Endogenous Regulator Of G Protein Signaling Proteins Research Articles

Differential Control of Opioid Antinociception to Thermal Stimuli in a Knock-In Mouse Expressing Regulator of G-Protein Signaling-Insensitive GαoProtein

Regulator of G-protein signaling (RGS) proteins classically function as negative modulators of G-protein-coupled receptor signaling. In vitro, RGS proteins have been shown to inhibit signaling by agonists at the μ-opioid receptor, including morphine. The goal of the present study was to evaluate the contribution of endogenous RGS proteins to the antinociceptive effects of morphine and other opioid agonists. To do this, a knock-in mouse that expresses an RGS-insensitive (RGSi) mutant Gαo protein, Gαo(G184S) (Gαo RGSi), was evaluated for morphine or methadone antinociception in response to noxious thermal stimuli. Mice expressing Gαo RGSi subunits exhibited a naltrexone-sensitive enhancement of baseline latency in both the hot-plate and warm-water tail-withdrawal tests. In the hot-plate test, a measure of supraspinal nociception, morphine antinociception was increased, and this was associated with an increased ability of opioids to inhibit presynaptic GABA neurotransmission in the periaqueductal gray. In contrast, antinociception produced by either morphine or methadone was reduced in the tail-withdrawal test, a measure of spinal nociception. In whole-brain and spinal cord homogenates from mice expressing Gαo RGSi subunits, there was a small loss of Gαo expression and an accompanying decrease in basal G-protein activity. Our results strongly support a role for RGS proteins as negative regulators of opioid supraspinal antinociception and also reveal a potential novel function of RGS proteins as positive regulators of opioid spinal antinociceptive pathways.

RGS proteins in heart: brakes on the vagus

It has been nearly a century since Otto Loewi discovered that acetylcholine (ACh) release from the vagus produces bradycardia and reduced cardiac contractility. It is now known that parasympathetic control of the heart is mediated by ACh stimulation of Gi/o-coupled muscarinic M2 receptors, which directly activate G protein-coupled inwardly rectifying potassium (GIRK) channels via Gβγ resulting in membrane hyperpolarization and inhibition of action potential (AP) firing. However, expression of M2R–GIRK signaling components in heterologous systems failed to recapitulate native channel gating kinetics. The missing link was identified with the discovery of regulator of G protein signaling (RGS) proteins, which act as GTPase-activating proteins to accelerate the intrinsic GTPase activity of Gα resulting in termination of Gα- and Gβγ-mediated signaling to downstream effectors. Studies in mice expressing an RGS-insensitive Gαi2 mutant (G184S) implicated endogenous RGS proteins as key regulators of parasympathetic signaling in heart. Recently, two RGS proteins have been identified as critical regulators of M2R signaling in heart. RGS6 exhibits a uniquely robust expression in heart, especially in sinoatrial (SAN) and atrioventricular nodal regions. Mice lacking RGS6 exhibit increased bradycardia and inhibition of SAN AP firing in response to CCh as well as a loss of rapid activation and deactivation kinetics and current desensitization for ACh-induced GIRK current (IKACh). Similar findings were observed in mice lacking RGS4. Thus, dysregulation in RGS protein expression or function may contribute to pathologies involving aberrant electrical activity in cardiac pacemaker cells. Moreover, RGS6 expression was found to be up-regulated in heart under certain pathological conditions, including doxorubicin treatment, which is known to cause life-threatening cardiotoxicity and atrial fibrillation in cancer patients. On the other hand, increased vagal tone may be cardioprotective in heart failure where acetylcholinesterase inhibitors and vagal stimulation have been proposed as potential therapeutics. Together, these studies identify RGS proteins, especially RGS6, as new therapeutic targets for diseases such as sick sinus syndrome or other maladies involving abnormal autonomic control of the heart.

Inhibition of Gαi2 – RGS Protein Interactions Protects the Heart from Ischemic Injury

Signaling through Gαi-coupled receptors protects the heart from ischemic injury. The activity of Gαi is modulated by regulator of G protein signaling (RGS) proteins that terminate Gαi signaling by catalyzing the hydrolysis of Gαi-bound GTP. We used knock-in mice in which interactions between Gαi2 and RGS proteins are disrupted by a Gαi2G184S (GS) point mutation to determine whether interactions between Gαi2 and endogenous RGS proteins modulate Gαi-mediated protection from ischemic injury. Hearts isolated from wildtype (+/+) mice and mice homozygous (GS/GS) or heterozygous (GS/+) for Gαi2G184S were subjected to 30 minutes ischemia and 2 hours reperfusion using the Langendorff method. Infarcts in GS/GS and GS/+ hearts were significantly smaller than those of +/+ hearts. Expression of Gαi2G184S had no effect on preischemic contractile function. However, postischemic recovery of contractile function was significantly enhanced in GS/GS and GS/+ hearts compared to +/+ hearts. The cardioprotective phenotype of GS/+ and GS/GS hearts was reversed by 5-hydroxydecanoate indicating that inhibition of RGS- Gαi2 interactions protects the heart through a mechanism that requires mitochondrial K+ATP channels. These data suggest that RGS proteins may provide novel therapeutic targets to protect the heart from ischemic injury. (Supported by NIH R01 GM39561; AFPE, and the Bower, Bennet and Bennet Endowed Research Award).

RGS inhibition at G(alpha)i2 selectively potentiates 5-HT1A-mediated antidepressant effects.

Elevating serotonin (5-HT) levels with selective serotonin reuptake inhibitors (SSRIs) is the most widely used treatment for depression. However, current therapies are ineffective, have delayed benefit, or cause side effects in many patients. Here, we define a mechanism downstream of 5-HT1A receptors that mediates antidepressant-like behavior and is profoundly and selectively enhanced by genetic disruption of regulators of G protein signaling (RGS) activity at G(alpha)i2. Animals rendered insensitive to RGS protein regulation through a mutation in G(alpha)i2 (G184S) exhibited spontaneous antidepressant- and anxiolytic-like behaviors. Mice expressing RGS-insensitive G(alpha)i2 also exhibited increased cortical and hippocampal phosphorylation of glycogen synthase kinase-3beta, a constitutively active proapoptotic kinase that is inhibited through phosphorylation in response to serotonin, SSRIs, and 5-HT1 receptor agonists. Both behavioral and biochemical phenotypes were blocked by treatment with WAY 100635, a 5-HT1A-selective antagonist. RGS-insensitive mice were also 5-10 times more responsive to the antidepressant-like effects of the SSRI fluvoxamine and 5-HT1A-selective agonist 8-hydroxy-2-dipropylaminotetralin. In contrast, the antidepressant potency of agents acting through nonserotonergic mechanisms was unchanged as was 5-HT1A action on body temperature. The findings point to a critical role for endogenous RGS proteins to suppress the antidepressant-like effects of 5-HT1A receptor activation. By selectively enhancing the beneficial effects of serotonin, inhibition of RGS proteins represents a therapeutic approach for the treatment of mood disorders.

RSBP-1 is a membrane-targeting subunit required by the Galpha(q)-specific but not the Galpha(o)-specific R7 regulator of G protein signaling in Caenorhabditis elegans.

Regulator of G protein signaling (RGS) proteins inhibit G protein signaling by activating Galpha GTPase activity, but the mechanisms that regulate RGS activity are not well understood. The mammalian R7 binding protein (R7BP) can interact with all members of the R7 family of RGS proteins, and palmitoylation of R7BP can target R7 RGS proteins to the plasma membrane in cultured cells. However, whether endogenous R7 RGS proteins in neurons require R7BP or membrane localization for function remains unclear. We have identified and knocked out the only apparent R7BP homolog in Caenorhabditis elegans, RSBP-1. Genetic studies show that loss of RSBP-1 phenocopies loss of the R7 RGS protein EAT-16, but does not disrupt function of the related R7 RGS protein EGL-10. Biochemical analyses find that EAT-16 coimmunoprecipitates with RSBP-1 and is predominantly plasma membrane-associated, whereas EGL-10 does not coimmunoprecipitate with RSBP-1 and is not predominantly membrane-associated. Mutating the conserved membrane-targeting sequence in RSBP-1 disrupts both the membrane association and function of EAT-16, demonstrating that membrane targeting by RSBP-1 is essential for EAT-16 activity. Our analysis of endogenous R7 RGS proteins in C. elegans neurons reveals key differences in the functional requirements for membrane targeting between members of this protein family.

Endogenous RGS proteins attenuate Gαi‐mediated Lysophosphatidic Acid signaling pathways in Ovarian Cancer Cells

Lysophosphatidic acid (LPA) is the predominant growth factor driving the progression of ovarian cancer. Effects of LPA are mediated by cell surface G‐protein coupled receptors (GPCRs) that activate multiple heterotrimeric G‐proteins. G‐proteins are deactivated by Regulators of G‐protein Signaling (RGS) proteins. We hypothesized that RGS proteins may regulate G‐protein signaling pathways initiated by LPA in ovarian cancer cells. To determine the effect of endogenous RGS proteins on LPA signaling in ovarian cancer cells, we compared the activity of LPA in ovarian cancer cells expressing Gi subunit constructs that are either insensitive to RGS protein regulation (RGSi) or their RGS‐sensitive wild‐type (RGSwt) counterparts. LPA‐mediated inhibition of forskolin‐stimulated adenylyl cyclase activity was enhanced in cells expressing RGSi Gi proteins as compared to RGSwt Gi, suggesting that endogenous RGS proteins in ovarian cancer cells normally attenuate signaling by these G‐proteins. The enhanced Gi signaling reflected greater potency, efficacy, and duration of LPA effects in the absence of RGS regulation. Further, LPA‐stimulated ovarian cancer cell migration in a wound‐induced migration assay was enhanced in cells expressing Gai2 RGSi as compared to cells expressing Gai2 RGSwt. These data establish RGS proteins as important regulators of LPA signaling in ovarian cancer cells.

Endogenous Regulators of G Protein Signaling Differentially Modulate Full and Partial μ-Opioid Agonists at Adenylyl Cyclase as Predicted by a Collision Coupling Model

Regulator of G protein signaling (RGS) proteins accelerate the endogenous GTPase activity of Galpha(i/o) proteins to increase the rate of deactivation of active Galpha-GTP and Gbetagamma signaling molecules. Previous studies have suggested that RGS proteins are more effective on less efficiently coupled systems such as with partial agonist responses. To determine the role of endogenous RGS proteins in functional responses to mu-opioid agonists of different intrinsic efficacy, Galpha(i/o) subunits with a mutation at the pertussis toxin (PTX)-sensitive cysteine (C351I) and with or without a mutation at the RGS binding site (G184S) were stably expressed in C6 glioma cells expressing a mu-opioid receptor. Cells were treated overnight with PTX to inactivate endogenous G proteins. Maximal inhibition of forskolin-stimulated adenylyl cyclase by the low-efficacy partial agonists buprenorphine and nalbuphine was increased in cells expressing RGS-insensitive Galpha(o)(CIGS), Galpha(i2)(CIGS), or Galpha(i3)(CIGS) compared with their Galpha(CI) counterparts, but the RGS-insensitive mutation had little or no effect on the maximal inhibition by the higher efficacy agonists DAMGO and morphine. The potency of all the agonists to inhibit forskolin-stimulated adenylyl cyclase was increased in cells expressing RGS-insensitive Galpha(o)(CIGS), Galpha(i2)(CIGS), or Galpha(i3)(CIGS), regardless of efficacy. These data are comparable with predictions based on a collision coupling model. In this model, the rate of G protein inactivation, which is modulated by RGS proteins, and the rate of G protein activation, which is affected by agonist intrinsic efficacy, determine the maximal agonist response and potency at adenylyl cyclase under steady state conditions.

Endogenous RGS proteins attenuate Gα i-mediated lysophosphatidic acid signaling pathways in ovarian cancer cells

Lysophosphatidic acid is a bioactive phospholipid that is produced by and stimulates ovarian cancer cells, promoting proliferation, migration, invasion, and survival. Effects of LPA are mediated by cell surface G-protein coupled receptors (GPCRs) that activate multiple heterotrimeric G-proteins. G-proteins are deactivated by Regulator of G-protein Signaling (RGS) proteins. This led us to hypothesize that RGS proteins may regulate G-protein signaling pathways initiated by LPA in ovarian cancer cells. To determine the effect of endogenous RGS proteins on LPA signaling in ovarian cancer cells, we compared LPA activity in SKOV-3 ovarian cancer cells expressing G i subunit constructs that are either insensitive to RGS protein regulation (RGSi) or their RGS wild-type (RGSwt) counterparts. Both forms of the G-protein contained a point mutation rendering them insensitive to inhibition with pertussis toxin, and cells were treated with pertussis toxin prior to experiments to eliminate endogenous G i/o signaling. The potency and efficacy of LPA-mediated inhibition of forskolin-stimulated adenylyl cyclase activity was enhanced in cells expressing RGSi G i proteins as compared to RGSwt G i. We further showed that LPA signaling that is subject to RGS regulation terminates much faster than signaling thru RGS insensitive G-proteins. Finally, LPA-stimulated SKOV-3 cell migration, as measured in a wound-induced migration assay, was enhanced in cells expressing Gα i2 RGSi as compared to cells expressing Gα i2 RGSwt, suggesting that endogenous RGS proteins in ovarian cancer cells normally attenuate this LPA effect. These data establish RGS proteins as novel regulators of LPA signaling in ovarian cancer cells.

Endogenous RGS proteins modulate SA and AV nodal functions in isolated heart: implications for sick sinus syndrome and AV block

G protein-coupled receptors play a pivotal role in regulating cardiac automaticity. Their function is controlled by regulator of G protein signaling (RGS) proteins acting as GTPase-activating proteins for Galpha subunits to suppress Galpha(i) and Galpha(q) signaling. Using knock-in mice in which Galpha(i2)-RGS binding and negative regulation are disrupted by a genomic Galpha(i2)G184S (GS) point mutation, we recently (Fu Y, Huang X, Zhong H, Mortensen RM, D'Alecy LG, Neubig RR. Circ Res 98: 659-666, 2006) showed that endogenous RGS proteins suppress muscarinic receptor-mediated bradycardia. To determine whether this was due to direct regulation of cardiac pacemakers or to alterations in the central nervous system or vascular responses, we examined isolated, perfused hearts. Isoproterenol-stimulated beating rates of heterozygote (+/GS) and homozygote (GS/GS) hearts were significantly more sensitive to inhibition by carbachol than were those of wild type (+/+). Even greater effects were seen in the absence of isoproterenol; the potency of muscarinic-mediated bradycardia was enhanced fivefold in GS/GS and twofold in +/GS hearts compared with +/+. A(1)-adenosine receptor-mediated bradycardia was unaffected. In addition to effects on the sinoatrial node, +/GS and GS/GS hearts show significantly increased carbachol-induced third-degree atrioventricular (AV) block. Atrial pacing studies demonstrated an increased PR interval and AV effective refractory period in GS/GS hearts compared with +/+. Thus loss of the inhibitory action of endogenous RGS proteins on Galpha(i2) potentiates muscarinic inhibition of cardiac automaticity and conduction. The severe carbachol-induced sinus bradycardia in Galpha(i2)G184S mice suggests a possible role for alterations of Galpha(i2) or RGS proteins in sick sinus syndrome and pathological AV block.

Selective Loss of Fine Tuning of Gq/11 Signaling by RGS2 Protein Exacerbates Cardiomyocyte Hypertrophy

Alterations in cardiac G protein-mediated signaling, most prominently G(q/11) signaling, are centrally involved in hypertrophy and heart failure development. Several RGS proteins that can act as negative regulators of G protein signaling are expressed in the heart, but their functional roles are still poorly understood. RGS expression changes have been described in hypertrophic and failing hearts. In this study, we report a marked decrease in RGS2 (but not other major cardiac RGS proteins (RGS3-RGS5)) that occurs prior to hypertrophy development in different models with enhanced G(q/11) signaling (transgenic expression of activated Galpha(q)(*) and pressure overload due to aortic constriction). To assess functional consequences of selective down-regulation of endogenous RGS2, we identified targeting sequences for effective RGS2 RNA interference and used lipid-based transfection to achieve uptake of fluorescently labeled RGS2 small interfering RNA in >90% of neonatal and adult ventricular myocytes. Endogenous RGS2 expression was dose-dependently suppressed (up to 90%) with no major change in RGS3-RGS5. RGS2 knockdown increased phenylephrine- and endothelin-1-induced phospholipase Cbeta stimulation in both cell types and exacerbated the hypertrophic effect (increase in cell size and radiolabeled protein) in neonatal myocytes, with no major change in G(q/11)-mediated ERK1/2, p38, or JNK activation. Taken together, this study demonstrates that endogenous RGS2 exerts functionally important inhibitory restraint on G(q/11)-mediated phospholipase Cbeta activation and hypertrophy in ventricular myocytes. Our findings point toward a potential pathophysiological role of loss of fine tuning due to selective RGS2 down-regulation in G(q/11)-mediated remodeling. Furthermore, this study shows the feasibility of effective RNA interference in cardiomyocytes using lipid-based small interfering RNA transfection.

Endogenous RGS Proteins and Gα Subtypes Differentially Control Muscarinic and Adenosine-Mediated Chronotropic Effects

Cardiac automaticity is controlled by G protein-coupled receptors, such as adrenergic, muscarinic, and adenosine receptors. The strength and duration of G protein signaling is attenuated by regulator of G protein signaling (RGS) proteins acting as GTPase-activating proteins for Galpha subunits; however, little is known about the role of endogenous RGS proteins in cardiac function. We created point mutations in Galpha subunits that disrupt Galpha-RGS binding and introduced them into embryonic stem (ES) cells by homologous recombination. Spontaneously contacting cardiocytes derived from the ES cells were used to evaluate the role of endogenous RGS proteins in chronotropic regulation. The RGS-insensitive GalphaoG184S homozygous knock-in (GalphaoGS/GS) cells demonstrated enhanced adenosine A1 and muscarinic M2 receptor-mediated bradycardic responses. In contrast, Galphai2GS/GS cells showed enhanced responses to M2 but not A1 receptors. Similarly M2 but not A1 bradycardic responses were dramatically enhanced in Galphai2GS/GS mice. Blocking G protein-coupled inward rectifying K+ (GIRK) channels largely abolished the mutation-induced enhancement of the M2 receptor-mediated response but had a minimal effect on A1 responses. The Galphas-dependent stimulation of beating rate by the beta2 adrenergic receptor agonist procaterol was significantly attenuated in GalphaoGS/GS and nearly abolished in Galphai2GS/GS cells because of enhanced signaling via a pertussis toxin sensitive mechanism. Thus, endogenous RGS proteins potently reduce the actions of Galpha(i/o)-linked receptors on cardiac automaticity. Furthermore, M2 and A1 receptors differentially use Galphai2 and Galphao and associated downstream effectors. Thus, alterations in RGS function may play a role in pathophysiological processes and RGS proteins could represent novel cardiovascular therapeutic targets.

Endogenous Regulator of G-Protein Signaling Proteins Regulate the Kinetics of Gαq/11-Mediated Modulation of Ion Channels in Central Nervous System Neurons

Slow synaptic potentials are generated when metabotropic G-protein-coupled receptors activate heterotrimeric G-proteins, which in turn modulate ion channels. Many neurons generate excitatory postsynaptic potentials mediated by G-proteins of the Galphaq/11 family, which in turn activate phospholipase C-beta. Accessory GTPase-activating proteins (GAPs) are thought to be required to accelerate GTP hydrolysis and rapidly turn off G-proteins, but the involvement of GAPs in neuronal Galphaq/11 signaling has not been examined. Here, we show that regulator of G-protein signaling (RGS) proteins provide necessary GAP activity at neuronal Galphaq/11 subunits. We reconstituted inhibition of native 2-pore domain potassium channels in cerebellar granule neurons by expressing chimeric Galpha subunits that are activated by Galphai/o-coupled receptors, thus bypassing endogenous Galphaq/11 subunits. RGS-insensitive variants of these chimeras mediated inhibition of potassium channels that developed and recovered more slowly than inhibition mediated by RGS-sensitive (wild-type) chimeras or native Galphaq/11 subunits. These changes were not accompanied by a change in agonist sensitivity, as might be expected if RGS proteins acted primarily as effector antagonists. The slowed recovery from potassium channel inhibition was largely reversed by an additional mutation that mimics the RGS-bound state. These results suggest that endogenous RGS proteins regulate the kinetics of rapid Galphaq/11-mediated signals in central nervous system neurons by providing GAP activity.

Endogenous regulator of g protein signaling proteins reduce {mu}-opioid receptor desensitization and down-regulation and adenylyl cyclase tolerance in C6 cells.

Chronic exposure of cells to mu-opioid agonists leads to tolerance which can be measured by a reduced ability to activate signaling pathways in the cell. Cell signaling through inhibitory G proteins is negatively regulated by RGS (regulator of G protein signaling) proteins. Here we examine the hypothesis that the GTPase accelerating activity of RGS proteins, by altering the lifetime of Galpha and Gbetagamma, plays a role in the development of cellular tolerance to mu-opioids. C6 glioma cells were stably transfected with mu-opioid receptor and pertussis toxin (PTX)-insensitive Galpha(o) that was either sensitive or insensitive to endogenous RGS proteins. Cells were treated with PTX to uncouple endogenous Galpha proteins followed by exposure to the mu-opioid agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) or morphine. Receptor desensitization as measured by agonist-stimulated [(35)S]GTPgammaS binding and receptor down-regulation as measured by [(3)H]diprenorphine binding were increased in cells expressing RGS-insensitive Galpha(o). Exposure to high concentrations of morphine or the peptidic mu-opioid agonist DAMGO led to a tolerance to inhibit adenylyl cyclase activity in both cell types with a rapid (30 min) and a slower component. Using a submaximal concentration of DAMGO to induce a reduced level of tolerance, a shift in the concentration-effect curve for DAMGO to inhibit adenylyl cyclase activity was seen in the cells expressing RGS-insensitive Galpha(o), but not in the cells expressing RGS-sensitive Galpha(o), which can be partly explained by an increased supersensitization of the adenylyl cyclase response. The results show that RGS proteins endogenously expressed in C6 cells reduce agonist-induced mu-opioid receptor desensitization, down-regulation, and sensitivity to tolerance to inhibit adenylyl cyclase activity.

Endogenous regulator of G protein signaling proteins suppress Galphao-dependent, mu-opioid agonist-mediated adenylyl cyclase supersensitization.

Chronic mu-opioid agonist treatment leads to dependence with withdrawal on removal of agonist. At the cellular level withdrawal is accompanied by a supersensitization of adenylyl cyclase, an effect that requires inhibitory Galpha proteins. Inhibitory Galpha protein action is modulated by regulator of G protein signaling (RGS) proteins that act as GTPase activating proteins and reduce the lifetime of Galpha-GTP. In this article, we use C6 glioma cells expressing the rat mu-opioid receptor (C6mu) to examine the hypothesis that Galphao alone can mediate mu-opioid agonist induced adenylyl cyclase supersensitivity and that endogenous RGS proteins serve to limit the extent of this supersensitization. C6mu cells were stably transfected with pertussis toxin (PTX)-insensitive Galphao that was either sensitive or insensitive to endogenous RGS proteins. Cells were treated with PTX to uncouple endogenous Galpha proteins followed by exposure to the mu-opioid agonists [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin or morphine. Supersensitization was observed in cells expressing wild-type Galpha, but this was lost on PTX treatment. In cells expressing PTX-insensitive Galphao supersensitization was recovered, confirming that Galphao alone can support supersensitization. In cells expressing the RGS-insensitive mutant Galphao, there was a greater degree of supersensitization and the concentration of micro-agonist needed to achieve half-maximal supersensitization was reduced by 10-fold. The amount of supersensitization seen did not directly relate to the degree of acute inhibition of adenylyl cyclase. These results demonstrate a role for Galphao in adenylyl cyclase supersensitization after mu-agonist exposure and show that this action is modulated by endogenous RGS proteins.

Endogenous RGS Proteins Regulate Presynaptic and Postsynaptic Function: Functional Expression of RGS-Insensitive Gα Subunits in Central Nervous System Neurons

Regulators of G-protein signaling (RGS)-insensitive (RGSi) G-protein alpha subunits can be used to indirectly determine the function of endogenous RGS proteins in native cells. This article describes the application of RGSi Galpha subunits to the study of endogenous RGS function in central nervous system (CNS) neurons. Presynaptic inhibition of neurotransmitter release was reconstituted in primary neurons using RGSi Galpha(i/o) subunits, whereas postsynaptic regulation of potassium channels was reconstituted using RGSi chimeras of Galpha(q) and Galpha(i). These studies have shown that endogenous RGS proteins are essential for the rapid termination of some G-protein-mediated signals in CNS neurons, whereas these proteins are much less important for the regulation of other signals. Together, these techniques have helped reveal the complexity of RGS regulation of CNS function.

Use of RGS-Insensitive Gα Subunits to Study Endogenous RGS Protein Action on G-Protein Modulation of N-Type Calcium Channels in Sympathetic Neurons

Regulators of G-protein signaling (RGS) proteins are a large family of signaling proteins that control both the magnitude and temporal characteristics of heterotrimeric G-protein-mediated signaling. A current challenge is to define how endogenous RGS protein function impacts G-protein modulation of ionic channels in mammalian neurons. The experimental strategy described here utilizes distinct mutations in Galpha subunits that confer Bordetella pertussis toxin (PTX) and RGS protein insensitivity. The native signaling pathway in rat sympathetic neurons that mediates voltage-dependent modulation of N-type Ca2+ channels is ablated by PTX treatment and the signaling is reconstituted by expressing a PTX/RGS-insensitive Galpha mutant along with Gbeta and Ggamma subunits. As neurons are resistant to conventional transfection modalities, heterologous expression is accomplished by the direct microinjection of plasmids into the nucleus of the neuron. An advantage of this approach is that knowledge of the specific RGS subtypes participating in the pathway is not required. From the resulting alterations in the kinetics and pharmacology of G-protein-coupled receptor modulation of N-type Ca2+ channels, we can infer the role endogenous RGS proteins play in the signaling pathway.

Assays for G-Protein-Coupled Receptor Signaling Using RGS-Insensitive Gα Subunits

Regulators of G-protein signaling (RGS) proteins, by their action on Galpha(i/o) proteins, may enhance receptor-effector signaling by physical or kinetic scaffolding mechanisms. However, more than 30 mammalian proteins with RGS activity have been identified so it is difficult to determine which RGS protein is most relevant to a particular receptor system and in any particular cell. To avoid this problem, one approach is to examine agonist-stimulated second messenger signaling in cells expressing Galpha proteins that are insensitive to the GTPase accelerating property of all RGS proteins. This article describes protocols for the preparation and analysis of C6 rat glioma cells stably expressing RGS- and pertussis toxin-insensitive Galpha subunits; pertussis toxin treatment uncouples endogenous Galpha(i/o) proteins and allows for the determination of the expressed RGS-insensitive Galpha activity. Methods to determine signaling at the level of adenylyl cyclase, the extracellular signal-regulated kinase (ERK1/2) mitogen-activated protein kinase pathway, and intracellular Ca2+ levels are described. As a typical G-protein-coupled receptor, we have used the micro-opioid receptor expressed in C6 cells together with RGS-insensitive Galpha(o). In these cells, agonist inhibition of adenylyl cyclase and stimulation of ERK1/2 phosphorylation were enhanced markedly. In contrast, increases in intracellular calcium were less affected. The altered signaling in cells expressing RGS-insensitive Galpha(o) subunits allows for determination of the role of endogenous RGS proteins to limit and/or direct signaling.

Receptor-selective Effects of Endogenous RGS3 and RGS5 to Regulate Mitogen-activated Protein Kinase Activation in Rat Vascular Smooth Muscle Cells

Regulators of G protein signaling (RGS) proteins compose a highly diverse protein family best known for inhibition of G protein signaling by enhancing GTP hydrolysis by Galpha subunits. Little is known about the function of endogenous RGS proteins. In this study, we used synthetic ribozymes targeted to RGS2, RGS3, RGS5, and RGS7 to assess their function. After demonstrating the specificity of in vitro cleavage by the RGS ribozymes, rat aorta smooth muscle cells were used for transient transfection with the RGS-specific ribozymes. RGS3 and RGS5 ribozymes differentially enhanced carbachol- and angiotensin II-induced MAP kinase activity, respectively, whereas RGS2 and RGS7 ribozymes had no effect. This enhancement was pertussis toxin-insensitive. Thus RGS3 is a negative modulator of muscarinic m3 receptor signaling, and RGS5 is a negative modulator of angiotensin AT1a receptor signaling through G(q/11). Also, RGS5 ribozyme enhanced angiotensin-stimulated inositol phosphate release. These results indicate the feasibility of using the ribozyme technology to determine the functional role of endogenous RGS proteins in signaling pathways and to define novel receptor-selective roles of endogenous RGS3 and RGS5 in modulating MAP kinase responses to either carbachol or angiotensin.

Endogenous Regulator of G-Protein Signaling Proteins Modify N-Type Calcium Channel Modulation in Rat Sympathetic Neurons

Experiments using heterologous overexpression indicate that regulator of G-protein signaling (RGS) proteins play important roles in Gbetagamma-mediated ion channel modulation. However, the roles subserved by endogenous RGS proteins have not been extensively examined because tools for functionally inhibiting natively expressed RGS proteins are lacking. To address this void, we used a strategy in which Galpha(oA) was rendered insensitive to pertussis toxin (PTX) and RGS proteins by site-directed mutagenesis. Either PTX-insensitive (PTX-i) or both PTX- and RGS-insensitive (PTX/RGS-i) mutants of Galpha(oA) were expressed along with Gbeta(1) and Ggamma(2) subunits in rat sympathetic neurons. After overnight treatment with PTX to suppress natively expressed Galpha subunits, voltage-dependent Ca(2+) current inhibition by norepinephrine (NE) (10 microm) was reconstituted in neurons expressing either PTX-i or PTX/RGS-i Galpha(oA). When compared with neurons expressing PTX-i Galpha(oA), the steady-state concentration-response relationships for NE-induced Ca(2+) current inhibition were shifted to lower concentrations in neurons expressing PTX/RGS-i Galpha(oA). In addition to an increase in agonist potency, the expression of PTX/RGS-i Galpha(oA) dramatically retarded the current recovery after agonist removal. Interestingly, the alteration in current recovery was accompanied by a slowing in the onset of current inhibition. Together, our data suggest that endogenous RGS proteins contribute to membrane-delimited Ca(2+) channel modulation by regulating agonist potency and kinetics of G-protein-mediated signaling in neuronal cells.

Regulators of G Protein Signaling Proteins as Determinants of the Rate of Desensitization of Presynaptic Calcium Channels

Norepinephrine inhibits omega-conotoxin GVIA-sensitive presynaptic Ca2+ channels in chick dorsal root ganglion neurons through two pathways, one mediated by Go and the other by Gi. These pathways desensitize at different rates. We have found that recombinant Galpha interacting protein (GAIP) and regulators of G protein signaling (RGS)4 selectively accelerate the rate of desensitization of Go- and Gi-mediated pathways, respectively. Blockade of endogenous RGS proteins using antibodies raised against Galpha interacting protein and RGS4 slows the rate of desensitization of these pathways in a selective manner. These results demonstrate that different RGS proteins may interact with Gi and Go selectively, giving rise to distinct time courses of transmitter-mediated effects.