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John R. Hepler - One of the best experts on this subject based on the ideXlab platform.
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rgs14 modulates locomotor behavior and erk signaling induced by environmental novelty and cocaine within discrete limbic structures
Psychopharmacology, 2021Co-Authors: Stephanie L Foster, John R. Hepler, Daniel Lustberg, Nicholas H Harbin, Sara N Bramlett, David WeinshenkerAbstract:In rodents, exposure to novel environments or psychostimulants promotes locomotion. Indeed, locomotor reactivity to novelty strongly predicts behavioral responses to psychostimulants in animal models of addiction. RGS14 is a plasticity-restricting protein with unique functional domains that enable it to suppress ERK-dependent signaling as well as regulate G protein activity. Although recent studies show that RGS14 is expressed in multiple limbic regions implicated in psychostimulant- and novelty-induced hyperlocomotion, its function has been examined mostly in the context of hippocampal physiology and memory. We investigated whether RGS14 modulates novelty- and cocaine-induced locomotion (NIL and CIL, respectively) and neuronal activity. We assessed Rgs14 knockout (RGS14 KO) mice and wild-type (WT) littermate controls using NIL and CIL behavioral tests, followed by quantification of c-fos and phosphorylated ERK (pERK) induction in limbic regions that normally express RGS14. RGS14 KO mice were less active than WT controls in the NIL test, driven by avoidance of the center of the novel environment. By contrast, RGS14 KO mice demonstrated augmented peripheral locomotion in the CIL test conducted in either a familiar or novel environment. RGS14 KO mice exhibited increased thigmotaxis, as well as greater c-fos and pERK induction in the central amygdala and dorsal hippocampus, when cocaine and novelty were paired. RGS14 KO mice exhibited anti-correlated locomotor responses to novelty and cocaine, but displayed increased thigmotaxis in response to either stimuli which was augmented by their combination. Our findings also suggest RGS14 may reduce neuronal activity in limbic subregions by inhibiting ERK-dependent signaling.
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rgs14 regulation of post synaptic signaling and spine plasticity in brain
International Journal of Molecular Sciences, 2021Co-Authors: Nicholas H Harbin, Sara N Bramlett, Carolina Montanezmiranda, Gizem Terzioglu, John R. HeplerAbstract:The regulator of G-protein signaling 14 (RGS14) is a multifunctional signaling protein that regulates post synaptic plasticity in neurons. RGS14 is expressed in the brain regions essential for learning, memory, emotion, and stimulus-induced behaviors, including the basal ganglia, limbic system, and cortex. Behaviorally, RGS14 regulates spatial and object memory, female-specific responses to cued fear conditioning, and environmental- and psychostimulant-induced locomotion. At the cellular level, RGS14 acts as a scaffolding protein that integrates G protein, Ras/ERK, and calcium/calmodulin signaling pathways essential for spine plasticity and cell signaling, allowing RGS14 to naturally suppress long-term potentiation (LTP) and structural plasticity in hippocampal area CA2 pyramidal cells. Recent proteomics findings indicate that RGS14 also engages the actomyosin system in the brain, perhaps to impact spine morphogenesis. Of note, RGS14 is also a nucleocytoplasmic shuttling protein, where its role in the nucleus remains uncertain. Balanced nuclear import/export and dendritic spine localization are likely essential for RGS14 neuronal functions as a regulator of synaptic plasticity. Supporting this idea, human genetic variants disrupting RGS14 localization also disrupt RGS14’s effects on plasticity. This review will focus on the known and unexplored roles of RGS14 in cell signaling, physiology, disease and behavior.
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rgs14 modulates locomotor behavior and erk signaling induced by environmental novelty and cocaine within discrete limbic structures
bioRxiv, 2021Co-Authors: Stephanie L Foster, John R. Hepler, Daniel Lustberg, Nicholas H Harbin, Sara N Bramlett, David WeinshenkerAbstract:Rationale: In rodents, exposure to novel environments or psychostimulants promotes locomotor activity. Indeed, locomotor reactivity to novelty strongly predicts behavioral responses to psychostimulants in animal models of addiction. RGS14 is a plasticity restricting protein with unique functional domains that enable it to suppress ERK-dependent signaling as well as regulate G protein activity. Although recent studies show that RGS14 is expressed in multiple limbic regions implicated in psychostimulant- and novelty-induced hyperlocomotion, its function has been studied almost entirely in the context of hippocampal physiology and hippocampus-dependent behaviors. Objective: We sought to determine whether RGS14 modulates novelty- and psychostimulant-induced locomotion and neuronal activity. Methods: We assessed Rgs14 knockout (RGS14 KO) mice and wild-type (WT) littermate controls using novelty-induced locomotion (NIL) and cocaine-induced locomotion (CIL) behavioral tests with subsequent quantification of c-fos and phosphorylated ERK (pERK) induction in limbic regions that express RGS14. Results: Compared to WT controls, RGS14 KO mice exhibited attenuated locomotor responses in the NIL test, driven by avoidance of the center of the novel environment. By contrast, RGS14 KO mice demonstrated augmented peripheral locomotion in the CIL test conducted in either a familiar or novel environment. The absence of RGS14 enhanced induction of c-fos and pERK in the central amygdala and hippocampus (areas CA1 and CA2) when cocaine was administered in a novel environment. Conclusions: RGS14 regulates novelty- and psychostimulant-induced hyperlocomotion, particularly with respect to thigmotaxis. Further, our findings suggest RGS14 may reduce neuronal activity in discrete limbic subregions by inhibiting ERK-dependent signaling and transcription.
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genetic variants disrupt human rgs14 binding to nherf1 and regulation of npt2a mediated phosphate transport
bioRxiv, 2019Co-Authors: Peter A Friedman, Katherine E. Squires, Tatyana Mamonova, Clara E Magyar, Bruce W Sneddon, David R Emlet, John R. HeplerAbstract:RGS14 is a multifunctional scaffolding protein that integrates G protein, MAPK, and Ca++/CaM signaling pathways. RGS14 contains an RGS domain, tandem Ras/Rap-binding domains, and a G protein regulatory motif. Human/primate RGS14 differ from rodent proteins by possessing a carboxy-terminal extension encoding a Type I PDZ ligand. RGS14 has been implicated in disordered phosphate metabolism. The human RGS14 gene is adjacent to SLC34A1 that encodes the NPT2A sodium-phosphate cotransporter. Hormone-regulated NPT2A requires the PDZ protein NHERF1 which contains two PDZ domains (PDZ1 and PDZ2). NHERF1 binds the PDZ ligand carboxy tail of NPT2A to regulate phosphate uptake, and this NPT2A:NHERF1 complex is inhibited by parathyroid hormone (PTH). Studies here define roles for RGS14 in NHERF1-dependent, PTH-sensitive phosphate transport. We found that RGS14 binds to NHERF1 via the PDZ2 domain. PTH inhibits NPT2A-mediated phosphate transport and RGS14 blocked this action. Several rare human mutations have been reported in the RGS14 PDZ ligand located at residues 563 (D563N, D563G) and 565 (A565S, A565V). D563N disrupted RGS14 binding to NHERF1 and did not interfere with PTH action, whereas D563G, A565S, and A565V bound NHERF1 and were functionally equivalent to wild-type RGS14. Computational analysis and molecular dynamics modeling of NHERF1 PDZ2 binding to the RGS14 C-terminal PDZ ligands refined the structural determinants of this interaction. Additional studies demonstrated that RGS14 is expressed in human kidney proximal and distal tubule cells. Together, our findings are consistent with the view that RGS14 contributes to PTH-sensitive phosphate transport in humans. RGS14 coding variants may cause disordered phosphate metabolism.
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14 3 3γ binds regulator of g protein signaling 14 rgs14 at distinct sites to inhibit the rgs14 gαi alf4 signaling complex and rgs14 nuclear localization
Journal of Biological Chemistry, 2018Co-Authors: Kyle J Gerber, Katherine E. Squires, John R. HeplerAbstract:Regulator of G protein signaling 14 (RGS14) is a multifunctional brain scaffolding protein that integrates G protein and Ras/ERK signaling pathways. It is also a nucleocytoplasmic shuttling protein. RGS14 binds active Gαi/o via its RGS domain, Raf and active H-Ras–GTP via its R1 Ras-binding domain (RBD), and inactive Gαi1/3 via its G protein regulatory (GPR) domain. RGS14 suppresses long-term potentiation (LTP) in the CA2 region of the hippocampus, thereby regulating hippocampally based learning and memory. The 14-3-3 family of proteins is necessary for hippocampal LTP and associative learning and memory. Here, we show direct interaction between RGS14 and 14-3-3γ at two distinct sties, one phosphorylation-independent and the other phosphorylation-dependent at Ser-218 that is markedly potentiated by signaling downstream of active H-Ras. Using bioluminescence resonance energy transfer (BRET), we show that the pSer-218–dependent RGS14/14-3-3γ interaction inhibits active Gαi1–AlF4− binding to the RGS domain of RGS14 but has no effect on active H-Ras and inactive Gαi1–GDP binding to RGS14. By contrast, the phosphorylation-independent binding of 14-3-3 has no effect on RGS14/Gαi interactions but, instead, inhibits (directly or indirectly) RGS14 nuclear import and nucleocytoplasmic shuttling. Together, our findings describe a novel mechanism of negative regulation of RGS14 functions, specifically interactions with active Gαi and nuclear import, while leaving the function of other RGS14 domains intact. Ongoing studies will further elucidate the physiological function of this interaction between RGS14 and 14-3-3γ, providing insight into the functions of both RGS14 and 14-3-3 in their roles in modulating synaptic plasticity in the hippocampus.
David P Siderovski - One of the best experts on this subject based on the ideXlab platform.
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A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation.
PloS one, 2019Co-Authors: Adam B. Schroer, Melinda D. Willard, Emily A. Oestreich, Junaith S. Mohamed, Vincent Setola, David P SiderovskiAbstract:Regulators of G Protein Signaling (RGS proteins) inhibit G protein-coupled receptor (GPCR) signaling by accelerating the GTP hydrolysis rate of activated Gα subunits. Some RGS proteins exert additional signal modulatory functions, and RGS12 is one such protein, with five additional, functional domains: a PDZ domain, a phosphotyrosine-binding domain, two Ras-binding domains, and a Gα·GDP-binding GoLoco motif. RGS12 expression is temporospatially regulated in developing mouse embryos, with notable expression in somites and developing skeletal muscle. We therefore examined whether RGS12 is involved in the skeletal muscle myogenic program. In the adult mouse, RGS12 is expressed in the tibialis anterior (TA) muscle, and its expression is increased early after cardiotoxin-induced injury, suggesting a role in muscle regeneration. Consistent with a potential role in coordinating myogenic signals, RGS12 is also expressed in primary myoblasts; as these cells undergo differentiation and fusion into myotubes, RGS12 protein abundance is reduced. Myoblasts isolated from mice lacking Rgs12 expression have an impaired ability to differentiate into myotubes ex vivo, suggesting that RGS12 may play a role as a modulator/switch for differentiation. We also assessed the muscle regenerative capacity of mice conditionally deficient in skeletal muscle Rgs12 expression (via Pax7-driven Cre recombinase expression), following cardiotoxin-induced damage to the TA muscle. Eight days post-damage, mice lacking RGS12 in skeletal muscle had attenuated repair of muscle fibers. However, when mice lacking skeletal muscle expression of Rgs12 were cross-bred with mdx mice (a model of human Duchenne muscular dystrophy), no increase in muscle degeneration was observed over time. These data support the hypothesis that RGS12 plays a role in coordinating signals during the myogenic program in select circumstances, but loss of the protein may be compensated for within model syndromes of prolonged bouts of muscle damage and repair.
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the effect of rgs12 on pdgfβ receptor signalling to p42 p44 mitogen activated protein kinase in mammalian cells
Cellular Signalling, 2006Co-Authors: Balwinder Sambi, David P Siderovski, Francis S. Willard, Melinda D. Hains, Catherine M. Waters, Michelle Connell, Adam J. Kimple, Susan Pyne, Nigel J. PyneAbstract:We have previously shown that the PDGFβ receptor uses a classical GPCR-mediated pathway in order to induce efficient activation of p42/p44 MAPK in response to PDGF. We therefore, considered the possibility that GTPase accelerating proteins (RGS proteins), which regulate GPCR signalling, modulate PDGFβ receptor-mediated signal transmission. Several lines of evidence were obtained to support functional interaction between the PDGFβ receptor and RGS12 in HEK 293 and airway smooth muscle cells. Firstly, the over-expression of the RGS12 PDZ/PTB domain N-terminus or RGS12 PTB domain reduced the PDGF-induced activation of p42/p44 MAPK. Secondly, the RGS12 PDZ/PTB domain N-terminus and RGS12 PDZ domain can form a complex with the PDGFβ receptor. Therefore, the results presented here provide the first evidence to support the concept that the PDZ/PTB domain N-terminus and/or the PTB domain of RGS12 may modulate PDGFβ receptor signalling. In airway smooth muscle cells, over-expressed recombinant RGS12 and the isolated PDZ/PTB domain N-terminus co-localised with PDGFβ receptor in cytoplasmic vesicles. To provide additional evidence for a role of the PDZ/PTB domain N-terminus, we used RGS14. RGS14 has the same C-terminal domain architecture of an RGS box, tandem Ras-binding domains (RBDs) and GoLoco motif as RGS12, but lacks the PDZ/PTB domain N-terminus. In this regard, RGS14 exhibited a different sub-cellular distribution compared with RGS12, being diffusely distributed in ASM cells. These findings suggest that RGS12 via its PDZ/PTB domain N-terminus may regulate trafficking of the PDGFβ receptor in ASM cells.
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Return of the GDI: The GoLoco Motif in Cell Division
Annual review of biochemistry, 2004Co-Authors: Francis S. Willard, Randall J Kimple, David P SiderovskiAbstract:The GoLoco motif is a 19-amino-acid sequence with guanine nucleotide dissociation inhibitor activity against G-alpha subunits of the adenylyl-cyclase-inhibitory subclass. The GoLoco motif is present as an independent element within multidomain signaling regulators, such as Loco, RGS12, RGS14, and Rap1GAP, as well as in tandem arrays in proteins, such as AGS3, G18, LGN, Pcp-2/L7, and Partner of Inscuteable (Pins/Rapsynoid). Here we discuss the biochemical mechanisms of GoLoco motif action on G-alpha subunits in light of the recent crystal structure of G-alpha-i1 bound to the RGS14 GoLoco motif. Currently, there is sparse evidence for GoLoco motif regulation of canonical G-protein-coupled receptor signaling. Rather, studies of asymmetric cell division in Drosophila and Caenorhabditis elegans, as well as mammalian mitosis, implicate GoLoco proteins, such as Pins, GPR-1/GPR-2, LGN, and RGS14, in mitotic spindle organization and force generation. We discuss potential mechanisms by which GoLoco/Galpha complexes might modulate spindle dynamics.
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Purification and in vitro functional analyses of RGS12 and RGS14 GoLoco motif peptides
Methods in enzymology, 2004Co-Authors: Randall J Kimple, Francis S. Willard, David P SiderovskiAbstract:The GoLoco motif is a short polypeptide sequence that binds to heterotrimeric G-protein alpha subunits of the adenylyl cyclase-inhibitory (Galpha(i/o)) subclass in a nucleotide-dependent manner (i.e., solely to the GDP-bound ground state). This article describes methods used for the expression, purification, and in vitro evaluation of membrane-permeant tag fusion peptides derived from the GoLoco motif regions of "regulator of G-protein signaling" proteins type 12 (RGS12) and 14 (RGS14) and a consensus GoLoco sequence from the multiple GoLoco motif protein AGS3. Three different fluorescence-based assays are described for evaluating the in vitro function of these GoLoco peptides as guanine nucleotide dissociation inhibitors, including measurements of GTPgammaS binding and Galpha subunit activation by the planar ion aluminum tetrafluoride.
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rgs12 and rgs14 goloco motifs are g alpha i interaction sites with guanine nucleotide dissociation inhibitor activity
Journal of Biological Chemistry, 2001Co-Authors: Randall J Kimple, Helene Tronchere, Cynthia I Behe, Rebecca A Morris, Marilyn Gist Farquhar, Luc De Vries, David P SiderovskiAbstract:Abstract The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein α subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single Gαi/o-Loco (GoLoco) motif predicted to represent a second Gα interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with Gαi1, Gαi2, and Gαi3 in their GDP-bound forms. In GTPγS binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by Gαi1. Both regions also stabilize Gαi1 in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF . Our results indicate that both RGS12 and RGS14 harbor two distinctly different Gα interaction sites: a previously recognized N-terminal RGS box possessing Gαi/o GAP activity and a C-terminal GoLoco region exhibiting Gαi GDI activity. The presence of two, independent Gα interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple Gα GAP activity.
Shelley B Hooks - One of the best experts on this subject based on the ideXlab platform.
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regulator of g protein signaling 10 rgs10 expression is transcriptionally silenced in activated microglia by histone deacetylase activity
Molecular Pharmacology, 2017Co-Authors: Mohammed Alqinyah, Ercan Cacan, Susanna F Greer, Mourad W. Ali, Nagini Maganti, Ruchi Yadav, Mei Gao, Hanrong Weng, Shelley B HooksAbstract:RGS10 has emerged as a key regulator of proinflammatory cytokine production in microglia, functioning as an important neuroprotective factor. Although RGS10 is normally expressed in microglia at high levels, expression is silenced in vitro following activation of TLR4 receptor. Given the ability of RGS10 to regulate inflammatory signaling, dynamic regulation of RGS10 levels in microglia may be an important mechanism to tune inflammatory responses. The goals of the current study were to confirm that RGS10 is suppressed in an in vivo inflammatory model of microglial activation and to determine the mechanism for activation-dependent silencing of Rgs10 expression in microglia. We demonstrate that endogenous RGS10 is present in spinal cord microglia, and RGS10 protein levels are suppressed in the spinal cord in a nerve injury-induced neuropathic pain mouse model. We show that the histone deacetylase (HDAC) enzyme inhibitor trichostatin A blocks the ability of lipopolysaccharide (LPS) to suppress Rgs10 transcription in BV-2 and primary microglia, demonstrating that HDAC enzymes are required for LPS silencing of Rgs10 Furthermore, we used chromatin immunoprecipitation to demonstrate that H3 histones at the Rgs10 proximal promoter are deacetylated in BV-2 microglia following LPS activation, and HDAC1 association at the Rgs10 promoter is enhanced following LPS stimulation. Finally, we have shown that sphingosine 1-phosphate, an endogenous microglial signaling mediator that inhibits HDAC activity, enhances basal Rgs10 expression in BV-2 microglia, suggesting that Rgs10 expression is dynamically regulated in microglia in response to multiple signals.
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inhibition of hdac1 and dnmt1 modulate rgs10 expression and decrease ovarian cancer chemoresistance
PLOS ONE, 2014Co-Authors: Ercan Cacan, Nathaniel H Boyd, Susanna F Greer, Shelley B HooksAbstract:RGS10 is an important regulator of cell survival and chemoresistance in ovarian cancer. We recently showed that RGS10 transcript expression is suppressed during acquired chemoresistance in ovarian cancer. The suppression of RGS10 is due to DNA hypermethylation and histone deacetylation, two important mechanisms that contribute to silencing of tumor suppressor genes during cancer progression. Here, we fully investigate the molecular mechanisms of epigenetic silencing of RGS10 expression in chemoresistant A2780-AD ovarian cancer cells. We identify two important epigenetic regulators, HDAC1 and DNMT1, that exhibit aberrant association with RGS10 promoters in chemoresistant ovarian cancer cells. Knockdown of HDAC1 or DNMT1 expression, and pharmacological inhibition of DNMT or HDAC enzymatic activity, significantly increases RGS10 expression and cisplatin-mediated cell death. Finally, DNMT1 knock down also decreases HDAC1 binding to the RGS10 promoter in chemoresistant cells, suggesting HDAC1 recruitment to RGS10 promoters requires DNMT1 activity. Our results suggest that HDAC1 and DNMT1 contribute to the suppression of RGS10 during acquired chemoresistance and support inhibition of HDAC1 and DNMT1 as an adjuvant therapeutic approach to overcome ovarian cancer chemoresistance.
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regulators of g protein signaling rgs10 and rgs17 regulate chemoresistance in ovarian cancer cells
Molecular Cancer, 2010Co-Authors: Shelley B Hooks, Phillip Callihan, Molly K Altman, Jillian H Hurst, Mandi M. MurphAbstract:A critical therapeutic challenge in epithelial ovarian carcinoma is the development of chemoresistance among tumor cells following exposure to first line chemotherapeutics. The molecular and genetic changes that drive the development of chemoresistance are unknown, and this lack of mechanistic insight is a major obstacle in preventing and predicting the occurrence of refractory disease. We have recently shown that Regulators of G-protein Signaling (RGS) proteins negatively regulate signaling by lysophosphatidic acid (LPA), a growth factor elevated in malignant ascites fluid that triggers oncogenic growth and survival signaling in ovarian cancer cells. The goal of this study was to determine the role of RGS protein expression in ovarian cancer chemoresistance. In this study, we find that RGS2, RGS5, RGS10 and RGS17 transcripts are expressed at significantly lower levels in cells resistant to chemotherapy compared with parental, chemo-sensitive cells in gene expression datasets of multiple models of chemoresistance. Further, exposure of SKOV-3 cells to cytotoxic chemotherapy causes acute, persistent downregulation of RGS10 and RGS17 transcript expression. Direct inhibition of RGS10 or RGS17 expression using siRNA knock-down significantly reduces chemotherapy-induced cell toxicity. The effects of cisplatin, vincristine, and docetaxel are inhibited following RGS10 and RGS17 knock-down in cell viability assays and phosphatidyl serine externalization assays in SKOV-3 cells and MDR-HeyA8 cells. We further show that AKT activation is higher following RGS10 knock-down and RGS 10 and RGS17 overexpression blocked LPA mediated activation of AKT, suggesting that RGS proteins may blunt AKT survival pathways. Taken together, our data suggest that chemotherapy exposure triggers loss of RGS10 and RGS17 expression in ovarian cancer cells, and that loss of expression contributes to the development of chemoresistance, possibly through amplification of endogenous AKT signals. Our results establish RGS10 and RGS17 as novel regulators of cell survival and chemoresistance in ovarian cancer cells and suggest that their reduced expression may be diagnostic of chemoresistance.
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RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity.
The Journal of biological chemistry, 2003Co-Authors: Shelley B Hooks, Andrejs M Krumins, G L Waldo, James Corbitt, Erik T. Bodor, T. Kendall HardenAbstract:Abstract Regulator of G-protein signaling (RGS) proteins are GTPase activating proteins (GAPs) of heterotrimeric G-proteins that alter the amplitude and kinetics of receptor-promoted signaling. In this study we defined the G-protein α-subunit selectivity of purified Sf9 cell-derived R7 proteins, a subfamily of RGS proteins (RGS6, -7, -9, and -11) containing a Gγ-like (GGL) domain that mediates dimeric interaction with Gβ5. Gβ5/R7 dimers stimulated steady state GTPase activity of Gα-subunits of the Gi family, but not of Gαq or Gα11, when added to proteoliposomes containing M2 or M1 muscarinic receptor-coupled G-protein heterotrimers. Concentration effect curves of the Gβ5/R7 proteins revealed differences in potencies and efficacies toward Gα-subunits of the Gi family. Although all four Gβ5/R7 proteins exhibited similar potencies toward Gαo, Gβ5/RGS9 and Gβ5/RGS11 were more potent GAPs of Gαi1, Gαi2, and Gαi3 than were Gβ5/RGS6 and Gβ5/RGS7. The maximal GAP activity exhibited by Gβ5/RGS11 was 2- to 4-fold higher than that of Gβ5/RGS7 and Gβ5/RGS9, with Gβ5/RGS6 exhibiting an intermediate maximal GAP activity. Moreover, the less efficacious Gβ5/RGS7 and Gβ5/RGS9 inhibited Gβ5/RGS11-stimulated GTPase activity of Gαo. Therefore, R7 family RGS proteins are Gi family-selective GAPs with potentially important differences in activities.
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RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity.
The Journal of biological chemistry, 2003Co-Authors: Shelley B Hooks, Andrejs M Krumins, G L Waldo, James Corbitt, Erik T. Bodor, T. Kendall HardenAbstract:Regulator of G-protein signaling (RGS) proteins are GTPase activating proteins (GAPs) of heterotrimeric G-proteins that alter the amplitude and kinetics of receptor-promoted signaling. In this study we defined the G-protein alpha-subunit selectivity of purified Sf9 cell-derived R7 proteins, a subfamily of RGS proteins (RGS6, -7, -9, and -11) containing a Ggamma-like (GGL) domain that mediates dimeric interaction with Gbeta(5). Gbeta(5)/R7 dimers stimulated steady state GTPase activity of Galpha-subunits of the G(i) family, but not of Galpha(q) or Galpha(11), when added to proteoliposomes containing M2 or M1 muscarinic receptor-coupled G-protein heterotrimers. Concentration effect curves of the Gbeta(5)/R7 proteins revealed differences in potencies and efficacies toward Galpha-subunits of the G(i) family. Although all four Gbeta(5)/R7 proteins exhibited similar potencies toward Galpha(o), Gbeta(5)/RGS9 and Gbeta(5)/RGS11 were more potent GAPs of Galpha(i1), Galpha(i2), and Galpha(i3) than were Gbeta(5)/RGS6 and Gbeta(5)/RGS7. The maximal GAP activity exhibited by Gbeta(5)/RGS11 was 2- to 4-fold higher than that of Gbeta(5)/RGS7 and Gbeta(5)/RGS9, with Gbeta(5)/RGS6 exhibiting an intermediate maximal GAP activity. Moreover, the less efficacious Gbeta(5)/RGS7 and Gbeta(5)/RGS9 inhibited Gbeta(5)/RGS11-stimulated GTPase activity of Galpha(o). Therefore, R7 family RGS proteins are G(i) family-selective GAPs with potentially important differences in activities.
Russell W. Brown - One of the best experts on this subject based on the ideXlab platform.
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ontogenetic quinpirole treatment produces long lasting decreases in the expression of rgs9 but increases rgs17 in the striatum nucleus accumbens and frontal cortex
European Journal of Neuroscience, 2007Co-Authors: Amanda M Maple, Joshua P. Parlaman, Marla K. Perna, Gregg D. Stanwood, Russell W. BrownAbstract:Ontogenetic treatment of rats with the dopamine D 2 -like receptor agonist quinpirole produces a significant increase in dopamine D 2 receptor sensitivity that persists throughout the animal's lifetime, a phenomenon known as D 2 priming. The present study was designed to investigate the effects of priming of the D 2 receptor on the expression of three different members of the regulator of G-protein signaling (RGS) family: Rgs4, Rgs9 and Rgs17. Male offspring were ontogenetically treated with quinpirole or saline from postnatal days (P)1-21 and raised to adulthood. On ∼P65, animals were given an acute quinipirole injection (0.1 mg/kg) and the number of yawns was recorded for 1 h after the injection. Yawning has been shown to be a behavioural event mediated by the dopamine D 2 /D 3 receptor. Animals ontogenetically treated with quinpirole demonstrated a significant 2.5-fold increase in yawning as compared to controls. Rgs transcripts were analysed through in situ hybridization several weeks later. Rats ontogenetically treated with quinpirole demonstrated a significant decrease in Rgs9 expression in the frontal cortex, but a more robust decrease in the striatum and nucleus accumbens as compared to controls. Regarding Rgs17, ontogenetic quinpirole produced a modest but significant increase in expression in the same brain areas. There were no significant differences in Rgs4 expression produced by drug treatment in any of the brain regions analysed. This study demonstrates that ontogenetic quinpirole treatment, which results in priming of the D 2 receptor, results in significant decreases in Rgs9, which has been shown to regulate G-protein coupling to D 2 receptors.
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Ontogenetic quinpirole treatment produces long-lasting decreases in the expression of Rgs9, but increases Rgs17 in the striatum, nucleus accumbens and frontal cortex.
The European journal of neuroscience, 2007Co-Authors: Amanda M Maple, Joshua P. Parlaman, Marla K. Perna, Gregg D. Stanwood, Russell W. BrownAbstract:Ontogenetic treatment of rats with the dopamine D(2)-like receptor agonist quinpirole produces a significant increase in dopamine D(2) receptor sensitivity that persists throughout the animal's lifetime, a phenomenon known as D(2) priming. The present study was designed to investigate the effects of priming of the D(2) receptor on the expression of three different members of the regulator of G-protein signaling (RGS) family: Rgs4, Rgs9 and Rgs17. Male offspring were ontogenetically treated with quinpirole or saline from postnatal days (P)1-21 and raised to adulthood. On approximately P65, animals were given an acute quinipirole injection (0.1 mg/kg) and the number of yawns was recorded for 1 h after the injection. Yawning has been shown to be a behavioural event mediated by the dopamine D(2)/D(3) receptor. Animals ontogenetically treated with quinpirole demonstrated a significant 2.5-fold increase in yawning as compared to controls. Rgs transcripts were analysed through in situ hybridization several weeks later. Rats ontogenetically treated with quinpirole demonstrated a significant decrease in Rgs9 expression in the frontal cortex, but a more robust decrease in the striatum and nucleus accumbens as compared to controls. Regarding Rgs17, ontogenetic quinpirole produced a modest but significant increase in expression in the same brain areas. There were no significant differences in Rgs4 expression produced by drug treatment in any of the brain regions analysed. This study demonstrates that ontogenetic quinpirole treatment, which results in priming of the D(2) receptor, results in significant decreases in Rgs9, which has been shown to regulate G-protein coupling to D(2) receptors.
Randall J Kimple - One of the best experts on this subject based on the ideXlab platform.
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regulator of g protein signaling 14 rgs14 is a selective h ras effector
PLOS ONE, 2009Co-Authors: Francis S. Willard, Randall J Kimple, Adam J. Kimple, Melinda D. Willard, Emily A. Oestreich, M Soundararajan, Nathaniel A Sowa, D A Doyle, Channing J Der, Mark J ZylkaAbstract:Background: Regulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Ga-mediated GTP hydrolysis (‘‘GTPase-accelerating proteins’’ or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Ga GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras. Methodology/Principal Findings: Full-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co- transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor- mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling. Conclusions/Significance: In cells, RGS14 facilitates the formation of a selective Ras?GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras- binding domain architecture with RGS14.
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Return of the GDI: The GoLoco Motif in Cell Division
Annual review of biochemistry, 2004Co-Authors: Francis S. Willard, Randall J Kimple, David P SiderovskiAbstract:The GoLoco motif is a 19-amino-acid sequence with guanine nucleotide dissociation inhibitor activity against G-alpha subunits of the adenylyl-cyclase-inhibitory subclass. The GoLoco motif is present as an independent element within multidomain signaling regulators, such as Loco, RGS12, RGS14, and Rap1GAP, as well as in tandem arrays in proteins, such as AGS3, G18, LGN, Pcp-2/L7, and Partner of Inscuteable (Pins/Rapsynoid). Here we discuss the biochemical mechanisms of GoLoco motif action on G-alpha subunits in light of the recent crystal structure of G-alpha-i1 bound to the RGS14 GoLoco motif. Currently, there is sparse evidence for GoLoco motif regulation of canonical G-protein-coupled receptor signaling. Rather, studies of asymmetric cell division in Drosophila and Caenorhabditis elegans, as well as mammalian mitosis, implicate GoLoco proteins, such as Pins, GPR-1/GPR-2, LGN, and RGS14, in mitotic spindle organization and force generation. We discuss potential mechanisms by which GoLoco/Galpha complexes might modulate spindle dynamics.
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Purification and in vitro functional analyses of RGS12 and RGS14 GoLoco motif peptides
Methods in enzymology, 2004Co-Authors: Randall J Kimple, Francis S. Willard, David P SiderovskiAbstract:The GoLoco motif is a short polypeptide sequence that binds to heterotrimeric G-protein alpha subunits of the adenylyl cyclase-inhibitory (Galpha(i/o)) subclass in a nucleotide-dependent manner (i.e., solely to the GDP-bound ground state). This article describes methods used for the expression, purification, and in vitro evaluation of membrane-permeant tag fusion peptides derived from the GoLoco motif regions of "regulator of G-protein signaling" proteins type 12 (RGS12) and 14 (RGS14) and a consensus GoLoco sequence from the multiple GoLoco motif protein AGS3. Three different fluorescence-based assays are described for evaluating the in vitro function of these GoLoco peptides as guanine nucleotide dissociation inhibitors, including measurements of GTPgammaS binding and Galpha subunit activation by the planar ion aluminum tetrafluoride.
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rgs12 and rgs14 goloco motifs are g alpha i interaction sites with guanine nucleotide dissociation inhibitor activity
Journal of Biological Chemistry, 2001Co-Authors: Randall J Kimple, Helene Tronchere, Cynthia I Behe, Rebecca A Morris, Marilyn Gist Farquhar, Luc De Vries, David P SiderovskiAbstract:Abstract The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein α subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single Gαi/o-Loco (GoLoco) motif predicted to represent a second Gα interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with Gαi1, Gαi2, and Gαi3 in their GDP-bound forms. In GTPγS binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by Gαi1. Both regions also stabilize Gαi1 in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF . Our results indicate that both RGS12 and RGS14 harbor two distinctly different Gα interaction sites: a previously recognized N-terminal RGS box possessing Gαi/o GAP activity and a C-terminal GoLoco region exhibiting Gαi GDI activity. The presence of two, independent Gα interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple Gα GAP activity.
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RGS12 and RGS14 GoLoco Motifs Are GαiInteraction Sites with Guanine Nucleotide Dissociation Inhibitor Activity
The Journal of biological chemistry, 2001Co-Authors: Randall J Kimple, Helene Tronchere, Cynthia I Behe, Rebecca A Morris, Marilyn Gist Farquhar, Luc De Vries, David P SiderovskiAbstract:The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms. In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1). Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-). Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity. The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity.