RGS4 inhibits G-protein signaling in cardiomyocytes (original) (raw)
Related papers
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.
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.)
Tuning cardiomyocyte Gq signaling with RGS2
Journal of Molecular and Cellular Cardiology, 2006
Heterotrimeric G protein signals regulate cardiomyocyte contractile function, growth and survival. All 4 families of G proteins, including the G s , G i/o , G q/11 , and G 12/13 proteins, are expressed in cardiomyocytes [1]. Heterotrimeric G proteins are activated by cell surface seven transmembrane receptors that possess ligand-activated guanine nucleotide exchange factor activity, promoting the binding of GTP to the α subunit of the G protein [1]. GTP-bound α subunits dissociate from βγ subunits, and both moieties bind to various effectors, promoting signal transduction. The α subunits of G proteins possess intrinsic GTPase activity that leads to hydrolysis of GTP to GDP and the reformation of inactive heterotrimers. The rate of α subunit-mediated GTP hydrolysis observed in vitro is slow, but the rate in vivo is rapid, and this difference in GTPase rates led to the discovery a decade ago of a family of proteins called RGS, for Regulator of G protein Signaling [2-4]. RGS proteins dramatically accelerate the rate of GTP hydrolysis by stabilizing the transition state conformation of α subunits [5,6]. RGS proteins are characterized by the presence of a 120 amino acid RGS box that possesses GTPase-accelerating protein (GAP) activity [2]. There are over 30 RGS proteins present in mammalian organisms. Some RGS proteins, such as RGS2, RGS4, and RGS5, are relatively simple proteins, which consist mainly of an RGS box plus an N-terminal amphipathic membrane-targeting motif. Other RGS proteins contain additional protein domains, such as p115 rhoGEF, that contain an RGS box and a DH domain with activity GEF towards the small G protein rho [7]. Different RGS proteins have GAP activity towards different G protein families. RGS2, for example, is a relatively selective GAP for G q/11 proteins when tested in vitro under certain experimental conditions [8]. The RGS protein p115rhoGEF is a highly specific GAP for G 12/13 [7]. RGS4 has GAP activity towards both G q/11 and G i family members, but not towards G 12/13 or G s proteins when tested in vitro [9]. RGS-PX1 has been reported to have GAP activity for G s [10], although this finding has yet to be independently confirmed. Although RGS proteins promote the rapid deactivation of heterotrimeric G proteins, they also facilitate signaling in many situations. In order for a cell to respond to repeated rapid stimuli, prompt deactivation of a G protein is often required. For example, in the human retina, the extremely rapid deactivation of G t by RGS9 is required for the recovery phase of visual transduction, and is therefore necessary for the discrimination of low contrast or moving objects [11,12]. In addition, the
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.
FEBS Letters, 1998
Regulators of G-protein signalling (RGS) are recently identified proteins that shorten the lifetime of the activated G protein. We now show that rat cardiac myocytes express mRNA for at least 10 RGS. The mRNA for RGS-r is barely detectable in rat ventricles, but increases more than 20-fold during the 60-to 90-min process of isolating ventricular myocytes, and after 90 min of culture of atrial pieces in medium with Ca P+ . Both in myocytes and in atria, the rise in RGS-r is transient. The mRNA for cardiac RGS5, but not RGS-r, is developmentally regulated. These studies suggest that rapid regulation of RGS levels may be a new mechanism that governs how signals are transmitted across the cardiac cell membrane.
PLoS ONE, 2012
Cardiac hypertrophy is a well-established risk factor for cardiovascular morbidity and mortality. Activation of G q/11-mediated signaling is required for pressure overload-induced cardiomyocyte (CM) hypertrophy to develop. We previously showed that among Regulators of G protein Signaling, RGS2 selectively inhibits G q/11 signaling and its hypertrophic effects in isolated CM. In this study, we generated transgenic mice with CM-specific, conditional RGS2 expression (dTG) to investigate whether RGS2 overexpression can be used to attenuate G q/11-mediated signaling and hypertrophy in vivo. Transverse aortic constriction (TAC) induced a comparable rise in ventricular mass and ANF expression and corresponding hemodynamic changes in dTG compared to wild types (WT), regardless of the TAC duration (1-8 wks) and timing of RGS2 expression (from birth or adulthood). Inhibition of endothelin-1-induced G q/11-mediated phospholipase C b activity in ventricles and atrial appendages indicated functionality of transgenic RGS2. However, the inhibitory effect of transgenic RGS2 on G q/11mediated PLCb activation differed between ventricles and atria: (i) in sham-operated dTG mice the magnitude of the inhibitory effect was less pronounced in ventricles than in atria, and (ii) after TAC, negative regulation of G q/11 signaling was absent in ventricles but fully preserved in atria. Neither difference could be explained by differences in expression levels, including marked RGS2 downregulation after TAC in left ventricle and atrium. Counter-regulatory changes in other G q/11regulating RGS proteins (RGS4, RGS5, RGS6) and random insertion were also excluded as potential causes. Taken together, despite ample evidence for a role of RGS2 in negatively regulating G q/11 signaling and hypertrophy in CM, CM-specific RGS2 overexpression in transgenic mice in vivo did not lead to attenuate ventricular G q/11-mediated signaling and hypertrophy in response to pressure overload. Furthermore, our study suggests chamber-specific differences in the regulation of RGS2 functionality and potential future utility of the new transgenic model in mitigating G q/11 signaling in the atria in vivo.
RGS4 Reduces Contractile Dysfunction and Hypertrophic Gene Induction in Gα qOverexpressing Mice
Journal of Molecular and Cellular Cardiology, 2001
The intrinsic GTPase activity of G q is low, and RGS proteins which activate GTPase are expressed in the heart; however, their functional relevance in vivo is unknown. Transgenic mice with cardiac-specific overexpression of G q in myocardium exhibit cardiac hypertrophy, enhanced PKC membrane translocation, embryonic gene expression, and depressed cardiac contractility. We recently reported that transgenic mice with cardiac-specific expression of RGS4, a G q and G i GTPase activator, exhibit decreased left ventricular hypertrophy and ANF induction in response to pressure overload. To test the hypothesis that RGS4 can act as a G q-specific GTPase activating protein (GAP) in the in vivo heart, dual transgenic G q-40xRGS4 mice were generated to determine if RGS4 co-expression would ameliorate the G q-40 phenotype. At age 4 weeks, percent fractional shortening was normalized in dual transgenic mice as was left ventricular internal dimension and posterior and septal wall thicknesses. PKC membrane translocation and ANF and-skeletal actin mRNA levels were also normalized. Compound transgenic mice eventually developed depressed cardiac contractility that was evident by 9 weeks of age. These studies establish for the first time a role for RGS4 as a GAP for G q in the in vivo heart, and demonstrate that its regulated expression can have pathophysiologic consequences.
RGS3 Inhibits G Protein-Mediated Signaling via Translocation to the Membrane and Binding to Gα 11
Molecular and Cellular Biology, 1999
In the present study, we investigated the function and the mechanism of action of RGS3, a member of a family of proteins called regulators of G protein signaling (RGS). Polyclonal antibodies against RGS3 were produced and characterized. An 80-kDa protein was identified as RGS3 by immunoprecipitation and immunoblotting with anti-RGS3 antibodies in a human mesangial cell line (HMC) stably transfected with RGS3 cDNA. Coimmunoprecipitation experiments in RGS3-overexpressing cell lysates revealed that RGS3 bound to aluminum fluoride-activated Gα 11 and to a lesser extent to Gα i3 and that this binding was mediated by the RGS domain of RGS3. A role of RGS3 in postreceptor signaling was demonstrated by decreased calcium responses and mitogen-activated protein (MAP) kinase activity induced by endothelin-1 in HMC stably overexpressing RGS3. Moreover, depletion of endogenous RGS3 by transfection of antisense RGS3 cDNA in NIH 3T3 cells resulted in enhanced MAP kinase activation induced by endo...
Cardiotonic Steroids Stabilize Regulator of G Protein Signaling 2 Protein Levels
Molecular Pharmacology, 2012
Regulator of G protein signaling 2 (RGS2), a G(q)-specific GTPase-activating protein, is strongly implicated in cardiovascular function. RGS2(-/-) mice are hypertensive and prone to heart failure, and several rare human mutations that accelerate RGS2 degradation have been identified among patients with hypertension. Therefore, pharmacological up-regulation of RGS2 protein levels might be beneficial. We used a β-galactosidase complementation method to screen several thousand compounds with known pharmacological functions for those that increased RGS2 protein levels. Several cardiotonic steroids (CTSs), including ouabain and digoxin, increased RGS2 but not RGS4 protein levels. CTSs increased RGS2 protein levels through a post-transcriptional mechanism, by slowing protein degradation. RGS2 mRNA levels in primary vascular smooth muscle cells were unaffected by CTS treatment, whereas protein levels were increased 2- to 3-fold. Na(+)/K(+)-ATPase was required for the increase in RGS2 protein levels, because the effect was lost in Na(+)/K(+)-ATPase-knockdown cells. Furthermore, we demonstrated that CTS-induced increases in RGS2 levels were functional and reduced receptor-stimulated, G(q)-dependent, extracellular signal-regulated kinase phosphorylation. Finally, we showed that in vivo treatment with digoxin led to increased RGS2 protein levels in heart and kidney. CTS-induced increases in RGS2 protein levels and function might modify several deleterious mechanisms in hypertension and heart failure. This novel CTS mechanism might contribute to the beneficial actions of low-dose digoxin treatment in heart failure. Our results support the concept of small-molecule modulation of RGS2 protein levels as a new strategy for cardiovascular therapy.
RGS4 causes increased mortality and reduced cardiac hypertrophy in response to pressure overload
Journal of Clinical Investigation, 1999
RGS family members are GTPase-activating proteins (GAPs) for heterotrimeric G proteins. There is evidence that altered RGS gene expression may contribute to the pathogenesis of cardiac hypertrophy and failure. We investigated the ability of RGS4 to modulate cardiac physiology using a transgenic mouse model. Overexpression of RGS4 in postnatal ventricular tissue did not affect cardiac morphology or basal cardiac function, but markedly compromised the ability of the heart to adapt to transverse aortic constriction (TAC). In contrast to wild-type mice, the transgenic animals developed significantly reduced ventricular hypertrophy in response to pressure overload and also did not exhibit induction of the cardiac "fetal" gene program. TAC of the transgenic mice caused a rapid decompensation in most animals characterized by left ventricular dilatation, depressed systolic function, and increased postoperative mortality when compared with nontransgenic littermates. These results implicate RGS proteins as a crucial component of the signaling pathway involved in both the cardiac response to acute ventricular pressure overload and the cardiac hypertrophic program.