RGS3 and RGS4 are GTPase Activating Proteins in the Heart (original) (raw)
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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 ...
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.)
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.
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.
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
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.
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.
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.
Overexpression of Gs alpha protein in the hearts of transgenic mice
Journal of Clinical Investigation, 1995
Alterations in fi-adrenergic receptor-G.-adenylyl cyclase coupling underlie the reduced catecholamine responsiveness that is a hallmark of human and animal models of heart failure. To study the effect of altered expression of Gs., we overexpressed the short isoform of Gs,, in the hearts of transgenic mice, using a rat a-myosin heavy chain promoter. G.. mRNA levels were increased selectively in the hearts of transgenic mice, with a level 38 times the control. Despite this marked increase in mRNA, Western blotting identified only a 2.8-fold increase in the content of the Gs,, short isoform, whereas G, activity was increased by 88%. The discrepancy between Gs. mRNA and Gs,, protein levels suggests that the membrane content of Gsa is posttranscriptionally regulated. The steady-state adenylyl cyclase catalytic activity was not altered under either basal or stimulated conditions (GTP + isoproterenol, GTPyS, NaF, or forskolin). However, progress curve studies did show a significant decrease in the lag period necessary for GppNHp to stimulate adenylyl cyclase activity. Furthermore, the relative number of /3-adrenergic receptors binding agonist with high affinity was significantly increased. Our data demonstrate that a relatively small increase in the amount of the coupling protein Gs. can modify the rate of catalyst activation and the formation of agonist high affinity receptors. (J.
Cellular Signalling, 2014
The protective effect of Regulator of G protein Signaling 2 (RGS2) in cardiac hypertrophy is thought to occur through its ability to inhibit the chronic GPCR signaling that promotes pathogenic growth both in vivo and in cultured cardiomyocytes. However, RGS2 is known to have additional functions beyond its activity as a GTPase accelerating protein, such as the ability to bind to eukaryotic initiation factor, eIF2B, and inhibit protein synthesis. The RGS2 eIF2B-interacting domain (RGS2 eb) was examined for its ability to regulate hypertrophy in neonatal ventricular myocytes. Both full-length RGS2 and RGS2 eb were able to inhibit agonist-induced cardiomyocyte hypertrophy, but RGS2 eb had no effect on receptor-mediated inositol phosphate production, cAMP production, or ERK 1/2 activation. These results suggest that the protective effects of RGS2 in cardiac hypertrophy may derive at least in part from its ability to govern protein synthesis.