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• W2292601314 abstract "Regulators of G protein Signaling (RGS) promote deactivation of heterotrimeric G proteins thus controlling the magnitude and kinetics of responses mediated by G protein-coupled receptors (GPCR). In the nervous system, RGS7 and RGS9–2 play essential role in vision, reward processing, and movement control. Both RGS7 and RGS9–2 belong to the R7 subfamily of RGS proteins that form macromolecular complexes with R7-binding protein (R7BP). R7BP targets RGS proteins to the plasma membrane and augments their GTPase-accelerating protein (GAP) activity, ultimately accelerating deactivation of G protein signaling. However, it remains unclear if R7BP serves exclusively as a membrane anchoring subunit or further modulates RGS proteins to increase their GAP activity. To directly answer this question, we utilized a rapidly reversible chemically induced protein dimerization system that enabled us to control RGS localization independent from R7BP in living cells. To monitor kinetics of Gα deactivation, we coupled this strategy with measuring changes in the GAP activity by bioluminescence resonance energy transfer-based assay in a cellular system containing μ-opioid receptor. This approach was used to correlate changes in RGS localization and activity in the presence or absence of R7BP. Strikingly, we observed that RGS activity is augmented by membrane recruitment, in an orientation independent manner with no additional contributions provided by R7BP. These findings argue that the association of R7 RGS proteins with the membrane environment provides a major direct contribution to modulation of their GAP activity. Regulators of G protein Signaling (RGS) promote deactivation of heterotrimeric G proteins thus controlling the magnitude and kinetics of responses mediated by G protein-coupled receptors (GPCR). In the nervous system, RGS7 and RGS9–2 play essential role in vision, reward processing, and movement control. Both RGS7 and RGS9–2 belong to the R7 subfamily of RGS proteins that form macromolecular complexes with R7-binding protein (R7BP). R7BP targets RGS proteins to the plasma membrane and augments their GTPase-accelerating protein (GAP) activity, ultimately accelerating deactivation of G protein signaling. However, it remains unclear if R7BP serves exclusively as a membrane anchoring subunit or further modulates RGS proteins to increase their GAP activity. To directly answer this question, we utilized a rapidly reversible chemically induced protein dimerization system that enabled us to control RGS localization independent from R7BP in living cells. To monitor kinetics of Gα deactivation, we coupled this strategy with measuring changes in the GAP activity by bioluminescence resonance energy transfer-based assay in a cellular system containing μ-opioid receptor. This approach was used to correlate changes in RGS localization and activity in the presence or absence of R7BP. Strikingly, we observed that RGS activity is augmented by membrane recruitment, in an orientation independent manner with no additional contributions provided by R7BP. These findings argue that the association of R7 RGS proteins with the membrane environment provides a major direct contribution to modulation of their GAP activity. In the nervous system, signaling through transmembrane G protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCRG protein-coupled receptorGAPGTPase-activating proteinRGSregulator of G protein signalingR7BPR7-binding proteinMORμ-opioid receptor. plays a crucial role in a number of fundamental processes including differentiation, neurotransmission, and synaptic plasticity (1.Wettschureck N. Offermanns S. Mammalian G proteins and their cell type specific functions.Physiol. Rev. 2005; 85: 1159-1204Crossref PubMed Scopus (830) Google Scholar). Upon binding to an extracellular ligand, GPCRs undergo a conformational change that leads to activation of intracellular heterotrimeric Gαβγ proteins. This process involves dissociation of G proteins into GαGTP and Gβγ subunits freeing them for the interaction with downstream effectors (2.Neer E.J. Heterotrimeric G proteins: organizers of transmembrane signals.Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1287) Google Scholar). The magnitude of the response depends on the amount of time G proteins spend in their activated state. Therefore, timely deactivation of G proteins is crucial for determining the overall extent of signaling. G protein-coupled receptor GTPase-activating protein regulator of G protein signaling R7-binding protein μ-opioid receptor. Deactivation of G proteins is controlled by Regulators of G protein signaling (RGS) proteins which act as GTPase-activating proteins (GAPs) accelerating the rate of GTP hydrolysis on the Gα subunit thereby promoting return of G protein to its inactive Gαβγ heterotrimeric state. It is currently well accepted that the RGS-catalyzed G protein deactivation is essential for determining timing, extent and sensitivity of G protein signaling in a number of GPCR pathways (3.Ross E.M. Wilkie T.M. GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins.Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (929) Google Scholar, 4.Hollinger S. Hepler J.R. Cellular regulation of RGS proteins: modulators and integrators of G protein signaling.Pharmacol. Rev. 2002; 54: 527-559Crossref PubMed Scopus (600) Google Scholar). Among more than 30 known RGS proteins in mammalian genomes, critical roles for the signaling in the nervous system has been attributed to the R7 family that includes RGS6, RGS7, RGS9, and RGS11. Two members of the R7 family in particular, RGS7 and long splice isoform of RGS9: RGS9–2, ensure timely Gα deactivation required in key neuronal processes including vision, motor control, reward behavior, and nociception (5.Anderson G.R. Posokhova E. Martemyanov K.A. The R7 RGS protein family: multi-subunit regulators of neuronal G protein signaling.Cell Biochem. Biophys. 2009; 54: 33-46Crossref PubMed Scopus (107) Google Scholar). For example, the broadly expressed RGS7 has been shown to play key role in synaptic communication of ON-bipolar neurons in the retina (6.Cao Y. Pahlberg J. Sarria I. Kamasawa N. Sampath A.P. Martemyanov K.A. Regulators of G protein signaling RGS7 and RGS11 determine the onset of the light response in ON bipolar neurons.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7905-7910Crossref PubMed Scopus (67) Google Scholar), regulation GABA neurotransmission in hippocampus (7.Ostrovskaya O. Xie K. Masuho I. Fajardo-Serrano A. Lujan R. Wickman K. Martemyanov K.A. RGS7/Gbeta5/R7BP complex regulates synaptic plasticity and memory by modulating hippocampal GABABR-GIRK signaling.eLife. 2014; 3: e02053Crossref PubMed Scopus (46) Google Scholar), and control of morphine reward through μ-opioid receptor (MOR) signaling in the nucleus accumbens (8.Sutton L.P. Ostrovskaya O. Dao M. Xie K. Orlandi C. Smith R. Wee S. Martemyanov K.A. Regulator of G-Protein Signaling 7 Regulates Reward Behavior by Controlling Opioid Signaling in the Striatum.Biol. Psychiatry. 2015; (pii:S0006–3223(15)00653-8)10.1016/j.biopsych.2015.07.026PubMed Google Scholar). RGS9–2 expression is restricted to the striatum where it also regulates signaling through MOR (9.Psigfogeorgou K. Terzi D. Papachatzaki M.M. Varidaki A. Ferguson D. Gold S.J. Zachariou V. A unique role of RGS9–2 in the striatum as a positive or negative regulator of opiate analgesia.J. Neurosci. 2011; 31: 5617-5624Crossref PubMed Scopus (54) Google Scholar) and D2 dopamine receptors (10.Cabrera-Vera T.M. Hernandez S. Earls L.R. Medkova M. Sundgren-Andersson A.K. Surmeier D.J. Hamm H.E. RGS9–2 modulates D2 dopamine receptor-mediated Ca2+ channel inhibition in rat striatal cholinergic interneurons.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16339-16344Crossref PubMed Scopus (85) Google Scholar) and is involved in reward signaling, motor control and neuropathic pain (11.Mitsi V. Terzi D. Purushothaman I. Manouras L. Gaspari S. Neve R.L. Stratinaki M. Feng J. Shen L. Zachariou V. RGS9–2-controlled adaptations in the striatum determine the onset of action and efficacy of antidepressants in neuropathic pain states.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: E5088-E5097Crossref PubMed Scopus (25) Google Scholar, 12.Gold S.J. Hoang C.V. Potts B.W. Porras G. Pioli E. Kim K.W. Nadjar A. Qin C. LaHoste G.J. Li Q. Bioulac B.H. Waugh J.L. Gurevich E. Neve R.L. Bezard E. RGS9–2 negatively modulates L-3,4-dihydroxyphenylalanine-induced dyskinesia in experimental Parkinson's disease.J. Neurosci. 2007; 27: 14338-14348Crossref PubMed Scopus (109) Google Scholar, 13.Kovoor A. Seyffarth P. Ebert J. Barghshoon S. Chen C.K. Schwarz S. Axelrod J.D. Cheyette B.N. Simon M.I. Lester H.A. Schwarz J. D2 dopamine receptors colocalize regulator of G-protein signaling 9–2 (RGS9–2) via the RGS9 DEP domain, and RGS9 knock-out mice develop dyskinesias associated with dopamine pathways.J. Neurosci. 2005; 25: 2157-2165Crossref PubMed Scopus (144) Google Scholar, 14.Rahman Z. Schwarz J. Gold S.J. Zachariou V. Wein M.N. Choi K.H. Kovoor A. Chen C.K. DiLeone R.J. Schwarz S.C. Selley D.E. Sim-Selley L.J. Barrot M. Luedtke R.R. Self D. Neve R.L. Lester H.A. Simon M.I. Nestler E.J. RGS9 modulates dopamine signaling in the basal ganglia.Neuron. 2003; 38: 941-952Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 15.Zachariou V. Georgescu D. Sanchez N. Rahman Z. DiLeone R. Berton O. Neve R.L. Sim-Selley L.J. Selley D.E. Gold S.J. Nestler E.J. Essential role for RGS9 in opiate action.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13656-13661Crossref PubMed Scopus (203) Google Scholar, 16.Anderson G.R. Cao Y. Davidson S. Truong H.V. Pravetoni M. Thomas M.J. Wickman K. Giesler Jr., G.J. Martemyanov K.A. R7BP complexes with RGS9–2 and RGS7 in the striatum differentially control motor learning and locomotor responses to cocaine.Neuropsychopharmacology. 2010; 35: 1040-1050Crossref PubMed Scopus (39) Google Scholar). In neurons, the function of RGS7 and RGS9–2 is regulated by their association with several binding partners. Both RGS proteins form an obligatory heterodimer with type 5 G protein β-subunit (Gβ5), which facilitates their folding and provides proteolytic stability (17.Chen C.K. Eversole-Cire P. Zhang H. Mancino V. Chen Y.J. He W. Wensel T.G. Simon M.I. Instability of GGL domain-containing RGS proteins in mice lacking the G protein beta-subunit Gbeta5.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6604-6609Crossref PubMed Scopus (175) Google Scholar, 18.Cabrera J.L. De Freitas F. Satpaev D.K. Slepak V.Z. Identification of the G*5-RGS7 protein complex in the retina.Biochem. Biophys. Res. Commun. 1998; 249: 898-902Crossref PubMed Scopus (114) Google Scholar, 19.Makino E.R. Handy J.W. Li T.S. Arshavsky V.Y. The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein * subunit.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 1947-1952Crossref PubMed Scopus (194) Google Scholar). In addition, both RGS7/Gβ5 and RGS9–2/Gβ5 form complexes with small palmitoylated protein R7-binding protein (R7BP), which plays essential role in controlling their membrane localization and stability (20.Martemyanov K.A. Yoo P.J. Skiba N.P. Arshavsky V.Y. R7BP, a novel neuronal protein interacting with RGS proteins of the R7 family.J. Biol. Chem. 2005; 280: 5133-5136Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 21.Drenan R.M. Doupnik C.A. Jayaraman M. Buchwalter A.L. Kaltenbronn K.M. Huettner J.E. Linder M.E. Blumer K.J. R7BP augments the function of RGS7*Gβ5 complexes by a plasma membrane-targeting mechanism.J. Biol. Chem. 2006; 281: 28222-28231Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 22.Anderson G.R. Lujan R. Semenov A. Pravetoni M. Posokhova E.N. Song J.H. Uversky V. Chen C.K. Wickman K. Martemyanov K.A. Expression and localization of RGS9–2/G 5/R7BP complex in vivo is set by dynamic control of its constitutive degradation by cellular cysteine proteases.J. Neurosci. 2007; 27: 14117-14127Crossref PubMed Scopus (54) Google Scholar, 23.Posokhova E. Uversky V. Martemyanov K.A. Proteomic identification of Hsc70 as a mediator of RGS9–2 degradation by in vivo interactome analysis.J. Proteome Res. 2010; 9: 1510-1521Crossref PubMed Scopus (12) Google Scholar, 24.Drenan R.M. Doupnik C.A. Boyle M.P. Muglia L.J. Huettner J.E. Linder M.E. Blumer K.J. Palmitoylation regulates plasma membrane-nuclear shuttling of R7BP, a novel membrane anchor for the RGS7 family.J. Cell Biol. 2005; 169: 623-633Crossref PubMed Scopus (116) Google Scholar, 25.Anderson G.R. Semenov A. Song J.H. Martemyanov K.A. The membrane anchor R7BP controls the proteolytic stability of the striatal specific RGS protein, RGS9–2.J. Biol. Chem. 2007; 282: 4772-4781Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Another binding partner, the orphan receptor GPR158, is a selective membrane anchor for RGS7, which is similarly critical for RGS7 expression and membrane localization in the nervous system (26.Orlandi C. Xie K. Masuho I. Fajardo-Serrano A. Lujan R. Martemyanov K.A. Orphan receptor GPR158 is an allosteric modulator of RGS7 catalytic activity with an essential role in dictating its expression and localization in the brain.J. Biol. Chem. 2015; 290: 13622-13639Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 27.Orlandi C. Posokhova E. Masuho I. Ray T.A. Hasan N. Gregg R.G. Martemyanov K.A. GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes.J. Cell Biol. 2012; 197: 711-719Crossref PubMed Scopus (72) Google Scholar). Targeting to the plasma membrane is a consistent theme in regulation of RGS7/Gβ5 and RGS9–2/Gβ5 function. Both RGS7/Gβ5 and RGS9–2/Gβ5 are largely cytoplasmic when expressed in heterologous system (28.Song J.H. Waataja J.J. Martemyanov K.A. Subcellular targeting of RGS9–2 is controlled by multiple molecular determinants on its membrane anchor, R7BP.J. Biol. Chem. 2006; 281: 15361-15369Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 29.Liapis E. Sandiford S. Wang Q. Gaidosh G. Motti D. Levay K. Slepak V.Z. Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells.J. Neurochem. 2012; 122: 568-581Crossref PubMed Scopus (11) Google Scholar, 30.Zhang J.H. Barr V.A. Mo Y. Rojkova A.M. Liu S. Simonds W.F. Nuclear localization of G protein β5 and regulator of G protein signaling 7 in neurons and brain.J. Biol. Chem. 2001; 276: 10284-10289Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 31.Bouhamdan M. Michelhaugh S.K. Calin-Jageman I. Ahern-Djamali S. Bannon M.J. Brain-specific RGS9–2 is localized to the nucleus via its unique proline-rich domain.Biochim. Biophys. Acta. 2004; 1691: 141-150Crossref PubMed Scopus (26) Google Scholar), although a fraction of RGS7 has been shown to undergo palmitoylation thought to promote its membrane localization (32.Rose J.J. Taylor J.B. Shi J. Cockett M.I. Jones P.G. Hepler J.R. RGS7 is palmitoylated and exists as biochemically distinct forms.J. Neurochem. 2000; 75: 2103-2112Crossref PubMed Scopus (71) Google Scholar, 33.Takida S. Fischer C.C. Wedegaertner P.B. Palmitoylation and plasma membrane targeting of RGS7 are promoted by alpha o.Mol. Pharmacol. 2005; 67: 132-139Crossref PubMed Scopus (27) Google Scholar). The RGS7/Gβ5 and RGS9–2/Gβ5 complexes can be effectively recruited to the plasma membrane via interaction with their substrate, activated Gαo (33.Takida S. Fischer C.C. Wedegaertner P.B. Palmitoylation and plasma membrane targeting of RGS7 are promoted by alpha o.Mol. Pharmacol. 2005; 67: 132-139Crossref PubMed Scopus (27) Google Scholar) or membrane anchors R7BP (20.Martemyanov K.A. Yoo P.J. Skiba N.P. Arshavsky V.Y. R7BP, a novel neuronal protein interacting with RGS proteins of the R7 family.J. Biol. Chem. 2005; 280: 5133-5136Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 24.Drenan R.M. Doupnik C.A. Boyle M.P. Muglia L.J. Huettner J.E. Linder M.E. Blumer K.J. Palmitoylation regulates plasma membrane-nuclear shuttling of R7BP, a novel membrane anchor for the RGS7 family.J. Cell Biol. 2005; 169: 623-633Crossref PubMed Scopus (116) Google Scholar) and GPR158 (27.Orlandi C. Posokhova E. Masuho I. Ray T.A. Hasan N. Gregg R.G. Martemyanov K.A. GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes.J. Cell Biol. 2012; 197: 711-719Crossref PubMed Scopus (72) Google Scholar). In the case of R7BP, this membrane recruitment has been reported to result in augmentation of RGS9–2 and RGS7 GAP activity toward its substrates Gαi and Gαo, accelerating termination of the G protein driven responses (21.Drenan R.M. Doupnik C.A. Jayaraman M. Buchwalter A.L. Kaltenbronn K.M. Huettner J.E. Linder M.E. Blumer K.J. R7BP augments the function of RGS7*Gβ5 complexes by a plasma membrane-targeting mechanism.J. Biol. Chem. 2006; 281: 28222-28231Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 34.Masuho I. Xie K. Martemyanov K.A. Macromolecular composition dictates receptor and G protein selectivity of regulator of G protein signaling (RGS) 7 and 9–2 protein complexes in living cells.J. Biol. Chem. 2013; 288: 25129-25142Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The mechanism behind this stimulatory effect is unclear. On the one hand, membrane recruitment of RGS proteins may facilitate G protein deactivation by increasing the proximity to of RGS proteins to their membrane-bound substrate Gα-GTP (21.Drenan R.M. Doupnik C.A. Jayaraman M. Buchwalter A.L. Kaltenbronn K.M. Huettner J.E. Linder M.E. Blumer K.J. R7BP augments the function of RGS7*Gβ5 complexes by a plasma membrane-targeting mechanism.J. Biol. Chem. 2006; 281: 28222-28231Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 27.Orlandi C. Posokhova E. Masuho I. Ray T.A. Hasan N. Gregg R.G. Martemyanov K.A. GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes.J. Cell Biol. 2012; 197: 711-719Crossref PubMed Scopus (72) Google Scholar, 34.Masuho I. Xie K. Martemyanov K.A. Macromolecular composition dictates receptor and G protein selectivity of regulator of G protein signaling (RGS) 7 and 9–2 protein complexes in living cells.J. Biol. Chem. 2013; 288: 25129-25142Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). On the other hand, R7BP has been also proposed to act as an allosteric modulator of RGS proteins (35.Zhou H. Chisari M. Raehal K.M. Kaltenbronn K.M. Bohn L.M. Mennerick S.J. Blumer K.J. GIRK channel modulation by assembly with allosterically regulated RGS proteins.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 19977-19982Crossref PubMed Scopus (32) Google Scholar, 36.Narayanan V. Sandiford S.L. Wang Q. Keren-Raifman T. Levay K. Slepak V.Z. Intramolecular interaction between the DEP domain of RGS7 and the Gβ5 subunit.Biochemistry. 2007; 46: 6859-6870Crossref PubMed Scopus (29) Google Scholar). However, the impact of membrane association relative to possible direct effects of R7BP is unknown. In this study we examined the contribution of membrane localization of RGS7 and RGS9–2 in regulation of MOR receptor signaling. We implemented a reversible-chemical dimerization system to control RGS localization independent of binding partners and evaluated real-time changes in GAP activity with and without R7BP in live cells. Our results show that membrane recruitment of RGS is sufficient for attaining the full potentiation of their GAP activity. This finding suggests that augmentation of RGS activity by R7BP may result from positioning R7 RGS proteins in the context of lipid environment. The cloning of R7BP, Gβ5S, and RGS9–2 has been described (20.Martemyanov K.A. Yoo P.J. Skiba N.P. Arshavsky V.Y. R7BP, a novel neuronal protein interacting with RGS proteins of the R7 family.J. Biol. Chem. 2005; 280: 5133-5136Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 28.Song J.H. Waataja J.J. Martemyanov K.A. Subcellular targeting of RGS9–2 is controlled by multiple molecular determinants on its membrane anchor, R7BP.J. Biol. Chem. 2006; 281: 15361-15369Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). RGS7 was obtained from Missouri S&T cDNA Resource Center. The KRas membrane targeting construct (37.Lan T.H. Liu Q. Li C. Wu G. Lambert N.A. Sensitive and high resolution localization and tracking of membrane proteins in live cells with BRET.Traffic. 2012; 13: 1450-1456Crossref PubMed Scopus (65) Google Scholar) as well as the BRET biosensors Venus155–239-Gβ1, Venus1–155-Gγ2, were provided by Nevin A. Lambert (Medical College of Georgia, Augusta, GA) (38.Hollins B. Kuravi S. Digby G.J. Lambert N.A. The c-terminus of GRK3 indicates rapid dissociation of G protein heterotrimers.Cell Signal. 2009; 21: 1015-1021Crossref PubMed Scopus (94) Google Scholar). Cloning of masGRK3ctNluc reporter has been described (7.Ostrovskaya O. Xie K. Masuho I. Fajardo-Serrano A. Lujan R. Wickman K. Martemyanov K.A. RGS7/Gbeta5/R7BP complex regulates synaptic plasticity and memory by modulating hippocampal GABABR-GIRK signaling.eLife. 2014; 3: e02053Crossref PubMed Scopus (46) Google Scholar). The In-Fusion HD Cloning Kit (Clontech, Mountain View, CA) was used to generate the following constructs used in this study (all in pcDNA3.1): mSNAPf, RGS7-P2A-Gβ5, mCherry-RGS7-P2A-Gβ5, FKBP-mCherry-RGS7-P2A-Gβ5, mCherry-RGS7-FKBP-P2A-Gβ5, RGS9–2-P2A-Gβ5, mCherry-RGS9–2-P2A-Gβ5, FKBP-mCherry-RGS9–2-P2A-Gβ5, mCherry-RGS9–2-FKBP-P2A-Gβ5, sR7BP, sR7BP-FKBP, Venus-sR7BP-FKBP, and Venus-R7BP. mCherry was cloned from pmCherry-N1 (Clontech), SNAPf was cloned from pENTR4-SNAPf (provided by Eric Campeau/Addgene plasmid # 29652), and FKBP was cloned from Lyn-FKBP-FKBP-CFP (provided by Tobias Meyer/Addgene plasmid # 20149). Detailed cloning strategies and a list of primer sequences are available upon request. NG108–15 cells were cultured in DMEM supplemented with 10% fetal bovine serum, sodium hypoxanthine (0.1 mm), aminopterin (0.4 μm), thymidine (16 μm), penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C in a 5% CO2 humidified incubator. Cells were transfected at ∼80% confluence in 35-mm plates using Lipofectamine PLUS (2.5 μl) and LTX (4 μl) reagents for Bioluminescence Resonance Energy Transfer assay as previously described with modification (34.Masuho I. Xie K. Martemyanov K.A. Macromolecular composition dictates receptor and G protein selectivity of regulator of G protein signaling (RGS) 7 and 9–2 protein complexes in living cells.J. Biol. Chem. 2013; 288: 25129-25142Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). MOR, GαoA, Venus155–239-Gβ1, Venus1–155-Gγ2, masGRK3ct-Nluc, and SNAPf-KRas constructs were transfected at a 1:2:1:1:1:1 ratio. Experiments using RGS7, RGS9–2, or R7BP constructs were used at a 1:0.5:1 ratio relative to MOR. A total of 2.5 μg was transfected in each experiment using an empty vector to normalize the total amount of plasmid DNA. rCD1 and FK506 were purchased from Sirius Fine Chemicals (Bremen, Germany) and used at a final concentration of 1 μm. Cells were grown on poly-l-lysine coated coverslips, handled as stated above, fixed in a 4% paraformaldehyde/2% glucose solution for 10 min at room temperature, and mounted on slides with DAPI Fluoromount-G (SouthernBiotech). Confocal images were obtained using a Zeiss LSM 880 under a 20× objective (Carl Zeiss). DAPI and mCherry channels were overlaid using ImageJ software. As we have previously described (34.Masuho I. Xie K. Martemyanov K.A. Macromolecular composition dictates receptor and G protein selectivity of regulator of G protein signaling (RGS) 7 and 9–2 protein complexes in living cells.J. Biol. Chem. 2013; 288: 25129-25142Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), cells were detached in 5 mm EDTA in PBS at room temperature, centrifuged at 750 × g for 5 min, and resuspended in PBS containing 0.5 mm MgCl2 and 0.1% glucose. Approximately 75,000 cells were added to a 96-well plate followed by an equal volume of Nano-Glo Luciferase Assay Substrate (Promega, Madison, WI). BRET measurements were recorded at room temperature on a POLARstar Omega micro plate reader (BMG Labtech, Cary, NC) utilizing two emission photomultiplier tubes enabling simultaneous detection of light from masGRK3ct-Nluc (475 nm) and Gβ1γ2-Venus (535 nm) with a resolution of 50 milliseconds for every data point. BRET signal was calculated as the ratio of raw 535 nm intensity divided by the raw 475 nm intensity, which was then normalized by subtracting the baseline BRET ratio prior to agonist application and expressed as a percent of maximal BRET signal. Following BRET assay samples were centrifuged and resuspended in PBS supplemented with 150 mm NaCl, 1% Triton X-100, and Complete protease inhibitor mixture (Roche, Indianapolis, IN). Cells were lysed by sonication, centrifuged at 14,000 rpm for 20 min at 4 °C, and total protein concentration of the supernatant was determined using the Pierce 660 nm Protein Assay Reagent. Samples were denatured in SDS/urea sample buffer, resolved on PAGEr Gold Gels (Lonza, Basel, Switzerland), and transferred to PVDF membranes for detection with the following primary antibodies: sheep anti-RGS9–2 (20.Martemyanov K.A. Yoo P.J. Skiba N.P. Arshavsky V.Y. R7BP, a novel neuronal protein interacting with RGS proteins of the R7 family.J. Biol. Chem. 2005; 280: 5133-5136Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), rabbit anti-RGS7 (39.Rojkova A.M. Woodard G.E. Huang T.C. Combs C.A. Zhang J.H. Simonds W.F. Gγ subunit-selective G protein β5 mutant defines regulators of G protein signaling protein binding requirement for nuclear localization.J. Biol. Chem. 2003; 278: 12507-12512Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), rabbit anti-R7BP (40.Nini L. Waheed A.A. Panicker L.M. Czapiga M. Zhang J.H. Simonds W.F. R7-binding protein targets the G protein β 5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain.BMC Biochem. 2007; 8: 18Crossref PubMed Scopus (12) Google Scholar), mouse anti-GAPDH (Millipore; AB2302). Species-specific HRP-conjugated secondary antibodies and SuperSignal West Pico ECL (Pierce) was used to capture the signal on film. Western blots were quantified using ImageJ software. The relative expression of RGS was determined by subtracting the band density from cells only expressing endogenous RGS proteins. For each BRET experiment a single exponential fit, 1/τ (s−1), was obtained from the deactivation phase of the curve. A kGAP rate constant was determined by subtracting the basal deactivation rate of cells only expressing endogenous RGS proteins. To compare GAP activity across experiments, kGAP values were then normalized to RGS expression from Western analysis and presented as kGAP/expression (s−1). A minimum of three biological replicates were performed for each experiment. To separate the contribution of protein-protein interactions from the effects of changes in the subcellular localization we sought to develop a system where we can measure RGS GAP activity as we change its association with the plasma membrane. To achieve this, we applied a recently described chemically induced dimerization system that allows controlling protein-protein interactions by a set of small molecule drugs in a rapid and completely reversible manner (41.Feng S. Laketa V. Stein F. Rutkowska A. MacNamara A. Depner S. Klingmüller U. Saez-Rodriguez J. Schultz C. A rapidly reversible chemical dimerizer system to study lipid signaling in living cells.Angew Chem. Int. Ed Engl. 2014; 53: 6720-6723Crossref PubMed Scopus (56) Google Scholar). The system utilizes interaction between two proteins, SNAPf and FKBP, which dimerize upon binding to a cell permeable small molecule rCD1. This interaction is rapidly disrupted by addition of a second small molecule FK506 that serves as a competitive antagonist. We positioned SNAPf to the plasma membrane, by appending it to the C-terminal membrane-targeting domain of K-Ras, which contains a polybasic cluster of amino acids followed by a site for prenylation, calling the resulting construct mSNAPf. The matching counterpart, FKBP was fused with the fluorescent protein mCherry and added to the N terminus of RGS7 and RGS9–2 (Fig. 1, A and B). When expressed in NG108–15 together with Gβ5 both FKBP-mCherry-RGS7 and FKBP-mCherry-RGS9–2 localized exclusively to the cytoplasm (Fig. 1A). This localization was no different than distribution of RGS7/Gβ5 and RGS9–2/Gβ5 complexes tagged only with mCherry but not FKBP (Fig. 1A) and matched previously documented localization of these proteins in transfected cells (28.Song J.H. Waataja J.J. Martemyanov K.A. Subcellular targeting of RGS9–2 is controlled by multiple molecular determinants on its membrane anchor, R7BP.J. Biol. Chem. 2006; 281: 15361-15369Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 29.Liapis E. Sandiford S. Wang Q. Gaidosh G. Motti D. Levay K. Slepak V.Z. Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells.J. Neurochem. 2012; 122: 568-581Crossref PubMed Scopus (11) Google Scholar, 31.Bouhamdan M. Michelhaugh S.K. Calin-Jageman I. Ahern-Djamali S. Bannon M.J. Brain-specific RGS9–2 is localized to the nucleus via its unique proline-rich domain.Biochim. Biophys. Acta. 2004; 1691: 141-150Crossref PubMed Scopus (26) Google Scholar). Furthermore, FKBP-mCherry-RGS7 or FKBP-mCherry-RGS9–2 complexes with Gβ5 retained their ability to be completely recruited to the plasma membrane upon co-expression with R7BP (Fig. 1A). Co-expression of FKBP-mCherry-RGS7 or FKBP-mCher" @default.
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• W2292601314 title "Association with the Plasma Membrane Is Sufficient for Potentiating Catalytic Activity of Regulators of G Protein Signaling (RGS) Proteins of the R7 Subfamily" @default.
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• W2292601314 doi "https://doi.org/10.1074/jbc.m115.713446" @default.
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