots. Analysis of agonist/antagonist competition data for dopamine D(1) receptors using the two-state dimer receptor model has also been performed. Although fitting to the two-state dimer receptor model was similar to the fitting to the "two-independent-site receptor model", the former is simpler, and a discrimination test selects the two-state dimer receptor model as the best. This model was also very robust in fitting data of estrogen binding to the estrogen receptor, for which Scatchard plots are concave upward. On the one hand, the model would predict the already demonstrated existence of estrogen receptor dimers. On the other hand, the model would predict that concave upward Scatchard plots reflect positive cooperativity, which can be neither predicted nor explained by assuming the existence of two different affinity states. In summary, the two-state dimer receptor model is good for fitting data of binding to dimeric receptors displaying either linear, concave upward, or concave downward Scatchard plots.

Characterization of 12 GnRH peptide agonists - a kinetic perspective.

PubMed

Nederpelt, Indira; Georgi, Victoria; Schiele, Felix; Nowak-Reppel, Katrin; Fernández-Montalván, Amaury E; IJzerman, Adriaan P; Heitman, Laura H

2016-01-01

Drug-target residence time is an important, yet often overlooked, parameter in drug discovery. Multiple studies have proposed an increased residence time to be beneficial for improved drug efficacy and/or longer duration of action. Currently, there are many drugs on the market targeting the gonadotropin-releasing hormone (GnRH) receptor for the treatment of hormone-dependent diseases. Surprisingly, the kinetic receptor-binding parameters of these analogues have not yet been reported. Therefore, this project focused on determining the receptor-binding kinetics of 12 GnRH peptide agonists, including many marketed drugs. A novel radioligand-binding competition association assay was developed and optimized for the human GnRH receptor with the use of a radiolabelled peptide agonist, [(125) I]-triptorelin. In addition to radioligand-binding studies, a homogeneous time-resolved FRET Tag-lite™ method was developed as an alternative assay for the same purpose. Two novel competition association assays were successfully developed and applied to determine the kinetic receptor-binding characteristics of 12 high-affinity GnRH peptide agonists. Results obtained from both methods were highly correlated. Interestingly, the binding kinetics of the peptide agonists were more divergent than their affinities with residence times ranging from 5.6 min (goserelin) to 125 min (deslorelin). Our research provides new insights by incorporating kinetic, next to equilibrium, binding parameters in current research and development that can potentially improve future drug discovery targeting the GnRH receptor. © 2015 The British Pharmacological Society.

Characterization of 12 GnRH peptide agonists – a kinetic perspective

PubMed Central

Nederpelt, Indira; Georgi, Victoria; Schiele, Felix; Nowak‐Reppel, Katrin; Fernández‐Montalván, Amaury E.; IJzerman, Adriaan P.

2015-01-01

Background and Purpose Drug‐target residence time is an important, yet often overlooked, parameter in drug discovery. Multiple studies have proposed an increased residence time to be beneficial for improved drug efficacy and/or longer duration of action. Currently, there are many drugs on the market targeting the gonadotropin‐releasing hormone (GnRH) receptor for the treatment of hormone‐dependent diseases. Surprisingly, the kinetic receptor‐binding parameters of these analogues have not yet been reported. Therefore, this project focused on determining the receptor‐binding kinetics of 12 GnRH peptide agonists, including many marketed drugs. Experimental Approach A novel radioligand‐binding competition association assay was developed and optimized for the human GnRH receptor with the use of a radiolabelled peptide agonist, [125I]‐triptorelin. In addition to radioligand‐binding studies, a homogeneous time‐resolved FRET Tag‐lite™ method was developed as an alternative assay for the same purpose. Key Results Two novel competition association assays were successfully developed and applied to determine the kinetic receptor‐binding characteristics of 12 high‐affinity GnRH peptide agonists. Results obtained from both methods were highly correlated. Interestingly, the binding kinetics of the peptide agonists were more divergent than their affinities with residence times ranging from 5.6 min (goserelin) to 125 min (deslorelin). Conclusions and Implications Our research provides new insights by incorporating kinetic, next to equilibrium, binding parameters in current research and development that can potentially improve future drug discovery targeting the GnRH receptor. PMID:26398856

An elevation in physical coupling of type 1 inositol 1,4,5-trisphosphate (IP3) receptors to transient receptor potential 3 (TRPC3) channels constricts mesenteric arteries in genetic hypertension.

PubMed

Adebiyi, Adebowale; Thomas-Gatewood, Candice M; Leo, M Dennis; Kidd, Michael W; Neeb, Zachary P; Jaggar, Jonathan H

2012-11-01

Hypertension is associated with an elevation in agonist-induced vasoconstriction, but mechanisms involved require further investigation. Many vasoconstrictors bind to phospholipase C-coupled receptors, leading to an elevation in inositol 1,4,5-trisphosphate (IP(3)) that activates sarcoplasmic reticulum IP(3) receptors. In cerebral artery myocytes, IP(3) receptors release sarcoplasmic reticulum Ca(2+) and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP(3) receptors control vascular contractility in systemic arteries and IP(3)R involvement in elevated agonist-induced vasoconstriction during hypertension. Total and plasma membrane-localized TRPC3 protein was ≈2.7- and 2-fold higher in mesenteric arteries of spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP(3)R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-SHRs and WKY rats. Control, IP(3)-induced and endothelin-1 (ET-1)-induced fluorescence resonance energy transfer between IP3R1 and TRPC3 was higher in SHR than WKY myocytes. IP3-induced cation current was ≈3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and calmodulin and IP(3) receptor binding domain peptide, an IP(3)R-TRP physical coupling inhibitor, reduced IP(3)-induced cation current and ET-1-induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a sarcoplasmic reticulum Ca(2+)-ATPase blocker, did not alter ET-1-stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP(3)R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP(3)R1 to TRPC3 channel molecular coupling augments ET-1-induced vasoconstriction during hypertension.

[18F]F15599, a novel 5-HT1A receptor agonist, as a radioligand for PET neuroimaging.

PubMed

Lemoine, Laëtitia; Verdurand, Mathieu; Vacher, Bernard; Blanc, Elodie; Le Bars, Didier; Newman-Tancredi, Adrian; Zimmer, Luc

2010-03-01

The serotonin-1A (5-HT(1A)) receptor is implicated in the pathophysiology of major neuropsychiatric disorders. Thus, the functional imaging of 5-HT(1A) receptors by positron emission tomography (PET) may contribute to the understanding of its role in those pathologies and their therapeutics. These receptors exist in high- and low-affinity states and it is proposed that agonists bind preferentially to the high-affinity state of the receptor and therefore could provide a measure of the functional 5-HT(1A) receptors. Since all clinical PET 5-HT(1A) radiopharmaceuticals are antagonists, it is of great interest to develop a( 18)F labelled agonist. F15599 (3-chloro-4-fluorophenyl-(4-fluoro-4{[(5-methyl-pyrimidin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl)-methanone) is a novel ligand with high affinity and selectivity for 5-HT(1A) receptors and is currently tested as an antidepressant. In pharmacological tests in rat, it exhibits preferential agonist activity at post-synaptic 5-HT(1A) receptors in cortical brain regions. Here, its nitro-precursor was synthesised and radiolabelled via a fluoronucleophilic substitution. Radiopharmacological evaluations included in vitro and ex vivo autoradiography in rat brain and PET scans on rats and cats. Results were compared with simultaneous studies using [(18)F]MPPF, a validated 5-HT(1A) antagonist radiopharmaceutical. The chemical and radiochemical purities of [(18)F]F15599 were >98%. In vitro [(18)F]F15599 binding was consistent with the known 5-HT(1A) receptors distribution (hippocampus, dorsal raphe nucleus, and notably cortical areas) and addition of Gpp(NH)p inhibited [(18)F]F15599 binding, consistent with a specific binding to G protein-coupled receptors. In vitro binding of [(18)F]F15599 was blocked by WAY100635 and 8-OH-DPAT, respectively, prototypical 5-HT(1A) antagonist and agonist. The ex vivo and in vivo studies demonstrated that the radiotracer readily entered the rat and the cat brain and generated few brain radioactive metabolites. Remarkably, in microPET studies, [(18)F]F15599 notably displayed a pattern of brain labelling that did not correlate with in vitro observations. Thus, in cat, the highest binding was observed in dorsal raphe and cingulate cortex with little binding in other cortical regions and none in hippocampus. In vivo binding was abolished by WAY100635, indicating specific labelling of 5-HT(1A) receptors. [(18)F]F15599 is a radiofluorinated agonist presenting interesting characteristics for probing in vitro and in vivo the high-affinity states of the 5-HT(1A) receptors. Its differential labelling of 5-HT(1A) receptors in vitro and in vivo may result from its reported preferential interaction with receptors coupled to specific G-protein subtypes.

A constitutively active G protein-coupled acetylcholine receptor regulates motility of larval Schistosoma mansoni.

PubMed

MacDonald, Kevin; Kimber, Michael J; Day, Tim A; Ribeiro, Paula

2015-07-01

The neuromuscular system of helminths controls a variety of essential biological processes and therefore represents a good source of novel drug targets. The neuroactive substance, acetylcholine controls movement of Schistosoma mansoni but the mode of action is poorly understood. Here, we present first evidence of a functional G protein-coupled acetylcholine receptor in S. mansoni, which we have named SmGAR. A bioinformatics analysis indicated that SmGAR belongs to a clade of invertebrate GAR-like receptors and is related to vertebrate muscarinic acetylcholine receptors. Functional expression studies in yeast showed that SmGAR is constitutively active but can be further activated by acetylcholine and, to a lesser extent, the cholinergic agonist, carbachol. Anti-cholinergic drugs, atropine and promethazine, were found to have inverse agonist activity towards SmGAR, causing a significant decrease in the receptor's basal activity. An RNAi phenotypic assay revealed that suppression of SmGAR activity in early-stage larval schistosomulae leads to a drastic reduction in larval motility. In sum, our results provide the first molecular evidence that cholinergic GAR-like receptors are present in schistosomes and are required for proper motor control in the larvae. The results further identify SmGAR as a possible candidate for antiparasitic drug targeting. Copyright © 2015 Elsevier B.V. All rights reserved.

Targeting human Mas-related G protein-coupled receptor X1 to inhibit persistent pain

PubMed Central

Li, Zhe; Tseng, Pang-Yen; Tiwari, Vinod; Xu, Qian; He, Shao-Qiu; Wang, Yan; Zheng, Qin; Han, Liang; Wu, Zhiping; Blobaum, Anna L.; Cui, Yiyuan; Tiwari, Vineeta; Sun, Shuohao; Cheng, Yingying; Huang-Lionnet, Julie H. Y.; Geng, Yixun; Xiao, Bo; Peng, Junmin; Hopkins, Corey; Raja, Srinivasa N.; Guan, Yun; Dong, Xinzhong

2017-01-01

Human Mas-related G protein-coupled receptor X1 (MRGPRX1) is a promising target for pain inhibition, mainly because of its restricted expression in nociceptors within the peripheral nervous system. However, constrained by species differences across Mrgprs, drug candidates that activate MRGPRX1 do not activate rodent receptors, leaving no responsive animal model to test the effect on pain in vivo. Here, we generated a transgenic mouse line in which we replaced mouse Mrgprs with human MrgprX1. This humanized mouse allowed us to characterize an agonist [bovine adrenal medulla 8–22 (BAM8–22)] and a positive allosteric modulator (PAM), ML382, of MRGPRX1. Cellular studies suggested that ML382 enhances the ability of BAM8–22 to inhibit high-voltage-activated Ca2+ channels and attenuate spinal nociceptive transmission. Importantly, both BAM8–22 and ML382 effectively attenuated evoked, persistent, and spontaneous pain without causing obvious side effects. Notably, ML382 by itself attenuated both evoked pain hypersensitivity and spontaneous pain in MrgprX1 mice after nerve injury without acquiring coadministration of an exogenous agonist. Our findings suggest that humanized MrgprX1 mice provide a promising preclinical model and that activating MRGPRX1 is an effective way to treat persistent pain. PMID:28223516

Activation of muscle nicotinic acetylcholine receptor channels by nicotinic and muscarinic agonists

PubMed Central

Akk, Gustav; Auerbach, Anthony

1999-01-01

The dose-response parameters of recombinant mouse adult neuromuscular acetylcholine receptor channels (nAChR) activated by carbamylcholine, nicotine, muscarine and oxotremorine were measured. Rate constants for agonist association and dissociation, and channel opening and closing, were estimated from single-channel kinetic analysis.The dissociation equilibrium constants were (mM): ACh (0.16)carbamylcholine (5.1)>oxotremorine M (0.6)>nicotine (0.5)>muscarine (0.15).Rat neuronal α4β2 nAChR can be activated by all of the agonists. However, detailed kinetic analysis was impossible because the recordings lacked clusters representing the activity of a single receptor complex. Thus, the number of channels in the patch was unknown and the activation rate constants could not be determined.Considering both receptor affinity and agonist efficacy, muscarine and oxotremorine are significant agonists of muscle-type nAChR. The results are discussed in terms of structure-function relationships at the nAChR transmitter binding site. PMID:10602325

Cyclic AMP agonist inhibition increases at low levels of histamine release from human basophils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tung, R.S.; Lichtenstein, L.M.

1981-09-01

The relationship between the intensity of the signal for antigen-induced immunoglobulin E-mediated histamine release from human basophils and the concentration of agonist needed to inhibit release has been determined. The agonists, prostaglandin E1, dimaprit, fenoterol, isobutylmethylxanthine and dibutyryl cyclic AMP, all act by increasing the cyclic AMP level. Each agonist was 10- to 1000-fold more potent (relative ID50) at low levels of histamine release (5-10% of total histamine) than at high levels (50-80%). Thus, the inhibitory potential of a drug is a function of the concentration of antigen used to initiate the response. Our results are now more in accordmore » with the inhibitory profile of these drugs in human lung tissue. It is suggested that in vivo release is likely to be low and that this is the level at which to evaluate drugs in vitro.« less

A DFT approach to discriminate the antagonist and partial agonist activity of ligands binding to the NMDA receptor

NASA Astrophysics Data System (ADS)

Haslak, Zeynep Pinar; Bozkurt, Esra; Dutagaci, Bercem; De Proft, Frank; Aviyente, Viktorya; De Vleeschouwer, Freija

2018-02-01

The activation of N-methyl-D-aspartate receptors is found to be intimately associated with neurodegenerative diseases which make them promising therapeutic targets. Despite the significantly increasing multidisciplinary interests centred on this ionotropic channel, design of new ligands with intended functional activity remains a great challenge. In this article, a computational study based on density functional theory is presented to understand the structural factors of ligands determining their function as antagonists and partial agonists. With this aim, the GluN1 subunit is chosen as being one of the essential components in the activation mechanism, and quantum chemical calculations are implemented for 30 antagonists and 30 partial agonists known to bind to this subunit with different binding affinities. Several quantum chemical descriptors are investigated which might unlock the difference between antagonists and partial agonists.

Synthesis, biological evaluation and molecular modeling of 2-amino-2-phenylethanol derivatives as novel β2-adrenoceptor agonists.

PubMed

Ge, Xinyue; Mo, Yongmei; Xing, Gang; Ji, Lei; Zhao, Haiyan; Chen, Jianfang; He, Bin; Chen, Xuyao; Xing, Ruijuan; Li, Xiaoqiang; Zhao, Ying; Li, Jinyan; Yan, Haining; Woo, Anthony Yiu-Ho; Zhang, Yuyang; Lin, Bin; Pan, Li; Cheng, Maosheng

2018-04-26

A novel series of 2-amino-2-phenylethanol derivatives were developed as β 2 -adrenoceptor agonists. Among them, 2-amino-3-fluoro-5-(2-hydroxy-1-(isopropylamino)ethyl)benzonitrile (compound 2f) exhibited the highest activity (EC 50 = 0.25 nM) in stimulating β 2 -adrenoceptor-mediated cellular cAMP production with a 763.6-fold selectivity over the β 1 -adrenoceptor. The (S)-isomer of 2f was subsequently found to be 8.5-fold more active than the (R)-isomer. Molecular docking was performed to determine the putative binding modes of this new class of β 2 -adrenoceptor agonists. Taken together, these data show that compound 2f is a promising lead compound worthy of further study for the development of β 2 -adrenoceptor agonists. Copyright © 2018 Elsevier Inc. All rights reserved.

Lupinalbin A as the most potent estrogen receptor α- and aryl hydrocarbon receptor agonist in Eriosema laurentii de Wild. (Leguminosae).

PubMed

Ateba, Sylvin Benjamin; Njamen, Dieudonné; Medjakovic, Svjetlana; Zehl, Martin; Kaehlig, Hanspeter; Jungbauer, Alois; Krenn, Liselotte

2014-08-09

Eriosema laurentii De Wild. (Leguminosae) is a plant used in Cameroon against infertility and gynecological or menopausal complaints. In our previous report, a methanol extract of its aerial parts was shown to exhibit estrogenic and aryl hydrocarbon receptor agonistic activities in vitro and to prevent menopausal symptoms in ovariectomized Wistar rats. In order to determine the major estrogen receptor α (ERα) agonists in the extract, an activity-guided fractionation was performed using the ERα yeast screen. To check whether the ERα active fractions/compounds also accounted for the aryl hydrocarbon receptor (AhR) agonistic activity of the crude methanol extract, they were further tested on the AhR yeast screen. This study led to the identification of 2'-hydroxygenistein, lupinalbin A and genistein as major estrogenic principles of the extract. 2'-hydroxygenistein and lupinalbin A were, for the first time, also shown to possess an AhR agonistic activity, whereas genistein was not active in this assay. In addition, it was possible to deduce structure-activity relationships. These results suggest that the identified compounds are the major active principles responsible for the estrogenic and AhR agonistic activities of the crude methanol extract of the aerial parts of Eriosema laurentii.

Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery.

PubMed

Hu, Jianping; Feng, Zhiwei; Ma, Shifan; Zhang, Yu; Tong, Qin; Alqarni, Mohammed Hamed; Gou, Xiaojun; Xie, Xiang-Qun

2016-06-27

Cannabinoid receptor 2 (CB2), a G protein-coupled receptor (GPCR), is a promising target for the treatment of neuropathic pain, osteoporosis, immune system, cancer, and drug abuse. The lack of an experimental three-dimensional CB2 structure has hindered not only the development of studies of conformational differences between the inactive and active CB2 but also the rational discovery of novel functional compounds targeting CB2. In this work, we constructed models of both inactive and active CB2 by homology modeling. Then we conducted two comparative 100 ns molecular dynamics (MD) simulations on the two systems-the active CB2 bound with both the agonist and G protein and the inactive CB2 bound with inverse agonist-to analyze the conformational difference of CB2 proteins and the key residues involved in molecular recognition. Our results showed that the inactive CB2 and the inverse agonist remained stable during the MD simulation. However, during the MD simulations, we observed dynamical details about the breakdown of the "ionic lock" between R131(3.50) and D240(6.30) as well as the outward/inward movements of transmembrane domains of the active CB2 that bind with G proteins and agonist (TM5, TM6, and TM7). All of these results are congruent with the experimental data and recent reports. Moreover, our results indicate that W258(6.48) in TM6 and residues in TM4 (V164(4.56)-L169(4.61)) contribute greatly to the binding of the agonist on the basis of the binding energy decomposition, while residues S180-F183 in extracellular loop 2 (ECL2) may be of importance in recognition of the inverse agonist. Furthermore, pharmacophore modeling and virtual screening were carried out for the inactive and active CB2 models in parallel. Among all 10 hits, two compounds exhibited novel scaffolds and can be used as novel chemical probes for future studies of CB2. Importantly, our studies show that the hits obtained from the inactive CB2 model mainly act as inverse agonist(s) or neutral antagonist(s) at low concentration. Moreover, the hit from the active CB2 model also behaves as a neutral antagonist at low concentration. Our studies provide new insight leading to a better understanding of the structural and conformational differences between two states of CB2 and illuminate the effects of structure on virtual screening and drug design.

Traction in smooth muscle cells varies with cell spreading

NASA Technical Reports Server (NTRS)

Tolic-Norrelykke, Iva Marija; Wang, Ning

2005-01-01

Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell-substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.

Reversal of inhibition of putative dopaminergic neurons of the ventral tegmental area: Interaction of GABAB and D2 receptors

PubMed Central

Nimitvilai, Sudarat; Arora, Devinder S.; McElvain, Maureen A.; Brodie, Mark S.

2012-01-01

Neurons of the ventral tegmental area (VTA) are critical in the rewarding and reinforcing properties of drugs of abuse. Desensitization of VTA neurons to moderate extracellular concentrations of dopamine (DA) is dependent on protein kinase C (PKC) and intracellular calcium levels. This desensitization is called DA inhibition reversal (DIR), as it requires concurrent activation of D2 and D1-like receptors; activation of D2 receptors alone does not result in desensitization. Activation of other G-protein linked receptors can substitute for D1 activation. Like D2 receptors, GABAB receptors in the VTA are coupled to G-protein-linked potassium channels. In the present study, we examined interactions between a GABAB agonist, baclofen, and dopamine agonists, dopamine and quinpirole, to determine whether there was some interaction in the processes of desensitization of GABAB and D2 responses. Long-duration administration of baclofen alone produced reversal of the baclofen-induced inhibition indicative of desensitization, and this desensitization persisted for at least 60 min after baclofen washout. Desensitization to baclofen was dependent on protein kinase C. Dopamine inhibition was reduced for 30 min after baclofen-induced desensitization and conversely, the magnitude of baclofen inhibition was reduced for 30 min by long-duration application of dopamine, but not quinpirole. These results indicate that D2 and GABAB receptors share some protein kinase C-dependent mechanisms of receptor desensitization. PMID:22986166

Proteolytic degradation of regulator of G protein signaling 2 facilitates temporal regulation of Gq/11 signaling and vascular contraction.

PubMed

Kanai, Stanley M; Edwards, Alethia J; Rurik, Joel G; Osei-Owusu, Patrick; Blumer, Kendall J

2017-11-24

Regulator of G protein signaling 2 (RGS2) controls signaling by receptors coupled to the G q/11 class heterotrimeric G proteins. RGS2 deficiency causes several phenotypes in mice and occurs in several diseases, including hypertension in which a proteolytically unstable RGS2 mutant has been reported. However, the mechanisms and functions of RGS2 proteolysis remain poorly understood. Here we addressed these questions by identifying degradation signals in RGS2, and studying dynamic regulation of G q/11 -evoked Ca 2+ signaling and vascular contraction. We identified a novel bipartite degradation signal in the N-terminal domain of RGS2. Mutations disrupting this signal blunted proteolytic degradation downstream of E3 ubiquitin ligase binding to RGS2. Analysis of RGS2 mutants proteolyzed at various rates and the effects of proteasome inhibition indicated that proteolytic degradation controls agonist efficacy by setting RGS2 protein expression levels, and affecting the rate at which cells regain agonist responsiveness as synthesis of RGS2 stops. Analyzing contraction of mesenteric resistance arteries supported the biological relevance of this mechanism. Because RGS2 mRNA expression often is strikingly and transiently up-regulated and then down-regulated upon cell stimulation, our findings indicate that proteolytic degradation tightly couples RGS2 transcription, protein levels, and function. Together these mechanisms provide tight temporal control of G q/11 -coupled receptor signaling in the cardiovascular, immune, and nervous systems. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Persistent aryl hydrocarbon receptor inducers increase with altitude, and estrogen-like disrupters are low in soils of the Alps.

PubMed

Levy, Walkiria; Henkelmann, Bernhard; Bernhöft, Silke; Bovee, Toine; Buegger, Franz; Jakobi, Gert; Kirchner, Manfred; Bassan, Rodolfo; Kräuchi, Norbert; Moche, Wolfgang; Offenthaler, Ivo; Simončič, Primoz; Weiss, Peter; Schramm, Karl-Werner

2011-01-01

Soil samples from remote Alpine areas were analyzed for polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans and polychlorinated biphenyls by high-resolution gas chromatography/high-resolution gas spectrometry. Additionally, the EROD micro-assay and a genetically modified yeast estrogen bioassay were carried out to determine persistent aryl hydrocarbon receptors (AhR) and estrogen receptors (ER) agonists, respectively. Regarding the AhR agonists, the toxicity equivalents of analytical and EROD determined values were compared, targeting both altitude of samples and their soil organic content. The ratio between bioassay derived equivalents and analytical determinations suggested no significant contribution of unknown AhR inducers in these sampling sites and some antagonism in soils with relatively high PCB loading. More CYP1A1 expression was induced at the highest sites or about 1400-1500 m a.s.l. along the altitude profiles. Surprisingly, no clear tendencies with the soil organic content were found for dioxin-like compounds. Mean values obtained in the present study were for ER agonists, 2: 0.37±0.12ng 17ß-estradiol EQ g-1 dry soil [corrected] and 6.1 ± 4.2 pg TCDD-EQ g⁻¹ dry soil for AhR agonists. Low bioassay responses with a higher relative amount of ER disrupters than AhR inducers were detected,indicating the higher abundance of estrogen-like than persistent dioxin-like compounds in these forested areas [corrected].

Lorcaserin, a novel selective human 5-hydroxytryptamine2C agonist: in vitro and in vivo pharmacological characterization.

PubMed

Thomsen, William J; Grottick, Andrew J; Menzaghi, Frederique; Reyes-Saldana, Hazel; Espitia, Stephen; Yuskin, Diane; Whelan, Kevin; Martin, Michael; Morgan, Michael; Chen, Weichao; Al-Shamma, Hussien; Smith, Brian; Chalmers, Derek; Behan, Dominic

2008-05-01

5-Hydroxytryptamine (5-HT)(2C) receptor agonists hold promise for the treatment of obesity. In this study, we describe the in vitro and in vivo characteristics of lorcaserin [(1R)-8-chloro-2,3,4,5-tetrahydro-1-methyl-1H-3 benzazepine], a selective, high affinity 5-HT(2C) full agonist. Lorcaserin bound to human and rat 5-HT(2C) receptors with high affinity (K(i) = 15 +/- 1 nM, 29 +/- 7 nM, respectively), and it was a full agonist for the human 5-HT(2C) receptor in a functional inositol phosphate accumulation assay, with 18- and 104-fold selectivity over 5-HT(2A) and 5-HT(2B) receptors, respectively. Lorcaserin was also highly selective for human 5-HT(2C) over other human 5-HT receptors (5-HT(1A), 5-HT(3), 5-HT(4C), 5-HT5(5A), 5-HT(6), and 5-HT(7)), in addition to a panel of 67 other G protein-coupled receptors and ion channels. Lorcaserin did not compete for binding of ligands to serotonin, dopamine, and norepinephrine transporters, and it did not alter their function in vitro. Behavioral observations indicated that unlike the 5-HT(2A) agonist (+/-)-1-(2,5-dimethoxy-4-phenyl)-2-aminopropane, lorcaserin did not induce behavioral changes indicative of functional 5-HT(2A) agonist activity. Acutely, lorcaserin reduced food intake in rats, an effect that was reversed by pretreatment with the 5-HT(2C)-selective antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl-carbamoyl]indoline (SB242,084) but not the 5-HT(2A) antagonist (R)-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol (MDL 100,907), demonstrating mediation by the 5-HT(2C) receptor. Chronic daily treatment with lorcaserin to rats maintained on a high fat diet produced dose-dependent reductions in food intake and body weight gain that were maintained during the 4-week study. Upon discontinuation, body weight returned to control levels. These data demonstrate lorcaserin to be a potent, selective, and efficacious agonist of the 5-HT(2C) receptor, with potential for the treatment of obesity.

Comparative Analysis of the Cardioprotective Properties of Opioid Receptor Agonists in a Rat Model of Myocardial Infarction

PubMed Central

Maslov, Leonid N.; Lishmanov, Yury B.; Oeltgen, Peter R.; Barzakh, Eva I.; Krylatov, Andrey V.; Naryzhnaya, Natalia V.; Pei, Jian-Ming; Brown, Stephen A.

2010-01-01

Objectives This study was conducted to test the hypothesis that opioid receptor (OR) mediated cardioprotection is agonist-specific when administered prior to coronary artery occlusion and reperfusion in a rat model. Methods Anesthetized open-chest male Wistar rats were subjected to 45 minutes of left coronary artery occlusion and 2 hours of reperfusion. Opioid agonists were infused 15 minutes prior to coronary artery occlusion. Two control groups and 15 opioid treated groups were studied. Controls were infused with either saline alone (n = 16) or dimethyl sulfoxide (DMSO) plus hydroxypropyl-β-cyclodextrin in saline (n = 19). The μ selective agonist DAMGO was infused at either 150 nmol/kg (n = 15) or 1500 nmol/kg (n = 14), and Dermorphin-H was infused at 150 nmol/kg (n = 14). The δ1 selective agonist D-Pen2,5 Enkephalin (DPDPE) was infused at 150 nmol/kg (n = 16) or 1500 nmol/kg (n = 14). The δ2 selective agonists Deltorphin II (n = 16), Deltorphin-Dvariant (n = 15) and Deltorphin-E (n = 14) were infused at 150 nmol/kg. The selective κ1 opioid agonist U-50488 was infused at 240 nmol/kg (n = 14), 1500 nmol/kg (n = 14), and 2,400 nmol/kg (n = 14). The selective κ2 opioid agonist GR-89696 was infused at 150 nmol/kg (n = 14) and 1500 nmol/kg (n = 15). Orphinan FQ (Nociceptin), also referred to as OR Ligand1 (ORL1), was infused at 220 nmol/kg (n = 15) and 1500 nmol/kg (n = 15). The infarct size/area at risk (IS/AAR) ratio was determined after reperfusion by negative staining with patent blue violet dye. Hemodynamic parameters including heart rate, mean arterial blood pressure (MAP), and rate pressure product (RPP) were determined. Results Pretreatment with the δ2 OR agonist Deltorphin II (150 nmol/kg) significantly reduced the IS/AAR ratio, while Deltorphin-Dvariant and Deltorphin-E did not exhibit an infarct sparing effect at that treatment dose. Activation of δ1 OR by DPDPE, κ1 OR by U-50488, κ2 OR by U-50488, μ OR by DAMGO, Dermophin-H, and Nociceptin had no effect on the IS/AAR ratio. U-50488 at 2,400 nmol/l induced a bradycardic effect. All other opioids had no effect on hemodynamic parameters at the doses tested. Conclusions Peripheral δ2 OR activation by Deltorphin II induces infarct size reduction in this animal model. Agonists of μ, δ1, κ1, κ2, and Nociceptin receptors at the doses tested did not induce cardiac tolerance to ischemia/reperfusion injury in vivo. PMID:21175523

Flow-injection chemiluminescence method to detect a β2 adrenergic agonist.

PubMed

Zhang, Guangbin; Tang, Yuhai; Shang, Jian; Wang, Zhongcheng; Yu, Hua; Du, Wei; Fu, Qiang

2015-02-01

A new method for the detection of β2 adrenergic agonists was developed based on the chemiluminescence (CL) reaction of β2 adrenergic agonist with potassium ferricyanide-luminol CL. The effect of β2 adrenergic agonists including isoprenaline hydrochloride, salbutamol sulfate, terbutaline sulfate and ractopamine on the CL intensity of potassium ferricyanide-luminol was discovered. Detection of the β2 adrenergic agonist was carried out in a flow system. Using uniform design experimentation, the influence factors of CL were optimized. The optimal experimental conditions were 1 mmol/L of potassium ferricyanide, 10 µmol/L of luminol, 1.2 mmol/L of sodium hydroxide, a flow speed of 2.6 mL/min and a distance of 1.2 cm from 'Y2 ' to the flow cell. The linear ranges and limit of detection were 10-100 and 5 ng/mL for isoprenaline hydrochloride, 20-100 and 5 ng/mL for salbutamol sulfate, 8-200 and 1 ng/mL for terbutaline sulfate, 20-100 and 4 ng/mL for ractopamine, respectively. The proposed method allowed 200 injections/h with excellent repeatability and precision. It was successfully applied to the determination of three β2 adrenergic agonists in commercial pharmaceutical formulations with recoveries in the range of 96.8-98.5%. The possible CL reaction mechanism of potassium ferricyanide-luminol-β2 adrenergic agonist was discussed from the UV/vis spectra. Copyright © 2014 John Wiley & Sons, Ltd.

Gastrin-releasing peptide receptor-induced internalization, down-regulation, desensitization, and growth: possible role for cyclic AMP.

PubMed

Benya, R V; Fathi, Z; Kusui, T; Pradhan, T; Battey, J F; Jensen, R T

1994-08-01

Stimulation of the gastrin-releasing peptide receptor (GRP-R) in Swiss 3T3 cells resembles that of a number of other recently described G protein-coupled receptors, insofar as both the phospholipase C and adenylyl cyclase signal transduction pathways are activated. GRP-R activation induces numerous alterations in both the cell and the receptor, but because two signal transduction pathways are activated it is difficult to determine the specific contributions of either pathway. We have found that BALB/3T3 fibroblasts transfected with the coding sequence for the GRP-R are pharmacologically indistinguishable from native receptor-expressing cells and activate phospholipase C in a manner similar to that of the native receptor but fail to increase cAMP in response to bombesin; thus, they may be useful cells to explore the role of activation of each pathway in altering cell and receptor function. Swiss 3T3 cells and GRP-R-transfected BALB/3T3 cells expressed identically glycosylated receptors that bound various agonists and antagonists similarly. G protein activation, as determined by evaluation of agonist-induced activation of phospholipase C and by analysis of the effect of guanosine-5'-(beta,gamma-imido)triphosphate on GRP-R binding affinity, was indistinguishable. Agonist stimulation of GRP-R caused similar receptor changes (internalization and down-regulation) and homologous desensitization in both cell types. Bombesin stimulation of Swiss 3T3 cells that had been preincubated with forskolin increased cAMP levels 9-fold, but no bombesin-specific increase in cAMP levels was detected in transfected cells, even though forskolin and cholera toxin increased cAMP levels in these cells. Quiescent Swiss 3T3 cells treated with bombesin rapidly increased c-fos mRNA levels and [3H]thymidine incorporation, whereas both effects were potentiated by forskolin. The specific protein kinase A inhibitor H-89 blocked increases in c-fos levels and [3H]thymidine incorporation induced by low concentrations of bombesin. GRP-R-transfected BALB/3T3 cells increased c-fos mRNA levels and [3H]thymidine incorporation with the addition of serum but not bombesin. These data suggest that bombesin-stimulated increases in cellular levels of cAMP appear not to be an important mediator of GRP-R internalization, down-regulation, or desensitization but do play an important role in bombesin-induced mitogenesis.

Estrogenic and serotonergic butenolides from the leaves of Piper hispidum Swingle (Piperaceae)

PubMed Central

Michel, Joanna L; Chen, Yegao; Zhang, Hongjie; Huang, Yue; Krunic, Alecjev; Orjala, Jimmy; Veliz, Mario; Soni, Kapil K.; Soejarto, Djaja Doel; Caceres, Armando; Perez, Alice; Mahady, Gail B

2010-01-01

Ethnopharmacological relevance Our previous work has demonstrated that several plants in the Piperaceae family are commonly used by the Q’eqchi Maya of Livingston, Guatemala to treat amenorrhea, dysmenorrhea, and pain. Extracts of Piper hispidum Swingle (Piperaceae), bound to the estrogen (ER) and serotonin (5-HT7) receptors. Aim of the study To investigate the estrogenic and serotonergic activities of P. hispidum extracts in functionalized assays, identify the active chemical constituents in the leaf extract, and test these compounds as agonists or antagonists of ER and 5-HT7. Materials and methods The effects of the P. hispidum leaf extracts were investigated in estrogen reporter gene and endogenous gene assays in MCF-7 cells to determine if the extracts acted as an estrogen agonist or antagonist. In addition, the active compounds were isolated using ER- and 5-HT7 receptor bioassay-guided fractionation. The structures of the purified compounds were identified using high-resolution LC-MS and NMR spectroscopic methods. The ER- and 5-HT7-agonist effects of the purified chemical constituents were tested in a 2ERE-reporter gene assay in MCF-7 cells and in serotonin binding and functionalized assays. Results Three butenolides including one new compound (1) were isolated from the leaves of P. hispidum, and their structures were determined. Compound 1 bound to the serotonin receptor 5-HT7 with IC50 values of 16.1 and 8.3 μM, respectively, and using GTP shift assays, compound 1 was found to be a partial agonist of the 5-HT7 receptor. The P. hispidum leaf extracts, as well as compounds 2 and 3 enhanced the expression of estrogen responsive reporter and endogenous genes in MCF-7 cells, demonstrating estrogen agonist effects. Conclusions Extracts of P. hispidum act as agonists of the ER and 5-HT7 receptors. Compound 1, a new natural product, identified as 9, 10-methylenedioxy-5,6-Z-fadyenolide, was isolated as the 5-HT7 agonist. Compounds 2 and 3 are reported for the first time in P. hispidum, and identified as the estrogen agonists. No inhibition of CYP450 was observed for any of these compounds in concentrations up to 1 μM. These activities are consistent with the Q’eqchi traditional use of the plant for the treatment of disorders associated with the female reproductive cycle. PMID:20304039

Estrogenic and serotonergic butenolides from the leaves of Piper hispidum Swingle (Piperaceae).

PubMed

Michel, Joanna L; Chen, Yegao; Zhang, Hongjie; Huang, Yue; Krunic, Aleksej; Orjala, Jimmy; Veliz, Mario; Soni, Kapil K; Soejarto, Djaja Doel; Caceres, Armando; Perez, Alice; Mahady, Gail B

2010-05-27

Our previous work has demonstrated that several plants in the Piperaceae family are commonly used by the Q'eqchi Maya of Livingston, Guatemala to treat amenorrhea, dysmenorrhea, and pain. Extracts of Piper hispidum Swingle (Piperaceae), bound to the estrogen (ER) and serotonin (5-HT7) receptors. To investigate the estrogenic and serotonergic activities of Piper hispidum extracts in functionalized assays, identify the active chemical constituents in the leaf extract, and test these compounds as agonists or antagonists of ER and 5-HT7. The effects of the Piper hispidum leaf extracts were investigated in estrogen reporter gene and endogenous gene assays in MCF-7 cells to determine if the extracts acted as an estrogen agonist or antagonist. In addition, the active compounds were isolated using ER- and 5-HT7 receptor bioassay-guided fractionation. The structures of the purified compounds were identified using high-resolution LC-MS and NMR spectroscopic methods. The ER- and 5-HT7-agonist effects of the purified chemical constituents were tested in a 2ERE-reporter gene assay in MCF-7 cells and in serotonin binding and functionalized assays. Three butenolides including one new compound (1) were isolated from the leaves of Piper hispidum, and their structures were determined. Compound 1 bound to the serotonin receptor 5-HT(7) with IC(50) values of 16.1 and 8.3 microM, respectively, and using GTP shift assays, Compound 1 was found to be a partial agonist of the 5-HT(7) receptor. The Piper hispidum leaf extracts, as well as Compounds 2 and 3 enhanced the expression of estrogen responsive reporter and endogenous genes in MCF-7 cells, demonstrating estrogen agonist effects. Extracts of Piper hispidum act as agonists of the ER and 5-HT(7) receptors. Compound 1, a new natural product, identified as 9,10-methylenedioxy-5,6-Z-fadyenolide, was isolated as the 5-HT(7) agonist. Compounds 2 and 3 are reported for the first time in Piper hispidum, and identified as the estrogen agonists. No inhibition of CYP450 was observed for any of these compounds in concentrations up to 1 microM. These activities are consistent with the Q'eqchi traditional use of the plant for the treatment of disorders associated with the female reproductive cycle. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Modification of postural response caused by footwear conditions.

PubMed

Maejima, H; Kamoda, C; Takayanagi, K; Hosoda, M; Kobayashi, R; Minematsu, A; Sasaki, H; Matsuda, Y; Tanaka, Y; Matsuo, A; Kanemura, N; Ueda, T; Yoshimura, O

2000-01-01

The purpose of this study was to clarify the effect of changing footwear conditions on postural response against postural perturbation. Twenty-three healthy subjects participated in this study. Postural response was induced by moving a platform forward, hereafter referred to as forward-perturbation of a platform. The center of pressure (COP) from the force plate and the electromyograms (EMG) of the tibialis anterior (TA) and quadriceps femoris (QUAD), which are both agonists of the response, were measured. The effect of plantar material and shape of footwear on postural response was examined as footwear condition. Changing plantar materials had an effect on integrated EMG of the agonists (IEMG) but not on the response pattern. On the other hand, the shape of footwear had an effect on the response pattern but not on IEMG. It was supposed from this result that changes in somatosensory input, caused by coupling of plantar material and shape of footwear, modifies postural response variously.

Tyramine and β-phenylethylamine, from fermented food products, as agonists for the human trace amine-associated receptor 1 (hTAAR1) in the stomach.

PubMed

Ohta, Hiroto; Takebe, Youhei; Murakami, Yuka; Takahama, Yusei; Morimura, Shigeru

2017-05-01

The aromatic amines tyramine and β-phenylethylamine are abundant in fermented foods. Recently, a family of human trace amine-associated receptors (hTAARs) was discovered that responds to these compounds. This study examined the expression of hTAAR genes in five human organs. Among them, the stomach expressed hTAAR1 and hTAAR9. Interestingly, more hTAAR1 was expressed in the pylorus than in the other stomach regions. The CRE-SEAP reporter assay revealed that only hTAAR1 functioned as a G s -coupled receptor in response to tyramine and β-phenylethylamine stimulation. The β-phenylethylamine-mediated hTAAR1 activity could be potentiated using 3-isobutyl-1-methylxanthine. These data suggest that tyramine and β-phenylethylamine in fermented foods act at hTAAR1 as agonists in the pylorus of stomach.

Oxytrex: an oxycodone and ultra-low-dose naltrexone formulation.

PubMed

Webster, Lynn R

2007-08-01

Oxytrex (Pain Therapeutics, Inc.) is an oral opioid that combines a therapeutic amount of oxycodone with an ultra-low dose of the antagonist naltrexone. Animal data indicate that this combination minimizes the development of physical dependence and analgesic tolerance while prolonging analgesia. Oxytrex is in late-stage clinical development by Pain Therapeutics for the treatment of moderate-to-severe chronic pain. To evaluate the safety and efficacy of the oxycodone/naltrexone combination, three clinical studies have been conducted, one in healthy volunteers and the other two in patients with chronic pain. The putative mechanism of ultra-low-dose naltrexone is to prevent an alteration in G-protein coupling by opioid receptors that is associated with opioid tolerance and dependence. Opioid agonists are initially inhibitory but become excitatory through constant opioid receptor activity. The agonist/antagonist combination of Oxytrex may reduce the conversion from an inhibitory to an excitatory receptor, thereby decreasing the development of tolerance and physical dependence.

Structural basis for molecular recognition at serotonin receptors.

PubMed

Wang, Chong; Jiang, Yi; Ma, Jinming; Wu, Huixian; Wacker, Daniel; Katritch, Vsevolod; Han, Gye Won; Liu, Wei; Huang, Xi-Ping; Vardy, Eyal; McCorvy, John D; Gao, Xiang; Zhou, X Edward; Melcher, Karsten; Zhang, Chenghai; Bai, Fang; Yang, Huaiyu; Yang, Linlin; Jiang, Hualiang; Roth, Bryan L; Cherezov, Vadim; Stevens, Raymond C; Xu, H Eric

2013-05-03

Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs.

Development of novel ligands for peptide GPCRs.

PubMed

Moran, Brian M; McKillop, Aine M; O'Harte, Finbarr Pm

2016-12-01

Incretin based glucagon-like peptide-1 receptor (GLP-1R) agonists which target a G-protein coupled receptor (GPCR) are currently used in the treatment of type 2 diabetes. This review focuses on GPCRs from pancreatic β-cells, including GLP-1, glucose-dependent insulinotropic polypeptide (GIP), glucagon, somatostatin, pancreatic polypeptide (PP), cholecystokinin (CCK), peptide YY (PYY), oxyntomodulin (OXM) and ghrelin receptors. In addition, fatty acids GPCRs are thought to have an increasing role in regulating peptide secretions namely short fatty acids GPCR (GPR41, GPR43), medium chain fatty acid GPCR (GPR84), long chain fatty acid GPCR (GPR40, GPR120) and cannabinoid-like GPCR (GPR55, GPR119). Several pre-clinical and clinical trials are currently ongoing in peptide GPCR based therapies, including dual and triple agonist peptides which activate two or more GPCRs simultaneously. Copyright © 2016 Elsevier Ltd. All rights reserved.

The amphiphilic peptide adenoregulin enhances agonist binding to A1-adenosine receptors and [35S]GTP gamma S to brain membranes.

PubMed

Moni, R W; Romero, F S; Daly, J W

1995-08-01

1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog, Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brain membranes. 2. The maximal enhancement of agonist binding, and in parentheses, the concentration of adenoregulin affording maximal enhancement were as follows: 60% (20 microM) for A1-adenosine receptors, 30% (100 microM) for A2a-adenosine receptors, 20% (2 microM) for alpha 2-adrenergic receptors, and 30% (10 microM) for 5HT1A receptors. High affinity agonist binding for A1-, alpha 2-, and 5HT1A-receptors was virtually abolished by GTP gamma S in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elicited by adenoregulin. 3. The effect of adenoregulin on binding of N6-cyclohexyladenosine ([3H]CHA) to A1-receptors was relatively slow and was irreversible. Adenoregulin increased the Bmax value for [3H]CHA binding sites, and the proportion of high affinity states, and slowed the rate of [3H]CHA dissociation. Binding of the A1-selective antagonist, [3H]DPCPX, was maximally enhanced by only 13% at 2 microM adenoregulin. Basal and A1-adenosine receptor-stimulated binding of [35S]GTP gamma S were maximally enhanced 45% and 23%, respectively, by 50 microM adenoregulin. In CHAPS-solubilized membranes from rat cortex, the binding of both [3H]CHA and [3H]DPCPX were enhanced by adenoregulin. Binding of [3H]CHA to membranes from DDT1 MF-2 cells was maximally enhanced 17% at 20 microM adenoregulin. In intact DDT1 MF-2 cells, 20 microM adenoregulin did not potentiate the inhibition of cyclic AMP accumulation mediated via the adenosine A1 receptor. 4. It is proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with guanyl nucleotide-free G-protein.

Constitutive activity of the δ-opioid receptor expressed in C6 glioma cells: identification of non-peptide δ-inverse agonists

PubMed Central

Neilan, Claire L; Akil, Huda; Woods, James H; Traynor, John R

1999-01-01

G-protein coupled receptors can exhibit constitutive activity resulting in the formation of active ternary complexes in the absence of an agonist. In this study we have investigated constitutive activity in C6 glioma cells expressing either the cloned δ-(OP1) receptor (C6δ), or the cloned μ-(OP3) opioid receptor (C6μ).Constitutive activity was measured in the absence of Na+ ions to provide an increased signal. The degree of constitutive activity was defined as the level of [35S]-GTPγS binding that could be inhibited by pre-treatment with pertussis toxin (PTX). In C6δ cells the level of basal [35S]-GTPγS binding was reduced by 51.9±6.1 fmols mg−1 protein, whereas in C6μ and C6 wild-type cells treatment with PTX reduced basal [35S]-GTPγS binding by only 10.0±3.5 and 8.6±3.1 fmols mg−1 protein respectively.The δ-antagonists N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864), 7-benzylidenenaltrexone (BNTX) and naltriben (NTB), in addition to clocinnamox (C-CAM), acted as δ-opioid receptor inverse agonists. Naloxone, buprenorphine, and naltrindole were neutral antagonists. Furthermore, naltrindole blocked the reduction in [35S]-GTPγS binding caused by the inverse agonists. The inverse agonists did not inhibit basal [35S]-GTPγS binding in C6μ or C6 wild-type cell membranes.Competition binding assays in C6δ cell membranes revealed a leftward shift in the displacement curve of [3H]-naltrindole by ICI 174,864 and C-CAM in the presence of NaCl and the GTP analogue, GppNHp. There was no change in the displacement curve for BNTX or NTB under these conditions.These data confirm the presence of constitutive activity associated with the δ-opioid receptor and identify three novel, non-peptide, δ-opioid inverse agonists. PMID:10516632

Olodaterol Attenuates Citric Acid-Induced Cough in Naïve and Ovalbumin-Sensitized and Challenged Guinea Pigs

PubMed Central

Wex, Eva; Bouyssou, Thierry

2015-01-01

Excessive coughing is a common feature of airway diseases. Different G-protein coupled receptors, including β2-adrenergic receptors (β2-AR), have been implicated in the molecular mechanisms underlying the cough reflex. However, the potential antitussive property of β2-AR agonists in patients with respiratory disease is a matter of ongoing debate. The aim of our study was to test the efficacy of the long-acting β2-AR agonist olodaterol with regard to its antitussive property in a pre-clinical model of citric acid-induced cough in guinea pigs and to compare the results to different clinically relevant β2-AR agonists. In our study β2-AR agonists were intratracheally administered, as dry powder, into the lungs of naïve or ovalbumin-sensitized guinea pigs 15 minutes prior to induction of cough by exposure to citric acid. Cough events were counted over 15 minutes during the citric acid exposure. Olodaterol dose-dependently inhibited the number of cough events in naïve and even more potently and with a greater maximal efficacy in ovalbumin-sensitized guinea pigs (p < 0.01). Formoterol and salmeterol showed a trend towards reducing cough. On the contrary, indacaterol demonstrated pro-tussive properties as it significantly increased the number of coughs, both in naïve and ovalbumin-sensitized animals (p < 0.001). In conclusion, olodaterol, at doses eliciting bronchodilation, showed antitussive properties in a model of citric acid-induced cough in naïve and ovalbumin-sensitized guinea pigs. This is in agreement with pre-clinical and clinical studies showing antitussive efficacy of β2-AR agonists. Indacaterol increased the number of coughs in this model, which concurs with clinical data where a transient cough has been observed after indacaterol inhalation. While the antitussive properties of β2-AR agonists can be explained by their ability to lead to the cAMP-induced hyperpolarization of the neuron membrane thereby inhibiting sensory nerve activation and the cough reflex, the mechanism underlying the pro-tussive property of indacaterol is not known. PMID:25781609

Regulation of β1- and β3-adrenergic agonist-stimulated lipolytic response in hyperthyroid and hypothyroid rat white adipocytes

PubMed Central

Germack, Renée; Starzec, Anna; Perret, Gérard Y

2000-01-01

This study examined the effects of thyroid status on the lipolytic responses of rat white adipocytes to β-adrenoceptor (β-AR) stimulation. The β1- and β3-AR mRNAs and proteins were measured by Northern and saturation analyses, respectively. Glycerol production and adenyl cyclase (AC) activity induced by various non-selective and selective β1/β3-AR agonists and drugs which act distal to the receptor in the signalling cascade were measured in cells from untreated, tri-iodothyronine (T3)-treated and thyroidectomized rats. The β3-AR density was enhanced (72%) by T3-treatment and reduced (50%) by introduction of a hypothyroid state while β1-AR number remained unaffected. The β1- and β3-AR density was correlated with the specific mRNA level in all thyroid status. The lipolytic responses to isoprenaline, noradrenaline (β1/β3/β3-AR agonists) and BRL 37344 (β3-AR agonist) were potentiated by 48, 58 and 48%, respectively in hyperthyroidism and reduced by about 80% in hypothyroidism. T3-treatment increased the maximal lipolytic response to the partial β3-AR (CGP 12177) and β1-AR (xamoterol) agonists by 234 and 260%, respectively, increasing their efficacy (intrinsic activity: 0.95 versus 0.43 and 1.02 versus 0.42). The maximal AC response to these agonists was increased by 84 and 58%, respectively, without changing their efficacy. In the hypothyroid state, the maximal lipolytic and AC responses were decreased with CGP (0.17±0.03 versus 0.41±0.08 μmol glycerol/106 adipocytes; 0.048±0.005 versus 0.114±0.006 pmol cyclic AMP min−1 mg−1) but not changed with xamoterol. The changes in lipolytic responses to postreceptor-acting agents (forskolin, enprofylline and dibutenyl cyclic AMP, (Bu)2cAMP) suggest the modifications on receptor coupling and phosphodiesterase levels in both thyroid states. Thyroid status affects lipolysis by modifying β3-AR density and postreceptor events without changes in the β1-AR functionality. PMID:10711342

Suppression of atherosclerosis by synthetic REV-ERB agonist

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sitaula, Sadichha; Billon, Cyrielle; Kamenecka, Theodore M.

2015-05-08

The nuclear receptors for heme, REV-ERBα and REV-ERBβ, play important roles in the regulation of metabolism and inflammation. Recently it was demonstrated that reduced REV-ERBα expression in hematopoetic cells in LDL receptor null mice led to increased atherosclerosis. We sought to determine if synthetic REV-ERB agonists that we have developed might have the ability to suppress atherosclerosis in this model. A previously characterized synthetic REV-ERB agonist, SR9009, was used to determine if activation of REV-ERB activity would affect atherosclerosis in LDL receptor deficient mice. Atherosclerotic plaque size was significantly reduced (p < 0.05) in mice administered SR9009 (100 mg/kg) for seven weeks comparedmore » to control mice (n = 10 per group). SR9009 treatment of bone marrow-derived mouse macrophages (BMDM) reduced the polarization of BMDMs to proinflammatory M1 macrophage while increasing the polarization of BMDMs to anti-inflammatory M2 macrophages. Our results suggest that pharmacological targeting of REV-ERBs may be a viable therapeutic option for treatment of atherosclerosis. - Highlights: • Synthetic REV-ERB agonist treatment reduced atherosclerosis in a mouse model. • Pharmacological activation of REV-ERB decreased M1 macrophage polarization. • Pharmacological activation of REV-ERB increased M2 macrophage polarization.« less

In vivo functions of GPR30/GPER-1, a membrane receptor for estrogen: from discovery to functions in vivo.

PubMed

Mizukami, Yoichi

2010-01-01

G protein-coupled receptor 30/G protein-coupled estrogen receptor-1 (GPR30/GPER-1) was reported as a novel membrane receptor for estrogen in 2005. However, the research on GPR30 has produced conflicting reports with regard to its intracellular localization, the tissue distribution of its expression, and some its functions. Recently, in addition to the finding of G-1, a GPR30 agonist, GPR30 KO mice have been produced in laboratories, and this has significantly increased the confidence in the data. In this review, the intrinsic appearance of GPR30 is approached based mainly on data obtained in vivo.

PPARγ partial agonist GQ-16 strongly represses a subset of genes in 3T3-L1 adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Milton, Flora Aparecida; Genomic Medicine, Houston Methodist Research Institute, Houston, TX; Cvoro, Aleksandra

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists that improve insulin resistance but trigger side effects such as weight gain, edema, congestive heart failure and bone loss. GQ-16 is a PPARγ partial agonist that improves glucose tolerance and insulin sensitivity in mouse models of obesity and diabetes without inducing weight gain or edema. It is not clear whether GQ-16 acts as a partial agonist at all PPARγ target genes, or whether it displays gene-selective actions. To determine how GQ-16 influences PPARγ activity on a gene by gene basis, we compared effects of rosiglitazone (Rosi) and GQ-16 in mature 3T3-L1more » adipocytes using microarray and qRT-PCR. Rosi changed expression of 1156 genes in 3T3-L1, but GQ-16 only changed 89 genes. GQ-16 generally showed weak effects upon Rosi induced genes, consistent with partial agonist actions, but a subset of modestly Rosi induced and strongly repressed genes displayed disproportionately strong GQ-16 responses. PPARγ partial agonists MLR24 and SR1664 also exhibit disproportionately strong effects on transcriptional repression. We conclude that GQ-16 displays a continuum of weak partial agonist effects but efficiently represses some negatively regulated PPARγ responsive genes. Strong repressive effects could contribute to physiologic actions of GQ-16. - Highlights: • GQ-16 is an insulin sensitizing PPARγ ligand with reduced harmful side effects. • GQ-16 displays a continuum of weak partial agonist activities at PPARγ-induced genes. • GQ-16 exerts strong repressive effects at a subset of genes. • These inhibitor actions should be evaluated in models of adipose tissue inflammation.« less

Cigarette smoke exposure inhibits contact hypersensitivity via the generation of platelet activating factor agonists

PubMed Central

Sahu, Ravi P.; Petrache, Irina; Van Demark, Mary J; Rashid, Badri M.; Ocana, Jesus A.; Tang, Yuxuan; Yi, Qiaofang; Turner, Matthew J.; Konger, Raymond L.; Travers, Jeffrey B.

2013-01-01

Previous studies have established that pro-oxidative stressors suppress host immunity due to their ability to generate oxidized lipids with PAF-receptor (PAF-R) agonist activity. Although exposure to the pro-oxidative stressor cigarette smoke (CS) is known to exert immunomodulatory effects, little is known regarding the role of platelet-activating factor (PAF) in these events. The current studies sought to determine the role of PAF-R signaling in CS-mediated immunomodulatory effects. We demonstrate that CS exposure induces the generation of a transient PAF-R agonistic activity in the blood of mice. CS exposure inhibits contact hypersensitivity in a PAF-R-dependent manner as PAF-R-deficient mice were resistant to these effects. Blocking PAF-R agonist production either by systemic antioxidants or treatment with serum PAF-acetyl hydrolase enzyme blocked both the CS-mediated generation of PAF-R-agonists and PAF-R dependent inhibition of CHS reactions, indicating a role for oxidized glycerophosphocholines with PAF-R agonistic activity in this process. In addition, cyclooxygenase-2 (COX-2) inhibition did not block PAF-R agonist production but prevented CS-induced inhibition of CHS. This suggests that COX-2 acts downstream of the PAF-R in mediating CS-induced systemic immunosuppression. Moreover, CS-exposure induced a significant increase in the expression of the regulatory T cell reporter gene in FoxP3EGFP mice but not in FoxP3EGFP mice on a PAF-R-deficient background. Finally, Treg depletion via anti-CD25 antibodies blocked CS-mediated inhibition of CHS, indicating the potential involvement of Tregs in CS-mediated systemic immunosuppression. These studies provide the first evidence that the pro-oxidative stressor CS can modulate cutaneous immunity via the generation of PAF-R agonists produced through lipid oxidation. PMID:23355733

Suppression of atherosclerosis by synthetic REV-ERB agonist

PubMed Central

Sitaula, Sadichha; Billon, Cyrielle; Kamenecka, Theodore M.; Solt, Laura A.; Burris, Thomas P.

2015-01-01

The nuclear receptors for heme, REV-ERBα and REV-ERBβ, play important roles in the regulation of metabolism and inflammation. Recently it was demonstrated that reduced REV-ERBα expression in hematopoetic cells in LDL receptor null mice led to increased atherosclerosis. We sought to determine if synthetic REV-ERB agonists that we have developed might have the ability to suppress atherosclerosis in this model. A previously characterized synthetic REV-ERB agonist, SR9009, was used to determine if activation of REV-ERB activity would affect atherosclerosis in LDL receptor deficient mice. Atherosclerotic plaque size was significantly reduced (p < 0.05) in mice administered SR9009 (100 mg/kg) for seven weeks compared to control mice (n = 10 per group). SR9009 treatment of bone marrow-derived mouse macrophages (BMDM) reduced the polarization of BMDMs to proinflammatory M1 macrophage while increasing the polarization of BMDMs to anti-inflammatory M2 macrophages. Our results suggest that pharmacological targeting of REV-ERBs may be a viable therapeutic option for treatment of atherosclerosis. PMID:25800870

Targeting GPR30 in Abiraterone and MDV3100 Resistant Prostate Cancer

DTIC Science & Technology

2016-10-01

environment in vivo. We had previously demonstrated that G-1 inhibited growth in cell culture experiments and a hormone -independent PC-3Published by...coupled receptor 30 (GPR30) is a seven-transmembrane estrogen receptor and activation by its specific agonist G-1 inhibited growth in multiple...significantly inhibited the growth and extended the progression-free survival of patient-derived xenograft models that are sensitive (LuCaP 136CR, P

Comparison of the functional potencies of ropinirole and other dopamine receptor agonists at human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells

PubMed Central

Coldwell, Martyn C; Boyfield, Izzy; Brown, Tony; Hagan, Jim J; Middlemiss, Derek N

1999-01-01

The aim of the present study was to characterize functional responses to ropinirole, its major metabolites in man (SKF-104557 (4-[2-(propylamino)ethyl]-2-(3H) indolone), SKF-97930 (4-carboxy-2-(3H) indolone)) and other dopamine receptor agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) receptors separately expressed in Chinese hamster ovary cells using microphysiometry.All the receptor agonists tested (ropinirole, SKF-104557, SKF-97930, bromocriptine, lisuride, pergolide, pramipexole, talipexole, dopamine) increased extracellular acidification rate in Chinese hamster ovary clones expressing the human D2, D3 or D4 receptor. The pEC50s of ropinirole at hD2, hD3 and hD4 receptors were 7.4, 8.4 and 6.8, respectively. Ropinirole is therefore at least 10 fold selective for the human dopamine D3 receptor over the other D2 receptor family members.At the hD2 and hD3 dopamine receptors all the compounds tested were full agonists as compared to quinpirole. Talipexole and the ropinirole metabolite, SKF-104557, were partial agonists at the hD4 receptor.Bromocriptine and lisuride had a slow onset of agonist action which precluded determination of EC50s.The rank order of agonist potencies was dissimilar to the rank order of radioligand binding affinities at each of the dopamine receptor subtypes. Functional selectivities of the dopamine receptor agonists, as measured in the microphysiometer, were less than radioligand binding selectivities.The results show that ropinirole is a full agonist at human D2, D3 and D4 dopamine receptors. SKF-104557 the major human metabolite of ropinirole, had similar radioligand binding affinities to, but lower functional potencies than, the parent compound. PMID:10455328

Molecular modelling studies on the ORL1-receptor and ORL1-agonists

NASA Astrophysics Data System (ADS)

Bröer, Britta M.; Gurrath, Marion; Höltje, Hans-Dieter

2003-11-01

The ORL1 ( opioid receptor like 1)- receptor is a member of the family of rhodopsin-like G protein-coupled receptors (GPCR) and represents an interesting new therapeutical target since it is involved in a variety of biomedical important processes, such as anxiety, nociception, feeding, and memory. In order to shed light on the molecular basis of the interactions of the GPCR with its ligands, the receptor protein and a dataset of specific agonists were examined using molecular modelling methods. For that purpose, the conformational space of a very potent non-peptide ORL1-receptor agonist (Ro 64-6198) with a small number of rotatable bonds was analysed in order to derive a pharmacophoric arrangement. The conformational analyses yielded a conformation that served as template for the superposition of a set of related analogues. Structural superposition was achieved by employing the program FlexS. Using the experimental binding data and the superposition of the ligands, a 3D-QSAR analysis applying the GRID/GOLPE method was carried out. After the ligand-based modelling approach, a 3D model of the ORL1-receptor has been constructed using homology modelling methods based on the crystal structure of bovine rhodopsin. A representative structure of the model taken from molecular dynamics simulations was used for a manual docking procedure. Asp-130 and Thr-305 within the ORL1-receptor model served as important hydrophilic interaction partners. Furthermore, a hydrophobic cavity was identified stabilizing the agonists within their binding site. The manual docking results were supported using FlexX, which identified the same protein-ligand interaction points.

A combined ligand-based and target-based drug design approach for G-protein coupled receptors: application to salvinorin A, a selective kappa opioid receptor agonist

NASA Astrophysics Data System (ADS)

Singh, Nidhi; Chevé, Gwénaël; Ferguson, David M.; McCurdy, Christopher R.

2006-08-01

Combined ligand-based and target-based drug design approaches provide a synergistic advantage over either method individually. Therefore, we set out to develop a powerful virtual screening model to identify novel molecular scaffolds as potential leads for the human KOP (hKOP) receptor employing a combined approach. Utilizing a set of recently reported derivatives of salvinorin A, a structurally unique KOP receptor agonist, a pharmacophore model was developed that consisted of two hydrogen bond acceptor and three hydrophobic features. The model was cross-validated by randomizing the data using the CatScramble technique. Further validation was carried out using a test set that performed well in classifying active and inactive molecules correctly. Simultaneously, a bovine rhodopsin based "agonist-bound" hKOP receptor model was also generated. The model provided more accurate information about the putative binding site of salvinorin A based ligands. Several protein structure-checking programs were used to validate the model. In addition, this model was in agreement with the mutation experiments carried out on KOP receptor. The predictive ability of the model was evaluated by docking a set of known KOP receptor agonists into the active site of this model. The docked scores correlated reasonably well with experimental p K i values. It is hypothesized that the integration of these two independently generated models would enable a swift and reliable identification of new lead compounds that could reduce time and cost of hit finding within the drug discovery and development process, particularly in the case of GPCRs.

Quercetin acutely relaxes airway smooth muscle and potentiates β-agonist-induced relaxation via dual phosphodiesterase inhibition of PLCβ and PDE4

PubMed Central

Emala, Charles W.

2013-01-01

Asthma is a disease of the airways with symptoms including exaggerated airway narrowing and airway inflammation. Early asthma therapies used methylxanthines to relieve symptoms, in part, by inhibiting cyclic nucleotide phosphodiesterases (PDEs), the enzyme responsible for degrading cAMP. The classification of tissue-specific PDE subtypes and the clinical introduction of PDE-selective inhibitors for chronic obstructive pulmonary disease (i.e., roflumilast) have reopened the possibility of using PDE inhibition in the treatment of asthma. Quercetin is a naturally derived PDE4-selective inhibitor found in fruits, vegetables, and tea. We hypothesized that quercetin relaxes airway smooth muscle via cAMP-mediated pathways and augments β-agonist relaxation. Tracheal rings from male A/J mice were mounted in myographs and contracted with acetylcholine (ACh). Addition of quercetin (100 nM-1 mM) acutely and concentration-dependently relaxed airway rings precontracted with ACh. In separate studies, pretreatment with quercetin (100 μM) prevented force generation upon exposure to ACh. In additional studies, quercetin (50 μM) significantly potentiated isoproterenol-induced relaxations. In in vitro assays, quercetin directly attenuated phospholipase C activity, decreased inositol phosphate synthesis, and decreased intracellular calcium responses to Gq-coupled agonists (histamine or bradykinin). Finally, nebulization of quercetin (100 μM) in an in vivo model of airway responsiveness significantly attenuated methacholine-induced increases in airway resistance. These novel data show that the natural PDE4-selective inhibitor quercetin may provide therapeutic relief of asthma symptoms and decrease reliance on short-acting β-agonists. PMID:23873842

New functional activity of aripiprazole revealed: robust antagonism of D2 dopamine receptor-stimulated Gβγ signaling

PubMed Central

Brust, Tarsis F.; Hayes, Michael P.; Roman, David L.; Watts, Val J.

2014-01-01

The dopamine D2 receptor (DRD2) is a G protein-coupled receptor (GPCR) that is generally considered to be a primary target in the treatment of schizophrenia. First generation antipsychotic drugs (e.g. haloperidol) are antagonists of the DRD2, while second generation antipsychotic drugs (e.g. olanzapine) antagonize DRD2 and 5HT2A receptors. Notably, both these classes of drugs may cause side effects associated with D2 receptor antagonism (e.g. hyperprolactemia and extrapyramidal symptoms). The novel, “third generation” antipsychotic drug, aripiprazole is also used to treat schizophrenia, with the remarkable advantage that its tendency to cause extrapyramidal symptoms is minimal. Aripiprazole is considered a partial agonist of the DRD2, but it also has partial agonist/antagonist activity for other GPCRs. Further, aripiprazole has been reported to have a unique activity profile in functional assays with the DRD2. In the present study the molecular pharmacology of aripiprazole was further examined in HEK cell models stably expressing the DRD2 and specific isoforms of adenylyl cyclase to assess functional responses of Gα and Gβγ subunits. Additional studies examined the activity of aripiprazole in DRD2-mediated heterologous sensitization of adenylyl cyclase and cell-based dynamic mass redistribution (DMR). Aripiprazole displayed a unique functional profile for modulation of G proteins, being a partial agonist for Gαi/o and a robust antagonist for Gβγ signaling. Additionally, aripiprazole was a weak partial agonist for both heterologous sensitization and dynamic mass redistribution. PMID:25449598

PAR-2 agonists induce contraction of murine small intestine through neurokinin receptors.

PubMed

Zhao, Aiping; Shea-Donohue, Terez

2003-10-01

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor and is expressed throughout the gut. It is well known that PAR-2 participates in the regulation of gastrointestinal motility; however, the results are inconsistent. The present study investigated the effect and mechanism of PAR-2 activation on murine small intestinal smooth muscle function in vitro. Both trypsin and PAR-2-activating peptide SLIGRL induced a small relaxation followed by a concentration-dependent contraction. The sensitivity to trypsin was greater than that to SLIGRL (EC50 = 0.03 vs. 40 microM), but maximal responses were similar (12.3 +/- 1.6 vs. 13.7 +/- 1.3 N/cm2). Trypsin-evoked contraction (1 microM) exhibited a rapid desensitization, whereas the desensitization of response to SLIGRL was less even at high concentration (50 microM). Atropine had no effect on PAR-2 agonist-induced contractions. In contrast, TTX and capsaicin significantly attenuated those contractions, implicating a neurogenic mechanism that may involve capsaicin-sensitive sensory nerves. Furthermore, contractions induced by trypsin and SLIGRL were reduced by neurokinin receptor NK1 antagonist SR-140333 or NK2 antagonist SR-48968 alone or were further reduced by combined application of SR-140333 and SR-48968, indicating the involvement of neurokinin receptors. In addition, desensitizing neurokinin receptors with substance P and/or neurokinin A decreased the PAR-2 agonist-evoked contraction. We concluded that PAR-2 agonists induced a contraction of murine intestinal smooth muscle that was mediated by nerves. The excitatory effect is also dependent on sensory neural pathways and requires both NK1 and NK2 receptors.

Cholinergic abnormalities in autism: is there a rationale for selective nicotinic agonist interventions?

PubMed

Deutsch, Stephen I; Urbano, Maria R; Neumann, Serina A; Burket, Jessica A; Katz, Elionora

2010-05-01

The core dysfunctions of autism spectrum disorders, which include autistic disorder, Asperger disorder, and pervasive developmental disorder not otherwise specified, include deficits in socialization and communication and a need for the preservation of "sameness;" intellectual impairment and epilepsy are common comorbidities. Data suggest that pathological involvement of cholinergic nuclei and altered expression of acetylcholine receptors, particularly nicotinic acetylcholine receptors, occur in brain of persons with autistic disorder. However, many of these studies involved postmortem tissue from small samples of primarily adult persons. Thus, the findings may reflect compensatory changes and may relate more closely to intellectual impairment and the confounding effects of seizures and medications, as opposed to the core dysfunctions of autism. Nonetheless, because of the roles played by acetylcholine receptors in general, and nicotinic acetylcholine receptors in particular, in normal processes of attention, cognition, and memory, selective cholinergic interventions should be explored for possible therapeutic effects. Additionally, there are electrophysiological data that complement the clinical observations of frequent comorbid seizure disorders in these patients, suggesting a disturbance in the balance of excitatory and inhibitory tone in the brains of persons with autistic disorders. Conceivably, because the alpha7 nicotinic acetylcholine receptor is located on the surface of gamma-aminobutyric acid inhibitory neurons, selective stimulation of this receptor would promote gamma-aminobutyric acid's release and restore diminished inhibitory tone. The development of agonists and partial agonists for nicotinic acetylcholine receptors and positive allosteric modulators that enhance the efficiency of coupling between the binding of agonist and channel opening should facilitate consideration of clinical trials.

Endothelin

PubMed Central

Hyndman, Kelly A.; Dhaun, Neeraj; Southan, Christopher; Kohan, Donald E.; Pollock, Jennifer S.; Pollock, David M.; Webb, David J.; Maguire, Janet J.

2016-01-01

The endothelins comprise three structurally similar 21-amino acid peptides. Endothelin-1 and -2 activate two G-protein coupled receptors, ETA and ETB, with equal affinity, whereas endothelin-3 has a lower affinity for the ETA subtype. Genes encoding the peptides are present only among vertebrates. The ligand-receptor signaling pathway is a vertebrate innovation and may reflect the evolution of endothelin-1 as the most potent vasoconstrictor in the human cardiovascular system with remarkably long lasting action. Highly selective peptide ETA and ETB antagonists and ETB agonists together with radiolabeled analogs have accurately delineated endothelin pharmacology in humans and animal models, although surprisingly no ETA agonist has been discovered. ET antagonists (bosentan, ambrisentan) have revolutionized the treatment of pulmonary arterial hypertension, with the next generation of antagonists exhibiting improved efficacy (macitentan). Clinical trials continue to explore new applications, particularly in renal failure and for reducing proteinuria in diabetic nephropathy. Translational studies suggest a potential benefit of ETB agonists in chemotherapy and neuroprotection. However, demonstrating clinical efficacy of combined inhibitors of the endothelin converting enzyme and neutral endopeptidase has proved elusive. Over 28 genetic modifications have been made to the ET system in mice through global or cell-specific knockouts, knock ins, or alterations in gene expression of endothelin ligands or their target receptors. These studies have identified key roles for the endothelin isoforms and new therapeutic targets in development, fluid-electrolyte homeostasis, and cardiovascular and neuronal function. For the future, novel pharmacological strategies are emerging via small molecule epigenetic modulators, biologicals such as ETB monoclonal antibodies and the potential of signaling pathway biased agonists and antagonists. PMID:26956245

Efficacy of antipsychotic agents at human 5-HT(1A) receptors determined by [3H]WAY100,635 binding affinity ratios: relationship to efficacy for G-protein activation.

PubMed

Newman-Tancredi, A; Verrièle, L; Touzard, M; Millan, M J

2001-10-05

5-HT(1A) receptors are implicated in the aetiology of schizophrenia. Herein, the influence of 15 antipsychotics on the binding of the selective 'neutral' antagonist, [3H]WAY100,635 ([3H]N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-cyclo-hexanecarboxamide), was examined at human 5-HT(1A) receptors expressed in Chinese Hamster Ovary cells. In competition binding experiments, 5-HT displayed biphasic isotherms which were shifted to the right in the presence of the G-protein uncoupling agent, GTPgammaS (100 microM). In analogy, the isotherms of ziprasidone, quetiapine and S16924 (((R-2-[1-[2-(2,3-dihydro-benzo[1,4]dioxin-5-yloxy)-ethyl]-pyrrolidin-3yl]-1-(4-fluoro-phenyl)-ethanone), were displaced to the right by GTPgammaS, consistent with agonist actions. Binding of several other antipsychotics, such as ocaperidone, olanzapine and risperidone, was little influenced by GTPgammaS. Isotherms of the neuroleptics, haloperidol, chlorpromazine and thioridazine were shifted to the left in the presence of GTPgammaS, suggesting inverse agonist properties. For most ligands, the magnitude of affinity changes induced by GTPgammaS (alteration in pK(i) values) correlated well with their previously determined efficacies in [35S]GTPgammaS binding studies [Eur. J. Pharmacol. 355 (1998) 245]. In contrast, the affinity of the 'atypical' antipsychotic agent, clozapine, which is a known partial agonist at 5-HT(1A) receptors, was less influenced by GTPgammaS. When the ratio of high-/low-affinity values was plotted against efficacy, hyperbolic isotherms were obtained, consistent with a modified ternary complex model which assumes that receptors can adopt active conformations in the absence of agonist. In conclusion, modulation of [3H]-WAY100,635 binding by GTPgammaS differentiated agonist vs. inverse agonist properties of antipsychotics at 5-HT(1A) receptors. These may contribute to differing profiles of antipsychotic activity.

Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor.

PubMed

Bunzow, J R; Sonders, M S; Arttamangkul, S; Harrison, L M; Zhang, G; Quigley, D I; Darland, T; Suchland, K L; Pasumamula, S; Kennedy, J L; Olson, S B; Magenis, R E; Amara, S G; Grandy, D K

2001-12-01

The trace amine para-tyramine is structurally and functionally related to the amphetamines and the biogenic amine neurotransmitters. It is currently thought that the biological activities elicited by trace amines such as p-tyramine and the psychostimulant amphetamines are manifestations of their ability to inhibit the clearance of extracellular transmitter and/or stimulate the efflux of transmitter from intracellular stores. Here we report the discovery and pharmacological characterization of a rat G protein-coupled receptor that stimulates the production of cAMP when exposed to the trace amines p-tyramine, beta-phenethylamine, tryptamine, and octopamine. An extensive pharmacological survey revealed that psychostimulant and hallucinogenic amphetamines, numerous ergoline derivatives, adrenergic ligands, and 3-methylated metabolites of the catecholamine neurotransmitters are also good agonists at the rat trace amine receptor 1 (rTAR1). These results suggest that the trace amines and catecholamine metabolites may serve as the endogenous ligands of a novel intercellular signaling system found widely throughout the vertebrate brain and periphery. Furthermore, the discovery that amphetamines, including 3,4-methylenedioxymethamphetamine (MDMA; "ecstasy"), are potent rTAR1 agonists suggests that the effects of these widely used drugs may be mediated in part by this receptor as well as their previously characterized targets, the neurotransmitter transporter proteins.

Action of Molecular Switches in GPCRs - Theoretical and Experimental Studies

PubMed Central

Trzaskowski, B; Latek, D; Yuan, S; Ghoshdastider, U; Debinski, A; Filipek, S

2012-01-01

G protein coupled receptors (GPCRs), also called 7TM receptors, form a huge superfamily of membrane proteins that, upon activation by extracellular agonists, pass the signal to the cell interior. Ligands can bind either to extracellular N-terminus and loops (e.g. glutamate receptors) or to the binding site within transmembrane helices (Rhodopsin-like family). They are all activated by agonists although a spontaneous auto-activation of an empty receptor can also be observed. Biochemical and crystallographic methods together with molecular dynamics simulations and other theoretical techniques provided models of the receptor activation based on the action of so-called “molecular switches” buried in the receptor structure. They are changed by agonists but also by inverse agonists evoking an ensemble of activation states leading toward different activation pathways. Switches discovered so far include the ionic lock switch, the 3-7 lock switch, the tyrosine toggle switch linked with the nPxxy motif in TM7, and the transmission switch. The latter one was proposed instead of the tryptophan rotamer toggle switch because no change of the rotamer was observed in structures of activated receptors. The global toggle switch suggested earlier consisting of a vertical rigid motion of TM6, seems also to be implausible based on the recent crystal structures of GPCRs with agonists. Theoretical and experimental methods (crystallography, NMR, specific spectroscopic methods like FRET/BRET but also single-molecule-force-spectroscopy) are currently used to study the effect of ligands on the receptor structure, location of stable structural segments/domains of GPCRs, and to answer the still open question on how ligands are binding: either via ensemble of conformational receptor states or rather via induced fit mechanisms. On the other hand the structural investigations of homo- and heterodimers and higher oligomers revealed the mechanism of allosteric signal transmission and receptor activation that could lead to design highly effective and selective allosteric or ago-allosteric drugs. PMID:22300046

Action of molecular switches in GPCRs--theoretical and experimental studies.

PubMed

Trzaskowski, B; Latek, D; Yuan, S; Ghoshdastider, U; Debinski, A; Filipek, S

2012-01-01

G protein coupled receptors (GPCRs), also called 7TM receptors, form a huge superfamily of membrane proteins that, upon activation by extracellular agonists, pass the signal to the cell interior. Ligands can bind either to extracellular N-terminus and loops (e.g. glutamate receptors) or to the binding site within transmembrane helices (Rhodopsin-like family). They are all activated by agonists although a spontaneous auto-activation of an empty receptor can also be observed. Biochemical and crystallographic methods together with molecular dynamics simulations and other theoretical techniques provided models of the receptor activation based on the action of so-called "molecular switches" buried in the receptor structure. They are changed by agonists but also by inverse agonists evoking an ensemble of activation states leading toward different activation pathways. Switches discovered so far include the ionic lock switch, the 3-7 lock switch, the tyrosine toggle switch linked with the nPxxy motif in TM7, and the transmission switch. The latter one was proposed instead of the tryptophan rotamer toggle switch because no change of the rotamer was observed in structures of activated receptors. The global toggle switch suggested earlier consisting of a vertical rigid motion of TM6, seems also to be implausible based on the recent crystal structures of GPCRs with agonists. Theoretical and experimental methods (crystallography, NMR, specific spectroscopic methods like FRET/BRET but also single-molecule-force-spectroscopy) are currently used to study the effect of ligands on the receptor structure, location of stable structural segments/domains of GPCRs, and to answer the still open question on how ligands are binding: either via ensemble of conformational receptor states or rather via induced fit mechanisms. On the other hand the structural investigations of homoand heterodimers and higher oligomers revealed the mechanism of allosteric signal transmission and receptor activation that could lead to design highly effective and selective allosteric or ago-allosteric drugs.

Atypical Signaling and Functional Desensitization Response of MAS Receptor to Peptide Ligands

PubMed Central

Tirupula, Kalyan C.; Desnoyer, Russell; Speth, Robert C.; Karnik, Sadashiva S.

2014-01-01

MAS is a G protein-coupled receptor (GPCR) implicated in multiple physiological processes. Several physiological peptide ligands such as angiotensin-(1–7), angiotensin fragments and neuropeptide FF (NPFF) are reported to act on MAS. Studies of conventional G protein signaling and receptor desensitization upon stimulation of MAS with the peptide ligands are limited so far. Therefore, we systematically analyzed G protein signals activated by the peptide ligands. MAS-selective non-peptide ligands that were previously shown to activate G proteins were used as controls for comparison on a common cell based assay platform. Activation of MAS by the non-peptide agonist (1) increased intracellular calcium and D-myo-inositol-1-phosphate (IP1) levels which are indicative of the activation of classical Gαq-phospholipase C signaling pathways, (2) decreased Gαi mediated cAMP levels and (3) stimulated Gα12-dependent expression of luciferase reporter. In all these assays, MAS exhibited strong constitutive activity that was inhibited by the non-peptide inverse agonist. Further, in the calcium response assay, MAS was resistant to stimulation by a second dose of the non-peptide agonist after the first activation has waned suggesting functional desensitization. In contrast, activation of MAS by the peptide ligand NPFF initiated a rapid rise in intracellular calcium with very weak IP1 accumulation which is unlike classical Gαq-phospholipase C signaling pathway. NPFF only weakly stimulated MAS-mediated activation of Gα12 and Gαi signaling pathways. Furthermore, unlike non-peptide agonist-activated MAS, NPFF-activated MAS could be readily re-stimulated the second time by the agonists. Functional assays with key ligand binding MAS mutants suggest that NPFF and non-peptide ligands bind to overlapping regions. Angiotensin-(1–7) and other angiotensin fragments weakly potentiated an NPFF-like calcium response at non-physiological concentrations (≥100 µM). Overall, our data suggest that peptide ligands induce atypical signaling and functional desensitization of MAS. PMID:25068582

Partial agonist clonidine mediates alpha(2)-AR subtypes specific regulation of cAMP accumulation in adenylyl cyclase II transfected DDT1-MF2 cells.

PubMed

Limon-Boulez, I; Bouet-Alard, R; Gettys, T W; Lanier, S M; Maltier, J P; Legrand, C

2001-02-01

alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR > alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).

THE ROLE OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS IN CARCINOGENESIS AND CHEMOPREVENTION

PubMed Central

Peters, Jeffrey M.; Shah, Yatrik M.; Gonzalez, Frank J.

2012-01-01

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that are involved in regulating glucose and lipid homeostasis, inflammation, proliferation and differentiation. Although all of these functions might contribute to the influence of PPARs in carcinogenesis, there is a distinct need for a balanced review of the literature and additional experimentation to determine the potential for targeting PPARs for cancer therapy and cancer chemoprevention. As PPAR agonists include drugs used for the treatment of metabolic diseases, a more complete understanding of the roles of PPARs in cancer will aid in determining any increased cancer risk for patients undergoing therapy with PPAR agonists. PMID:22318237

Deficient production of IL-1 receptor antagonist and IL-6 coupled to oxidative stress in cryopyrin-associated periodic syndrome monocytes.

PubMed

Carta, Sonia; Tassi, Sara; Delfino, Laura; Omenetti, Alessia; Raffa, Salvatore; Torrisi, Maria Rosaria; Martini, Alberto; Gattorno, Marco; Rubartelli, Anna

2012-09-01

To determine whether dysregulated production of cytokines downstream of interleukin (IL)-1 participates in the pathophysiology of cryopyrin-associated periodic syndromes (CAPS). Primary monocytes from patients with CAPS, unstimulated or after stimulation with lipopolysaccharide (LPS) and other Toll-like receptor (TLR) agonists, were examined for signs of stress and production of IL-1β, IL-1 receptor antagonist (IL-1Ra) and IL-6 in comparison with monocytes from patients with autoimmune diseases and from healthy donors. Unstimulated CAPS monocytes showed mild signs of stress including elevated levels of reactive oxygen species and fragmented mitochondria. Stress signs were worsened by TLR stimulation and eventually led to protein synthesis inhibition with strong impairment of production of cytokines downstream of IL-1, such as IL-1Ra and IL-6. These defects were not detected in monocytes from autoimmune patients and healthy donors. The stress state of LPS-stimulated CAPS monocytes and the consequent inhibition of translation are likely to be responsible for the impaired production of IL-1Ra and IL-6. The deficient secretion of these cytokines coupled with increased IL-1β release explains the severity of the IL-1-related clinical manifestations and the predominant implication of innate immunity in CAPS.

Structural signatures of DRD4 mutants revealed using molecular dynamics simulations: Implications for drug targeting.

PubMed

Jatana, Nidhi; Thukral, Lipi; Latha, N

2016-01-01

Human Dopamine Receptor D4 (DRD4) orchestrates several neurological functions and represents a target for many psychological disorders. Here, we examined two rare variants in DRD4; V194G and R237L, which elicit functional alterations leading to disruption of ligand binding and G protein coupling, respectively. Using atomistic molecular dynamics (MD) simulations, we provide in-depth analysis to reveal structural signatures of wild and mutant complexes with their bound agonist and antagonist ligands. We constructed intra-protein network graphs to discriminate the global conformational changes induced by mutations. The simulations also allowed us to elucidate the local side-chain dynamical variations in ligand-bound mutant receptors. The data suggest that the mutation in transmembrane V (V194G) drastically disrupts the organization of ligand binding site and causes disorder in the native helical arrangement. Interestingly, the R237L mutation leads to significant rewiring of side-chain contacts in the intracellular loop 3 (site of mutation) and also affects the distant transmembrane topology. Additionally, these mutations lead to compact ICL3 region compared to the wild type, indicating that the receptor would be inaccessible for G protein coupling. Our findings thus reveal unreported structural determinants of the mutated DRD4 receptor and provide a robust framework for design of effective novel drugs.

The Mechanism of Interaction of Oximes with the Muscarinic-Cholinergic Complex in the Central Nervous System

DTIC Science & Technology

1983-11-03

ACh binding to the remaining sites. However, the affinity of oxotremorine to the high affinity agonist binding sites was reduced. The relative...when examined in the remaining sites in the washed membranes, were similar to those in control membranes. The affinity of the agonist oxotremorine ... oxotremorine was substituted for atropine. All determinations were carriid out in quadruplicate, each one varying by < 15%. Centrifugation assays

Melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 modified at the para position of the benzyl side chain (DPhe): importance for mouse melanocortin-3 receptor agonist versus antagonist activity.

PubMed

Proneth, Bettina; Pogozheva, Irina D; Portillo, Federico P; Mosberg, Henry I; Haskell-Luevano, Carrie

2008-09-25

The melanocortin-3 and -4 receptors (MC3R, MC4R) have been implicated in energy homeostasis and obesity. Whereas the physiological role of the MC4R is extensively studied, little is known about the MC3R. One caveat is the limited availability of ligands that are selective for the MC3R. Previous studies identified Ac-His-DPhe(p-I)-Arg-Trp-NH 2, which possessed partial agonist/antagonist pharmacology at the mMC3R while retaining full nanomolar agonist pharmacology at the mMC4R. These data allowed for the hypothesis that the DPhe position in melanocortin tetrapeptides can be used to examine ligand side-chain determinants important for differentiation of mMC3R agonist versus antagonist activity. A series of 15 DPhe (7) modified Ac-His-DPhe (7)-Arg-Trp-NH 2 tetrapeptides has been synthesized and pharmacologically characterized. Most notable results include the identification of modifications that resulted in potent antagonists/partial agonists at the mMC3R and full, potent agonists at the mMC4R. These SAR studies provide experimental evidence that the molecular mechanism of antagonism at the mMC3R differentiates this subtype from the mMC4R.

Melanocortin Tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 Modified at the Para Position of the Benzyl Side Chain (DPhe): Importance for Mouse Melanocortin-3 Receptor Agonist versus Antagonist Activity

PubMed Central

Proneth, Bettina; Pogozheva, Irina D.; Portillo, Federico P.; Mosberg, Henry I.; Haskell-Luevano, Carrie

2010-01-01

The melanocortin-3 and -4 receptors (MC3R, MC4R) have been implicated in energy homeostasis and obesity. Whereas the physiological role of the MC4R is extensively studied, little is known about the MC3R. One caveat is the limited availability of ligands that are selective for the MC3R. Previous studies identified Ac-His-DPhe(p-I)-Arg-Trp-NH2, which possessed partial agonist/antagonist pharmacology at the mMC3R while retaining full nanomolar agonist pharmacology at the mMC4R. These data allowed for the hypothesis that the DPhe position in melanocortin tetrapeptides can be used to examine ligand side-chain determinants important for differentiation of mMC3R agonist versus antagonist activity. A series of 15 DPhe7 modified Ac-His-DPhe7-Arg-Trp-NH2 tetrapeptides has been synthesized and pharmacologically characterized. Most notable results include the identification of modifications that resulted in potent antagonists/partial agonists at the mMC3R and full, potent agonists at the mMC4R. These SAR studies provide experimental evidence that the molecular mechanism of antagonism at the mMC3R differentiates this subtype from the mMC4R. PMID:18800761

The therapeutic potential of nicotinic acetylcholine receptor agonists for pain control.

PubMed

Decker, M W; Meyer, M D; Sullivan, J P

2001-10-01

Due to the limitations of currently available analgesics, a number of novel alternatives are currently under investigation, including neuronal nicotinic acetylcholine receptor (nAChR) agonists. During the 1990s, the discovery of the antinociceptive properties of the potent nAChR agonist epibatidine in rodents sparked interest in the analgesic potential of this class of compounds. Although epibatidine also has several mechanism-related toxicities, the identification of considerable nAChR diversity suggested that the toxicities and therapeutic actions of the compound might be mediated by distinct receptor subtypes. Consistent with this view, a number of novel nAChR agonists with antinociceptive activity and improved safety profiles in preclinical models have now been identified, including A-85380, ABT-594, DBO-83, SIB-1663 and RJR-2403. Of these, ABT-594 is the most advanced and is currently in Phase II clinical evaluation. Nicotinically-mediated antinociception has been demonstrated in a variety of rodent pain models and is likely mediated by the activation of descending inhibitory pathways originating in the brainstem with the predominant high-affinity nicotine site in brain, the alpha4beta2 subtype, playing a critical role. Thus, preclinical findings suggest that nAChR agonists have the potential to be highly efficacious treatments in a variety of pain states. However, clinical proof-of-principle studies will be required to determine if nAChR agonists are active in pathological pain.

Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a [35S]GTPgammaS binding study.

PubMed

Newman-Tancredi, A; Gavaudan, S; Conte, C; Chaput, C; Touzard, M; Verrièle, L; Audinot, V; Millan, M J

1998-08-21

Recombinant human (h) 5-HT1A receptor-mediated G-protein activation was characterised in membranes of transfected Chinese hamster ovary (CHO) cells by use of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS binding). The potency and efficacy of 21 5-HT receptor agonists and antagonists was determined. The agonists, 5-CT (carboxamidotryptamine) and flesinoxan displayed high affinity (subnanomolar Ki values) and high efficacy (Emax > 90%, relative to 5-HT = 100%). In contrast, ipsapirone, zalospirone and buspirone displayed partial agonist activity. EC50s for agonist stimulation of [35S]GTPgammaS binding correlated well with Ki values from competition binding (r = +0.99). Among the compounds tested for antagonist activity, methiothepin and (+)butaclamol exhibited 'inverse agonist' behaviour, inhibiting basal [35S]GTPgammaS binding. The actions of 17 antipsychotic agents were investigated. Clozapine and several putatively 'atypical' antipsychotic agents, including ziprasidone, quetiapine and tiospirone, exhibited partial agonist activity and marked affinity at h5-HT1A receptors, similar to their affinity at hD2 dopamine receptors. In contrast, risperidone and sertindole displayed low affinity at h5-HT1A receptors and behaved as 'neutral' antagonists, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Likewise the 'typical' neuroleptics, haloperidol, pimozide, raclopride and chlorpromazine exhibited relatively low affinity and 'neutral' antagonist activity at h5-HT1A receptors with Ki values which correlated with their respective Kb values. The present data show that (i) [35S]GTPgammaS binding is an effective method to evaluate the efficacy and potency of agonists and antagonists at recombinant human 5-HT1A receptors. (ii) Like clozapine, several putatively 'atypical' antipsychotic drugs display balanced serotonin h5-HT1A/dopamine hD2 receptor affinity and partial agonist activity at h5-HT1A receptors. (iii) Several 'typical' and some putatively 'atypical' antipsychotic agents displayed antagonist properties at h5-HT1A sites with generally much lower affinity than at hD2 dopamine receptors. It is suggested that agonist activity at 5-HT1A receptors may be of utility for certain antipsychotic agents.

Delta Opioid Receptor (DOR) Ligands and Pharmacology: Development of Indolo- and Quinolinomorphinan Derivatives Based on the Message-Address Concept.

PubMed

Saitoh, Akiyoshi; Nagase, Hiroshi

2016-10-28

The pharmacology of the delta opioid receptor (DOR) has lagged, mainly due to the lack of an agonist with high potency and selectivity in vivo. The DOR is now receiving increasing attention, and there has been progress in the synthesis of better novel ligands. The discovery of a selective receptor DOR antagonist, naltrindole (NTI), stimulated the design and synthesis of (±)TAN-67, which was designed based on the message-address concept and the accessory site theory. Intensive studies using (±)TAN-67 determined the DOR-mediated various pharmacological effects, such as antinociceptive effects for painful diabetic neuropathy and cardiovascular protective effects. We improved the agonist activity of TAN-67 to afford SN-28, which was modified to KNT-127, a novel compound that improved the blood-brain barrier permeability. In addition, KNT-127 showed higher selectivity for the DOR and had potent agonist activity following systemic administration. Interestingly, KNT-127 produced no convulsive effects, unlike prototype DOR agonists. The KNT-127 type derivatives with a quinolinomorphinan structure are expected to be promising candidates for the development of therapeutic DOR agonists.

A Physiological Neural Controller of a Muscle Fiber Oculomotor Plant in Horizontal Monkey Saccades

PubMed Central

Enderle, John D.

2014-01-01

A neural network model of biophysical neurons in the midbrain is presented to drive a muscle fiber oculomotor plant during horizontal monkey saccades. Neural circuitry, including omnipause neuron, premotor excitatory and inhibitory burst neurons, long lead burst neuron, tonic neuron, interneuron, abducens nucleus, and oculomotor nucleus, is developed to examine saccade dynamics. The time-optimal control strategy by realization of agonist and antagonist controller models is investigated. In consequence, each agonist muscle fiber is stimulated by an agonist neuron, while an antagonist muscle fiber is unstimulated by a pause and step from the antagonist neuron. It is concluded that the neural network is constrained by a minimum duration of the agonist pulse and that the most dominant factor in determining the saccade magnitude is the number of active neurons for the small saccades. For the large saccades, however, the duration of agonist burst firing significantly affects the control of saccades. The proposed saccadic circuitry establishes a complete model of saccade generation since it not only includes the neural circuits at both the premotor and motor stages of the saccade generator, but also uses a time-optimal controller to yield the desired saccade magnitude. PMID:24944832

Comparative Review of Approved Melatonin Agonists for the Treatment of Circadian Rhythm Sleep-Wake Disorders.

PubMed

Williams, Wilbur P Trey; McLin, Dewey E; Dressman, Marlene A; Neubauer, David N

2016-09-01

Circadian rhythm sleep-wake disorders (CRSWDs) are characterized by persistent or recurrent patterns of sleep disturbance related primarily to alterations of the circadian rhythm system or the misalignment between the endogenous circadian rhythm and exogenous factors that affect the timing or duration of sleep. These disorders collectively represent a significant unmet medical need, with a total prevalence in the millions, a substantial negative impact on quality of life, and a lack of studied treatments for most of these disorders. Activation of the endogenous melatonin receptors appears to play an important role in setting the circadian clock in the suprachiasmatic nucleus of the hypothalamus. Therefore, melatonin agonists, which may be able to shift and/or stabilize the circadian phase, have been identified as potential therapeutic candidates for the treatment of CRSWDs. Currently, only one melatonin receptor agonist, tasimelteon, is approved for the treatment of a CRSWD: non-24-hour sleep-wake disorder (or non-24). However, three additional commercially available melatonin receptor agonists-agomelatine, prolonged-release melatonin, and ramelteon-have been investigated for potential use for treatment of CRSWDs. Data indicate that these melatonin receptor agonists have distinct pharmacologic profiles that may help clarify their clinical use in CRSWDs. We review the pharmacokinetic and pharmacodynamic properties of these melatonin agonists and summarize their efficacy profiles when used for the treatment of CRSWDs. Further studies are needed to determine the therapeutic potential of these melatonin agonists for most CRSWDs. © 2016 Vanda Pharmaceuticals, Inc. Pharmacotherapy published by Wiley Periodicals, Inc. on behalf of Pharmacotherapy Publications, Inc.

High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments.

PubMed

Roubalova, Lenka; Vosahlikova, Miroslava; Brejchova, Jana; Sykora, Jan; Rudajev, Vladimir; Svoboda, Petr

2015-01-01

HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye. Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.

Mild Lipid Stress Induces Profound Loss of MC4R Protein Abundance and Function

PubMed Central

Cragle, Faith K.

2014-01-01

Food intake is controlled at the central level by the melanocortin pathway in which the agonist α-MSH binds to melanocortin 4 receptor (MC4R), a Gs-coupled G protein-coupled receptor expressed by neurons in the paraventricular nuclei of the hypothalamus, which signals to reduce appetite. Consumption of a high-fat diet induces hypothalamic accumulation of palmitate, endoplasmic reticulum (ER) stress, apoptosis, and unresponsiveness to prolonged treatment with MC4R agonists. Here we have modeled effects of lipid stress on MC4R by using mHypoE-42 immortalized hypothalamic neurons expressing endogenous MC4R and Neuro2A cells expressing a tagged MC4R reporter, HA-MC4R-GFP. In the hypothalamic neurons, exposure to elevated palmitate in the physiological range induced splicing of X-box binding protein 1, but it did not activate C/EBP-homologous protein or induce increased levels of cleaved caspase-3, indicating mild ER stress. Such mild ER stress coexisted with a minimal loss of MC4R mRNA and yet a profound loss of cAMP signaling in response to incubation with the agonist. These findings were mirrored in the Neuro2A cells expressing HA-MC4R-GFP, in which protein abundance of the tagged receptor was decreased, whereas the activity per receptor number was maintained. The loss of cAMP signaling in response to α-MSH by elevated palmitate was corrected by treatment with a chemical chaperone, 4-phenylbutyrate in both mHypoE-42 hypothalamic neurons and in Neuro2A cells in which protein abundance of HA-MC4R-GFP was increased. The data indicate that posttranscriptional decrease of MC4R protein contribute to lower the response to α-MSH in hypothalamic neurons exposed to even a mild level of lipid stress and that a chemical chaperone corrects such a defect. PMID:24506538

Tamoxifen regulation of bone growth and endocrine function in the ovariectomized rat: discrimination of responses involving estrogen receptor α/estrogen receptor β, G protein-coupled estrogen receptor, or estrogen-related receptor γ using fulvestrant (ICI 182780).

PubMed

Fitts, James M; Klein, Robert M; Powers, C Andrew

2011-07-01

Tamoxifen is a selective estrogen receptor (ER) modulator, but it is also a deactivating ligand for estrogen-related receptor-γ (ERRγ) and a full agonist for the G protein-coupled estrogen receptor (GPER). Fulvestrant is a selective ER down-regulator that lacks agonist effects on ERα/ERβ, is inactive on ERRγ, but acts as a full agonist on GPER. Fulvestrant effects on tamoxifen actions on uterine and somatic growth, bone, the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis, and pituitary prolactin were analyzed to pharmacologically discriminate tamoxifen effects that may be mediated by ERα/ERβ versus ERRγ versus GPER. Ovariectomized rats received tamoxifen (0.6 mg/kg/daily) plus fulvestrant at 0, 3, 6, or 12 mg/kg/daily for 5 weeks; controls received vehicle or 6 mg/kg fulvestrant daily. Tamoxifen effects to increase uterine weight, decrease serum IGF-I, increase pituitary prolactin, and increase bone mineral density could be fully blocked by fulvestrant, indicating mediation by ERα/ERβ. Tamoxifen effects to decrease pituitary GH, tibia length, and body weight were only partially blocked by fulvestrant, indicating involvement of mechanisms unrelated to ERα/ERβ. Fulvestrant did not inhibit tamoxifen actions to reduce total pituitary protein, again indicating effects not mediated by ERα/ERβ. Tamoxifen actions to reduce serum GH were mimicked rather than inhibited by fulvestrant, pharmacological features consistent with GPER involvement. However, fulvestrant alone increased IGF-I and also blocked tamoxifen-evoked IGF-I decreases; thus fulvestrant effects on serum GH might reflect increased IGF-I feedback inhibition. Fulvestrant alone had no effect on the other parameters. The findings indicate that mechanisms unrelated to ERα/ERβ contribute to tamoxifen effects on body weight, bone growth, and pituitary function.

Cross-talk between Carboxypeptidase M and the Kinin B1 Receptor Mediates a New Mode of G Protein-coupled Receptor Signaling*

PubMed Central

Zhang, Xianming; Tan, Fulong; Brovkovych, Viktor; Zhang, Yongkang; Skidgel, Randal A.

2011-01-01

G protein-coupled receptor (GPCR) signaling is affected by formation of GPCR homo- or heterodimers, but GPCR regulation by other cell surface proteins is not well understood. We reported that the kinin B1 receptor (B1R) heterodimerizes with membrane carboxypeptidase M (CPM), facilitating receptor signaling via CPM-mediated conversion of bradykinin or kallidin to des-Arg kinin B1R agonists. Here, we found that a catalytically inactive CPM mutant that still binds substrate (CPM-E264Q) also facilitates efficient B1R signaling by B2 receptor agonists bradykinin or kallidin. This response required co-expression of B1R and CPM-E264Q in the same cell, was disrupted by antibody that dissociates CPM from B1R, and was not found with a CPM-E264Q-B1R fusion protein. An additional mutation that reduced the affinity of CPM for C-terminal Arg and increased the affinity for C-terminal Lys inhibited the B1R response to bradykinin (with C-terminal Arg) but generated a response to Lys9-bradykinin. CPM-E264Q-mediated activation of B1Rs by bradykinin resulted in increased intramolecular fluorescence resonance energy transfer (FRET) in a B1R FRET construct, similar to that generated directly by a B1R agonist. In cytokine-treated human lung microvascular endothelial cells, disruption of B1R-CPM heterodimers inhibited B1R-dependent NO production stimulated by bradykinin and blocked the increased endothelial permeability caused by treatment with bradykinin and pyrogallol (a superoxide generator). Thus, CPM and B1Rs on cell membranes form a critical complex that potentiates B1R signaling. Kinin peptide binding to CPM causes a conformational change in the B1R leading to intracellular signaling and reveals a new mode of GPCR activation by a cell surface peptidase. PMID:21454694

Biphasic Regulation of Yes-associated Protein (YAP) Cellular Localization, Phosphorylation, and Activity by G Protein-coupled Receptor Agonists in Intestinal Epithelial Cells: A NOVEL ROLE FOR PROTEIN KINASE D (PKD).

PubMed

Wang, Jia; Sinnett-Smith, James; Stevens, Jan V; Young, Steven H; Rozengurt, Enrique

2016-08-19

We examined the regulation of Yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in intestinal epithelial cells. Our results show that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II, a potent mitogen for these cells, induced rapid translocation of YAP from the nucleus to the cytoplasm (within 15 min) and a concomitant increase in YAP phosphorylation at Ser(127) and Ser(397) Angiotensin II elicited YAP phosphorylation and cytoplasmic accumulation in a dose-dependent manner (ED50 = 0.3 nm). Similar YAP responses were provoked by stimulation with vasopressin or serum. Treatment of the cells with the protein kinase D (PKD) family inhibitors CRT0066101 and kb NB 142-70 prevented the increase in YAP phosphorylation on Ser(127) and Ser(397) via Lats2, YAP cytoplasmic accumulation, and increase in the mRNA levels of YAP/TEAD-regulated genes (Ctgf and Areg). Furthermore, siRNA-mediated knockdown of PKD1, PKD2, and PKD3 markedly attenuated YAP nuclear-cytoplasmic shuttling, phosphorylation at Ser(127), and induction of Ctgf and Areg expression in response to GPCR activation. These results identify a novel role for the PKD family in the control of biphasic localization, phosphorylation, and transcriptional activity of YAP in intestinal epithelial cells. In turn, YAP and TAZ are necessary for the stimulation of the proliferative response of intestinal epithelial cells to GPCR agonists that act via PKD. The discovery of interaction between YAP and PKD pathways identifies a novel cross-talk in signal transduction and demonstrates, for the first time, that the PKDs feed into the YAP pathway. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Rapid effects of dorsal hippocampal G-protein coupled estrogen receptor on learning in female mice.

PubMed

Lymer, Jennifer; Robinson, Alana; Winters, Boyer D; Choleris, Elena

2017-03-01

Through rapid mechanisms of action, estrogens affect learning and memory processes. It has been shown that 17β-estradiol and an Estrogen Receptor (ER) α agonist enhances performance in social recognition, object recognition, and object placement tasks when administered systemically or infused in the dorsal hippocampus. In contrast, systemic and dorsal hippocampal ERβ activation only promote spatial learning. In addition, 17β-estradiol, the ERα and the G-protein coupled estrogen receptor (GPER) agonists increase dendritic spine density in the CA1 hippocampus. Recently, we have shown that selective systemic activation of the GPER also rapidly facilitated social recognition, object recognition, and object placement learning in female mice. Whether activation the GPER specifically in the dorsal hippocampus can also rapidly improve learning and memory prior to acquisition is unknown. Here, we investigated the rapid effects of infusion of the GPER agonist, G-1 (dose: 50nM, 100nM, 200nM), in the dorsal hippocampus on social recognition, object recognition, and object placement learning tasks in home cage. These paradigms were completed within 40min, which is within the range of rapid estrogenic effects. Dorsal hippocampal administration of G-1 improved social (doses: 50nM, 200nM G-1) and object (dose: 200nM G-1) recognition with no effect on object placement. Additionally, when spatial cues were minimized by testing in a Y-apparatus, G-1 administration promoted social (doses: 100nM, 200nM G-1) and object (doses: 50nM, 100nM, 200nM G-1) recognition. Therefore, like ERα, the GPER in the hippocampus appears to be sufficient for the rapid facilitation of social and object recognition in female mice, but not for the rapid facilitation of object placement learning. Thus, the GPER in the dorsal hippocampus is involved in estrogenic mediation of learning and memory and these effects likely occur through rapid signalling mechanisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inverse agonism at the P2Y12 receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor.

PubMed

Aungraheeta, Riyaad; Conibear, Alexandra; Butler, Mark; Kelly, Eamonn; Nylander, Sven; Mumford, Andrew; Mundell, Stuart J

2016-12-08

Ticagrelor is a potent antagonist of the P2Y 12 receptor (P2Y 12 R) and consequently an inhibitor of platelet activity effective in the treatment of atherothrombosis. Here, we sought to further characterize its molecular mechanism of action. Initial studies showed that ticagrelor promoted a greater inhibition of adenosine 5'-diphosphate (ADP)-induced Ca 2+ release in washed platelets vs other P2Y 12 R antagonists. This additional effect of ticagrelor beyond P2Y 12 R antagonism was in part as a consequence of ticagrelor inhibiting the equilibrative nucleoside transporter 1 (ENT1) on platelets, leading to accumulation of extracellular adenosine and activation of G s -coupled adenosine A 2A receptors. This contributed to an increase in basal cyclic adenosine monophosphate (cAMP) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P). In addition, ticagrelor increased platelet cAMP and VASP-P in the absence of ADP in an adenosine receptor-independent manner. We hypothesized that this increase originated from a direct effect on basal agonist-independent P2Y 12 R signaling, and this was validated in 1321N1 cells stably transfected with human P2Y 12 R. In these cells, ticagrelor blocked the constitutive agonist-independent activity of the P2Y 12 R, limiting basal G i -coupled signaling and thereby increasing cAMP levels. These data suggest that ticagrelor has the pharmacological profile of an inverse agonist. Based on our results showing insurmountable inhibition of ADP-induced Ca 2+ release and forskolin-induced cAMP, the mode of antagonism of ticagrelor also appears noncompetitive, at least functionally. In summary, our studies describe 2 novel modes of action of ticagrelor, inhibition of platelet ENT1 and inverse agonism at the P2Y 12 R that contribute to its effective inhibition of platelet activation. © 2016 by The American Society of Hematology.

Unexpected opioid activity profiles of analogues of the novel peptide kappa opioid receptor ligand CJ-15,208.

PubMed

Aldrich, Jane V; Kulkarni, Santosh S; Senadheera, Sanjeewa N; Ross, Nicolette C; Reilley, Kate J; Eans, Shainnel O; Ganno, Michelle L; Murray, Thomas F; McLaughlin, Jay P

2011-09-05

An alanine scan was performed on the novel κ opioid receptor (KOR) peptide ligand CJ-15,208 to determine which residues contribute to the potent in vivo agonist activity observed for the parent peptide. These cyclic tetrapeptides were synthesized by a combination of solid-phase peptide synthesis of the linear precursors, followed by cyclization in solution. Like the parent peptide, each of the analogues exhibited agonist activity and KOR antagonist activity in an antinociceptive assay in vivo. Unlike the parent peptide, the agonist activity of the potent analogues was mediated predominantly, if not exclusively, by μ opioid receptors (MOR). Thus analogues 2 and 4, in which one of the phenylalanine residues was replaced by alanine, exhibited both potent MOR agonist activity and KOR antagonist activity in vivo. These peptides represent novel lead compounds for the development of peptide-based opioid analgesics. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stoichiometry for α-bungarotoxin block of α7 acetylcholine receptors

NASA Astrophysics Data System (ADS)

Dacosta, Corrie J. B.; Free, Chris R.; Sine, Steven M.

2015-08-01

α-Bungarotoxin (α-Btx) binds to the five agonist binding sites on the homopentameric α7-acetylcholine receptor, yet the number of bound α-Btx molecules required to prevent agonist-induced channel opening remains unknown. To determine the stoichiometry for α-Btx blockade, we generate receptors comprised of wild-type and α-Btx-resistant subunits, tag one of the subunit types with conductance mutations to report subunit stoichiometry, and following incubation with α-Btx, monitor opening of individual receptor channels with defined subunit stoichiometry. We find that a single α-Btx-sensitive subunit confers nearly maximal suppression of channel opening, despite four binding sites remaining unoccupied by α-Btx and accessible to the agonist. Given structural evidence that α-Btx locks the agonist binding site in an inactive conformation, we conclude that the dominant mechanism of antagonism is non-competitive, originating from conformational arrest of the binding sites, and that the five α7 subunits are interdependent and maintain conformational symmetry in the open channel state.

Neural correlate of resting-state functional connectivity under α2 adrenergic receptor agonist, medetomidine.

PubMed

Nasrallah, Fatima A; Lew, Si Kang; Low, Amanda Si-Min; Chuang, Kai-Hsiang

2014-01-01

Correlative fluctuations in functional MRI (fMRI) signals across the brain at rest have been taken as a measure of functional connectivity, but the neural basis of this resting-state MRI (rsMRI) signal is not clear. Previously, we found that the α2 adrenergic agonist, medetomidine, suppressed the rsMRI correlation dose-dependently but not the stimulus evoked activation. To understand the underlying electrophysiology and neurovascular coupling, which might be altered due to the vasoconstrictive nature of medetomidine, somatosensory evoked potential (SEP) and resting electroencephalography (EEG) were measured and correlated with corresponding BOLD signals in rat brains under three dosages of medetomidine. The SEP elicited by electrical stimulation to both forepaws was unchanged regardless of medetomidine dosage, which was consistent with the BOLD activation. Identical relationship between the SEP and BOLD signal under different medetomidine dosages indicates that the neurovascular coupling was not affected. Under resting state, EEG power was the same but a depression of inter-hemispheric EEG coherence in the gamma band was observed at higher medetomidine dosage. Different from medetomidine, both resting EEG power and BOLD power and coherence were significantly suppressed with increased isoflurane level. Such reduction was likely due to suppressed neural activity as shown by diminished SEP and BOLD activation under isoflurane, suggesting different mechanisms of losing synchrony at resting-state. Even though, similarity between electrophysiology and BOLD under stimulation and resting-state implicates a tight neurovascular coupling in both medetomidine and isoflurane. Our results confirm that medetomidine does not suppress neural activity but dissociates connectivity in the somatosensory cortex. The differential effect of medetomidine and its receptor specific action supports the neuronal origin of functional connectivity and implicates the mechanism of its sedative effect. © 2013. Published by Elsevier Inc. All rights reserved.

Interaction between protein kinase C and protein kinase A can modulate transmitter release at the rat neuromuscular synapse.

PubMed

Santafé, M M; Garcia, N; Lanuza, M A; Tomàs, M; Tomàs, J

2009-02-15

We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release.

Proceedings of the International Symposium on Subtypes of Muscarinic Receptors (4th), Held in Wiesbaden (Germany, F.R.) on 20-22 July 1989

DTIC Science & Technology

1990-06-15

C. However, primary astrocytes is not stimulated by oxotremorine , a this coupling is much less efficient than that mediated muscarinic agonist which...and oxotremorine -M, possessed suffi- modest improvement in affinity but without the antici- cient intrnsic efficacy to maximally stimulate cortical PI...other cholino- Animal studies mimetics i.e. oxotremorine , in different animal spe- Antinociceptive studies in animals cies s . Centrally administered

Elucidating Ligand-Modulated Conformational Landscape of GPCRs Using Cloud-Computing Approaches.

PubMed

Shukla, Diwakar; Lawrenz, Morgan; Pande, Vijay S

2015-01-01

G-protein-coupled receptors (GPCRs) are a versatile family of membrane-bound signaling proteins. Despite the recent successes in obtaining crystal structures of GPCRs, much needs to be learned about the conformational changes associated with their activation. Furthermore, the mechanism by which ligands modulate the activation of GPCRs has remained elusive. Molecular simulations provide a way of obtaining detailed an atomistic description of GPCR activation dynamics. However, simulating GPCR activation is challenging due to the long timescales involved and the associated challenge of gaining insights from the "Big" simulation datasets. Here, we demonstrate how cloud-computing approaches have been used to tackle these challenges and obtain insights into the activation mechanism of GPCRs. In particular, we review the use of Markov state model (MSM)-based sampling algorithms for sampling milliseconds of dynamics of a major drug target, the G-protein-coupled receptor β2-AR. MSMs of agonist and inverse agonist-bound β2-AR reveal multiple activation pathways and how ligands function via modulation of the ensemble of activation pathways. We target this ensemble of conformations with computer-aided drug design approaches, with the goal of designing drugs that interact more closely with diverse receptor states, for overall increased efficacy and specificity. We conclude by discussing how cloud-based approaches present a powerful and broadly available tool for studying the complex biological systems routinely. © 2015 Elsevier Inc. All rights reserved.

G Protein-Coupled Estrogen Receptor-Selective Ligands Modulate Endometrial Tumor Growth

PubMed Central

Petrie, Whitney K.; Dennis, Megan K.; Dai, Donghai; Arterburn, Jeffrey B.; Smith, Harriet O.; Hathaway, Helen J.; Prossnitz, Eric R.

2013-01-01

Endometrial carcinoma is the most common cancer of the female reproductive tract. GPER/GPR30 is a 7-transmembrane spanning G protein-coupled receptor that has been identified as the third estrogen receptor, in addition to ERα and ERβ. High GPER expression is predictive of poor survival in endometrial and ovarian cancer, but despite this, the estrogen-mediated signaling pathways and specific estrogen receptors involved in endometrial cancer remain unclear. Here, employing ERα-negative Hec50 endometrial cancer cells, we demonstrate that GPER mediates estrogen-stimulated activation of ERK and PI3K via matrix metalloproteinase activation and subsequent transactivation of the EGFR and that ER-targeted therapeutic agents (4-hydroxytamoxifen, ICI182,780/fulvestrant, and Raloxifene), the phytoestrogen genistein, and the “ERα-selective” agonist propylpyrazole triol also function as GPER agonists. Furthermore, xenograft tumors of Hec50 cells yield enhanced growth with G-1 and estrogen, the latter being inhibited by GPER-selective pharmacologic antagonism with G36. These results have important implications with respect to the use of putatively ER-selective ligands and particularly for the widespread long-term use of “ER-targeted” therapeutics. Moreover, our findings shed light on the potential mechanisms of SERM/SERD side effects reported in many clinical studies. Finally, our results provide the first demonstration that pharmacological inhibition of GPER activity in vivo prevents estrogen-mediated tumor growth. PMID:24379833

G protein-coupled receptor 30 regulates trophoblast invasion and its deficiency is associated with preeclampsia.

PubMed

Tong, Chao; Feng, Xiang; Chen, Jun; Qi, Xingchen; Zhou, Liyuan; Shi, Shuming; Kc, Kamana; Stanley, Joanna L; Baker, Philip N; Zhang, Hua

2016-04-01

Preeclampsia is known to be associated with reduced circulating levels of estrogen. The effects of estrogen in preeclampsia are normally mediated by the classical estrogen receptors. Intriguingly, a novel estrogen receptor, G protein-coupled receptor 30 (GPR30), has been recently found to play an important role in several estrogenic effects. However, the mechanisms by which GPR30 may mediate the development of preeclampsia remain unknown. We observed that the expression of GPR30 in placental trophoblast cells is lower in preeclamptic placentas compared with normotensive controls. We then investigated the role of GPR30 in trophoblast cell invasion by utilizing placental explants and the immortalized human trophoblast cell line (HTR8/SVneo). The selective GPR30 agonist G1 and a general estrogen receptors agonist 17-β-estradiol (E2) both improved trophoblast cells invasion by upregulating MMP9 expression and the PI3K-Akt signaling pathway. This effect was abolished by a selective GPR30 inhibitor G15, implying that GPR30 may be involved in regulating trophoblast invasion, and that down-regulation of this receptor may result in the development of preeclampsia. The present study suggests that GPR30 is a critical regulator of trophoblast cell invasion, and as such may be a potential therapeutic interventional target for preeclampsia and other pregnancy complications resulting from impaired trophoblast invasion.

G protein-coupled estrogen receptor-selective ligands modulate endometrial tumor growth.

PubMed

Petrie, Whitney K; Dennis, Megan K; Hu, Chelin; Dai, Donghai; Arterburn, Jeffrey B; Smith, Harriet O; Hathaway, Helen J; Prossnitz, Eric R

2013-01-01

Endometrial carcinoma is the most common cancer of the female reproductive tract. GPER/GPR30 is a 7-transmembrane spanning G protein-coupled receptor that has been identified as the third estrogen receptor, in addition to ERα and ERβ. High GPER expression is predictive of poor survival in endometrial and ovarian cancer, but despite this, the estrogen-mediated signaling pathways and specific estrogen receptors involved in endometrial cancer remain unclear. Here, employing ERα-negative Hec50 endometrial cancer cells, we demonstrate that GPER mediates estrogen-stimulated activation of ERK and PI3K via matrix metalloproteinase activation and subsequent transactivation of the EGFR and that ER-targeted therapeutic agents (4-hydroxytamoxifen, ICI182,780/fulvestrant, and Raloxifene), the phytoestrogen genistein, and the "ERα-selective" agonist propylpyrazole triol also function as GPER agonists. Furthermore, xenograft tumors of Hec50 cells yield enhanced growth with G-1 and estrogen, the latter being inhibited by GPER-selective pharmacologic antagonism with G36. These results have important implications with respect to the use of putatively ER-selective ligands and particularly for the widespread long-term use of "ER-targeted" therapeutics. Moreover, our findings shed light on the potential mechanisms of SERM/SERD side effects reported in many clinical studies. Finally, our results provide the first demonstration that pharmacological inhibition of GPER activity in vivo prevents estrogen-mediated tumor growth.

Activation of G protein coupled estrogen receptor (GPER) promotes the migration of renal cell carcinoma via the PI3K/AKT/MMP-9 signals.

PubMed

Guan, Bao-Zhang; Yan, Rui-Ling; Huang, Jian-Wei; Li, Fo-Lan; Zhong, Ying-Xue; Chen, Yu; Liu, Fan-Na; Hu, Bo; Huang, Si-Bo; Yin, Liang-Hong

2018-03-04

Renal cell carcinoma (RCC) is the third most frequent malignancy within urological oncology. However, the mechanisms responsible for RCC metastasis are still needed further illustration. Our present study revealed that a seven-transmembrane receptor G-protein coupled estrogen receptor (GPER) was highly detected in various RCC cell lines such as ACHN, OS-RC-2 and SW839. The activation of GPER by its specific agonist G-1 significantly promoted the in vitro migration and invasion of ACHN and OS-RC-2 cells. G-1 also up regulated the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9. The inhibitor of MMP-9 (Cat-444278), but not MMP-2 (Sc-204092), abolished G-1 induced cell migration, which suggested that MMP-9 is the key molecule mediating G-1 induced RCC progression. Further, G-1 treatment resulted in phosphorylation of AKT and ERK in RCC cells. PI3K/AKT inhibitor (LY294002), while not ERK inhibitor (PD98059), significantly abolished G-1 induced up regulation of MMP-9 in both AHCN and OS-RC-2 cells. Generally, our data revealed that activation of GPER by its specific agonist G-1 promoted the metastasis of RCC cells through PI3K/AKT/MMP-9 signals, which might be a promising new target for drug discovery of RCC patients.

Discovery and Characterization of a Novel Small-Molecule Agonist for Medium-Chain Free Fatty Acid Receptor G Protein-Coupled Receptor 84.

PubMed

Zhang, Qing; Yang, Hui; Li, Jing; Xie, Xin

2016-05-01

G protein-coupled receptor 84 (GPR84) is a free fatty acid receptor activated by medium-chain free fatty acids with 9-14 carbons. It is expressed mainly in the immune-related tissues, such as spleen, bone marrow, and peripheral blood leukocytes. GPR84 plays significant roles in inflammatory processes and may represent a novel drug target for the treatment of immune-mediated diseases. However, the lack of potent and specific ligands for GPR84 hindered the study of its functions and the development of potential clinical applications. Here, we report the screen of 160,000 small-molecule compounds with a calcium mobilization assay using a human embryonic kidney 293 cell line stably expressing GPR84 and Gα16, and the identification of 2-(hexylthio)pyrimidine-4,6-diol (ZQ-16) as a potent and selective agonist of GPR84 with a novel structure. ZQ-16 activates several GPR84-mediated signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, phosphorylation of extracellular signal-regulated protein kinase 1/2, receptor desensitization and internalization, and receptor-β-arrestin interaction. This compound may be a useful tool to study the functions of GPR84 and a potential candidate for further structural optimization. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca(2+) regulation in airway smooth muscle (ASM).

PubMed

Delmotte, Philippe; Sieck, Gary C

2015-02-01

Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca(2+) ([Ca(2+)]cyt) responses to agonist stimulation and Ca(2+) sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca(2+)]cyt induced by agonists leads to a transient increase in mitochondrial Ca(2+) ([Ca(2+)]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca(2+)]mito is blunted despite enhanced [Ca(2+)]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion-ER/SR coupling, decreased mitochondrial Ca(2+) buffering, mitochondrial fragmentation, and increased cell proliferation.

Dopamine D4 Receptor Counteracts Morphine-Induced Changes in μ Opioid Receptor Signaling in the Striosomes of the Rat Caudate Putamen

PubMed Central

Suárez-Boomgaard, Diana; Gago, Belén; Valderrama-Carvajal, Alejandra; Roales-Buján, Ruth; Van Craenenbroeck, Kathleen; Duchou, Jolien; Borroto-Escuela, Dasiel O.; Medina-Luque, José; de la Calle, Adelaida; Fuxe, Kjell; Rivera, Alicia

2014-01-01

The mu opioid receptor (MOR) is critical in mediating morphine analgesia. However, prolonged exposure to morphine induces adaptive changes in this receptor leading to the development of tolerance and addiction. In the present work we have studied whether the continuous administration of morphine induces changes in MOR protein levels, its pharmacological profile, and MOR-mediated G-protein activation in the striosomal compartment of the rat CPu, by using immunohistochemistry and receptor and DAMGO-stimulated [35S]GTPγS autoradiography. MOR immunoreactivity, agonist binding density and its coupling to G proteins are up-regulated in the striosomes by continuous morphine treatment in the absence of changes in enkephalin and dynorphin mRNA levels. In addition, co-treatment of morphine with the dopamine D4 receptor (D4R) agonist PD168,077 fully counteracts these adaptive changes in MOR, in spite of the fact that continuous PD168,077 treatment increases the [3H]DAMGO Bmax values to the same degree as seen after continuous morphine treatment. Thus, in spite of the fact that both receptors can be coupled to Gi/0 protein, the present results give support for the existence of antagonistic functional D4R-MOR receptor-receptor interactions in the adaptive changes occurring in MOR of striosomes on continuous administration of morphine. PMID:24451133

Mixed nicotinic and muscarinic features of cholinergic receptor coupled to secretion in bovine chromaffin cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shirvan, M.H.; Pollard, H.B.; Heldman, E.

Acetylcholine evokes release from cultured bovine chromaffin cells by a mechanism that is believed to be classically nicotinic. However, the authors found that the full muscarinic agonist oxotremorine-M (Oxo-M) induced a robust catecholamine (CA) secretion. By contrast, muscarine, pilocarpine, bethanechol, and McN-A-343 did not elicit any secretory response. Desensitization of the response to nicotine by Oxo-M and desensitization of the response to Oxo-M by nicotine suggest that both nicotine and Oxo-M were acting at the same receptor. Additional experiments supporting this conclusion show that nicotine-induced secretion and Oxo-M-induced secretion were similarly blocked by various muscarinic and nicotinic antagonists. Moreover, secretionmore » induced by nicotine and Oxo-M were Ca{sup 2+} dependent, and both agonists induced {sup 45}Ca{sup 2+} uptake. Equilibrium binding studies showed that ({sup 3}H)Oxo-M bound to chromaffin cell membranes with a K{sub d} value of 3.08 {times} 10{sup {minus}8}M and a Hill coefficient of 1.00, suggesting one binding site for this ligand. Nicotine inhibited Oxo-M binding in a noncompetitive manner, suggesting that both ligands bind at two different sites on the same receptor. They propose that the receptor on bovine chromaffin cells that is coupled to secretion represents an unusual cholinergic receptor that has both nicotinic and muscarinic features.« less

Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca2+ regulation in airway smooth muscle (ASM)1

PubMed Central

Delmotte, Philippe; Sieck, Gary C.

2015-01-01

Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca2+ ([Ca2+]cyt) responses to agonist stimulation and Ca2+ sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca2+]cyt induced by agonists leads to a transient increase in mitochondrial Ca2+ ([Ca2+]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca2+]mito is blunted despite enhanced [Ca2+]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion–ER/SR coupling, decreased mitochondrial Ca2+ buffering, mitochondrial fragmentation, and increased cell proliferation. PMID:25506723

Effect of Sarizotan, a 5-HT1a and D2-like receptor agonist, on respiration in three mouse models of Rett syndrome.

PubMed

Abdala, Ana P; Lioy, Daniel T; Garg, Saurabh K; Knopp, Sharon J; Paton, Julian F R; Bissonnette, John M

2014-06-01

Disturbances in respiration are common and debilitating features of Rett syndrome (RTT). A previous study showed that the 5-HT1a receptor agonist (R)-(+)-8-hydroxy-dipropyl-2-aminotetralin hydrobromide (8-OH-DPAT) significantly reduced the incidence of apnea and the irregular breathing pattern in a mouse model of the disorder. 8-OH-DPAT, however, is not available for clinical practice. Sarizotan, a full 5-HT1a agonist and a dopamine D2-like agonist/partial agonist, has been used in clinical trials for the treatment of l-dopa-induced dyskinesia. The purpose of this study was to evaluate the effects of sarizotan on respiration and locomotion in mouse models of RTT. Studies were performed in Bird and Jaenisch strains of methyl-CpG-binding protein 2--deficient heterozygous female and Jaenisch strain Mecp2 null male mice and in knock-in heterozygous female mice of a common nonsense mutation (R168X). Respiratory pattern was determined with body plethysmography, and locomotion was determined with open-field recording. Sarizotan or vehicle was administered 20 minutes before a 30-minute recording of respiratory pattern or motor behavior. In separate studies, a crossover design was used to administer the drug for 7 and for 14 days. Sarizotan reduced the incidence of apnea in all three RTT mouse models to approximately 15% of their pretreatment levels. The irregular breathing pattern was corrected to that of wild-type littermates. When administered for 7 or 14 days, apnea decreased to 25 to 33% of the incidence seen with vehicle. This study indicates that the clinically approved drug sarizotan is an effective treatment for respiratory disorders in mouse models of RTT.

Effect of Sarizotan, a 5-HT1a and D2-Like Receptor Agonist, on Respiration in Three Mouse Models of Rett Syndrome

PubMed Central

Abdala, Ana P.; Lioy, Daniel T.; Garg, Saurabh K.; Knopp, Sharon J.; Paton, Julian F. R.

2014-01-01

Disturbances in respiration are common and debilitating features of Rett syndrome (RTT). A previous study showed that the 5-HT1a receptor agonist (R)-(+)-8-hydroxy-dipropyl-2-aminotetralin hydrobromide (8-OH-DPAT) significantly reduced the incidence of apnea and the irregular breathing pattern in a mouse model of the disorder. 8-OH-DPAT, however, is not available for clinical practice. Sarizotan, a full 5-HT1a agonist and a dopamine D2–like agonist/partial agonist, has been used in clinical trials for the treatment of l-dopa–induced dyskinesia. The purpose of this study was to evaluate the effects of sarizotan on respiration and locomotion in mouse models of RTT. Studies were performed in Bird and Jaenisch strains of methyl-CpG–binding protein 2-–deficient heterozygous female and Jaenisch strain Mecp2 null male mice and in knock-in heterozygous female mice of a common nonsense mutation (R168X). Respiratory pattern was determined with body plethysmography, and locomotion was determined with open-field recording. Sarizotan or vehicle was administered 20 minutes before a 30-minute recording of respiratory pattern or motor behavior. In separate studies, a crossover design was used to administer the drug for 7 and for 14 days. Sarizotan reduced the incidence of apnea in all three RTT mouse models to approximately 15% of their pretreatment levels. The irregular breathing pattern was corrected to that of wild-type littermates. When administered for 7 or 14 days, apnea decreased to 25 to 33% of the incidence seen with vehicle. This study indicates that the clinically approved drug sarizotan is an effective treatment for respiratory disorders in mouse models of RTT. PMID:24351104

Comparative molecular field analysis of fenoterol derivatives interacting with an agonist-stabilized form of the β₂-adrenergic receptor.

PubMed

Plazinska, Anita; Pajak, Karolina; Rutkowska, Ewelina; Jimenez, Lucita; Kozocas, Joseph; Koolpe, Gary; Tanga, Mary; Toll, Lawrence; Wainer, Irving W; Jozwiak, Krzysztof

2014-01-01

The β₂-adrenergic receptor (β₂-AR) agonist [(3)H]-(R,R')-methoxyfenoterol was employed as the marker ligand in displacement studies measuring the binding affinities (Ki values) of the stereoisomers of a series of 4'-methoxyfenoterol analogs in which the length of the alkyl substituent at α' position was varied from 0 to 3 carbon atoms. The binding affinities of the compounds were additionally determined using the inverse agonist [(3)H]-CGP-12177 as the marker ligand and the ability of the compounds to stimulate cAMP accumulation, measured as EC₅₀ values, were determined in HEK293 cells expressing the β₂-AR. The data indicate that the highest binding affinities and functional activities were produced by methyl and ethyl substituents at the α' position. The results also indicate that the Ki values obtained using [(3)H]-(R,R')-methoxyfenoterol as the marker ligand modeled the EC₅₀ values obtained from cAMP stimulation better than the data obtained using [(3)H]-CGP-12177 as the marker ligand. The data from this study was combined with data from previous studies and processed using the Comparative Molecular Field Analysis approach to produce a CoMFA model reflecting the binding to the β₂-AR conformation probed by [(3)H]-(R,R')-4'-methoxyfenoterol. The CoMFA model of the agonist-stabilized β₂-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the β₂-AR is governed to a greater extend by steric effects than binding to the [(3)H]-CGP-12177-stabilized conformation(s) in which electrostatic interactions play a more predominate role. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparative Molecular Field Analysis of fenoterol derivatives interacting with an agonist-stabilized form of the β2-adrenergic receptor

PubMed Central

Plazinska, Anita; Pajak, Karolina; Rutkowska, Ewelina; Jimenez, Lucita; Kozocas, Joseph; Koolpe, Gary; Tanga, Mary; Toll, Lawrence; Wainer, Irving W.; Jozwiak, Krzysztof

2014-01-01

The β2-adrenergic receptor (β2-AR) agonist [3H]-(R,R′)-methoxyfenoterol was employed as the marker ligand in displacement studies measuring the binding affinities (Ki values) of the stereoisomers of a series of 4′-methoxyfenoterol analogs in which the length of the alkyl substituent at α′ position was varied from 0 to 3 carbon atoms. The binding affinities of the compounds were additionally determined using the inverse agonist [3H]-CGP-12177 as the marker ligand and the ability of the compounds to stimulate cAMP accumulation, measured as EC50 values, were determined in HEK293 cells expressing the β2-AR. The data indicate that the highest binding affinities and functional activities were produced by methyl and ethyl substituents at the α′ position. The results also indicate that the Ki values obtained using [3H]-(R,R′)-methoxyfenoterol as the marker ligand modeled the EC50 values obtained from cAMP stimulation better than the data obtained using [3H]-CGP-12177 as the marker ligand. The data from this study was combined with data from previous studies and processed using the Comparative Molecular Field Analysis approach to produce a CoMFA model reflecting the binding to the β2-AR conformation probed by [3H]-(R,R′)-4′-methoxyfenoterol. The CoMFA model of the agonist-stabilized β2-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the β2-AR is governed to a greater extend by steric effects than binding to the [3H]-CGP-12177-stabilized conformation(s) in which electrostatic interactions play a more predominate role. PMID:24326276

Agonist-induced β2-adrenoceptor desensitization and downregulation enhance pro-inflammatory cytokine release in human bronchial epithelial cells.

PubMed

Oehme, Susanne; Mittag, Anja; Schrödl, Wieland; Tarnok, Attila; Nieber, Karen; Abraham, Getu

2015-02-01

It is not clear whether increased asthma severity associated with long-term use of β2-adrenoceptor (β2-AR) agonists can be attributed to receptor degradation and increased inflammation. We investigated the cross-talk between β-AR agonist-mediated effects on β2-AR function and expression and cytokine release in human bronchial epithelial cells. In 16HBE14o(-) cells grown in the presence and absence of β-AR agonists and/or antagonists, the β2-AR density was assessed by radioligand binding; the receptor protein and mRNA was determined using laser scanning cytometer and RT-PCR; cAMP generation, the cytokines IL-6 and IL-8 release were determined using AlphaScreen Assay and ELISA, respectively. Isoprenaline (ISO) and salbutamol (Salbu) induced a concentration- and time-dependent significant decrease in β2-AR density. Both Salbu and ISO reduced cAMP generation in a concentration-dependent manner while in same cell culture the IL-6 and IL-8 release was significantly enhanced. These effects were antagonized to a greater extent by ICI 118.551 than by propranolol, but CGP 20712A had no effect. Reduction of the β2-AR protein and mRNA could be seen when cells were treated with ISO for 24 h. Our findings indicate a direct link between cytokine release and altered β2-AR expression and function in airway epithelial cells. β2-AR desensitization and downregulation induced by long-term treatment with β2-AR agonists during asthma may account for adverse reactions also due to enhanced release of pro-inflammatory mediators and should, thus, be considered in asthma therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Gonadotrophin-releasing hormone antagonists for assisted conception.

PubMed

Al-Inany, H G; Abou-Setta, A M; Aboulghar, M

2006-07-19

Gonadotrophin-releasing hormone antagonists produce immediate suppression of gonadotrophin secretion, hence, they can be given after starting gonadotrophin administration. This has resulted in dramatic reduction in the duration of treatment cycle. Two different regimes have been described. The multiple-dose protocol involves the administration of 0.25 mg cetrorelix (or ganirelix) daily from day six to seven of stimulation, or when the leading follicle is 14 to15 mm, until human chorionic gonadotrophin (HCG) administration and the single-dose protocol involves the single administration of 3 mg cetrorelix on day seven to eight of stimulation. Assuming comparable clinical outcome, these benefits would justify a change from the standard long protocol of GnRH agonists to the new GnRH antagonist regimens. To evaluate the evidence regarding the efficacy of gonadotrophin-releasing hormone (GnRH) antagonists with the standard long protocol of GnRH agonists for controlled ovarian hyperstimulation in assisted conception. We searched Cochrane Menstrual Disorders and Subfertility Group's Specialised Register, MEDLINE and EMBASE databases from 1987 to February 2006, and handsearched bibliographies of relevant publications and reviews, and abstracts of scientific meetings. We also contacted manufacturers in the field. Randomized controlled studies comparing different protocols of GnRH antagonists with GnRH agonists in assisted conception cycles were included in this review. Two authors independently assessed trial quality and extracted data. If relevant data were missing or unclear, the authors have been consulted Twenty seven RCTs comparing the GnRH antagonist to the long protocol of GnRH agonist fulfilled the inclusion criteria. Clinical pregnancy rate was significantly lower in the antagonist group. (OR = 0.84, 95% CI = 0.72 - 0.97). The ongoing pregnancy/ live-birth rate showed the same significant lower pregnancy in the antagonist group (P = 0.03; OR 0.82, 95% CI 0.69 to 0.98).However, there was statistically significant reduction in incidence of severe OHSS with antagonist protocol. The relative risk ratio was (P = 0.01; RR 0.61, 95% CI 0.42 to 0.89). In addition, interventions to prevent OHSS (e.g. coasting, cycle cancellation) were administered more frequently in the agonist group (P = 0.03; OR 0.44, 95% CI 0.21 to 0.93). GnRH antagonist protocol is a short and simple protocol with good clinical outcome with significant reduction in incidence of severe ovarian hyperstimulation syndrome and amount of gonadotrophins but the lower pregnancy rate compared to the GnRH agonist long protocol necessitates counseling subfertile couples before recommending change from GnRH agonist to antagonist..

Metabolic effects of orally administered small-molecule agonists of GPR55 and GPR119 in multiple low-dose streptozotocin-induced diabetic and incretin-receptor-knockout mice.

PubMed

McKillop, Aine M; Moran, Brian M; Abdel-Wahab, Yasser H A; Gormley, Noella M; Flatt, Peter R

2016-12-01

Abnormal cannabidiol (Abn-CBD) and AS-1269574 are potent selective agonists for GPR55 and GPR119, respectively. The present study evaluated the actions and ability of these small-molecule agonists to counteract experimental diabetes in mice. Diabetes was induced in NIH Swiss mice by five consecutive daily intraperitoneal injections of 40 mg/(kg body weight) streptozotocin. Diabetic mice received daily oral administration of Abn-CBD or AS-1269574 (0.1 μmol/kg) or saline vehicle (0.9% wt/vol. NaCl) over 28 days. Body weight, food intake, fluid intake, plasma glucose, insulin, glucose tolerance, insulin release, lipid profile and pancreatic morphology were examined. Mechanism of action of agonists was assessed in acute studies using incretin-receptor-knockout mice. Abn-CBD and AS-1269574 decreased plasma glucose (20-26%, p < 0.05) and increased circulating insulin (47-48%, p < 0.05) by 10-28 days, compared with saline-treated diabetic controls. Food intake and polydipsia were reduced by both agonists (21-23%, p < 0.05 and 33-35%, p < 0.01, respectively). After 28 days of treatment, plasma glucagon concentrations were reduced (p < 0.01) and glucose tolerance was enhanced by 19-44% by Abn-CBD (p < 0.05 or p < 0.001) and AS-1269574 (p < 0.05 to p < 0.001). Plasma insulin responses were improved (p < 0.01) and insulin resistance was decreased (p < 0.05 or p < 0.01) in both Abn-CBD- and AS-1269574-treated groups. Triacylglycerols were decreased by 19% with Abn-CBD (p < 0.05) and 32% with AS-1269574 (p < 0.01) while total cholesterol was reduced by 17% (p < 0.01) and 15% (p < 0.05), respectively. Both agonists enhanced beta cell proliferation (p < 0.001) although islet area was unchanged. Acute studies in Gipr- and Glp1r-knockout mice revealed an important role for the glucagon-like peptide 1 (GLP-1) receptor in the actions of both agonists, with the glucose-lowering effects of Abn-CBD also partly mediated through the glucose-dependent insulinotropic peptide (GIP) receptor. These data highlight the potential for fatty acid G-protein-coupled receptor-based therapies as novel insulinotropic and glucose-lowering agents acting partly through the activation of incretin receptors.

Substance P-induced trafficking of beta-arrestins. The role of beta-arrestins in endocytosis of the neurokinin-1 receptor.

PubMed

McConalogue, K; Déry, O; Lovett, M; Wong, H; Walsh, J H; Grady, E F; Bunnett, N W

1999-06-04

Agonist-induced redistribution of G-protein-coupled receptors (GPCRs) and beta-arrestins determines the subsequent cellular responsiveness to agonists and is important for signal transduction. We examined substance P (SP)-induced trafficking of beta-arrestin1 and the neurokinin-1 receptor (NK1R) in KNRK cells in real time using green fluorescent protein. Green fluorescent protein did not alter function or localization of the NK1R or beta-arrestin1. SP induced (a) striking and rapid (<1 min) translocation of beta-arrestin1 from the cytosol to the plasma membrane, which preceded NK1R endocytosis; (b) redistribution of the NK1R and beta-arrestin1 into the same endosomes containing SP and the transferrin receptor (2-10 min); (c) prolonged colocalization of the NK1R and beta-arrestin1 in endosomes (>60 min); (d) gradual resumption of the steady state distribution of the NK1R at the plasma membrane and beta-arrestin1 in the cytosol (4-6 h). SP stimulated a similar redistribution of immunoreactive beta-arrestin1 and beta-arrestin2. In contrast, SP did not affect Galphaq/11 distribution, which remained at the plasma membrane. Expression of the dominant negative beta-arrestin319-418 inhibited SP-induced endocytosis of the NK1R. Thus, SP induces rapid translocation of beta-arrestins to the plasma membrane, where they participate in NK1R endocytosis. beta-Arrestins colocalize with the NK1R in endosomes until the NK1R recycles and beta-arrestins return to the cytosol.

The selective V1a receptor agonist selepressin (FE 202158) blocks vascular leak in ovine severe sepsis

PubMed Central

Wiśniewska, Halina; Traber, Lillian D.; Lin, ChiiDean; Fan, Juanjuan; Hawkins, Hal K.; Cox, Robert A.; Wiśniewski, Kazimierz; Schteingart, Claudio D.; Landry, Donald W.; Rivière, Pierre J.-M.; Traber, Daniel L.

2014-01-01

Objective To determine if the selective vasopressin type 1a receptor (V1aR) agonist selepressin (FE 202158) is as effective as the mixed V1a/V2 receptor (V1aR/V2R) agonist vasopressor hormone arginine vasopressin (AVP) when used as a titrated first-line vasopressor therapy in an ovine model of Pseudomonas aeruginosa pneumonia-induced severe sepsis. Design Prospective, randomized, controlled laboratory experiment. Setting University animal research facility. Subjects Forty-five chronically instrumented sheep. Interventions Sheep were anesthetized, insufflated with cooled cotton smoke via tracheostomy, and P. aeruginosa were instilled into their airways. They were then placed on assisted ventilation, awakened, and resuscitated with lactated Ringer's solution titrated to maintain hematocrit ± 3% from baseline levels. If, despite fluid management, mean arterial pressure (MAP) fell by > 10 mm Hg from baseline levels, a continuous i.v. infusion of AVP or selepressin was titrated to raise and maintain MAP within 10 mm Hg of baseline. Effects of combination treatment of selepressin with the selective V2R agonist desmopressin were similarly investigated. Measurements and Main Results In septic sheep, MAP fell by ~30 mm Hg, systemic vascular resistance index (SVRI) decreased by ~50%, and ~7 L of fluid were retained over 24 h; this fluid accumulation was partially reduced by AVP and almost completely blocked by selepressin; combined infusion of selepressin and desmopressin increased fluid accumulation to levels similar to AVP treatment. Conclusions Resuscitation with the selective V1aR agonist selepressin blocked vascular leak more effectively than the mixed V1aR/V2R agonist AVP because of its lack of agonist activity at the V2R. PMID:24674922

Central nervous system neuropeptide Y signaling via the Y1 receptor partially dissociates feeding behavior from lipoprotein metabolism in lean rats.

PubMed

Rojas, Jennifer M; Stafford, John M; Saadat, Sanaz; Printz, Richard L; Beck-Sickinger, Annette G; Niswender, Kevin D

2012-12-15

Elevated plasma triglyceride (TG) levels contribute to an atherogenic dyslipidemia that is associated with obesity, diabetes, and metabolic syndrome. Numerous models of obesity are characterized by increased central nervous system (CNS) neuropeptide Y (NPY) tone that contributes to excess food intake and obesity. Previously, we demonstrated that intracerebroventricular (icv) administration of NPY in lean fasted rats also elevates hepatic production of very low-density lipoprotein (VLDL)-TG. Thus, we hypothesize that elevated CNS NPY action contributes to not only the pathogenesis of obesity but also dyslipidemia. Here, we sought to determine whether the effects of NPY on feeding and/or obesity are dissociable from effects on hepatic VLDL-TG secretion. Pair-fed, icv NPY-treated, chow-fed Long-Evans rats develop hypertriglyceridemia in the absence of increased food intake and body fat accumulation compared with vehicle-treated controls. We then modulated CNS NPY signaling by icv injection of selective NPY receptor agonists and found that Y1, Y2, Y4, and Y5 receptor agonists all induced hyperphagia in lean, ad libitum chow-fed Long-Evans rats, with the Y2 receptor agonist having the most pronounced effect. Next, we found that at equipotent doses for food intake NPY Y1 receptor agonist had the most robust effect on VLDL-TG secretion, a Y2 receptor agonist had a modest effect, and no effect was observed for Y4 and Y5 receptor agonists. These findings, using selective agonists, suggest the possibility that the effect of CNS NPY signaling on hepatic VLDL-TG secretion may be relatively dissociable from effects on feeding behavior via the Y1 receptor.

A pharmacological study of NK1 and NK2 tachykinin receptor characteristics in the rat isolated urinary bladder.

PubMed Central

Hall, J. M.; Flowers, J. M.; Morton, I. K.

1992-01-01

1. We have estimated potencies of tachykinin receptor agonist and antagonist analogues in order to determine the recognition characteristics of tachykinin receptors mediating phasic contractile responses of the rat isolated urinary bladder in vitro. 2. The NK1-selective synthetic agonists, substance P methyl ester and GR73632, the synthetic NK2-selective agonists [beta-Ala8]-NKA(4-10) and GR64349, and the mammalian tachykinins, neurokinin A and neurokinin B, were assayed relative to substance P and were found to be approximately equipotent. The NK3-selective agonist, senktide, was inactive (10 microM). 3. Potencies of all these agonists were not significantly different (P > 0.05) when experiments were carried out in the presence of the neutral endopeptidase inhibitor, phosphoramidon, and the kininase II inhibitor, enalaprilat (both 1 microM). 4. The NK1-selective antagonist, GR82334, inhibited responses to substance P methyl ester in a competitive manner in the rat urinary bladder and the rat ileum, and also in the guinea-pig ileum. Markedly different pKB estimates were obtained in the rat bladder (6.38) and rat ileum (6.56) compared to the guinea-pig ileum (7.42). GR82334 (3 microM) was inactive against responses of the rat bladder to [beta-Ala8]-NKA(4-10). 5. The NK1-selective antagonist (+/-)-CP-96,345 also inhibited responses of the rat bladder and guinea-pig ileum to substance P methyl ester; however, in the rat bladder at 1 microM, this antagonist reversibly inhibited responses both to the NK2-selective agonist [beta-Ala8]-NKA(4-10) and to the muscarinic agonist carbachol (P < or = 0.01), thus showing evidence of some non-selective depressant actions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1282072

The β2 agonist terbutaline specifically decreases pulmonary arterial pressure under normoxia and hypoxia via α adrenoceptor antagonism.

PubMed

Neumann, Vanessa; Knies, Ralf; Seidinger, Alexander; Simon, Annika; Lorenz, Kristina; Matthey, Michaela; Breuer, Johannes; Wenzel, Daniela

2018-05-01

Pulmonary hypertension is a severe, incurable disease with a poor prognosis. Although treatment regimens have improved during the last 2 decades, current pharmacologic strategies are limited and focus on the modulation of only a few pathways related to endothelin, NO, and prostacyclin signaling. Therefore, the identification of novel molecular targets is urgently needed. We found that the β 2 adrenoceptor (AR) agonists terbutaline (TER) and salbutamol induced a dose-dependent vasorelaxation in large pulmonary arteries but not aortas of mouse. This effect was found to be independent of β ARs and the endothelium but was determined by the type of the preconstrictor. Vasodilation by β 2 AR agonists occurred after pretreatment of pulmonary arteries with phenylephrine and serotonin, both agonists of α 1 ARs, but was absent after preconstriction with the thromboxane analog U46619. These data indicated α-adrenolytic activity of β 2 AR agonists, which was confirmed by a right shift of the phenylephrine dose-response curve by TER. This effect was physiologically relevant because TER also relaxed small intrapulmonary arteries in lung slices and diminished pulmonary arterial pressure in an isolated perfused lung model under normoxia and hypoxia. Finally, TER applied as an aerosol also selectively decreased pulmonary arterial pressure without effects on systemic blood pressure and heart rate in mouse in vivo. Thus, β 2 AR agonists display α-adrenolytic activity in pulmonary arteries ex vivo and in vivo, and may provide a novel option to reduce pulmonary arterial pressure in pulmonary hypertension.-Neumann, V., Knies, R., Seidinger, A., Simon, A., Lorenz, K., Matthey, M., Breuer, J., Wenzel, D. The β 2 agonist terbutaline specifically decreases pulmonary arterial pressure under normoxia and hypoxia via α adrenoceptor antagonism.

Enhancement of Attentional Performance by Selective Stimulation of α4β2* nAChRs: Underlying Cholinergic Mechanisms

PubMed Central

Howe, William M; Ji, Jinzhao; Parikh, Vinay; Williams, Sarah; Mocaër, Elisabeth; Trocmé-Thibierge, Caryn; Sarter, Martin

2010-01-01

Impairments in attention are a major component of the cognitive symptoms of neuropsychiatric and neurodegenerative disorders. Using an operant sustained attention task (SAT), including a distractor condition (dSAT), we assessed the putative pro-attentional effects of the selective α4β2* nicotinic acetylcholine receptor (nAChR) agonist S 38232 in comparison with the non-selective agonist nicotine. Neither drug benefited SAT performance. However, in interaction with the increased task demands implemented by distractor presentation, the selective agonist, but not nicotine, enhanced the detection of signals during the post-distractor recovery period. This effect is consistent with the hypothesis that second-long increases in cholinergic activity (‘transients') mediate the detection of cues and that nAChR agonists augment such transients. Electrochemical recordings of prefrontal cholinergic transients evoked by S 38232 and nicotine indicated that the α4β2* nAChR agonist evoked cholinergic transients that were characterized by a faster rise time and more rapid decay than those evoked by nicotine. Blockade of the α7 nAChR ‘sharpens' nicotine-evoked transients; therefore, we determined the effects of co-administration of nicotine and the α7 nAChR antagonist methyllycaconitine on dSAT performance. Compared with vehicle and nicotine alone, this combined treatment significantly enhanced the detection of signals. These results indicate that compared with nicotine, α4β2* nAChR agonists significantly enhance attentional performance and that the dSAT represents a useful behavioral screening tool. The combined behavioral and electrochemical evidence supports the hypothesis that nAChR agonist-evoked cholinergic transients, which are characterized by rapid rise time and fast decay, predict robust drug-induced enhancement of attentional performance. PMID:20147893

Ligand requirements for involvement of PKCε in synergistic analgesic interactions between spinal μ and δ opioid receptors.

PubMed

Schuster, D J; Metcalf, M D; Kitto, K F; Messing, R O; Fairbanks, C A; Wilcox, G L

2015-01-01

We recently found that PKCε was required for spinal analgesic synergy between two GPCRs, δ opioid receptors and α2 A adrenoceptors, co-located in the same cellular subpopulation. We sought to determine if co-delivery of μ and δ opioid receptor agonists would similarly result in synergy requiring PKCε. Combinations of μ and δ opioid receptor agonists were co-administered intrathecally by direct lumbar puncture to PKCε-wild-type (PKCε-WT) and -knockout (PKCε-KO) mice. Antinociception was assessed using the hot-water tail-flick assay. Drug interactions were evaluated by isobolographic analysis. All agonists produced comparable antinociception in both PKCε-WT and PKCε-KO mice. Of 19 agonist combinations that produced analgesic synergy, only 3 required PKCε for a synergistic interaction. In these three combinations, one of the agonists was morphine, although not all combinations involving morphine required PKCε. Morphine + deltorphin II and morphine + deltorphin I required PKCε for synergy, whereas a similar combination, morphine + deltorphin, did not. Additionally, morphine + oxymorphindole required PKCε for synergy, whereas a similar combination, morphine + oxycodindole, did not. We discovered biased agonism for a specific signalling pathway at the level of spinally co-delivered opioid agonists. As the bias is only revealed by an appropriate ligand combination and cannot be accounted for by a single drug, it is likely that the receptors these agonists act on are interacting with each other. Our results support the existence of μ and δ opioid receptor heteromers at the spinal level in vivo. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British Pharmacological Society.

β2-Agonist Induced cAMP Is Decreased in Asthmatic Airway Smooth Muscle Due to Increased PDE4D

PubMed Central

Trian, Thomas; Burgess, Janette K.; Niimi, Kyoko; Moir, Lyn M.; Ge, Qi; Berger, Patrick; Liggett, Stephen B.; Black, Judith L.; Oliver, Brian G.

2011-01-01

Background and Objective Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known. Objective To characterize the potential defect in β-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism. Methods We examined β2-adrenergic (β2AR) receptor expression and basal β-agonist and forskolin (direct activator of adenylyl cyclase) stimulated cAMP production in asthmatic cultured ASM (n = 15) and non-asthmatic ASM (n = 22). Based on these results, PDE activity, PDE4D expression and cell proliferation were determined. Results In the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ∼50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ∼2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM. Conclusion Decreased β-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal β-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be “hard-wired” into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed. PMID:21611147

In silico characterization of binding mode of CCR8 inhibitor: homology modeling, docking and membrane based MD simulation study.

PubMed

Gadhe, Changdev G; Balupuri, Anand; Cho, Seung Joo

2015-01-01

Human CC-chemokine receptor 8 (CCR8) is a crucial drug target in asthma that belongs to G-protein-coupled receptor superfamily, which is characterized by seven transmembrane helices. To date, there is no X-ray crystal structure available for CCR8; this hampers active research on the target. Molecular basis of interaction mechanism of antagonist with CCR8 remains unclear. In order to provide binding site information and stable binding mode, we performed modeling, docking and molecular dynamics (MD) simulation of CCR8. Docking study of biaryl-ether-piperidine derivative (13C) was performed inside predefined CCR8 binding site to get the representative conformation of 13C. Further, MD simulations of receptor and complex (13C-CCR8) inside dipalmitoylphosphatidylcholine lipid bilayers were performed to explore the effect of lipids. Results analyses showed that the Gln91, Tyr94, Cys106, Val109, Tyr113, Cys183, Tyr184, Ser185, Lys195, Thr198, Asn199, Met202, Phe254, and Glu286 were conserved in both docking and MD simulations. This indicated possible role of these residues in CCR8 antagonism. However, experimental mutational studies on these identified residues could be effective to confirm their importance in CCR8 antagonism. Furthermore, calculated Coulombic interactions represented the crucial roles of Glu286, Lys195, and Tyr113 in CCR8 antagonism. Important residues identified in this study overlap with the previous non-peptide agonist (LMD-009) binding site. Though, the non-peptide agonist and currently studied inhibitor (13C) share common substructure, but they differ in their effects on CCR8. So, to get more insight into their agonist and antagonist effects, further side-by-side experimental studies on both agonist (LMD-009) and antagonist (13C) are suggested.

The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway.

PubMed

Jacobsen, Stine Engesgaard; Ammendrup-Johnsen, Ina; Jansen, Anna Mai; Gether, Ulrik; Madsen, Kenneth Lindegaard; Bräuner-Osborne, Hans

2017-04-28

The class C G protein-coupled receptor GPRC6A is a putative nutrient-sensing receptor and represents a possible new drug target in metabolic disorders. However, the specific physiological role of this receptor has yet to be identified, and the mechanisms regulating its activity and cell surface availability also remain enigmatic. In the present study, we investigated the trafficking properties of GPRC6A by use of both a classical antibody feeding internalization assay in which cells were visualized using confocal microscopy and a novel internalization assay that is based on real-time measurements of fluorescence resonance energy transfer. Both assays revealed that GPRC6A predominantly undergoes constitutive internalization, whereas the agonist-induced effects were imperceptible. Moreover, postendocytic sorting was investigated by assessing the co-localization of internalized GPRC6A with selected Rab protein markers. Internalized GPRC6A was mainly co-localized with the early endosome marker Rab5 and the long loop recycling endosome marker Rab11 and to a much lesser extent with the late endosome marker Rab7. This suggests that upon agonist-independent internalization, GPRC6A is recycled via the Rab11-positive slow recycling pathway, which may be responsible for ensuring a persistent pool of GPRC6A receptors at the cell surface despite chronic agonist exposure. Distinct trafficking pathways have been reported for several of the class C receptors, and our results thus substantiate that non-canonical trafficking mechanisms are a common feature for the nutrient-sensing class C family that ensure functional receptors in the cell membrane despite prolonged agonist exposure. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway

PubMed Central

Jacobsen, Stine Engesgaard; Ammendrup-Johnsen, Ina; Jansen, Anna Mai; Gether, Ulrik; Madsen, Kenneth Lindegaard; Bräuner-Osborne, Hans

2017-01-01

The class C G protein-coupled receptor GPRC6A is a putative nutrient-sensing receptor and represents a possible new drug target in metabolic disorders. However, the specific physiological role of this receptor has yet to be identified, and the mechanisms regulating its activity and cell surface availability also remain enigmatic. In the present study, we investigated the trafficking properties of GPRC6A by use of both a classical antibody feeding internalization assay in which cells were visualized using confocal microscopy and a novel internalization assay that is based on real-time measurements of fluorescence resonance energy transfer. Both assays revealed that GPRC6A predominantly undergoes constitutive internalization, whereas the agonist-induced effects were imperceptible. Moreover, postendocytic sorting was investigated by assessing the co-localization of internalized GPRC6A with selected Rab protein markers. Internalized GPRC6A was mainly co-localized with the early endosome marker Rab5 and the long loop recycling endosome marker Rab11 and to a much lesser extent with the late endosome marker Rab7. This suggests that upon agonist-independent internalization, GPRC6A is recycled via the Rab11-positive slow recycling pathway, which may be responsible for ensuring a persistent pool of GPRC6A receptors at the cell surface despite chronic agonist exposure. Distinct trafficking pathways have been reported for several of the class C receptors, and our results thus substantiate that non-canonical trafficking mechanisms are a common feature for the nutrient-sensing class C family that ensure functional receptors in the cell membrane despite prolonged agonist exposure. PMID:28280242

Selective κ receptor partial agonist HS666 produces potent antinociception without inducing aversion after i.c.v. administration in mice.

PubMed

Spetea, Mariana; Eans, Shainnel O; Ganno, Michelle L; Lantero, Aquilino; Mairegger, Michael; Toll, Lawrence; Schmidhammer, Helmut; McLaughlin, Jay P

2017-08-01

The κ receptor has a central role in modulating neurotransmission in central and peripheral neuronal circuits that subserve pain and other behavioural responses. Although κ receptor agonists do not produce euphoria or lead to respiratory suppression, they induce dysphoria and sedation. We hypothesized that brain-penetrant κ receptor ligands possessing biased agonism towards G protein signalling over β-arrestin2 recruitment would produce robust antinociception with fewer associated liabilities. Two new diphenethylamines with high κ receptor selectivity, HS665 and HS666, were assessed following i.c.v. administration in mouse assays of antinociception with the 55°C warm-water tail withdrawal test, locomotor activity in the rotorod and conditioned place preference. The [ 35 S]-GTPγS binding and β-arrestin2 recruitment in vitro assays were used to characterize biased agonism. HS665 (κ receptor agonist) and HS666 (κ receptor partial agonist) demonstrated dose-dependent antinociception after i.c.v. administration mediated by the κ receptor. These highly selective κ receptor ligands displayed varying biased signalling towards G protein coupling in vitro, consistent with a reduced liability profile, reflected by reduced sedation and absence of conditioned place aversion for HS666. HS665 and HS666 activate central κ receptors to produce potent antinociception, with HS666 displaying pharmacological characteristics of a κ receptor analgesic with reduced liability for aversive effects correlating with its low efficacy in the β-arrestin2 signalling pathway. Our data provide further understanding of the contribution of central κ receptors in pain suppression, and the prospect of dissociating the antinociceptive effects of HS665 and HS666 from κ receptor-mediated adverse effects. © 2017 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.