Modulation of Endothelin-A Receptor,Gα Subunit,and RGS2 Expression during H9c2 Cardiomyoblast Differentiation

In cardiac myocytes, growth responses depend on activation of G protein-coupled receptors interacting with G_q/11 protein subfamily members. Endothelin receptors of the ET_A subtype belong to this receptor group inducing hypertrophic responses. To understand the role of ET_A receptors and signal transduction proteins in modulating cell growth, we analyzed the pharmacological profile of this receptor, its level of expression together with those of Gα subunits and the RGS2 protein in cardiomyoblasts differentiating into the cardiac phenotype. H9c2 rat cardiomyoblasts were grown in the presence of 10% fetal bovine serum (FBS) or 1% FBS plus all-trans-retinoic acid to induce the cardiac phenotype. The pharmacological properties of ET_A receptors were investigated by competition-binding experiments, whereas the protein expression profile was analyzed by immunoblot and immunocytochemistry. The pharmacological profile of ET_A receptors changed during differentiation of cardiomyoblasts into cardiomyocytes, and the amount of expressed receptor appeared to increase. Immunocytochemistry also showed a marked increase of receptor expression on cell membranes of differentiated cardiomyocytes. Among the other signaling proteins examined, both Gα_q/11 and RGS2 expression decreased in cells with the cardiac phenotype. Our results demonstrate that the expression of key proteins (ET_A receptor, Gα_q/11, and RGS2) involved in signal transduction of hypertrophic stimuli is modulated during cell differentiation and correlates with the cardiac phenotype.     

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.     

GRK2 is an endogenous protein inhibitor of the insulin signaling pathway for glucose transport stimulation

G protein-coupled receptor kinases (GRKs) represent a class of proteins that classically phosphorylate agonist-activated G protein-coupled receptors, leading to uncoupling of the receptor from further G protein activation. Recently, we have reported that the heterotrimeric G protein alpha-subunit, Galphaq/11, can mediate insulin-stimulated glucose transport. GRK2 contains a regulator of G protein signaling (RGS) domain with specificity for Galphaq/11. Therefore, we postulated that GRK2 could be an inhibitor of the insulin signaling cascade leading to glucose transport in 3T3-L1 adipocytes. In this study, we demonstrate that microinjection of anti-GRK2 antibody or siRNA against GRK2 increased insulin-stimulated insulin-responsive glucose transporter 4 (GLUT4) translocation, while adenovirus-mediated overexpression of wild-type or kinase-deficient GRK2 inhibited insulin-stimulated GLUT4 translocation as well as 2-deoxyglucose uptake. Importantly, a mutant GRK2 lacking the RGS domain was without effect. Taken together, these results indicate that through its RGS domain endogenous GRK2 functions as a negative regulator of insulin-stimulated glucose transport by interfering with Galphaq/11 signaling to GLUT4 translocation. Furthermore, inhibitors of GRK2 can lead to enhanced insulin sensitivity.     

Reconstituted slow muscarinic inhibition of neuronal (Ca(v)1.2c) L-type Ca2+ channels

Ca(2+) influx through L-type channels is critical for numerous physiological functions. Relatively little is known about modulation of neuronal L-type Ca(2+) channels. We studied modulation of neuronal Ca(V)1.2c channels heterologously expressed in HEK293 cells with each of the known muscarinic acetylcholine receptor subtypes. Galphaq/11-coupled M1, M3, and M5 receptors each produced robust inhibition of Ca(V)1.2c, whereas Galphai/o-coupled M2 and M4 receptors were ineffective. Channel inhibition through M1 receptors was studied in detail and was found to be kinetically slow, voltage-independent, and pertussis toxin-insensitive. Slow inhibition of Ca(V)1.2c was blocked by coexpressing RGS2 or RGS3T or by intracellular dialysis with antibodies directed against Galphaq/11. In contrast, inhibition was not reduced by coexpressing betaARK1ct or Galphat. These results indicate that slow inhibition required signaling by Galphaq/11, but not Gbetagamma, subunits. Slow inhibition did not require Ca(2+) transients or Ca(2+) influx through Ca(V)1.2c channels. Additionally, slow inhibition was insensitive to pharmacological inhibitors of phospholipases, protein kinases, and protein phosphatases. Intracellular BAPTA prevented slow inhibition via a mechanism other than Ca(2+) chelation. The cardiac splice-variant of Ca(V)1.2 (Ca(V)1.2a) and a splice-variant of the neuronal/neuroendocrine Ca(V)1.3 channel also appeared to undergo slow muscarinic inhibition. Thus, slow muscarinic inhibition may be a general characteristic of L-type channels having widespread physiological significance.     

Differential contribution of GTPase activation and effector antagonism to the inhibitory effect of RGS proteins on Gq-mediated signaling in vivo

RGS proteins act as negative regulators of G protein signaling by serving as GTPase-activating proteins (GAP) for alpha subunits of heterotrimeric G proteins (Galpha), thereby accelerating G protein inactivation. RGS proteins can also block Galpha-mediated signal production by competing with downstream effectors for Galpha binding. Little is known about the relative contribution of GAP and effector antagonism to the inhibitory effect of RGS proteins on G protein-mediated signaling. By comparing the inhibitory effect of RGS2, RGS3, RGS5, and RGS16 on Galpha(q)-mediated phospholipase Cbeta (PLCbeta) activation under conditions where GTPase activation is possible versus nonexistent, we demonstrate that members of the R4 RGS subfamily differ significantly in their dependence on GTPase acceleration. COS-7 cells were transiently transfected with either muscarinic M3 receptors, which couple to endogenous Gq protein and mediate a stimulatory effect of carbachol on PLCbeta, or constitutively active Galphaq*, which is inert to GTP hydrolysis and activates PLCbeta independent of receptor activation. In M3-expressing cells, all of the RGS proteins significantly blunted the efficacy and potency of carbachol. In contrast, Galphaq* -induced PLCbeta activation was inhibited by RGS2 and RGS3 but not RGS5 and RGS16. The observed differential effects were not due to changes in M3, Galphaq/Galphaq*, PLCbeta, or RGS expression, as shown by receptor binding assays and Western blots. We conclude that closely related R4 RGS family members differ in their mechanism of action. RGS5 and RGS16 appear to depend on G protein inactivation, whereas GAP-independent mechanisms (such as effector antagonism) are sufficient to mediate the inhibitory effect of RGS2 and RGS3.     

Identification of a novel alternative splicing variant of RGS5 mRNA in human ocular tissues

Regulator of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for Galpha subunits and negatively regulate G protein-coupled receptor signaling. Using RGS5 gene-specific RT-PCR, we have identified a novel alternative splicing variant of RGS5 mRNA in human ocular tissues. The alternative splicing of RGS5 mRNA occurred at position +44 (GenBank NM_003617), spliced out 174 bp (+44 to +218 bp) of the coding region, and encoded an RGS5s protein with a 108 amino acid N-terminal deletion. This study is the first to document alternative splicing of an RGS5 gene. We therefore studied RGS5 and RGS5s mRNA distribution in human tissues. In the eye, RGS5s was found to be highly expressed in the ciliary body and trabecular meshwork. It was also expressed in the kidney, brain, spleen, skeletal muscle and small intestine, but was not detectable in the liver, lung, heart. RGS5s was not found in monkey and rat ocular tissues, indicating species specificity for the eye. Comparing the recombinant RGS5 and RGS5s expression in HEK293/EBNA cells, RGS5s was present almost exclusively in the cytosolic fraction, whereas RGS5 was present in both membrane and cytosolic fractions. The data suggest that the N-terminal of RGS5 may be important for protein translocation to the cell membrane. Both RGS5 and RGS5s antagonized the rapid phosphorylation of p44/42 MAP kinase induced by Galphai coupled cannibinoid receptor-1 activation. RGS5, but not RGS5s, inhibited the Ca2+ signaling initiated by activation of Galphaq coupled angiotensin II receptors (AT1) and prostaglandin FP receptors. Cotransfection of RGS5s with RGS5 resulted in the blockade of RGS5 actions with respect to inhibition of the signal transduction initiated by activation of both AT1 and FP receptor, suggesting that RGS5s may contain functional domains that compete with RGS5 in the regulation of the Galphaq coupled AT1 and FP receptors. The unique expression pattern, cellular localization and functions of RGS5s suggest that RGS5s may play a critical role in the regulation of intracellular signaling pathways.     

G Protein-coupled receptor kinase 2 regulator of G protein signaling homology domain binds to both metabotropic glutamate receptor 1a and Galphaq to attenuate signaling

Heterotrimeric guanine nucleotide-binding (G) protein-coupled receptor kinases (GRKs) are cytosolic proteins that contribute to the adaptation of G protein-coupled receptor signaling. The canonical model for GRK-dependent receptor desensitization involves GRK-mediated receptor phosphorylation to promote the binding of arrestin proteins that sterically block receptor coupling to G proteins. However, GRK-mediated desensitization, in the absence of phosphorylation and arrestin binding, has been reported for metabotropic glutamate receptor 1 (mGluR1) and gamma-aminobutyric acid B receptors. Here we show that GRK2 mutants impaired in Galphaq/11 binding (R106A, D110A, and M114A), bind effectively to mGluR1a, but do not mediate mGluR1a adaptation. Galphaq/11 is immunoprecipitated as a complex with mGluR1a in the absence of agonist, and either agonist treatment or GRK2 overexpression promotes the dissociation of the receptor/Galphaq/11 complex. However, these mGluR1a/Galphaq/11 interactions are not antagonized by the overexpression of either GRK2 mutants defective in Galphaq/11 binding or RGS4. We have also identified a GRK2-D527A mutant that binds Galphaq/11 in an AlF4(-)-dependent manner but is unable to either bind mGluR1a or attenuate mGluR1a signaling. We conclude that the mechanism underlying GRK2 phosphorylation-independent attenuation of mGluR1a signaling is RH domain-dependent, requiring the binding of GRK2 to both Galphaq/11 and mGluR1a. This serves to coordinate GRK2 interactions with Galphaq/11 and to disrupt receptor/Galphaq/11 complexes. Our findings indicate that GRK2 regulates receptor/G protein interactions, in addition to its traditional role as a receptor kinase.     

5-HT1A receptor-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) is modulated by regulator of G protein signaling protein 19

The 5-HT1A receptor is a G protein coupled receptor (GPCR) that activates G proteins of the Gαi/o family. 5-HT1A receptors expressed in the raphe, hippocampus and prefrontal cortex are implicated in the control of mood and are targets for anti-depressant drugs. Regulators of G protein signaling (RGS) proteins are members of a large family that play important roles in signal transduction downstream of G protein coupled receptors (GPCRs). The main role of RGS proteins is to act as GTPase accelerating proteins (GAPs) to dampen or negatively regulate GPCR-mediated signaling. We have shown that a mouse expressing Gαi2 that is insensitive to all RGS protein GAP activity has an anti-depressant-like phenotype due to increased signaling of postsynaptic 5-HT1A receptors, thus implicating the 5-HT1A receptor–Gαi2 complex as an important target. Here we confirm that RGS proteins act as GAPs to regulate signaling to adenylate cyclase and the mitogen-activated protein kinase (MAPK) pathway downstream of the 5-HT1A receptor, using RGS-insensitive Gαi2 protein expressed in C6 cells. We go on to use short hairpin RNA (shRNA) to show that RGS19 is responsible for the GAP activity in C6 cells and also that RGS19 acts as a GAP for 5-HT1A receptor signaling in human neuroblastoma SH-SY5Y cells and primary hippocampal neurons. In addition, in both cell types the synergy between 5-HT1A receptor and the fibroblast growth factor receptor 1 in stimulating the MAPK pathway is enhanced following shRNA reduction of RGS19 expression. Thus RGS19 may be a viable new target for anti-depressant medications.     

RGS2 is upregulated by and attenuates the hypertrophic effect of alpha1-adrenergic activation in cultured ventricular myocytes

Regulator of G protein signaling (RGS) proteins counter the effects of G protein-coupled receptors (GPCRs) by limiting the abilities of G proteins to propagate signals, although little is known concerning their role in cardiac pathophysiology. We investigated the potential role of RGS proteins on alpha1-adrenergic receptor signals associated with hypertrophy in primary cultures of neonatal rat cardiomyocytes. Levels of mRNA encoding RGS proteins 1-5 were examined, and the alpha1-adrenergic agonist phenylephrine (PE) significantly increased RGS2 gene expression but had little or no effect on the others. The greatest changes in RGS2 mRNA occurred within the first hour of agonist addition. We next investigated the effects of RGS2 overexpression produced by infecting cells with an adenovirus encoding RGS2-cDNA on cardiomyocyte responses to PE. As expected, PE increased cardiomyocyte size and also significantly upregulated alpha-skeletal actin and ANP expression, the markers of hypertrophy, as well as the Na-H exchanger 1 isoform. These effects were blocked in cells infected with the adenovirus expressing RGS2. We also examined hypertrophy-associated MAP kinase pathways, and RGS2 overexpression completely prevented the activation of ERK by PE. In contrast, the activation of both JNK and p38 unexpectedly were increased by RGS2, although the ability of PE to further activate the p38 pathway was reduced. These results indicate that RGS2 is an important negative-regulatory factor in cardiac hypertrophy produced by alpha1-adrenergic receptor stimulation through complex mechanisms involving the modulation of mitogen-activated protein kinase signaling pathways.     

Differential effects of regulator of G protein signaling (RGS) proteins on serotonin 5-HT1A, 5-HT2A, and dopamine D2 receptor-mediated signaling and adenylyl cyclase activity

Regulator of G protein signaling (RGS) proteins function as GTPase accelerating proteins (GAP) for Galpha subunits, attenuating G-protein-coupled receptor signal transduction. The present study tested the ability of members of different subfamilies of RGS proteins to modulate both G-protein-dependent and -independent signaling in mammalian cells. RGS4, RGS10, and RGSZ1 significantly attenuated Galphai-mediated signaling by 5-HT1A, but not by dopamine D2, receptor-expressing cells. Additionally, RGS4 and RGS10 significantly inhibited forskolin-stimulated cAMP production in both cell lines. In contrast, RGS2, RGS7, and RGSZ1 had no effect on forskolin-stimulated cAMP production in these cells. RGS2 and RGS7 significantly decreased Galphaq-mediated signaling by 5-HT2A receptors, confirming that the RGS4 and RGS10 effects on forskolin-stimulated cAMP production were specific, and not simply due to overexpression. Interestingly, similar expression levels of RGS4 protein resulted in greater inhibition of G-protein-independent cAMP production compared to G-protein-dependent GAP activity. Our results suggest specificity and selectivity of RGS proteins on G-protein-dependent and -independent signaling in mammalian cells.     

Regulation of G protein-mediated signal transduction by RGS proteins

RGS proteins form a new family of regulatory proteins of G protein signaling. They contain homologous core domains (RGS domains) of about 120 amino acids. RGS domains interact with activated Galpha subunits. Several RGS proteins have been shown biochemically to act as GTPase activating proteins (GAPs) for their interacting Galpha subunits. Other than RGS domains, RGS proteins differ significantly in size, amino acid sequences, and tissue distribution. In addition, many RGS proteins have other protein-protein interaction motifs involved in cell signaling. We have shown that p115RhoGEF, a newly identified GEF(guanine nucleotide exchange factor) for RhoGTPase, has a RGS domain at its N-terminal region and this domain acts as a specific GAP for Galpha12 and Galpha13. Furthermore, binding of activated Galpha13 to this RGS domain stimulated GEF activity of p115RhoGEF. Activated Galpha12 inhibited Galpha13-stimulated GEF activity. Thus p115RhoGEF is a direct link between heterotrimeric G protein and RhoGTPase and it functions as an effector for Galpha12 and Galpha13 in addition to acting as their GAP. We also found that RGS domain at N-terminal regions of G protein receptor kinase 2 (GRK2) specifically interacts with Galphaq/11 and inhibits Galphaq-mediated activation of PLC-beta, apparently through sequestration of activated Galphaq. However, unlike other RGS proteins, this RGS domain did not show significant GAP activity to Galphaq. These results indicate that RGS proteins have far more diverse functions than acting simply as GAPs and the characterization of function of each RGS protein is crucial to understand the G protein signaling network in cells.     

Ecdysone-based system for controlled inducible expression of metabotropic glutamate receptor subtypes 2, 5, and 8

Stable and inducible expression of human metabotropic glutamate receptor types 2, 5, and 8 was achieved in HEK293 cells using the ecdysone inducible system. Treatment of the respective cell lines with ponasterone A resulted in time and concentration-dependent induction of receptor expression. In all cases, the functional activation of receptors was determined by measuring increases in intracellular calcium. The physiologically GalphaI-coupled receptors mGluR2 and mGluR8 were successfully coupled to phospholipase C activation using the chimeric G protein Galphaq/o. The pharmacological properties of recombinant receptors were characterized and proved to be similar to native receptors. Our data suggest that the ecdysone system has a number of characteristics that make it well suited for expressing mGluRs and that the combined use of this system and chimeric G proteins allows receptors to be characterized using a rapid and straightforward Ca2+ assay.     

Characterization of a novel C. elegans RGS protein with a C2 domain: evidence for direct association between C2 domain and Galphaq subunit

RGS (regulator of G protein signaling) proteins are GTPase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits and negatively regulate G protein-mediated signal transduction. In this study, we determined the cDNA sequence of a novel Caenorhabditis elegans (C. elegans) RGS protein. The predicted protein, termed C2-RGS, consists of 782 amino acids, and contains a C2 domain and an RGS domain. C2 domains are typically known to be Ca(2+) and phospholipid binding sites, found in many proteins involved in membrane traffic or signal transduction, and most of their biological roles are not identified. To study the function of C2-RGS protein, a series of six truncated versions of C2-RGS were constructed. When the full-length protein of C2-RGS was expressed transiently in AT1a-293T cells, ET-1-induced Ca(2+) responses were strongly suppressed. When each of the mutants with either RGS domain or C2 domain was expressed, the Ca(2+) responses were suppressed moderately. Furthermore, we found that C2 domain of PLC-beta1 also had a similar moderate inhibitory effect. RGS domain of C2-RGS bound to mammalian and C. elegans Galphai/o and Galphaq subunits only in the presence of GDP/AlF(4)(-), and had GAP activity to Galphai3. On the other hand, C2 domains of C2-RGS and PLC-beta1 also bound strongly to Galphaq subunit, in the presence of GDP, GDP/AlF(4)(-), and GTPgammaS, suggesting the stable persistent association between these C2 domains and Galphaq subunit at any stage during GTPase cycle. These results indicate that both the RGS domain and the C2 domain are responsible for the inhibitory effect of the full-length C2-RGS protein on Galphaq-mediated signaling, and suggest that C2 domains of C2-RGS and PLC-beta1 may act as a scaffold module to organize Galphaq and the respective whole protein molecule in a stable signaling complex, both in the absence and presence of stimulus.     

RGS2 inhibits β-adrenergic receptor-induced cardiomyocyte hypertrophy

The chronic stimulation of certain G protein-coupled receptors promotes cardiomyocyte hypertrophy and thus plays a pivotal role in the development of human heart failure. The beta-adrenergic receptors (β-AR) are unique among these in that they signal via Gs, whereas others, such as the alpha1-adrenergic (α1-AR) and endothelin-1 (ET-1) receptors, predominantly act through Gq. In this study, we investigated the potential role of regulator of G protein signalling 2 (RGS2) in modulating the hypertrophic effects of the β-AR agonist isoproterenol (ISO) in rat neonatal ventricular cardiomyocytes. We found that ISO-induced hypertrophy in rat neonatal ventricular myocytes was accompanied by the selective upregulation of RGS2 mRNA, with little or no change in RGS1, RGS3, RGS4 or RGS5. The adenylyl cyclase activator forskolin had a similar effect suggesting that it was mediated through cAMP production. To study the role of RGS2 upregulation in β-AR-dependent hypertrophy, cardiomyocytes were infected with adenovirus encoding RGS2 and assayed for cell growth, markers of hypertrophy, and β-AR signalling. ISO-induced increases in cell surface area were virtually eliminated by the overexpression of RGS2, as were increases in α-skeletal actin and atrial natriuretic peptide. RGS2 overexpression also significantly attenuated ISO-induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt activation, which may account for, or contribute to, its observed antihypertrophic effects. In contrast, RGS2 overexpression significantly activated JNK MAP kinase, while decreasing the potency but not the maximal effect of ISO on cAMP accumulation. In conclusion, the present results suggest that RGS2 negatively regulates hypertrophy induced by β-AR activation and thus may play a protective role in cardiac hypertrophy.     

A functional enhanced green fluorescent protein (EGFP)-tagged angiotensin II at(1a) receptor recruits the endogenous Galphaq/11 protein to the membrane and induces its specific internalization independently of receptor-g protein coupling in HEK-293 cells

The angiotensin II (Ang II) AT(1A) receptor was tagged at its C terminus with the enhanced green fluorescent protein (EGFP), and the corresponding chimeric cDNA was expressed in HEK-293 cells. This tagged receptor presents wild-type pharmacological and signaling properties and can be immunodetected by Western blotting and immunoprecipitation using EGFP antibodies. Therefore, this EGFP-tagged AT(1A) receptor is the perfect tool for analyzing in parallel the subcellular distributions of the receptor and its interacting G protein and their trafficking using confocal microscopy. Morphological observation of both the fluorescent receptor and its cognate Galphaq/11 protein, identified by indirect immunofluorescence, and the development of a specific software for digital image analysis together allow examination and quantification of the cellular distribution of these proteins before and after the binding of different agonist or antagonist ligands. These observations result in several conclusions: 1) Expression of increasing amounts of the AT(1A) receptor at the cell surface is associated with a progressive recruitment of the cytosolic Galphaq/11 protein at the membrane; 2) Internalization of the EGFP-tagged AT(1A) induced by peptide ligands but not nonpeptide ligands is accompanied by a Galphaq/11 protein intracellular translocation, which presents a similar kinetic pattern but occurs predominantly in a different compartment; and 3) This Galphaq/11 protein cellular translocation is dependent on receptor internalization process, but not G protein coupling and signal transduction mechanisms, as assessed by pharmacological data using agonists and antagonists and the characterization of AT(1A) receptor mutants (D(74)N and Delta329) for which the coupling and internalization functions are modified.     

G beta 5.RGS7 inhibits G alpha q-mediated signaling via a direct protein-protein interaction

A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Ggamma-like domain through which they form obligatory dimers with the G protein subunit Gbeta5 in vivo. In Caenorhabditis elegans, orthologs of Gbeta5.RGS dimers are implicated in regulating both Galphai and Galphaq signaling, and in cell-based assays these dimers regulate Galphai/o- and Galphaq/11-mediated pathways. However, initial studies with purified Gbeta5.RGS6 or Gbeta5.RGS7 showed that they only serve as GTPase activating proteins for Galphao. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Galphao or Galphaq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7.Gbeta5 complex binds to Galphaq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gbeta5, and Galpha subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gbeta5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7.Gbeta5 complex and cyan fluorescence protein-tagged Galphaq, indicating a direct interaction between these molecules.     

一氧化氮在防止心肌肥厚反应中的作用及其机制

本工作从整体和细胞水平探讨一氧化氮（ＮＯ）在防止心肌肥厚反应中的作用及其机制。压力超负荷心肌肥厚大鼠左心室肌ＮＯ含量减少。内源性ＮＯ可能通过非ｃＧＭＰ依赖机制减轻压力超负荷引起的心肌肥厚。在培养的新生大鼠心肌细胞中血管紧张素Ⅱ（ＡⅡ）、内皮素－１（ＥＴ－１）和去甲肾上腺素（ＮＥ）通过各自的受体和偶连的Ｇ蛋白,一方面引起心肌细胞肥大;另一方面抑制一氧化氮合酶（ＮＯＳ）活性和ＮＯ生成。心肌细胞和非心肌     

G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes.

We evaluated the role of the G alpha-q (Galphaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Galphaq function by single cell microinjection of anti-Galphaq/11 antibody or RGS2 protein (a GAP protein for Galphaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Galphaq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Galphaq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Galphaq (WT-Galphaq) or a constitutively active Galphaq mutant (Q209L-Galphaq) by using an adenovirus expression vector. In the basal state, Q209L-Galphaq expression stimulated 2-deoxy-D-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Galphaq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Galphaq stimulates PI3-kinase activity in p110alpha and p110gamma immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110alpha by 10-fold. Nevertheless, only microinjection of anti-p110alpha (and not p110gamma) antibody inhibited both insulin- and Q209L-Galphaq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Galphaq are dependent on the p110alpha subunit of PI3-kinase. In summary, (i) Galphaq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Galphaq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Galphaq can transmit signals from the insulin receptor to the p110alpha subunit of PI3-kinase, which leads to GLUT4 translocation.     

Difluoromethylornithine inhibits hypertrophic, pro-fibrotic and pro-apoptotic actions of aldosterone in cardiac cells

Recent studies have shown that aldosterone may play a critical role in the transition to heart failure and that heart is a direct target of the action of aldosterone, which can provoke hypertrophy and apoptosis of isolated cardiomyocytes and also increase the expression of genes that favor tissue fibrosis. Early work from this and other laboratories has established a link between the aliphatic polyamines and cardiac hypertrophy, while more recently an involvement of polyamines even in cell death and survival has emerged. In the present study we have treated cardiac cells, i.e. rat H9c2 cardiomyoblasts and neonatal cardiomyocytes, with (d,l)-2-(difluoromethyl)ornithine, a specific inhibitor of polyamine biosynthesis, to investigate the effects of polyamines in relation to the hypertrophic, pro-fibrotic and pro-apoptotic actions of aldosterone. The results indicate that inhibition of polyamine biosynthesis may prevent or attenuate the adverse actions of aldosterone, by modulating the expression of genes related to cardiac hypertrophy and fibrosis, as well as the levels of proteins and the activities of enzymes that control apoptosis.