Regulator of G Protein Signaling 5

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(Trends Cardiovasc Med2009;19:26–30)n2009,Elsevier Inc.A Critical Role of Regulators ofG Protein Signaling Proteins inG Protein-Coupled ReceptorSignalingG protein-coupled receptors(GPCRs)areessential regulators of biologic processes.In the cardiovascular system alone,sev-eral hundred GPCRs are expressed andregulate key functions such as vasculartone and heart rate(Tang and Insel2004).Coupled to GPCRs are heterotri-meric G proteins that act as signaltransducers and are classified into fourcategories based on the functional andstructural similarities of theαsubunit;the Gαs subfamily stimulates adenylylcyclase(AC)activity;Gαi/o proteinsinhibit AC,as well as voltage-dependentcalcium channels;Gαq proteins stimu-lateβisoforms of phospholipase C;andG12/13signaling leads to GTPase Rhoactivation.In their active forms,bothGTP-bound Gαand the Gβγdimer dis-associate and signal to various intracel-lular effectors.Subsequently,G-proteindeactivation occurs by the intrinsicGTPase activity(GAP)of the Gαsubunit,which involves the hydrolysis of the Gα-bound GTP back to GDP,resulting in aconformational change that promotesthe reassociation of the Gβγsubunitand,thus,terminates the initial trigger.G protein-coupled receptor signalingis rapidly activated and deactivated inresponse to environmental cues.Although activation of G proteins israpid,self-catalyzed hydrolysis of GαGTPto GαGDP is slow and relies on externalGAP activity to sustain physiologicallymeaningful rates.Such GAP activity isprovided by the family of regulators of Gprotein signaling(RGS)molecules.Although RGS proteins were originallyidentified as negative regulators of Gproteins,it is now understood that theyare multifunctional proteins that gener-ally modulate G protein signaling.Todate,there are more than30RGSproteins that have been identified inmammals(Willars2006).All contain ahighly conserved carboxyl-terminaldomain of approximately125aminoacids(RGS domain or RGS box),whichconfers the unifying catalytic function ofRGS rger RGS moleculeshave additional domains enabling themto form multiprotein complexes.Severalreviews have excellently detailed thestructure,expression,and classificationof RGS proteins(Hendriks-Balk et al. Mitali Manzur and Ruth Ganss are at theWestern Australian Institute for MedicalResearch,The University of Western AustraliaCentre for Medical Research,Perth,WesternAustralia6000,Australia.⁎Address correspondence to:Ruth Ganss,Western Australian Institute for MedicalResearch,Rear,50Murray St.,MRF Building,Level5,Perth,WA6000,Australia.Tel.:(+61)892240354;fax:(+61)892240322.;e-mail:ganss@.au.©2009,Elsevier Inc.All rights reserved.1050-1738/09/$-see front matter2008,Willars2006).The following dis-cussion will focus on some members of the B/R4(RGS1-5,8,13,16,18,21) subfamily of RGS molecules,in particu-lar,RGS2,4,and5,and discuss their implications in cardiovascular function. An Emerging Role of RGS Mole-cules in Cardiovascular Function Recently,there has been an exponential increase in studies on RGS molecules and their signaling activities.However, these data are mainly derived from reconstituted in vitro systems,where RGS molecules are overexpressed along with GPCRs and very little is known of their physiologic relevance in vivo.In view of this,RGS knockout and trans-genic mouse models are now being developed and provide us with new insights into complex systems,including their role in cardiovascular function (Table1).For instance,knockout studies have revealed an important role for RGS2in vascular homeostasis in vivo. RGS2-deficient mice are hypertensive and exhibit prolonged vasoconstriction by attenuating signaling via Gαq-coupled receptors(Heximer et al.2003).Interest-ingly,RGS2itself is regulated by nitric oxide(NO)and,thus,is involved in a cross-talk between Gαq and NO signaling to mainly regulate peripheral vascular mechanisms(Sun et al.2005,Tang et al. 2003).Preliminary studies in humans further support an involvement of RGS2 in hypertension(Semplicini et al.2006, Yang et al.2005).Cardiac hypertrophy was not observed in RGS2knockout mice.However,decreased RGS2mRNA levels have been described in the hearts of Gαq-overexpressing mice before the development of cardiac hypertrophy (Zhang et al.2006).More recently,Takimoto et al.(2009)reported thatRGS2deficiency exacerbates hypertro-phy under chronic Gαq stimulation,thus,demonstrating a critical role of RGS2inearly cardiac stress in response to pres-sure overload.Another RGS molecule,RGS4,whoseexpression is enriched in the heart andcentral nervous system(Cifelli et al.2008,Erdely et al.2004,Rogers et al.2001),has been correlated to cardiachypertrophy in both mice(Rogers et al.1999)and humans(Owen et al.2001).RGS4is upregulated in failing humanhearts as a result of cardiomyopathy(Mittmann et al.2002,Owen et al.2001).Evidence is increasing that theenhanced RGS4expression counter-reg-ulates Gαq-induced signaling caused byhypertrophic stimuli(Rogers et al.2001,Tokudome et al.2008).Thus,RGS4,incontrast to RGS2,may have a protectiveeffect in late stages of heart failure.Moreover,RGS4is required for attenua-tion of parasympathetic-dependent Gprotein signaling and heart rate controlin correlation with its high expression inthe sinoatrial node(Cifelli et al.2008).These findings remarkably demonstratethe exquisite role of RGS modulators incardiac adaptation and the implicationsof their spatiotemporal expression.Changes in RGS expression have beenobserved in a variety of cardiovasculardiseases,and the relative importance andcell/organ specificity of different RGSsubtypes is only beginning to emerge(Hendriks-Balk et al.2008).A Newly Discovered Role of RGS5inVascular RemodelingRGS5,another member of the B/R4family,is expressed in most majororgans,including the heart,lungs,skele-tal muscle,brain,kidneys,and placenta,as well as in highly specialized cell typessuch as vascular smooth muscle cells,glomerular mesangial cells of the kidney,and cardiac myocytes(Seki et al.1998).Interestingly,gene expression studiesrevealed that RGS5expression is dyna-mically regulated in vivo,which is in linewith RGS5being a regulator of adaptiveprocesses and vascular remodeling.Forinstance,RGS5expression is downregu-lated in the brain capillaries and choroidplexus of stroke-prone hypertensive rats(Kirsch et al.2001).Gene profiling alsoidentified RGS5as a marker for arterialsmooth muscle cells over vein(Adams etal.2000,Li et al.2004).Interestingly,itsexpression is dramatically downregu-lated in atherosclerotic plaque-derivedsmooth muscle cells(SMCs)from pri-mates(Li et al.2004)and the fibrous capof advanced atherosclerotic plaques inhumans(Adams et al.2006).A gene profiling experiment is alsohow RGS5initially came to our ownattention(Berger et al.2005).RGS5wasfound to be highly expressed in angio-genic tumor vessels,whereas being sig-nificantly downregulated in a lessangiogenic vasculature in regressingtumors under therapy(Ganss et al.2002).Moreover,RGS5mRNA was upre-gulated during ovarian angiogenesis andin skin wounds during the formation ofgranulation tissue,before subsiding asthe wound closes(Berger et al.2005).Wesubsequently localized RGS5expressionto pericytes,close relatives of vascularSMCs(vSMC)that wrap aroundendothelial cells and are an integral partof the blood vessel wall(Armulik et al.2005).Thus,RGS5was identified as thefirst marker for angiogenic pericytes atsites of vessel remodeling in the tumormicroenvironment,during wound heal-ing and ovulation(Berger et al.2005).To functionally analyze RGS5intumors,we generated RGS5-deficientmice that were crossed with transgenic,tumor-prone RIP1-Tag5mice.Thesetransgenic mice express the oncogeneSV40large T antigen(Tag)under thecontrol of the rat insulin gene promoterinβcells of the pancreas and developpancreatic islet cell carcinomas.Bloodvessels in insulinomas are typicallydilated and tortuous,with an overallchaotic appearance(Ryschich et al.2002).However,in the absence ofTable1.Cardiovascular studies in mouse models in vivo involving RGS2, 4,and5RGS subtype Cardiovascular function ReferencesRGS2Blood pressure regulation Heximer et al.(2003)Tang et al.(2003)Cardiac adaptation Gross et al.(2005)Obst et al.(2006)Takimoto et al.(2009) RGS4Cardiac adaptation Rogers et al.(2001)Tokudome et al.(2008)Cifelli et al.(2008)RGS5Blood pressure regulation Cho et al.(2008)Nisancioglu et al.(2008)RGS5,tumors arise with a more homo-geneous vasculature that closely resem-bles vessels in normal organs(Hamzah et al.2008).Interestingly,wild-type tumor pericytes expressed high levels of plate-let-derived growth factor receptorβ(PDGFRβ)and low levels ofαsmooth muscle actin,an expression pattern typi-cal of immature pericytes(Song et al. 2005).In contrast,RGS5-deficient tumor pericytes displayed very low levels of PDGFRβand high levels ofαsmooth muscle actin expression,representing a mature pericyte population.This finding strongly implies that RGS5negatively impacts on pericyte maturation and is actively involved in vessel normalization. Importantly,these changes in vessel morphology correlated with alterations in the tumor environment such as reduced vascular leakiness and tumor hypoxia and significantly improved anti-tumor immunotherapy(reviewed in Manzur et al.(2008)).RGS5is also expressed in pericytes surrounding brain capillaries(Bondjers et al.2003), and deletion of RGS5reduced edema after transient cerebral ischemia,demon-strating reduced vascular permeability similar to our observation inânormali-zedâRGS5-deficient tumor vessels (Hamzah et al.2008).Currently,it is notclear how RGS5,via its intrinsic GAPactivity or functions outside the RGSbox,controls vascular maturation andvessel remodeling.These complex pro-cesses may involve communication withunderlying endothelial cells and GPCR-mediated adhesion and migration signals(Cho et al.2003).RGS5as a Regulator ofCardiovascular FunctionPericytes have been associated withhemodynamic functions,similar toSMC of larger vessels,and can controlvasoconstriction and vasodilation withincapillary beds(Bergers and Song2005).Thus,although it is possible that peri-cytes also play a role in blood pressurehomeostasis,they represent a heteroge-neous cell population,and a conclusivelink to RGS5is yet to be made.Interest-ingly,however,RGS5is expressed earlyduring arterial development(Bondjers etal.2003,Cho et al.2003)and is a markerof arterial SMC in adults(Adams et al.2000,Li et al.2004),suggesting a role ofRGS5in arterial contraction and regula-tion of vascular tone.This was supportedby a human genomewide linkage andcandidate gene-based screen that linkedRGS5to blood pressure regulation(Chang et al.2007).Recently,two inde-pendent studies demonstrated that bothsystolic and diastolic blood pressure wassignificantly lower in RGS5-deficientmice compared with wild type(Cho etal.2008,Nisancioglu et al.2008).Thisfinding is somewhat unexpected becauseRGS5signaling has been mainly linkedto Gαi and Gαq proteins,both mediatorsof vasoconstriction in vSMC(Cho et al.2003,Wang et al.2002,2008).Currently,there is no evidence for a role of RGS5inendothelial cell-mediated vasorelaxationbecause of its specific expression inpericytes and vSMC.Indeed,furtherinsights into RGS-mediated cardiovascu-lar signaling and receptor selectivity arerequired to fully appreciate how the lackof RGS5induces hypotension,whereasRGS2-deficient mice are hypertensive(Heximer et al.2003).Regulatory Networks of RGS2and5in vSMCRGS5knockout mice have only recentlybeen generated,and therefore,mostsignaling studies have been conductedin cell culture.In contrast,the roleof Figure1.Potential roles of RGS2and RGS5in the regulation of vascular tone.Cellular messengers that modulate or are modulated by RGS2and RGS5either in vitro(segmented line)or in vivo(solid line).PLC indicates phospholipase C;GC,guanylate cyclase;PKA,protein kinase A;PKC, protein kinase C;PKG,protein kinase G;DAG,diacylglycerol;IP3,inositol phosphate3.RGS2in cardiovascular function has been analyzed in an elegant combination of in vivo and in vitro studies using gene knockout mice(Table1).RGS2prefer-entially interacts with Gαq(Heximer et al.1999)and attenuates signaling via Gαq-coupled GPCR receptors such as PAR1/thrombin,α-adrenergic receptor/ phenylephrine,and angiotensin II recep-tor type I/angiotensin,but not Gαi/ser-otonin(Heximer et al.2003,Tang et al. 2003).In addition,RGS2is phosphory-lated by cyclic guanosine monopho-sphate-dependent kinase I-α,which itself is activated by the NO–cyclic gua-nosine monophosphate pathway(Figure 1)(Takimoto et al.2009,Tang et al. 2003).The net effect of the cross-talk between Gαq and NO signaling is increased RGS2GAP activity,which terminates Gαq signaling.Loss of RGS2-mediated signaling induces hyper-tension in RGS2-deficient mice(Hexi-mer et al.2003).Moreover,RGS2also seems to interfere with Gαs-cyclic ade-nosine monophosphate(cAMP)signal-ing through direct inhibition of AC and Gαs proteins,which would be expected to enhance vasoconstriction(Roy et al. 2006,Sinnarajah et al.2001)(Figure1).RGS5is known to act as GAP for Gαq and Gαi proteins(Cho et al.2003,Wang et al.2008,Zhou et al.2001),and transient deletion of RGS5from vascular smooth muscle cells enhanced angio-tensin II signaling(Wang et al.2002) (Figure1).Protein kinase C phosphor-ylation of RGS5causes loss of inhibition of endothelin-1-Gαq signaling(Moroi et al.2007).This clearly contrasts with the in vivo finding of hypotension in RGS5-deficient mice.However,the in vivo scenario might be more complex.For instance,RGS5is a physiologic target for the NO-mediated N-end rule degradation pathway(Figure1)that functions through its ability to destroy specific regulatory proteins bearing an N-term-inal cysteine such as RGS4,5,and16(Hu et al.2005,Lee et al.2005).Moreover, RGS5was upregulated in cardiac tissue of mice that overexpressedβ2-adrenergic receptor(β2AR),as well as in hearts of rats that were chronically treated with theβAR agonist isoproterenol(Jean-Baptiste et al.2005).Interestingly,both β1AR andβ2AR signaling is usually coupled to Gαs proteins and increased cAMP levels,which ultimately lead to vessel relaxation.Therefore,RGS5may interfere with several regulatory path-ways,as does RGS2,however,with adifferent net outcome.More studies inphysiologically relevant disease modelswill be needed to determine dominantpathway(s)of RGS5in cardiovascularsignaling.Concluding RemarksCardiovascular diseases,includinghypertension,heart failure,and athero-sclerosis,remain one of the largestcauses of morbidity and mortality inWestern populations.GPCR-mediatedsignaling is critical for normal functionin the cardiovascular system and iscurrently the primary target for thepharmacologic treatment of disease.There is a growing interest in RGSmolecules because their role as specificGPCR modulators emerges.In particu-lar,RGS2and RGS4have been recog-nized as important regulators ofcardiovascular physiology and poten-tially novel drug targets.To date,animportant role for RGS5in tumor peri-cytes and neovascularization has beendemonstrated.These findings reveal newperspectives on vascular remodeling andhave significant implications for antic-ancer therapy.Furthermore,there isincreasing evidence for RGS5involve-ment in vascular pathologies.How thesediverse biologic functions are connectedand whether they share common signal-ing cascades remains to be determined.As the unique and specific profile ofRGS5is being discovered,new effectorfunctions beyond GAP activity may berevealed.AcknowledgmentsThe authors acknowledge support fortheir original work summarized in thisarticle from the National Health andMedical Research Council,Australia,the Western Australian Institute for Med-ical Research,and the University ofWestern Australia,Perth,WesternAustralia.ReferencesAdams LD,Geary RL,McManus B&SchwartzSM:2000.A comparison of aorta and venacava medial message expression by cDNAarray analysis identifies a set of68consis-tently differentially expressed genes,all 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