Cardiac myocytes express mRNA for ten RGS proteins: changes in RGS mRNA expression in ventricular myocytes and cultured atria (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 ...
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.
Comparative immunocytochemical demonstration of G proteins in rat heart tissue
Acta Histochemica, 1994
Localization of G proteins in the rat heart tissue was investigated using primary affinitypurified antibodies against synthetic peptides with amino acid sequences corresponding to <Xsubunits (<Xi coml1lon and <Xi L 2) of G proteins, Detection of immunoreactivity was performed with the peroxidase-anti-peroxidase complex (PAP), avidin-biotin complex (ABC) and fluorescein-labelled secondary antibodies for light microscopy and the protein A-gold technique for electron microscopy. In ventricles and atria, immunostaining for G proteins was detected in the sarcolemma and perinuclear space of cardiomyocytes, In endotheliocytes and fibroblasts, immunoreactivity was present also in the endoplasmic reticulum, All four immunocytochemical methods permit to demonstrate the same localization of G proteins in heart tissue, The ABC method and fluorescein labelled secondary antibodies technique showed the same sensitivity which is higher than that of the PAP method, Nomarski contrast microscopy enhanced the visualization of the final reaction product formed by the peroxidase reaction developed with diaminobenzidine in the ABC method, The results are discussed in terms of the role of G proteins in signal transduction via plasma membrane and membranes of the intracysternal space of the ceiL
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
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.
Frontiers in Physiology, 2012
Inhibitory heterotrimeric G proteins and the control of heart rate. The activation of cell signaling pathways involving inhibitory heterotrimeric G proteins acts to slow the heart rate via modulation of ion channels. A large number of Regulators of G protein signalings (RGSs) can act as GTPase accelerating proteins to inhibitory G proteins and thus it is important to understand the network of RGS\G-protein interaction. We will review our recent findings on in vivo heart rate control in mice with global genetic deletion of various inhibitory G protein alpha subunits. We will discuss potential central and peripheral contributions to the phenotype and the controversies in the literature.
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.
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.)
Small G Proteins in the Cardiovascular System: Physiological and Pathological Aspects
Physiological Reviews, 2013
Small G proteins exist in eukaryotes from yeast to human and constitute the Ras superfamily comprising more than 100 members. This superfamily is structurally classified into five families: the Ras, Rho, Rab, Arf, and Ran families that control a wide variety of cell and biological functions through highly coordinated regulation processes. Increasing evidence has accumulated to identify small G proteins and their regulators as key players of the cardiovascular physiology that control a large panel of cardiac (heart rhythm, contraction, hypertrophy) and vascular functions (angiogenesis, vascular permeability, vasoconstriction). Indeed, basal Ras protein activity is required for homeostatic functions in physiological conditions, but sustained overactivation of Ras proteins or spatiotemporal dysregulation of Ras signaling pathways has pathological consequences in the cardiovascular system. The primary object of this review is to provide a comprehensive overview of the current progress i...
Journal of Cellular Biochemistry, 2001
Membrane and cytosolic fractions prepared from ventricular myocardium of young (21-day-old) hypoor hyperthyroid rats and adult (84-day-old) previously hypo-or hyperthyroid rats were analyzed by immunoblotting with speci®c anti-G-protein antibodies for the relative content of G s a, G i a/G o a, G q a/G 11 a, and Gb. All tested G protein subunits were present not only in myocardial membranes but were at least partially distributed in the cytosol, except for G o a2, and G 11 a. Cytosolic forms of the individual G proteins represented about 5±60% of total cellular amounts of these proteins. The long (G s a-L) isoform of G s a prevailed over the short (G s a-S) isoform in both crude myocardial membranes and cytosol. The G s a-L/G s a-S ratio in membranes as well as in cytosol increased during maturation due to a substantial increase in G s a-L. Interestingly, whereas the amount of membrane-bound G i a/G o a and G q a/G 11 a proteins tend to lower during postnatal development, cytosolic forms of these G proteins mostly rise. Neonatal hypothyroidism reduced the amount of myocardial G s a and increased that of G i a/G o a proteins. By contrast, neonatal hyperthyroidism increased expression of G s a and decreased that of G i a and G 11 a in young myocardium. Changes in G protein content induced by neonatal hypo-and hyperthyroidism in young rat myocardium were restored in adulthood. Alterations in the membranecytosol balance of G protein subunits associated with maturation or induced by altered thyroid status indicate physiological importance of cytosolic forms of these proteins in the rat myocardium.