Cardiotonic Steroids Stabilize Regulator of G Protein Signaling 2 Protein Levels (original) (raw)
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Multi-Tasking RGS Proteins in the Heart: The Next Therapeutic Target?
Circulation Research, 2005
Regulator of G-protein-signaling (RGS) proteins play a key role in the regulation of G-protein-coupled receptor (GPCR) signaling. The characteristic hallmark of RGS proteins is a conserved Ϸ120-aa RGS region that confers on these proteins the ability to serve as GTPase-activating proteins (GAPs) for G ␣ proteins. Most RGS proteins can serve as GAPs for multiple isoforms of G ␣ and therefore have the potential to influence many cellular signaling pathways. However, RGS proteins can be highly regulated and can demonstrate extreme specificity for a particular signaling pathway. RGS proteins can be regulated by altering their GAP activity or subcellular localization; such regulation is achieved by phosphorylation, palmitoylation, and interaction with protein and lipid-binding partners. Many RGS proteins have GAP-independent functions that influence GPCR and non-GPCR-mediated signaling, such as effector regulation or action as an effector. Hence, RGS proteins should be considered multifunctional signaling regulators. GPCR-mediated signaling is critical for normal function in the cardiovascular system and is currently the primary target for the pharmacological treatment of disease. Alterations in RGS protein levels, in particular RGS2 and RGS4, produce cardiovascular phenotypes. Thus, because of the importance of GPCR-signaling pathways and the profound influence of RGS proteins on these pathways, RGS proteins are regulators of cardiovascular physiology and potentially novel drug targets as well. (Circ Res. 2005;96:401-411.)
RGS3 and RGS4 are GTPase Activating Proteins in the Heart
Journal of Molecular and Cellular Cardiology, 1998
RGS family members are regulatory molecules that act as GTPase activating proteins (GAPs) for G subunits of heterotrimeric G proteins. RGS proteins are able to deactivate G protein subunits of the G i , G o and G q subtypes when tested in vitro and in vivo. Although the function of RGS proteins in cardiac physiology is unknown, their ability to deactivate G subunits suggests that they may inhibit the action of muscarinic,-adrenergic, endothelin, and other agonists. To evaluate the role of RGS family members in the regulation of cardiac physiology, we investigated the expression pattern of two RGS genes in normal and diseased rat heart tissue. RGS3 and RGS4 mRNAs and proteins were detected in adult myocardium. RGS3 and RGS4 gene expression was markedly enhanced in two model systems of cardiac hypertrophy: growth factor-stimulated cultured neonatal rat cardiomyocytes and pulmonary artery-banded (PAB) mice. RGS3 and RGS4 mRNA levels were reduced in failing myocardium obtained from SHHF/Mcc-fa cp (SHHF) rats. These findings support the hypothesis that RGS gene expression is highly regulated in myocardium and imply that RGS family members play an important role in the regulation of cardiac function.
RGS4 inhibits G-protein signaling in cardiomyocytes
Circulation, 1999
Methods and ResultsWe investigated the ability of RGS proteins to block G-protein signaling in vivo by using a cultured cardiomyocyte transfection system. Endothelin-1, angiotensin II, and phenylephrine signal through G q or G i family members and promote the hypertrophy of ...
Rgs5 Targeting Leads to Chronic Low Blood Pressure and a Lean Body Habitus
Molecular and Cellular Biology, 2008
RGS5 is a potent GTPase-activating protein for G i␣ and G q␣ that is expressed strongly in pericytes and is present in vascular smooth muscle cells. To study the role of RGS5 in blood vessel physiology, we generated Rgs5-deficient mice. The Rgs5 ؊/؊ mice developed normally, without obvious defects in cardiovascular development or function. Surprisingly, Rgs5 ؊/؊ mice had persistently low blood pressure, lower in female mice than in male mice, without concomitant cardiac dysfunction, and a lean body habitus. The examination of the major blood vessels revealed that the aortas of Rgs5 ؊/؊ mice were dilated compared to those of control mice, without altered wall thickness. Isolated aortic smooth muscle cells from the Rgs5 ؊/؊ mice exhibited exaggerated levels of phosphorylation of vasodilator-stimulated phosphoprotein and extracellular signal-regulated kinase in response to stimulation with either sodium nitroprusside or sphingosine 1-phosphate. The results of this study, along with those of previous studies demonstrating that RGS5 stability is under the control of nitric oxide via the N-end rule pathway, suggest that RGS5 may balance vascular tone by attenuating vasodilatory signaling in vivo in opposition to RGS2, another RGS (regulator of G protein signaling) family member known to inhibit G protein-coupled receptor-mediated vasoconstrictor signaling. Blocking the function or the expression of RGS5 may provide an alternative approach to treat hypertension.
Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice
Journal of Clinical Investigation, 2003
Signaling by hormones and neurotransmitters that activate G protein-coupled receptors (GPCRs) maintains blood pressure within the normal range despite large changes in cardiac output that can occur within seconds. This implies that blood pressure regulation requires precise kinetic control of GPCR signaling. To test this hypothesis, we analyzed mice deficient in RGS2, a GTPase-activating protein that greatly accelerates the deactivation rate of heterotrimeric G proteins in vitro. Both rgs2 +/and rgs2-/mice exhibited a strong hypertensive phenotype, renovascular abnormalities, persistent constriction of the resistance vasculature, and prolonged response of the vasculature to vasoconstrictors in vivo. Analysis of P2Y receptor-mediated Ca 2+ signaling in vascular smooth muscle cells in vitro indicated that loss of RGS2 increased agonist potency and efficacy and slowed the kinetics of signal termination. These results establish that abnormally prolonged signaling by G protein-coupled vasoconstrictor receptors can contribute to the onset of hypertension, and they suggest that genetic defects affecting the function or expression of RGS2 may be novel risk factors for development of hypertension in humans.
NO-dependent blood pressure regulation in RGS2-deficient mice
AJP: Regulatory, Integrative and Comparative Physiology, 2005
The regulator of G protein signaling (RGS) 2, a GTPase-activating protein, is activated via the nitric oxide (NO)-cGMP pathway and thereby may influence blood pressure regulation. To test that notion, we measured mean arterial blood pressure (MAP) and heart rate (HR) with telemetry in N ω -nitro-Larginine methyl ester (L-NAME, 5 mg L-NAME/10 ml tap water)-treated RGS2-deficient (RGS2 −/− ) and RGS2-sufficient (RGS2 +/+ ) mice and assessed autonomic function. Without L-NAME, RGS2 −/− mice showed during day and night a similar increase of MAP compared with controls. L-NAME treatment increased MAP in both strains. nNOS is involved in this L-NAMEdependent blood pressure increase, since 7-nitroindazole increased MAP by 8 and 9 mmHg (P < 0.05) in both strains. The L-NAME-induced MAP increase of 14-15 mmHg during night was similar in both strains. However, the L-NAME-induced MAP increase during the day was smaller in RGS2 −/− than in RGS2 +/+ (11 ± 1 vs. 17 ± 2 mmHg; P < 0.05). Urinary norepinephrine and epinephrine excretion was higher in RGS2 −/− than in RGS2 +/+ mice. The MAP decrease after prazosin was more pronounced in L-NAME-RGS2 −/− . HR variability parameters [root mean square of successive differences (RMSSD), low-frequency (LF) power, and high-frequency (HF) power] and baroreflex sensitivity were increased in RGS2 −/− . Atropine and atropine plus metoprolol markedly reduced RMSSD, LF, and HF. Our data suggest an interaction between RGS2 and the NO-cGMP pathway. The blunted L-NAME response in RGS2 −/− during the day suggests impaired NO signaling. The MAP increases during the active phase in RGS2 −/− mice may be related to central sympathetic activation and increased vascular adrenergic responsiveness.
Journal of Biological Chemistry, 2005
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 G␣ 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 C 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 C 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.
Journal of Hypertension, 2006
Context RGS2 (regulators of G-protein signaling) is a negative regulator of G aq protein signaling, which mediates the action of several vasoconstrictors. RGS2-deficient mouse line exhibits a hypertensive phenotype and a prolonged response to vasoconstrictors. Objective To compare RGS2 expression in peripheral blood mononuclear cells (PBMs) and cultured fibroblasts from normotensive subjects and hypertensive patients. Methods PBMs were isolated from 100 controls and 150 essential hypertensives. Additionally, fibroblasts were isolated from skin biopsy of 11 normotensives and 12 hypertensives and cultured up to the third passage. Quantitative mRNA and protein RGS2 expression were performed by real-time quantitative reverse transcriptasepolymerase chain reaction and by immunoblotting, respectively. Free Ca 2R measurement was performed in monolayers of 24-h serum-deprived cells, using FURA-2 AM. Phosphorylation of the extracellular signal-regulated kinases ERK1/2 was measured by immunoblotting. Polymorphism (C1114G) in the 3 0 untranslated region of the RGS2 gene was investigated by direct sequencing and real-time polymerase chain reaction (PCR). Results RGS2 mRNA expression was significantly lower in PBM and in fibroblasts from hypertensives, in comparison to normotensives. C1114G polymorphism was associated with RGS2 expression, with the lowest values in GG hypertensives. The 1114G allele frequency was increased in hypertensives compared with normotensives. Angiotensin II-stimulated intracellular Ca 2R increase and ERK1/2 phosphorylation were higher in fibroblasts from hypertensive patients compared with control subjects, and in those with the G allele, independently of the blood pressure status. The angiotensin II-stimulated Ca 2R mobilization and ERK1/2 phosphorylation were negatively correlated with RGS2 mRNA expression. Conclusion Low expression of RGS2 contributes to increased G-protein-coupled signaling in hypertensive patients. The allele G is associated with low RGS2 expression and blood pressure increase in humans.
Small Molecule Disruption of G Signaling Inhibits the Progression of Heart Failure
Circulation Research, 2010
Rationale-Excess signaling through cardiac Gβγ subunits is an important component of heart failure (HF) pathophysiology. They recruit elevated levels of cytosolic G-protein coupled receptor kinase 2 (GRK2) to agonist-stimulated β-adrenergic receptors (β-ARs) in HF, leading to chronic β-AR desensitization and down-regulation; these events are all hallmarks of HF. Previous data suggested that inhibiting Gβγ signaling and its interaction with GRK2 could be of therapeutic value in HF.