The Experts below are selected from a list of 2352 Experts worldwide ranked by ideXlab platform
Richard R Neubig - One of the best experts on this subject based on the ideXlab platform.
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The role of regulator of G protein signaling 4 in delta-opioid receptor-mediated behaviors
Psychopharmacology, 2017Co-Authors: Isaac J. Dripps, Richard R Neubig, John R Traynor, Qin Wang, Kenner C. Rice, Emily M JutkiewiczAbstract:Rationale Regulator of G protein signaling (RGS) proteins act as negative modulators of G protein signaling. RGS4 has been shown to negatively modulate G protein signaling mediated by the delta opioid receptor (DOPr) in vitro. However, the role of RGS4 in modulating DOPr-mediated behaviors in vivo has not been elucidated. Objective The aim of this study was to compare the ability of the DOPr agonist SNC80 to induce DOPr-mediated antinociception, antihyperalgesia, antidepressant-like effects, and convulsions in wild-type and RGS4 knockout mice. Methods Antinociception was assessed in the acetic acid stretch assay. Antihyperalgesia was measured in a nitroglycerin-induced thermal hyperalgesia assay. Antidepressant-like effects were evaluated in the forced swim and tail suspension tests. Mice were also observed for convulsive activity post-SNC80 treatment. SNC80-induced phosphorylation of MAP kinase in striatal tissue from RGS4 wild-type and knockout mice was quantified by Western blot. DOPr number from forebrain tissue was measured using [^3H]DPDPE saturation binding. Results Elimination of RGS4 potentiated SNC80-induced antinociception and antihyperalgesia. SNC80-induced antidepressant-like effects were potentiated in RGS4 knockout mice in the forced swim test but not in the tail suspension test. Additionally, RGS4 knockout did not alter SNC80-induced convulsions. SNC80-induced phosphorylation of MAP kinase was potentiated in striatum from RGS4 knockout mice. Loss of RGS4 did not affect total DOPr number. Conclusions Overall, these findings demonstrate that reduction of RGS4 functionally may increase the therapeutic index of SNC80. These results provide the first evidence of differential regulation of DOPr-mediated behaviors by RGS proteins and G protein signaling pathways.
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RGS4 differentially regulates antidepressant and locomotor behaviors in vivo
The FASEB Journal, 2015Co-Authors: Cienna Nielsen, Richard R Neubig, John R Traynor, Babak Shirvani, Amanda Baun, John Devilbiss, Lilyann Nyangau, Lena Chung, Jeffery N TalbotAbstract:Evidence suggests the antidepressant actions of serotonin (5-HT) are modulated by regulators of G protein signaling (RGS) proteins. Specifically, we have shown that upregulation of cortical RGS4 (mRNA and protein) occurs in response to genetic disruption of RGS protein activity at the GTP-binding protein Gαi2 and following chronic administration of serotonin-selective reuptake inhibitors (SSRIs). Genetic deletion of RGS4 (RGS4(-/-)) had no effect on basal antidepressant-like behaviors or generalized locomotor activity. However, RGS4(-/-) mice exhibited decreased antidepressant-like responses to the SSRI fluoxetine, whereas desipramine, a selective inhibitor of norepinephrine reuptake, was fully efficacious. Conversely, the regulation of locomotor activity by desipramine was augmented in RGS4(-/-) animals relative to vehicle-treated controls. RGS4 expression had no effect on locomotor activity following fluoxetine administration. Overall, these data suggest altered expression of RGS4 differentially influen...
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nmr methods for detection of small molecule binding to RGS4
Methods in Enzymology, 2013Co-Authors: Andrew J Storaska, Richard R NeubigAbstract:Abstract The duration and amplitude of G-protein-coupled receptor (GPCR) signaling is controlled by regulator of G-protein signaling (RGS) proteins. The 20 RGS family members act as GTPase accelerating proteins through their interaction with the Gα subunit of the Gαβγ heterotrimer. Their influence over GPCR signaling has attracted many to these proteins as advantageous therapeutic targets. The nature of the RGS structure has proven to be difficult to target with small molecules using traditional high-throughput screening methods. This chapter describes NMR methods for studying small molecule interactions on RGS4. These methods can detect ligand binding without the requirement for an effect on protein function. Furthermore, the sensitivity of NMR permits detection of weaker protein–ligand interactions, such as those found with smaller fragment compounds. Fragment-based screening may be path forward to identifying a number of active small molecules toward RGS proteins. Methods and considerations for running a fragment-based screen on RGS4 using NMR are outlined in this section.
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a nanomolar potency small molecule inhibitor of regulator of g protein signaling proteins
Biochemistry, 2011Co-Authors: Levi L Blazer, Haoming Zhang, Emma M Casey, Stephen M Husbands, Richard R NeubigAbstract:Regulators of G-protein signaling (RGS) proteins are potent negative modulators of signal transduction through G-protein-coupled receptors. They function by binding to activated (GTP-bound) Gα subunits and accelerating the rate of GTP hydrolysis. Modulation of RGS activity by small molecules is an attractive mechanism for fine-tuning GPCR signaling for therapeutic and research purposes. Here we describe the pharmacologic properties and mechanism of action of CCG-50014, the most potent small molecule RGS inhibitor to date. It has an IC50 for RGS4 of 30 nM and is >20-fold selective for RGS4 over other RGS proteins. CCG-50014 binds covalently to the RGS, forming an adduct on two cysteine residues located in an allosteric regulatory site. It is not a general cysteine alkylator as it does not inhibit activity of the cysteine protease papain at concentrations >3000-fold higher than those required to inhibit RGS4 function. It is also >1000-fold more potent as an RGS4 inhibitor than are the cysteine alkylators N-...
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a covalent peptide inhibitor of RGS4 identified in a focused one bead one compound library screen
BMC Pharmacology, 2009Co-Authors: Rebecca A Roof, David L. Roman, Samuel T Clements, Katarzyna Sobczykkojiro, Levi L Blazer, Henry I Mosberg, Richard R NeubigAbstract:Background Regulators of G protein signaling (RGSs) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors (GPCRs). The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We recently described a focused one-bead, one-compound (OBOC) library screen to identify peptide inhibitors of RGS4. Here we extend our observations to include another peptide with a different mechanism of action.
Kirk M Druey - One of the best experts on this subject based on the ideXlab platform.
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RGS4 overexpression in lung attenuates airway hyperresponsiveness in mice
American Journal of Respiratory Cell and Molecular Biology, 2018Co-Authors: Laura A Madigan, Gordon S Wong, Elizabeth M Gordon, Weisheng Chen, Nariman Balenga, Cynthia J Koziolwhite, Reynold A Panettieri, Stewart J Levine, Kirk M DrueyAbstract:A cardinal feature of asthma is airway hyperresponsiveness (AHR) to spasmogens, many of which activate G protein–coupled receptors (GPCRs) on airway smooth muscle (ASM) cells. Asthma subtypes associated with allergy are characterized by eosinophilic inflammation in the lung due to the type 2 immune response to allergens and proinflammatory mediators that promote AHR. The degree to which intrinsic abnormalities of ASM contribute to this phenotype remains unknown. The regulators of G protein signaling (RGS) proteins are a large group of intracellular proteins that inhibit GPCR signaling pathways. RGS2- and RGS5-deficient mice develop AHR spontaneously. Although RGS4 is upregulated in ASM from patients with severe asthma, the effects of increased RGS4 expression on AHR in vivo are unknown. Here, we examined the impact of forced RGS4 overexpression in lung on AHR using transgenic (Tg) mice. Tg RGS4 was expressed in bronchial epithelium and ASM in vivo, and protein expression in lung was increased at least 4-f...
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R4 Regulator of G Protein Signaling (RGS) Proteins in Inflammation and Immunity
The AAPS Journal, 2016Co-Authors: Eunice C. Chan, Kirk M DrueyAbstract:G protein-coupled receptors (GPCRs) have important functions in both innate and adaptive immunity, with the capacity to bridge interactions between the two arms of the host responses to pathogens through direct recognition of secreted microbial products or the by-products of host cells damaged by pathogen exposure. In the mid-1990s, a large group of intracellular proteins was discovered, the regulator of G protein signaling (RGS) family, whose main, but not exclusive, function appears to be to constrain the intensity and duration of GPCR signaling. The R4/B subfamily—the focus of this review—includes RGS1–5, 8, 13, 16, 18, and 21, which are the smallest RGS proteins in size, with the exception of RGS3. Prominent roles in the trafficking of B and T lymphocytes and macrophages have been described for RGS1, RGS13, and RGS16, while RGS18 appears to control platelet and osteoclast functions. Additional G protein independent functions of RGS13 have been uncovered in gene expression in B lymphocytes and mast cell-mediated allergic reactions. In this review, we discuss potential physiological roles of this RGS protein subfamily, primarily in leukocytes having central roles in immune and inflammatory responses. We also discuss approaches to target RGS proteins therapeutically, which represents a virtually untapped strategy to combat exaggerated immune responses leading to inflammation.
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an RGS4 mediated phenotypic switch of bronchial smooth muscle cells promotes fixed airway obstruction in asthma
PLOS ONE, 2012Co-Authors: Gautam Damera, Kirk M Druey, Philip R Cooper, Vera P Krymskaya, Roy J Soberman, Yassine Amrani, Toshinori Hoshi, Christopher E Brightling, Reynold A PanettieriAbstract:In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.
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RGS4 and rgs2 bind coatomer and inhibit copi association with golgi membranes and intracellular transport
Molecular Biology of the Cell, 2000Co-Authors: Brandon M Sullivan, John H Kehrl, Dennis Brown, Kimberly J Harrisonlavoie, Vladimir Marshansky, D A Ausiello, Kirk M DrueyAbstract:COPI, a protein complex consisting of coatomer and the small GTPase ARF1, is an integral component of some intracellular transport carriers. The association of COPI with secretory membranes has been implicated in the maintenance of Golgi integrity and the normal functioning of intracellular transport in eukaryotes. The regulator of G protein signaling, RGS4, interacted with the COPI subunit β′-COP in a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound purified recombinant β′-COP in vitro. Endogenous cytosolic RGS4 from NG108 cells and RGS2 from HEK293T cells cofractionated with the COPI complex by gel filtration. Binding of β′-COP to RGS4 occurred through two dilysine motifs in RGS4, similar to those contained in some aminoglycoside antibiotics that are known to bind coatomer. RGS4 inhibited COPI binding to Golgi membranes independently of its GTPase-accelerating activity on Giα. In RGS4-transfected LLC-PK1 cells, the amount of COPI in the Golgi region was considerably reduced compared with that in wild-type cells, but there was no detectable difference in the amount of either Golgi-associated ARF1 or the integral Golgi membrane protein giantin, indicating that Golgi integrity was preserved. In addition, RGS4 expression inhibited trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and impaired secretion of placental alkaline phosphatase from HEK293T cells. The inhibitory effect of RGS4 in these assays was independent of GTPase-accelerating activity but correlated with its ability to bind COPI. Thus, these data support the hypothesis that these RGS proteins sequester coatomer in the cytoplasm and inhibit its recruitment onto Golgi membranes, which may in turn modulate Golgi–plasma membrane or intra-Golgi transport.
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the regulator of g protein signaling RGS4 selectively enhances α2a adreoreceptor stimulation of the gtpase activity of go1α and gi2α
Journal of Biological Chemistry, 2000Co-Authors: Antonella Cavalli, Kirk M Druey, Graeme MilliganAbstract:Abstract Agonist-stimulated high affinity GTPase activity of fusion proteins between the α2A-adrenoreceptor and the α subunits of forms of the G proteins Gi1, Gi2, Gi3, and Go1, modified to render them insensitive to the action of pertussis toxin, was measured following transient expression in COS-7 cells. Addition of a recombinant regulator of G protein signaling protein, RGS4, did not significantly affect basal GTPase activity nor agonist stimulation of the fusion proteins containing Gαi1and Gαi3 but markedly enhanced agonist-stimulation of the proteins containing Gαi2 and Gαo1. The effect of RGS4 on the α2A-adrenoreceptor-Gαo1 fusion protein was concentration-dependent with EC50 of 30 ± 3 nm and the potency of the receptor agonist UK14304 was reduced 3-fold by 100 nm RGS4. Equivalent reconstitution with Asn88-Ser RGS4 failed to enhance agonist function on the α2A-adrenoreceptor-Gαo1 or α2A-adrenoreceptor-Gαi2 fusion proteins. Enzyme kinetic analysis of the GTPase activity of the α2A-adrenoreceptor-Gαo1 and α2A-adrenoreceptor-Gαi2 fusion proteins demonstrated that RGS4 both substantially increased GTPaseV max and significantly increasedK m of the fusion proteins for GTP. The increase inK m for GTP was dependent upon RGS4 amount and is consistent with previously proposed mechanisms of RGS function. Agonist-stimulated GTPase turnover number in the presence of 100 nm RGS4 was substantially higher for α2A-adrenoreceptor-Gαo1 than for α2A-adrenoreceptor-Gαi2. These studies demonstrate that although RGS4 has been described as a generic stimulator of the GTPase activity of Gi-family G proteins, selectivity of this interaction and quantitative variation in its function can be monitored in the presence of receptor activation of the G proteins.
John R Traynor - One of the best experts on this subject based on the ideXlab platform.
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regulator of g protein signaling rgs protein modulation of opioid receptor signaling as a potential target for pain management
Frontiers in Molecular Neuroscience, 2020Co-Authors: Nicolas B Senese, Ram Kandasamy, Kelsey E Kochan, John R TraynorAbstract:: Opioid drugs are the gold standard for the management of pain, but their use is severely limited by dangerous and unpleasant side effects. All clinically available opioid analgesics bind to and activate the mu-opioid receptor (MOR), a heterotrimeric G-protein-coupled receptor, to produce analgesia. The activity of these receptors is modulated by a family of intracellular RGS proteins or regulators of G-protein signaling proteins, characterized by the presence of a conserved RGS Homology (RH) domain. These proteins act as negative regulators of G-protein signaling by serving as GTPase accelerating proteins or GAPS to switch off signaling by both the Gα and βγ subunits of heterotrimeric G-proteins. Consequently, knockdown or knockout of RGS protein activity enhances signaling downstream of MOR. In this review we discuss current knowledge of how this activity, across the different families of RGS proteins, modulates MOR activity, as well as activity of other members of the opioid receptor family, and so pain and analgesia in animal models, with particular emphasis on RGS4 and RGS9 families. We discuss inhibition of RGS proteins with small molecule inhibitors that bind to sensitive cysteine moieties in the RH domain and the potential for targeting this family of intracellular proteins as adjuncts to provide an opioid sparing effect or as standalone analgesics by promoting the activity of endogenous opioid peptides. Overall, we conclude that RGS proteins may be a novel drug target to provide analgesia with reduced opioid-like side effects, but that much basic work is needed to define the roles for specific RGS proteins, particularly in chronic pain, as well as a need to develop newer inhibitors.
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The role of regulator of G protein signaling 4 in delta-opioid receptor-mediated behaviors
Psychopharmacology, 2017Co-Authors: Isaac J. Dripps, Richard R Neubig, John R Traynor, Qin Wang, Kenner C. Rice, Emily M JutkiewiczAbstract:Rationale Regulator of G protein signaling (RGS) proteins act as negative modulators of G protein signaling. RGS4 has been shown to negatively modulate G protein signaling mediated by the delta opioid receptor (DOPr) in vitro. However, the role of RGS4 in modulating DOPr-mediated behaviors in vivo has not been elucidated. Objective The aim of this study was to compare the ability of the DOPr agonist SNC80 to induce DOPr-mediated antinociception, antihyperalgesia, antidepressant-like effects, and convulsions in wild-type and RGS4 knockout mice. Methods Antinociception was assessed in the acetic acid stretch assay. Antihyperalgesia was measured in a nitroglycerin-induced thermal hyperalgesia assay. Antidepressant-like effects were evaluated in the forced swim and tail suspension tests. Mice were also observed for convulsive activity post-SNC80 treatment. SNC80-induced phosphorylation of MAP kinase in striatal tissue from RGS4 wild-type and knockout mice was quantified by Western blot. DOPr number from forebrain tissue was measured using [^3H]DPDPE saturation binding. Results Elimination of RGS4 potentiated SNC80-induced antinociception and antihyperalgesia. SNC80-induced antidepressant-like effects were potentiated in RGS4 knockout mice in the forced swim test but not in the tail suspension test. Additionally, RGS4 knockout did not alter SNC80-induced convulsions. SNC80-induced phosphorylation of MAP kinase was potentiated in striatum from RGS4 knockout mice. Loss of RGS4 did not affect total DOPr number. Conclusions Overall, these findings demonstrate that reduction of RGS4 functionally may increase the therapeutic index of SNC80. These results provide the first evidence of differential regulation of DOPr-mediated behaviors by RGS proteins and G protein signaling pathways.
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RGS4 differentially regulates antidepressant and locomotor behaviors in vivo
The FASEB Journal, 2015Co-Authors: Cienna Nielsen, Richard R Neubig, John R Traynor, Babak Shirvani, Amanda Baun, John Devilbiss, Lilyann Nyangau, Lena Chung, Jeffery N TalbotAbstract:Evidence suggests the antidepressant actions of serotonin (5-HT) are modulated by regulators of G protein signaling (RGS) proteins. Specifically, we have shown that upregulation of cortical RGS4 (mRNA and protein) occurs in response to genetic disruption of RGS protein activity at the GTP-binding protein Gαi2 and following chronic administration of serotonin-selective reuptake inhibitors (SSRIs). Genetic deletion of RGS4 (RGS4(-/-)) had no effect on basal antidepressant-like behaviors or generalized locomotor activity. However, RGS4(-/-) mice exhibited decreased antidepressant-like responses to the SSRI fluoxetine, whereas desipramine, a selective inhibitor of norepinephrine reuptake, was fully efficacious. Conversely, the regulation of locomotor activity by desipramine was augmented in RGS4(-/-) animals relative to vehicle-treated controls. RGS4 expression had no effect on locomotor activity following fluoxetine administration. Overall, these data suggest altered expression of RGS4 differentially influen...
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differential modulation of μ and δ opioid receptor agonists by endogenous RGS4 protein in sh sy5y cells
Journal of Biological Chemistry, 2009Co-Authors: Qin Wang, Leeyuan Liuchen, John R TraynorAbstract:Regulator of G-protein signaling (RGS) proteins are a family of molecules that control the duration of G protein signaling. A variety of RGS proteins have been reported to modulate opioid receptor signaling. Here we show that RGS4 is abundantly expressed in human neuroblastoma SH-SY5Y cells that endogenously express μ- and δ-opioid receptors and test the hypothesis that the activity of opioids in these cells is modulated by RGS4. Endogenous RGS4 protein was reduced by ∼90% in SH-SY5Y cells stably expressing short hairpin RNA specifically targeted to RGS4. In these cells, the potency and maximal effect of δ-opioid receptor agonist (SNC80)-mediated inhibition of forskolin-stimulated cAMP accumulation was increased compared with control cells. This effect was reversed by transient transfection of a stable RGS4 mutant (HA-RGS4C2S). Furthermore, MAPK activation by SNC80 was increased in cells with knockdown of RGS4. In contrast, there was no change in the μ-opioid (morphine) response at adenylyl cyclase or MAPK. FLAG-tagged opioid receptors and HA-RGS4C2S were transiently expressed in HEK293T cells, and co-immunoprecipitation experiments showed that the δ-opioid receptor but not the μ-opioid receptor could be precipitated together with the stable RGS4. Using chimeras of the δ- and μ-opioid receptors, the C-tail and third intracellular domain of the δ-opioid receptor were suggested to be the sites of interaction with RGS4. The findings demonstrate a role for endogenous RGS4 protein in modulating δ-opioid receptor signaling in SH-SY5Y cells and provide evidence for a receptor-specific effect of RGS4.
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identification of small molecule inhibitors of RGS4 using a high throughput flow cytometry protein interaction assay
Molecular Pharmacology, 2007Co-Authors: David L. Roman, Roger K Sunahara, Jeffery N Talbot, Rebecca A Roof, John R Traynor, Richard R NeubigAbstract:Regulators of G-protein signaling (RGS) proteins are important components of signal transduction pathways initiated through G-protein-coupled receptors (GPCRs). RGS proteins accelerate the intrinsic GTPase activity of G-protein α-subunits (Gα) and thus shorten the time course and reduce the magnitude of G-protein α- and βγ-subunit signaling. Inhibiting RGS action has been proposed as a means to enhance the activity and specificity of GPCR agonist drugs, but pharmacological targeting of protein-protein interactions has typically been difficult. The aim of this project was to identify inhibitors of RGS4. Using a Luminex 96-well plate bead analyzer and a novel flow-cytometric protein interaction assay to assess Gα-RGS interactions in a high-throughput screen, we identified the first small-molecule inhibitor of an RGS protein. Of 3028 compounds screened, 1, methyl N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986), inhibited RGS4/Gαo binding with 3 to 5 μM potency. It binds to RGS4, inhibits RGS4 stimulation of Gαo GTPase activity in vitro, and prevents RGS4 regulation of μ-opioid-inhibited adenylyl cyclase activity in permeabilized cells. Furthermore, CCG-4986 is selective for RGS4 and does not inhibit RGS8. Thus, we demonstrate the feasibility of targeting RGS/Gα protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.
Yoshihisa Kurachi - One of the best experts on this subject based on the ideXlab platform.
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structural determinants at the m2 muscarinic receptor modulate the RGS4 girk response to pilocarpine by impairment of the receptor voltage sensitivity
Scientific Reports, 2017Co-Authors: Ishan Chen, Kazuharu Furutani, Yoshihisa KurachiAbstract:Membrane potential controls the response of the M2 muscarinic receptor to its ligands. Membrane hyperpolarization increases response to the full agonist acetylcholine (ACh) while decreasing response to the partial agonist pilocarpine. We previously have demonstrated that the regulator of G-protein signaling (RGS) 4 protein discriminates between the voltage-dependent responses of ACh and pilocarpine; however, the underlying mechanism remains unclear. Here we show that RGS4 is involved in the voltage-dependent behavior of the M2 muscarinic receptor-mediated signaling in response to pilocarpine. Additionally we revealed structural determinants on the M2 muscarinic receptor underlying the voltage-dependent response. By electrophysiological recording in Xenopus oocytes expressing M2 muscarinic receptor and G-protein-gated inwardly rectifying K+ channels, we quantified voltage-dependent desensitization of pilocarpine-induced current in the presence or absence of RGS4. Hyperpolarization-induced desensitization of the current required for RGS4, also depended on pilocarpine concentration. Mutations of charged residues in the aspartic acid-arginine-tyrosine motif of the M2 muscarinic receptor, but not intracellular loop 3, significantly impaired the voltage-dependence of RGS4 function. Thus, our results demonstrated that voltage-dependence of RGS4 modulation is derived from the M2 muscarinic receptor. These results provide novel insights into how membrane potential impacts G-protein signaling by modulating GPCR communication with downstream effectors.
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RGS4 regulates partial agonism of the m2 muscarinic receptor activated k currents
The Journal of Physiology, 2014Co-Authors: Ishan Chen, Atsushi Inanobe, Kazuharu Furutani, Yoshihisa KurachiAbstract:Partial agonists are used clinically to avoid overstimulation of receptor-mediated signalling, as they produce a submaximal response even at 100% receptor occupancy. The submaximal efficacy of partial agonists is due to conformational change of the agonist–receptor complex, which reduces effector activation. In addition to signalling activators, several regulators help control intracellular signal transductions. However, it remains unclear whether these signalling regulators contribute to partial agonism. Here we show that regulator of G-protein signalling (RGS) 4 is a determinant for partial agonism of the M2 muscarinic receptor (M2R). In rat atrial myocytes, pilocarpine evoked smaller G-protein-gated K+ inwardly rectifying (KG) currents than those evoked by ACh. In a Xenopus oocyte expression system, pilocarpine acted as a partial agonist in the presence of RGS4 as it did in atrial myocytes, while it acted like a full agonist in the absence of RGS4. Functional couplings within the agonist–receptor complex/G-protein/RGS4 system controlled the efficacy of pilocarpine relative to ACh. The pilocarpine–M2R complex suppressed G-protein-mediated activation of KG currents via RGS4. Our results demonstrate that partial agonism of M2R is regulated by the RGS4-mediated inhibition of G-protein signalling. This finding helps us to understand the molecular components and mechanism underlying the partial agonism of M2R-mediated physiological responses.
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RGS4 regulates partial agonism of the M2 muscarinic receptor‐activated K+ currents
The Journal of Physiology, 2014Co-Authors: Ishan Chen, Atsushi Inanobe, Kazuharu Furutani, Yoshihisa KurachiAbstract:Partial agonists are used clinically to avoid overstimulation of receptor-mediated signalling, as they produce a submaximal response even at 100% receptor occupancy. The submaximal efficacy of partial agonists is due to conformational change of the agonist–receptor complex, which reduces effector activation. In addition to signalling activators, several regulators help control intracellular signal transductions. However, it remains unclear whether these signalling regulators contribute to partial agonism. Here we show that regulator of G-protein signalling (RGS) 4 is a determinant for partial agonism of the M2 muscarinic receptor (M2R). In rat atrial myocytes, pilocarpine evoked smaller G-protein-gated K+ inwardly rectifying (KG) currents than those evoked by ACh. In a Xenopus oocyte expression system, pilocarpine acted as a partial agonist in the presence of RGS4 as it did in atrial myocytes, while it acted like a full agonist in the absence of RGS4. Functional couplings within the agonist–receptor complex/G-protein/RGS4 system controlled the efficacy of pilocarpine relative to ACh. The pilocarpine–M2R complex suppressed G-protein-mediated activation of KG currents via RGS4. Our results demonstrate that partial agonism of M2R is regulated by the RGS4-mediated inhibition of G-protein signalling. This finding helps us to understand the molecular components and mechanism underlying the partial agonism of M2R-mediated physiological responses.
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phosphatidylinositol 3 4 5 trisphosphate and ca2 calmodulin competitively bind to the regulators of g protein signalling rgs domain of RGS4 and reciprocally regulate its action
Biochemical Journal, 2005Co-Authors: Masaru Ishii, Satoru Fujita, Mitsuhiko Yamada, Yukio Hosaka, Yoshihisa KurachiAbstract:RGS (regulators of G-protein signalling) are a diverse group of proteins, which accelerate intrinsic GTP hydrolysis on heterotrimeric G-protein α subunits. They are involved in the control of a physiological behaviour known as ‘relaxation’ of G-protein-gated K+ channels in cardiac myocytes. The GTPase-accelerating activity of cardiac RGS proteins, such as RGS4, is inhibited by PtdIns(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) and this inhibition is cancelled by Ca2+/calmodulin (CaM) formed during membrane depolarization. G-protein-gated K+ channel activity decreases on depolarization owing to the facilitation of GTPase-activating protein activity by RGS proteins and vice versa on hyperpolarization. The molecular mechanism responsible for this reciprocal control of RGS action by PtdIns(3,4,5)P3 and Ca2+/CaM, however, has not been fully elucidated. Using lipid–protein co-sedimentation assay and surface plasmon resonance measurements, we show in the present study that the control of the GTPase-accelerating activity of the RGS4 protein is achieved through the competitive binding of PtdIns(3,4,5)P3 and Ca2+/CaM within its RGS domain. Competitive binding occurs exclusively within the RGS domain and involves a cluster of positively charged residues located on the surface opposite to the Gα interaction site. In the RGS proteins conserving these residues, the reciprocal regulation by PtdIns(3,4,5)P3 and Ca2+/CaM may be important for their physiological regulation of G-protein signalling.
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interaction between the rgs domain of RGS4 with g protein α subunits mediates the voltage dependent relaxation of the g protein gated potassium channel
The Journal of Physiology, 2001Co-Authors: Atsushi Inanobe, Masaru Ishii, Satoru Fujita, Yasunaka Makino, Kenji Matsushita, Motohiko Chachin, Yoshihisa KurachiAbstract:Acetylcholine (ACh)-induced deceleration of heart beat and the formation of slow inhibitory postsynaptic potentials by various inhibitory neurotransmitters are mediated by G protein-gated inwardly rectifying K+ (KG) channels (Yamada et al. 1998). The cardiac KG channel is composed of Kir3.1 and Kir3.4, while neuronal channels are mainly composed of Kir3.1 and Kir3.2. Both types of channel can be reconstituted by co-expression of their respective Kir3.0 subunits and pertussis toxin (PTX)-sensitive G protein-coupled receptors in Xenopus oocytes (Kubo et al. 1993; Duprat et al. 1994; Krapivinsky et al. 1995; Lesage et al. 1995). Activation, desensitization and deactivation of the reconstituted ACh-induced KG current are accelerated by regulators of G protein signalling (RGS) proteins, thus mimicking to some extent the properties of native KG currents (Doupnik et al. 1997; Saitoh et al. 1997, 1999; Chuang et al. 1998; Herlitze et al. 1999). Therefore, RGS proteins seem to be involved in the physiological control of the KG channel system. One of the characteristic features of ACh-induced KG current in cardiac myocytes is the property of voltage-dependent ‘relaxation’ (Noma & Trautwein 1978; Yamada et al. 1998). The cardiac KG current activated by ACh is composed of instantaneous and time-dependent components. The instantaneous component reflects the open probability of the KG channel at the holding potential. The time-dependent component reflects the gradual increase in channel open probability upon hyperpolarizing voltage steps (Yamada et al. 1998). The ratio between these components varies in an agonist concentration-dependent manner. Increasing the concentration of ACh ([ACh]) increases the proportion of the instantaneous component and decreases the proportion of the time-dependent component which is also accelerated (Fujita et al. 2000). In this way relaxation is a mechanism for reducing the open probability of KG channels at depolarized potentials at low [ACh]. It can therefore be important in the sino-atrial node for slowing the pacemaker depolarization without affecting the action potential configuration. We recently showed that co-expression of RGS4 restored the agonist concentration-dependent relaxation to the reconstituted KG current in Xenopus oocytes, in addition to the acceleration of activation and deactivation, although we could not reproduce the effect of RGS4 on short-term desensitization (Fujita et al. 2000). The induction of relaxation in the reconstituted KG channel is a newly identified effect of RGS proteins and replicates the ACh-induced voltage-dependent relaxation of the native cardiac KG channel. The molecular mechanism of this phenomenon, however, has not yet been determined. In this study, we examined the signalling pathway mediating the effect of RGS4 on KG current relaxation by heterologously expressing various combinations of membrane receptors, G proteins, Kir3.0 subunits and RGS4 mutants in Xenopus oocytes. We found that the RGS domain of RGS4 and PTX-sensitive G proteins were required for RGS4-modulation of the kinetics of the KG current.
David L. Roman - One of the best experts on this subject based on the ideXlab platform.
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modification and functional inhibition of regulator of g protein signaling 4 RGS4 by 4 hydroxy 2 nonenal
Chemical Research in Toxicology, 2013Co-Authors: Aaron C Monroy, Jonathan A Doorn, David L. RomanAbstract:Oxidative stress has been implicated as a component of various pathologies including ischemia/reperfusion injury (IRI) and neurodegenerative diseases such as Parkinson’s disease (PD) and schizophrenia. Similarly, regulator of G-protein signaling 4 (RGS4) has been implicated as an important player in each of these pathologies. RGS4, like other RGS proteins, is responsible for temporally regulating G-protein coupled receptor signaling by increasing the intrinsic GTPase activity of Gα subunit of the heterotrimeric signaling complex. In this study we evaluated whether modification by 4-hydroxy-2-nonenal (4HNE), a common lipid peroxidation product, inhibits RGS4. Using immunoprecipitation, we first determined RGS4 modification was occurring in cells at concentrations of 4HNE within reported physiological conditions. Following this determination, we evaluated modification of RGS4 by 4HNE by both Western blot and mass spectrometry (MS). Once it was established that covalent modification occurred only on cysteine...
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A High Throughput Screen for RGS Proteins Using Steady State Monitoring of Free Phosphate Formation
PLOS ONE, 2013Co-Authors: C. Aaron Monroy, Duncan I Mackie, David L. RomanAbstract:G-protein coupled receptors are a diverse group that are the target of over 50% of marketed drugs. Activation of these receptors results in the exchange of bound GDP for GTP in the Gα subunit of the heterotrimeric G-protein. The Gα subunit dissociates from the β/γ subunits and both proceed to affect downstream signaling targets. The signal terminates by the hydrolysis of GTP to GDP and is temporally regulated by Regulators of G-protein Signaling (RGS) proteins that act as GTPase Activating Proteins (GAPs). This makes RGS proteins potentially desirable targets for “tuning” the effects of current therapies as well as developing novel pharmacotherapies. Current methods for evaluating RGS activity depend on laborious and/or expensive techniques. In this study we developed a simple and inexpensive assay for the steady state analysis of RGS protein GAP activity, using RGS4, RGS8 and RGS17 as models. Additionally, we report the use of RGS4 as a model for high throughput assay development. After initial setup, this assay can be conducted in a highly parallel fashion with a read time of less than 8 minutes for a 1536-well plate. The assay exhibited a robust Z-factor of 0.6 in a 1536-well plate. We conducted a pilot screen for inhibitors using a small, 2320 compound library. From this screen, 13 compounds were identified as compounds for further analysis. The successful development of this assay for high-throughput screening provides a low cost, high speed, simple method for assessing RGS protein activity.
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a covalent peptide inhibitor of RGS4 identified in a focused one bead one compound library screen
BMC Pharmacology, 2009Co-Authors: Rebecca A Roof, David L. Roman, Samuel T Clements, Katarzyna Sobczykkojiro, Levi L Blazer, Henry I Mosberg, Richard R NeubigAbstract:Background Regulators of G protein signaling (RGSs) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors (GPCRs). The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We recently described a focused one-bead, one-compound (OBOC) library screen to identify peptide inhibitors of RGS4. Here we extend our observations to include another peptide with a different mechanism of action.
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novel peptide ligands of RGS4 from a focused one bead one compound library
Chemical Biology & Drug Design, 2008Co-Authors: Rebecca A Roof, David L. Roman, Richard R Neubig, Katarzyna Sobczykkojiro, Levi L Blazer, Anjanette J Turbiak, Irina D Pogozheva, Henry I MosbergAbstract:: Regulators of G protein signaling accelerate GTP hydrolysis by G alpha subunits and profoundly inhibit signaling by G protein-coupled receptors. The distinct expression patterns and pathophysiologic regulation of regulators of G protein signaling proteins suggest that inhibitors may have therapeutic potential. We previously reported the design, mechanistic evaluation, and structure-activity relationships of a disulfide-containing cyclic peptide inhibitor of RGS4, YJ34 (Ac-Val-Lys-c[Cys-Thr-Gly-Ile-Cys]-Glu-NH(2), S-S) (Roof et al., Chem Biol Drug Des, 67, 2006, 266). Using a focused one-bead, one-compound peptide library that contains features known to be necessary for the activity of YJ34, we now identify peptides that bind to RGS4. Six peptides showed confirmed binding to RGS4 by flow cytometry. Two analogs of peptide 2 (Gly-Thr-c[Cys-Phe-Gly-Thr-Cys]-Trp-NH(2), S-S with a free or acetylated N-terminus) inhibited RGS4-stimulated G alpha(o) GTPase activity at 25-50 microM. They selectively inhibit RGS4 but not RGS7, RGS16, and RGS19. Their inhibition of RGS4 does not depend on cysteine-modification of RGS4, as they do not lose activity when all cysteines are removed from RGS4. Peptide 2 has been modeled to fit in the same binding pocket predicted for YJ34 but in the reverse orientation.
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identification of small molecule inhibitors of RGS4 using a high throughput flow cytometry protein interaction assay
Molecular Pharmacology, 2007Co-Authors: David L. Roman, Roger K Sunahara, Jeffery N Talbot, Rebecca A Roof, John R Traynor, Richard R NeubigAbstract:Regulators of G-protein signaling (RGS) proteins are important components of signal transduction pathways initiated through G-protein-coupled receptors (GPCRs). RGS proteins accelerate the intrinsic GTPase activity of G-protein α-subunits (Gα) and thus shorten the time course and reduce the magnitude of G-protein α- and βγ-subunit signaling. Inhibiting RGS action has been proposed as a means to enhance the activity and specificity of GPCR agonist drugs, but pharmacological targeting of protein-protein interactions has typically been difficult. The aim of this project was to identify inhibitors of RGS4. Using a Luminex 96-well plate bead analyzer and a novel flow-cytometric protein interaction assay to assess Gα-RGS interactions in a high-throughput screen, we identified the first small-molecule inhibitor of an RGS protein. Of 3028 compounds screened, 1, methyl N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986), inhibited RGS4/Gαo binding with 3 to 5 μM potency. It binds to RGS4, inhibits RGS4 stimulation of Gαo GTPase activity in vitro, and prevents RGS4 regulation of μ-opioid-inhibited adenylyl cyclase activity in permeabilized cells. Furthermore, CCG-4986 is selective for RGS4 and does not inhibit RGS8. Thus, we demonstrate the feasibility of targeting RGS/Gα protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.