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CharacterizingtheRoleofRGS5intheRegulationofVascular SmoothMuscleCellFunction

By SamTirgari Athesissubmittedinconformitywiththerequirements forthedegreeofMastersofScience inPhysiologyatthe UniversityofToronto CopyrightbySamTirgari2009

CharacterizingtheRoleofRGS5intheRegulationofVascular SmoothMuscleCellFunction

SamTirgari MastersofScience inPhysiologyatthe UniversityofToronto 2009 ABSTRACT RegulatorsofGproteinsignaling(RGS)modulateGproteincoupledreceptor (GPCR)activityinvascularsmoothmusclecells(VSMCs).Onesuchprotein,RGS5,has beenshowntohaveselectiveexpressioninVSMCsandpericytes,andcaninhibit signalingfromGqandGisubunits.UsinganRGS5knockoutmodel,weassessedthe functionaleffectofRGS5intheconstrictionanddilationofresistancearterioles. Furthermore,weexaminedtheintracellularlipidinteractionofRGSproteinstoidentify thedeterminantsregulatingthebiologicfunctionofRGS5.Surprisingly,lossofRGS5 functioninmesentericarterioleshadnoeffectonconstrictionanddilationofresistance arterioles.CulturedVSMCsshowedincreasedbasalERK1/2phosphorylationand increasedVASPsignalinginresponsetoSNPtreatmentinRGS5KOVSMCsascompared towildtypecontrols,withnoeffectoncellproliferation.ThesedatasuggestRGS5may integratemultipleintracellularpathwayswithcompetingeffectsonVSMCcontraction.

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Acknowledgements Firstandforemost,atremendousandheartfeltthankyoutomysupervisor,

mentor,andfriend,Dr.ScottP.Heximer.WordscannotdescribehowmuchIappreciate allyoureffortandsupportoverthepasttwoyears.Thanksfortakingachance,andIam foreverinyourdebt.However,noneofthiswouldbepossiblewithouttheconstantlove andsupportofmyfamily.Theiradviceandencouragementhavebeenaguidingforcein allofmyendeavors,bothatschoolandotherwise.Thisisasmuchanaccomplishment forthemasitisforme. Nowtothelabgentlemen(andlady),withoutwhom,theselasttwoyears

wouldhaveseemedmuchlonger.Carlo,thanksforallthegreatadvice.Steve,thanksfor sufferingwithme,andforsnoring.Kaveesh,thanksfortheEatery.Guillaume,thanksfor yourlessonsinmulticulturalism.Jenny,thankyouforbeingatthesametimealabMom andagiftedsurgeonandmentor.Iwillneverwearasweatervestagainwithoutthinking fondlyofallourtimesinthelab. Finally,thankstoallthepeoplewhokeptmesaneoutsidethelab.Kian,

Alexandra,andalltheClaudieboysandgirls,youhaveallbeenfantasticfriendsand muchneededunbiasedadvicegiversoverthepastwhile,andalthoughIloveyouall,I willcompletelyunderstandifyouneverreadthisthesis.

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ExperimentalContributionsandAcknowledgements Unlessotherwisenotedbelow,allexperimentsanddatapresentedinthis

thesiswerecollectedandanalyzedbytheauthor. FortheircontributionstotheERK1/2westernimmunoblottingandcell

proliferationassays,IwouldliketothankKarenChanandArashGhashghai,respectively. ThankyoualsotoJennyZhang,StevenGuandCarloCifellifortheircontinuedassistance andadviceinnumerousaspectsofthisstudy,frommousebreedingtotroubleshooting apparatusissues. AtremendousthankyoutoDr.SteffenBolzandDr.GregHarefortheir

supportandmentorship.ThankyoualsototheBolzlabfortheirassistancebothin learningtheintricaciesofsmallvesselisolationaswellastheirgenerosityinsharingthe pressuremyographyapparatusandtheirassistancewithtroubleshootingthose experiments. IwouldalsoliketoacknowledgethehelpofMarynaGorelickfromthe

researchlaboratoryofAlanDavidsoninthedepartmentofBiochemistryatthe UniversityofTorontoforallheraidinthecirculardichroismandtryptophanfluorimetry studies. iv

TableofContents Precontent a. Abstractii b. Acknowledgementsiii c. TableofContentsv d. ListofFiguresvii e. ListofAbbreviationsix 1. Introduction a. Cardiovascularphysiologyandbloodpressureregulation1 i. Theroleoftheresistancevasculatureinbloodpressure4 ii. Assayingresistancearteryfunction5 b. VSMCcontractionandvasoconstriction6 c. HeterotrimericGproteinsinVSMCfunction8 i. Thebaylisseffect(myogenicresponse)andtheroleof11 Gproteins d. TheGTPasecycleofheterotrimericGproteins12 e. RegulatorsofGproteinsignalingsuperfamily14 i. R7subfamily15 ii. R12subfamily15 iii. Rzsubfamily16 iv. R4subfamily16 1. RGS517 2. R4subfamilyRGSproteinamphipathichelixdomain27 f. Rationale28 g. Hypothesis30 2. MaterialsandMethods a. RGS5Reporterknockoutmouse31 i. BreedingandgenotypingofRGS5KOmouse31 b. PressureMyography32 i. Myogenicresponse33 ii. PEdoseresponse34 iii. AChdoseresponse34 iv. S1Pdoseresponse35 c. Aorticsmoothmusclecellcultureexperiments35 i. ERKsignalingassay35 ii. VASPsignalingassay36 iii. Cellproliferationassay36 d. RGS2NTDalphahelixlipidinteractionassays36 i. TryptophanFluorimetry36 ii. CircularDichroism37 e. StatisticalMethods38 v

3. Results a. PressureMyography39 i. MyogenicResponse39 ii. Sphingosine1phosphate44 iii. Phenylepherine46 iv. Acetylcholine48 b. ERKsignalingassay50 c. VASPsignalingassay54 d. Cellproliferationassay56 e. CircularDichroism58 f. TryptophanFluorimetry61 4. Discussion a. RGS568 i. ThemyogenicresponseinRGS5knockoutvessels70 ii. CellularproliferationandmigratorysignalinginRGS5KOcells73 iii. Limitations75 iv. Futuredirections76 b. RGSAmphipathichelixstructureandfuturedirections79 5. References81

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ListofFigures FIGURE1:Physiologicalfactorsaffectingbloodpressure3 FIGURE2:BaselinebloodpressureofRGS5KOandwildtypemice21 FIGURE3:ExpressionofRGS5throughoutthearterialvasculature24 FIGURE4:SchematicdiagramofLacZNeocassetteinsertinRGS5exon226 FIGURE5:MyogenicresponseofRGS5KOandwildtyperesistancearterioles41 FIGURE6:PassivetoneofRGS5KOandwildtyperesistancearterioles42 FIGURE7:IntralumenaldiameterofRGS5KOandwildtyperesistancearterioles43 FIGURE8:S1PdoseresponseofRGS5KOandwildtyperesistancearterioles45 FIGURE9:PEdoseresponseofRGS5KOandwildtyperesistancearterioles47 FIGURE10:AChdoseresponseofRGS5KOandwildtyperesistancearterioles49 FIGURE11:ERK1/2phosphorylationassaysinculturedRGS5KOandwildtypeVSMCs52 FIGURE12:BasalERK1/2phosphorylationinculturedRGS5KOandwildtypeVSMCs53 FIGURE13:VASPphosphorylationassaysinculturedRGS5KOandwildtypeVSMCs55 FIGURE14:CellproliferationassayofculturedRGS5KOandwildtypeVSMCs57 FIGURE15:CirculardischroismanalysisofRGS2wildtypeandmutantNTDpeptides59 FIGURE16:TryptophanfluorimetryanalysisofRGS2wildtypeandmutantNTD vii

peptides63 FIGURE17:TFEadditiontoR44Hpeptidescauseshelixformationwithoutany associatedblueshiftinthetryptophanspectra65 FIGURE18:SequenceconservationamongR4familyNTDand"snorkeling"modelof peptidelipidinteraction67

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ListofAbbreviations ACE­Angiotensinconvertingenzyme ACh­Acetylcholine AngII­AngiotensinII AT1R­Angiotensinreceptortype1 AT2R­Angiotensinreceptortype2 BP­Bloodpressure CV­Cardiovascular CVD­Cardiovasculardisease DAG­Diacylglycerol GPCR­Gproteincoupledreceptor EDHF­Endothelialderivedhyperpolarizingfactor ERK1/2­Extracellularregulatedkinase1/2 ES­Embryonicstemcells ETA­EndothelinreceptortypeA ET1­Endothelin1 GAP­GTPaseactivatingprotein GDP­Guaninediphosphate GTP­Guaninetriphosphate IP3­1,4,5inositoltriphosphate

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KO­Knockout MLC­Myosinlightchain20 MLCK­Myosinlightchainkinase MLCP­Myosinlightchainphosphatase NO­Nitricoxide NTD­Nterminaldomain PE­Phenylepherine PIP2­Phosphatidylinosital4,5bisphosphate PKA­ProteinkinaseA PKC­ProteinkinaseC PKG­ProteinkinaseG PLCPhospholipase RGS­RegulatorofGproteinsignaling S1P­Sphingosine1phosphate S1PR­Sphingosine1phosphatereceptor SNP­Sodiumnitroferricyanide VASP­Vasodilatorstimulatedprotein VSMC­Vascularsmoothmusclecell WT­Wildtype

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Chapter1:Introduction 1.aCardiovascularphysiologyandbloodpressureregulation AmongthecurrenthealthanddiseaseparadigmsinCanadaandthedeveloped world,chronicCVdisease,andmorespecificallyheartdisease,isaleadingcauseof morbidityandmortality(Burt,Wheltonetal.1995;Whitworth2003).Persistentlyhigh bloodpressure(hypertension)isakeyriskfactorforthedevelopmentofheartand othercardiovasculardiseases.Hypertensionisdefinedasasystemicbloodpressure equaltoorexceeding140mmHg/90mmHg(systolic/diastolic).Asof2005,Statistics Canadareportedover1.7millionCanadiansovertheageof65werelivingwith hypertension­approximatelytwooutofthreeofwhomarefemale.Intotal,over4 millionCanadiansovertheageof12livewithhypertension.Inadditiontothis,the numberofCanadianswith"highnormal"bloodpressure(130139/8089)isrisingand everydaytheseindividualsareatriskofmovingintothehypertensivegroup.These numbersandtheburdentheycauseonthehealthcaresystemarestaggering,however, whatistrulyalarmingishowfastthesefiguresarerisingandhowfewofthesecasescan beexplainedandproperlytreated(Chobanian,Bakrisetal.2003).Approximately90 95%oftheclinicallypresentedcasesofhypertensionaretermedprimaryhypertension, whichgenerallyreferstohypertensionofunknownmedicalorigin(CarreteroandOparil 2000).Numerousstudieshavelinkedhypertensiontoincreasedbodymassindexand 1

age,withtreatmentstrategiesfocusingonbetteringlifestylefactorstocombatthe illness.Thesestrategiesincludereducingbodyweight,sodiumintake,andalcohol consumption,aswellasincreasingphysicalactivityandcessationofsmokingfor smokers(Chobanian,Bakrisetal.2003).Unfortunately,inmanyofthesecases,changes inlifestylefactorshavelimitedefficacyinreducingthesepatients'bloodpressures (MiuraandNakagawa2005),forcingphysicianstoemploypharmacologictherapies. Thesedatapointtotheimportanceofbasicresearchintounderstandingthe mechanismsthatarecriticalforcontrollingsystemicbloodpressure.

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FIGURE1

FIGURE1:Thephysiologicalfactorsaffectingbloodpressure.Normalbloodpressure maintenancerequirestheinterplayoftheshowncellular,tissueandorgansystems.The twoprimarycomponentsofthedeterminantsofbloodpressurecanbedividedinto cardiacandvascularmechanisms(leftandrightsidesrespectively),withthefocusofthis thesisbeingonthevascularaspect;specifically,ontheroleofvesseltoneandarteriole radiusonmaintenanceofsystemicbloodpressure.Thisdiagramalsoillustratesthe potentialforavarietyofcompensatorymechanismsfromfactorsdirectlyinvolvedin modulatingvesseltoneandintissuesentirelyindependentofthevasculature. 3

1.a.iTheresistancevasculatureinbloodpressurephysiology Integrativecardiovascularphysiology(CV)isabroadfieldrequiringadetailed understandingofthecommunicationbetweenawidenumberofvitalsystems,organs andtissues.Notwithstandingthesignificantcontributionofrenalandcentralnervous systemfunctiontothecontrolofsystemicbloodpressure,theperipheralvasculatureis oftentheendeffectororganofsuchphysiologicregulationandthereforealarge amountofworkhasbeendedicatedtounderstandingthesignalingpathwaysthat controlitsfunction(GrassiandHeistad2009;Leibovitz,Ebrahimianetal.2009;Resch, Wiestetal.2009).Arteriolesaretheprimarysiteofregulationofperipheralvascular tone.Theyaresituated,intermsofsizeaswellasanatomicallocation,betweenthe largeconduitarteries,suchastheaorta,carotidandfemoralarteries,andthefragile capillarybeds.Theresistancearteriolesareuniqueintheirmuralcomposition, containinglargemuscularmediallayersandrelativelyhighratiosofwallthicknessto lumensize.Thesedifferfromconduitarteries,whichhavelargeamountsofelastinin additiontoSMCsinthemediallayers,andthecapillaries,whichhavenosmoothmuscle cellmediallayersatall.Thisstructuralfeatureofcapillaries,asidefromfacilitatingrapid diffusionofnutrientsandwasteproductstoandfromthesurroundingtissue,leavesthe vasculaturesensitivetosuddenincreasesinflowandpressure(WestandMathieu Costello1995).Assuch,suddenincreasesordecreasesincardiacoutputwouldcause extensiveandpotentiallyirreversibledamagetothesedelicatevascularbeds.Therefore theroleoftheresistancevasculatureisbothtomaintainnormalflowratesto

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downstreamtissuesoverawiderangeofsystemicpressures,aswellasassistinthe establishmentandregulationofsystemicpressure. 1.a.iiAssayingresistancearteryfunction Todatetherehavebeennumerousstudiesexamininghowmodificationsin thesevesselsandtheirfunctioncaneffectoverallsystemicpressure(LeeandSmeda 1985;Laurant,Touyzetal.1997;Resch,Wiestetal.2009).Furthermore,anumberof currentandpotentialtreatmentstrategiesfordealingwithhypertensionaredirectedat adjustingtheresponsesofthesebloodvessels(Khan,Hemmelgarnetal.2009).Inorder toassesstheirfunction,anumberofassaysareemployed,andamongtheseisabroad groupofexperimentstermedmyography(McPherson1992;WrightandAngus2000). Briefly,amyographusesavarietyofstrategiesformeasuring(graph)thesmooth muscle(myo)dependantvasoconstrictionortheinterplayofsignalsbetween endotheliumandSMCthatleadstovasodilation.Therearevaryingtypesofmyographs, withmodificationsrangingfromfundamentalmeasuringparameterstosuperficial aestheticsoftheapparatusitself(AngusandWright2000).Theprimarymethodology usedinthisthesiswasthepressuremyograph.Inpressuremyography,isolatedintact bloodvesselsarecannulated,pressurizedandmaintainedatphysiologicalornear physiologicaltemperatures.Thevesselsaremaintainedusingphysiologicalbufferand arestretchedtonearphysiologicallengths.Assuch,alleffortsaretakentoreproducea nearcomparablephysiologicalenvironmentforthevessel.Conversely,inwire 5

myography,isolatedvesselringsaremountedbetweenwiresthatareconnectedto forcetransducers.Theseassaysmeasuretheforceofconstrictionfromisolatedrings andquantifythedataasameasureofvesselfunction.Unfortunately,thestateofthe vesselatthetimeofmeasurement,aswellasthetypeofforcebeingexerteduponit, arefarfromphysiological.Thischiefcriticismisthemainfactorbehindthechoicetouse pressuremyographyinourcase(Buus,VanBaveletal.1994).Pressuremyography, however,isbynomeansa"perfect"physiologicalassay.Thesystememployspressure withtheabsenceofflow,thusremovingimportantfactorssuchasshearandits downstreameffectsonvascularsignaling.Thiscriticismmakespressuremyographya limitedtool.Furthermore,ininstanceswherevesselsaremarkedlyshorter,suchas pulmonaryarterioles,andthusdifficulttocannulateontoadjacentmicropipettes,wire myographyispreferredasvesseldiscsegmentscanbesignificantlyshorterthanthose usedforpressuremyography. 1.b.VSMCcontractionandvasoconstriction Usingpressuremyographyitispossibletomeasureandquantify vasoconstrictionanddilation,andthroughthistoassayvascularsmoothmusclecell (VSMC)functionandphysiology.Briefly,themediallayerofresistancearterioles,as alreadystated,isrichinSMCs:aspecializedcellcapableofcontraction.Thiscontractile mechanismdiffersfromskeletalorcardiacmuscleinitsrelativelyslowerresponseto stimulusandabilitytomaintainalongeroverallperiodofcontraction.Itismediatedby anumberofintracellularproteins,howeverGqmediatedsignalingcomprisesthe

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majorityofthedrivingforcebehindSMCcontraction.ThesignalinginducedbyGq coupledreceptorsisacomplicatedinterconnectedseriesofpartneredproteinsand secondarymessengers.Briefly,uponactivationviaanextracellularagonist,Gqactivity leadstoactivationofthemembraneboundprotein,phospholipaseC(PLC).PLC propogatesthesignalthroughtheformationofinositol1,4,5triphosphate(IP3)and diacylglycerol(DAG)fromtheprecursorphosphatidylinositol4,5bisphosphate(PIP2).At thispoint,thesignalingbecomesfurthercomplicatedduetomultiplepathwaysbeing activatedintandem.IP3bindingtoreceptorsonthesarcoplasmicreticulummobilizes thecalciumstoredtherein,increasingintracellularcalciumlevels.Calciumbindsto Calmodulin,formingacomplex,andactivatesmyosinlightchainkinase(MLCK).MLCK phosphorylatesmyosinlightchain20(MLC),whichleadstocontractionofthesmooth musclecell.Occurringinparalleltotheabovepathway,accumulationofDAGleadsto activationofproteinkinaseC(PKC)andthroughthistheRho/RhoKsignalingpathways (ShimokawaandTakeshita2005).Thesesignalsultimatelyleadtotheinhibitionof myosinlightchainphosphatase(MLCP),theantagonisttoMLCKandcontractility.Asa result,GalphaqactivationleadstothephosphorylationofMLCandcontractionofthe SMCthroughtwodifferentpathways. Vasodilatorysignalingfromligandssuchasadenosine,catecholamines,and glucorticoidsalsooccurthroughGPCRs,andinarterioles,causeVSMCrelaxationboth directlyattheleveloftheVSMCandalsothroughtheendothelium.Thestudies presentedhereinfocusonthecontractileroleofVSMCs,theregulationofthat contractileresponse,andtheroleofthismechanismintheregulationofsystemic 7

vascularpressure.Furthermore,thestudiespresentedexaminetheprecisemechanisms behindtheregulationofthesecontractilesignalsandestablishesconnectionsbetween biochemicalabnormalitiesandclinicalimplications.Itshouldalsobenotedthat contractileactivityisnottheonlycomponentofVSMCfunction.SMCsareuniquein theirabilitytoshifttheirphenotypesfromoneofcontractiletoproliferativeand migratoryactivity(Owens1995).Thisshift,aswellasthemigrationofSMCsintothe intimallayer,isimplicatedinthedevelopmentofdiseasesinvolvingvascularlegions, suchasatherosclerosisandrestenosis(Ross1993).Infact,anumberofvascular agoniststhataffectcontractileresponse,havealsobeenshowntobeinvolvedinthe determinationofVSMCphenotype(SomlyoandSomlyo1994).Assuch,thepathways studiedbecomemuchmorecomplexandinterwoven,andrequirecarefulattentionto experimentalsetupandtheestablishmentofpropercontrols.Furthermore,the existenceofthesealternatefunctionscreatesyetanotherlayerofinquisitionintothe roleofVSMCspecificproteins.Ultimately,assessmentofvascularcontractileresponse tothesereceptorsmaypotentiallyleaveouttheimportanceofthis 1.cHeterotrimericGproteinsinVSMCfunction TheroleofGproteinsbothatthelevelofcardiac,vascularandrenaltissueshas beencontinuallycharacterizedoveryearsofinvestigation(Lefkowitz1996;Cho, Harrisonetal.2004;Callera,Tostesetal.2007).ChangesinGPCRsignalingmayresultin largesuddenshiftsincardiacoutput,andofparticularrelevancetothisthesis,changes 8

inVSMCcontractilefunctionandvascularresistance.Thesechangescangoontoaffect systemicpressureandthroughthat,leadtoanumberofclinicalcomplicationsas describedearlier. AlthoughtheroleofGproteinsinthenormalfunctionofthesesystemshasbeen showntobeessential,therestillremainaspectstotheirmolecularmechanismsthatare unclear.Onesuchregionofinterestisintheexactrolesforthenumberofgproteins andgproteincoupledreceptors(GPCRs)presentinboththeheartandthevasculature. Indeed,inthevasculature,GPCRsignalingisresponsibleforthebulkofcontractile activityofvascularsmoothmusclecells(VSMCs).Currently,oftheestablished vasoconstrictorsalargenumberofthemworkviaGPCRs.OftheseGPCRs,thelarge majoritysignalthroughGqandGi,bothtargetsoftheR4subfamilyofRGSproteins. Theseincludealphaadrenergicreceptors,angiotensin1receptors(AT1R,AT2R), endothelin1receptors(ETA),andsphingosine1phosphate(S1PR15)(Demoliou Mason1998;Waeber,Blondeauetal.2004).TheprevalenceofGqcoupledreceptors, theirpresenceonVSMCsandtheircouplingtoknownvasoconstrictorsestablishesthe importanceofthisfamilyofGproteinsinregulatingvascularpressure.Furthermore,the majorityofmedicationscurrentlyprescribedtotreathypertensiontargetGPCRs,aswell astheproductionofandbindingofGPCRagonists(Lefkowitz1996;CarreteroandOparil 2000;Chobanian,Bakrisetal.2003).Theseincludeangiotensinconvertingenzyme (ACE)inhibitors,angiotensinreceptor(AT1R,AT2R)blockers,aswellasalphaandbeta adrenergicreceptorblockers.TwokeyGPCRs,theAT1andAT2receptors,havebeen implicatedaskeyplayersinanumberofconditions,includinghypertension,cardiac 9

remodeling,andcardiachypertrophy(Zhang,Zhangetal.2003;Zhu,Zhuetal.2003). Furthermore,GPCRactivityhasbeenatargetfortheunderstandingandtreatmentof type2diabetesthroughrecentstudiesintheimpairedfunctionofpancreasisletcells (Ahren2009).Leukocytefunctionandmigration(IrukayamaTomobe,Tanakaetal. 2009),retinalphysiologyandrhodopsinfunction(Ahuja,Crockeretal.2009)and neuronalsignaling(Yu,Arttamangkuletal.2009)arebutafewexamplesofthemultiple diversefunctionsofGPCRsignaling.Inaddition,investigationintotheregulationof GPCRsignalingactivityhasrevealedthenecessityoftheseregulatorymechanisms (Gros,Benovicetal.1997;Heximer,Knutsenetal.2003). Themammalianvasculatureisahighlyactivephysiologicalplayerinthegreater schemeofoverallorganismhealth.Withinthevesselwall,theendothelium,vascular smoothmusclecellsandperivascularinnervationsareresponsibleforthebulkof vascularresponsesandsystemiceffectsthroughcomplexcommunicationandsignaling crosstalk.Theseinteractionshoweverarebeyondthescopeofthisthesis,whichwill insteadfocusonGPCRmediatedpathwayswithinvascularsmoothmusclecells (VSMCs).VSMCsareresponsibleforthecontractileordilatoryresponseofthevessel wall,andarefurthercharacterizedbytheabilitytoaltertheirphenotypesfromoneof modulatingvesseltone,toaproliferativeandangiogenicphenotype(Owens1995; ManabeandNagai2003).IthasbeendemonstratedthatGproteinsignaling,through GPCRs,isresponsibleforanumberofthesechangesincellularactivityandcontractility. Innormalquiescentarteries,thereisanequilibriumofsignalingbetweenthetwolayers leadingtoabasalleveloftonethroughoutthevasculature. 10

1.c.iBaylisseffect(myogenicresponse)andtheroleofGproteins/GPCRsasmediatorsof thiseffect Themyogenicresponseistheintrinsicabilityofresistancearteriolesto

contractinresponsetoincreasedtransmuralpressure(Bayliss1902;Falcone,Davisetal. 1991).ThisVSMCdependantmechanismisbelievedtoberesponsiblenotonlyforthe establishmentofrestingvesseltonebutalsorequiredforthemaintenanceofconstant flowoverarangeofpressurechanges(Schubert,Lidingtonetal.2008).Itisfromthis restingmembranetonethatfurthervasodilatorandvasoconstrictorsignalingact,thus renderingmaintenanceofthisvascularhomeostasisessentialfornormalcirculatory function.Currently,themechanismsinitiatingthemyogenicresponsearepoorly understoodduetothemultitudeofinteractingfactorsthatleadtopressureinduced contraction,aswellasvasoconstrictioningeneral.Whatisaccepted,however,isthat themyogenicresponseisaVSMCspecificcharacteristicandismostprevalentinthe arterialresistancevasculature,whichisrepletewithSMCs.Furthermore,S1Psignaling, ERKactivationandactivationoftheRho/RhokinasesignalingpathwayandMLCP inhibitionhavebeenestablishedasessentialmediatorsofthemyogenicresponse(Bolz, Vogeletal.2003;Schubert,Lidingtonetal.2008).TheseS1Preceptors(types2and3, onVSMCs)areGPCRsandactthroughavarietyofGsubunitsincludingGq,Giand G12/13.ExistingevidencesupportingspecificityofRGS5forthesereceptorsaswellas establishedspecificityforGqandGiprovidetherationaleforsignalingandfunctional assaysofS1PactivityinRGS5knockoutvesselsandprimaryculturedVSMCs(Cho, Harrisonetal.2003).Assuch,thearterialSMCspecificexpressionofRGS5,aswellas 11

S1PspecificityandgeneralcapacityforGqandGitargeting,suggestaroleforRGS5 inregulatingthemyogeniccontractileresponseinarterioles. 1.dTheGTPasecycleofHeterotrimericGproteins Atthemostfundamentallevel,signalsoutsidecellsmustbetransmittedinwards toelicitabiologicalresponse.Gproteincoupledreceptorsarethelargestfamilyof surfacereceptors,withover800currentlyidentifiedgenesinthehumangenometo date(CottonandClaing2009).Furthermore,Gproteinscanrespondtoanumberof agonists,rangingfromchemokines,hormonesandneurotransmitterstomechanical stretchandthemovementofmatrixproteins.Inthecaseofvasoactiveagonists,ligands bindtotheextracellulardomainofafamilyofreceptorproteinstermedseven transmembrane(7TM)proteins,whicharecoupledattheirintracellulardomainswitha heterotrimericGprotein,creatingaGproteincoupledreceptor(GPCR)(Bourne, Sandersetal.1990).Bindingoftheextracellularagonistinducesconformational changesinthe7TMreceptor,whichactivatesthecoupledheterotrimericGprotein.In theinactivatedstate,thethreesubunits(alpha,beta,gamma)oftheheterotrimericG proteinaretightlyboundandmaintaincloseproximitywiththe7TMreceptor,witha GDPoccupyingthebindingpocketoftheGsubunit.Uponbindingoftheextracellular ligandandconformationalchangeinthereceptor,theGDPisreplacedwithaGTP,and theGproteinsignalingbecomesactivated.Thesignalisthentransmittedintothecell throughtheactionoftheactivatedGproteinandit'sdownstreamsignalingpartners andsecondarymessengers.Morespecifically,itisthealphasubunitofthe

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heterotrimericGprotein(betaandgammabeingtheothertwosubunits)thatis responsibleforthevariationindownstreamsignaling.Uponactivation,GandG activateseparatesignalingpartnersresultinginachainofintracellularsignaling.This signalingisbroughttoanendbythehydrolysisoftheGTPboundtoGalpha.Invivothis signalingistightlycontrolledanddisplaysarapidON/OFFmotif,whiletheintrinsic GTPaseactivityoftheGsubunitistooslowtoallowthis.Intheabsenceoftherapid termination,thesignalcontinuesbeyondphysiologicallevels.Thoughinsomecasesthis maybeinnocuous,datafromHeximeretal(2003)hasshownahypertensivephenotype dueinparttothisoveractiveGproteinsignaling.Assuch,duetothehighnumberof vasoconstrictorssignalingthroughGPCRs,theGproteinregulatorymechanismsof VSMCsareessentialinmaintainingnormalsystemicpressure. Gsubunitsaredividedintofoursubtypes:Gi/0,Gq/11,Gs,andG12/13. EachGsubunitassociateswithanumberofdifferentdownstreamsignalingpartners andassuchaffectsavarietyofintracellularresponses.Thefocusofthisthesiswillbeon Gi/0andGq/11inVSMCs.VascularGPCRscoupledtoGqandGihavelongbeen characterizedasahighlyvasoactiveclassofreceptors.Specifically,Gqactivityleading sequentiallytoPLCactivation,intracellularcalciumrelease,andcontractionofVSMCsto producevasoconstrictionofarteries.Inaddition,theactivationofGiresultsinthe inhibitionofadenylylcyclaseandreductionofintracellularcAMPlevels.Inthe endothelium,GiiscoupledtoGPCRscoupledtoknownvasodilatorssuchasBradykinin (LiaoandHomcy1993).InVSMCs,thereductionincAMPlevelsresultsinreducedPKA activityandattenuationofGsmediatedvasodilatation. 13

1.eTheRGSsuperfamily SignalingbyneurotransmittersandhormonesthroughGproteincoupled receptorsisacommonmechanisminmostmammalianorgansystems.Thissignaling,as describedabove,isdependantonthehydrolysisofGTP,boundtotheGalphasubunit. AlphasubunitshaveintrinsicGTPaseactivitybutitisveryslow.Invivo,thehydrolysis ofthisGTPisknowntooccursignificantlyfasterthanthatwhichisobservedinvitro (Siderovski,Hesseletal.1996;Watson,Linderetal.1996).ThisGTPaseactivationis partlytheresultoftheactivityofafamilyofproteinstermedtheRegulatorsofG proteinSignaling(RGS).Thisfamilyofmorethan35proteins,characterizedbya120 aminoacidGTPaseactivatingdomain,isresponsibleforcatalyzingthehydrolysisofGTP bytheGsubunittospeedthisprocessupto2000fold(RossandWilkie2000).One possiblemanifestationofalackofRGSproteinactivityisinincreasedsensitivityofa GPCRtoitsassociatedagonist.Thisincreasedsensitivitywouldresultinaleftwardshift inthedoseresponsecurveforsaidagonist.Alternately,orinadditiontothis,the agonistmayinduceamorepronouncedcellularresponseatanygivenconcentration. Thiswouldbeevidentinanupwardshiftindoseresponsecurves,ormorelikely,a strongerresponseatpeakconcentrations. TheseGTPaseactivatingproteins(GAPs)formafamilyofnearly30different

peptides,withdiverseexpressionprofilesbetweenorgansystemsandtissues.Within thislargegroupofproteins,therearedistinctsubfamiliesofRGSproteinssharing 14

furthersequencehomology.Briefly,thesesubfamiliesaredistributedacrossawide varietyoftissuesandorgansystemsandassuchplayanumberofroles.Thesubfamilies includeRZ,R4,R7,andR12.IntheabsenceoftheseGAPs,thesignalingproducedfrom GPCRscanbegreatlyaffected(Heximer,Knutsenetal.2003;Cifelli,Roseetal.2008).In studyingthefunctionofanyoneRGSproteinitisimportanttounderstandthepotential expressionandfunctionaloverlapbetweenitandtheotherfamilymembers.Thus, providedbelowisabriefdescriptionofeachRGSsubfamily. 1.e.iRGS7like(R7)Subfamily ThisfamilyofRGSproteinscontainsRGS6,RGS7,RGS9andRGS11andalloftheir assortedsplicevariants.Theseproteinsareuniqueduetoacommon64aminoacid Ggammalike(GGL)domainaswellasaDishevelled/EGL10/Pleckstrindomain(DEP) (Snow,Kruminsetal.1998;Chatterjee,Liuetal.2003).Thesedistinctivedomainshave beenidentifiedaskeyfactorsintheregulatoryroleofRGSproteinsofthissubfamily, andareresponsibleforallowinganumberofintracellularsignalingactivitiesand proteinproteininteractions. 1.e.iiRGS12like(R12)Subfamily TheproteinsoftheR12subfamilyarisefromthemanysplicevariantsofthe Rgs10,Rgs12andRgs14genes.Theproteinsofthissubfamilyarelessunderstoodthan thoseofthepreviouslydescribedR7class.ThemembersoftheR12familyofproteins varygreatlyinsizewithRGS10containing173aminoacidsandRGS12containing1447 residues.Existingliteratureinthisfieldidentifiesanumberofdomainspecific 15

interactionsforRGS12andRGS14andGsubunitsandotherGTPases(Traver,Bidotet al.2000;Hollinger,Tayloretal.2001;Kimple,DeVriesetal.2001). 1.e.iiiRzlike(Rz)Subfamily ThemembersoftheRzsubfamilyareencodedbytheRgs17,Rgs19andRgs20 genes.AsisobservedwiththeotherRGSsubfamilies,awidevarietyofproteinsarise fromthesegenesasaresultofsplicevariants.Thefunctionoftheseproteinsvaries fromotherRGSsubfamiliesinthattheyarestronglyaffectedbyphosphorylationofboth themselvesandtheirbindingpartners(Glick,Meigsetal.1998;OgierDenis,Pattingreet al.2000). 1.e.ivRGS4like(R4)Subfamily ThemembersoftheR4familyarecomposedoflittlemorethananRGSbox domainandshortNterminusandCterminusextensions,makingtheseproteinsthe smallestandsimplestRGSproteinsintermsofstructure.Thissubfamilyisthelargestof theRGSsubgroupings,containingRGS1,2,3,4,5,8,13,and16,witheachofthese proteinstranscribedfromadistinctgene(Sierra,Gilbertetal.2002).Furthermore,with theexceptionofRGS2(Heximer,Watsonetal.1997),everymemberofthissubfamilyis abletobindandinhibitGiandGq/11signaling.Inaddition,withtheexceptionof RGS3,theRGSproteinsoftheR4subfamilyallcontainanNTDalphahelix(Bansal,Druey etal.2007).OfinteresttoourlaboratoryarethreespecificmembersoftheR4 subfamily:RGS2,RGS4,andRGS5.ThefocusofthisthesisisonRGS5,andtoalesser extentRGS2,bothofwhicharekeyproteinsidentifiedforthestudyofvascular 16

homeostasisandbloodpressureregulation(Grayson,Ohmsetal.2007).Indeed,there arephenotypespresentedfromgeneknockoutanimalmodelsforeachoftheabove proteins(hypertensiveRGS2knockout(Heximer,Knutsenetal.2003),andhypotensive RGS5knockout(Cho,Parketal.2008;Nisancioglu,Mahoneyetal.2008)).Theexisting literatureandpreliminarydatafromourgrouponmembersoftheR4groupare outlinedbelowandcomprisethemajorityoftherationaleforthestudiesdescribed herein. 1.e.iv.1RGS5 OurpreliminarydatashowsthatRGS5isthemosthighlyexpressedRGSprotein

inperipheralVSMCsandpericytes(ZhangandHeximer,unpublisheddata).RGS5has oftenbeencitedasapotentialtargetforantihypertensivetherapy(Grayson,Ohmset al.2007)andmorerecently,apotentialkeyplayerinthemodulationofvascular remodeling(ManzurandGanss2009).RGS5hasastrikingexpressionprofilethatis concentratedprimarilyintheVSMCsofarterialvasculatureinadultanimals.Briefly, rgs5geneexpressionpeaksintheembryonicperiod,andfallssubsequentlyinavascular bedspecificmannertomaintainingsignificantselectiveexpressionincertainblood vesselsofadultanimals(Cho,Kozasaetal.2003).Thisadultexpressionissignificantly higherinarterialSMCswhencomparedtoaccompanyingveins,suggestingarolefor RGS5intheregulationofarterialSMCsignalingactivity(Li,Adamsetal.2004).Data fromstudiesinmicelackingintactRGS2­anothermemberoftheR4subfamilyhas 17

revealedamarkedhypertensivephenotypewhencomparedtoagematchedwildtype controls(Heximer,Knutsenetal.2003).Thishypertensionispartlytheresultof prolongedvasoconstrictorsignalingthroughGqcoupledreceptors.Alsoofinterest,Li etal(2004)demonstratedthepresenceofRGS5inmostvascularbedsinnonhuman primates,includingiliac,mammaryandrenalarteries.Furthermore,examinationof peripheralatheroscleroticplaquesrevealedRGS5expressioninthemedialSMCs accompaniedbyapronounceddownregulationinSMCsoftheatheroscleroticplaque. RecentstudiesbyothergroupshavedemonstratedaninductionofRGS5duringtumor growth,andneovascularization.ThesefindingssuggestapotentialroleforRGS5asa regulatorofvascularremodeling,andapotentialcandidateforfurtherstudyasan intrinsicantiatheroscleroticfactor. Asdescribedearlier,GPCRmediatedcontractilepathwaysinVSMCsare

dependantonGqandGimediatedsignaling(Petitcolin,SpitzbarthRegrignyetal. 2001).RGS5hasbeendemonstratedtohavespecificityforbothofthesesubunits(Zhou, Moroietal.2001).Althoughthefocusofthisthesishasbeenthecontractilemachinery oftheVSMC,thisisnottheonlypathwayinwhichGqandGisignalingareactive. Numerouscellularactivitiesincludingproliferation,migrationandcellcelladhesionare mediatedbysignalingthroughthesesubunits(vanBiesen,Luttrelletal.1996;Ai,Kuzuya etal.2001).Briefly,GqactivatesPLCasdescribedearlier,howeverbothGqandGi havebeenshowntobeinvolvedinmitogenactivatedprotein(MAP)kinasepathways. ThesepathwaysarehighlyactiveinSMCsinvaryingfunctions,fromfocaladhesionsto Wntsignaling.Assuch,thespecificityofRGS5forbothsubunitssuggestsapotentialrole 18

inmanyoftheseprocesses.OnespecificVSMCfunctionthathasrecentlybeen identifiedasinvolvingERKsignaling,andisofparticularinteresttous,isthemyogenic response(Pitson,Morettietal.2003;Lidington,Peteretal.2009). Asstatedpreviously,thecurrentliteratureexaminingtheinvivoroleofRGS5

describesahypotensivephenotypeinRGS5knockoutmiceaswellasdilatedaorta tissuesections(bespecific),andalteredcellularsignaling(Cho,Parketal.2008; Nisancioglu,Mahoneyetal.2008).Specifically,Choetal(2008)reportincreasedbasal ERK1/2andbasalandstimulatedVASPphosphorylationinimmunoblotassaysof culturedsmoothmusclecellstreatedwithanumberofagonistsasapotential mechanismforthereportedbloodpressuredifferencesbetweenwtandKOanimals.It isimportanttomentionthattelemetricinvestigationofthebloodpressurephenotype inwakingRGS5KOmice(Figure2)couldnotconfirmthehypotensivephenotype, acquiredusingtailcuffmeasurementsofmousebloodpressure.Specifically,invasive bloodpressuremeasurementstakenusingcarotidcatheterizationandwireless telemetryrevealanormotensivephenotypeinRGS5knockoutmiceascomparedtowild typecontrols(Heximer,2009,unpublished).Thismethodofinvasivewakingblood pressuremeasurementissuperiortomeasurementsconductedusingatailcuff plethysmographyonanumberoflevels.Chiefamongthesefactorsistheuseofa catheterallowingdirectinternalmeasurementofmeanarterialpressurefromwithin thecarotidartery,asopposedtotailcuffmeasurements,whichmustbediscardedand repeatedintheeventofanimalmovementorweakpressurereadings.Moreover, carotidcathetertelemetryallowsanimalstobehavenormallyandmovefreelywhile 19

measurementsarebeingtaken,whileplethysmographyrequiresthemanual manipulationofanimalsintorestraints,asourceofagitationandsubsequent heightenedsympatheticnervousactivity.Itiscurrentlyunclearwhetherexternal factors,specificallystressorsdrivingsympatheticactivity,playaroleintheapparent discrepancybetweenoursandthepublishedresults.Thereispotentialforanaffected mechanismatthelevelofthenervousresponseintheRGS5KOmice,hinderingthe increaseinsympatheticactivityobservedinwildtypeanimals.However,examinations intothesesignalingpathwaysarebeyondthescopeofthisthesisandmustbe addressedinaseparatestudy.Furthermore,anumberofvascularspecificmechanisms maybeinvolvedinthemodulationofresistancevesseltoneandsystemicpressure, resultinginaglobalcompensatoryeffectandunchangedwholeanimalphenotype.

20

FIGURE2

FIGURE2:Thereisnodifferenceinmeanarterialpressurebetweenwildtypeand RGS5KOmice.WildtypeandRGS5KOmicewereimplantedwithacarotidcatheter telemetrydeviceallowingforcontinualmeasurementofmeanarterialpressureovera periodof48hrs.Thedatashownistheaveragemeanarterialpressuretakenfrom6 wildtypeand5knockoutanimals.(FigureisfromZhangetal,Unpublished) 21

Workdescribedhereinusespressuremyographytoidentifytheintracellular

pathwaysregulatedbyRGS5inVSMCsandtoexaminethepotentialdiscrepancy betweenthepublishedliteratureandourownphysiologicdata.Currentliteraturefrom studiesconductedonotherRGS5knockoutmousemodelsdescribeahypotensive phenotypeascomparedtowildtypecontrols.Inaddition,Choetalhavedescribed increasedVASPphosphorylationinassaysofintracellularsignalingconductedon culturedRGS5knockoutsmoothmusclecells.Theseresultshaveledtoaproposedrole forRGS5asanegativeregulatorofvasodilatorsignalinginthearterialvasculature. Acetylcholine(ACh),actingthroughendothelialcells,isanestablishedpotentdilatorof resistancearterioles.Briefly,theendothelialfactors(NO,EDHF,andProstaglandins)act onVSMCGPCRscoupledtoGssubunits.ThiscategoryofGsubunitactsthroughthe PKApathwayandthroughanumberofsignalingeffectssuchasinhibitionofMLCKand reductionofintracellularCalciumlevels.Sinceourmodelisawholeanimalknockout andtheseendothelialdependentprocessesoccurthroughGPCRsandGsubunits, thereisthepossibilitythatRGS5mayactatsomepointinthissignalingpathway.The majorityoftherationalefortheproposedroleofRGS5asamediatorofvasodilator signalingarisesfromthepublisheddatademonstratingincreasedVASPphosphorylation inRGS5knockoutVSMCs. VASP,orvasodilatorstimulatedphosphoprotein,isphosphorylatedinresponse

tovasodilatorsignalingandisoftenusedasareadoutofcyclicnucleotide(PKAandPKG activity(Chen,Daumetal.2004).Thesekinases,bothestablisheddownstreamsignaling partnersofGPCRsandGsignalinghavebeenlinkedtomanydifferentintracellular 22

signalingpathwaysinanumberofcelltypes.IncreasedVASPphosphorylationinthe RGS5knockouts,wouldpointtoapotentialroleofRGS5intheattenuationof vasodilatorsignaling.HowevertheEna/VASPfamilyofproteinsisinvolvedinanumber ofmatrixrelatedcellularprocesses(Reinhard,Jouvenaletal.1995)andassuch,itsrole asamarkerforvasodilatorsignalingactivitymaybeanunderestimateofittruebiologic potential.ModulationinVASPphosphorylationhasalsobeenconnectedtochangesin migratoryandcellcelladhesionactivityintissuecultureexperiments(Bear,Svitkinaet al.2002).

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FIGURE3

FIGURE3:SchematicfiguredepictingRGS5promoteractivityaswellasoriginof arterialtissue.Leftpanel,IllustratesthegradientofRGS5genepromoteractivity,which hasbeenassayedusingXGalstainingandrealtimequantitativeRTPCR(Zhangand Heximer,unpublished).Rightpanel,Displaysthecorrelationbetweenshiftsin embryonictissueoriginandpromoteractivityforeachportionofthearterial vasculature.

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RGS5reporter/knockoutmousemodel OurexperimentsutilizedisolatedresistancearteriesfromourRGS5 reporter/knockoutmouse(Deltagenlaboratories).Thisanimalmodelwasdeveloped usingaLacZneocassette,insertedintoexon2oftheRgs5gene(Figure3).Invivo,the endogenousRgs5promoterwilldriveLacZtranscription,whichwillallowforstaining andvisualizationofRGS5expressingcells.Thismodeliseffectiveasareporterofcells andtissueswhereinRGS5playsarole. 25

FIGURE4

FIGURE4:SchematicrepresentingLacZNeocassetteinsertintoRGS5gene.Insertion ofaLacZNeomycinresistancecassetteintoexon2disruptstranslationofthefunctional RGS5gene.Furthermore,thisstrategyallowstranscriptionoftheinserttobedrivenby theendogenouspromoter,invivo.Thisconstructisuniqueinitsinsertionofthegene constructintoexon2,whereexistingRGS5KOmousemodelsemployLacZinsertsinto exon1.The3'probesequenceusedforgenotypingoriginalEScelllinesisshownin blue. 26

1.e.iv.2RGSproteinamphipathichelixdomainstructureandfunction TheamphipathichelixofRGSproteinsisessentialforintracellularlocalizationand properfunction AllmembersoftheRGSsuperfamilycontainthecharacteristic120aminoacid

RGSboxdomain,howeveritisbecomingevidentthatadditionaldomainsinthe peptidesareessentialfornormalintracellulartraffickingandfunction.Amongthese additionaldomainsistheNterminalamphipathichelixpresentineveryR4familyRGS protein.Forexample,Heximeretal(2001)demonstratedtheimportantroleofthe RGS2Nterminusindeterminingsubcellularlocalizationandfunction(Heximer,Limet al.2001).Additionally,Zengetal(1998)havedemonstratedthenecessityoftheN terminaldomainofRGS4forpropereffectorassociationandGAPactivity(Zeng,Xuetal. 1998).BothofthesegroupsdemonstratedthenecessityoftheNterminaldomainfor properRGSfunction.ItwaslaterestablishedbyBernsteinetal(2000)thattheessential roleoftheNterminaldomainrequiredtheformationandbindingofanamphipathic alphahelix(Bernstein,Grilloetal.2000). TheimportanceoftheNTDamphipathichelixinRGSfunctionhasbeen

establishedovercontinuedstudy.Wehereusedbiochemicalassaystodeterminethe importanceofthisprototypicalamphipathichelixdomaininRGS2sothatwemight betterunderstanditsfunctioninRGS5andotherR4groupproteins.Arecentstudy conductedonahypertensiveJapanesecohortdemonstratedaconservedmutationin theRGS2gene(Yang,Kamideetal.2005).Briefly,asinglenucleotidepolymorphismof 27

1115G>A,resultinginamutationofarginine44tohistidine(R44H),wasidentifiedinthe abovementionedcohortofhypertensiveJapaneseindividuals.Weundertook investigationstoaddresshowthisaffectsNterminaldomain(NTD)structureand interactionwiththeplasmamembraneusingaseriesofspectroscopicanalyses. Previousstudiesusingthistechniqueweresuccessfulinidentifyingthesecondary structureoftheNTDofRGS4(Bernstein,Grilloetal.2000).Thesestudiesdemonstrated thattheassociationwiththeplasmamembrane,whichisnecessaryforproperfunction oftheRGSprotein,occurredthroughaNTDhelix. 1.fRationale RGS5asaregulatorvasculartoneandVSMCsignaling Ourlabandothershavedemonstratedavascularsmoothmusclecellspecific expressionpatternforRGS5.RGS5hasbeenshowntoinhibitGiandGqcoupled signaling,andmorespecificallywhenoverexpressedinVSMCsitcaninhibitsignalingvia thevasoconstrictiveagonistreceptorsforAngII,ET1andS1P.Surprisingly,however, recentstudiesbyChoetal(Cho,Parketal.2008)andNisanciogluetal(Nisancioglu, Mahoneyetal.2008)havedemonstratedhypotensioninRGS5knockoutmice, implicatingitspotentialroleasaninhibitorofvasodilatorysignaling.Todate,thereis noexplanationfortheapparentdiscrepancybetweenthebiochemicalactionsofRGS5

28

anditsbiologicroleinvivo.ThisworkaimstoinvestigateseveralVSMCspecific signalingpathwaysinRGS5KOmiceinordertodeterminethecontributionofRGS5to bloodpressureregulationinvivo. RGSproteinamphipathichelixdomainstructureandfunction Ourlabandothershavepreviouslydemonstratedthenecessityforintactamphipathic helixdomainstructureforproperfunctionofmembersoftheR4subfamilyofRGS proteins(Bernstein,Grilloetal.2000;Gu,Heetal.2007;Gu,Tirgarietal.2008). Furthermore,duetotheheavysequenceconservationintheNTDhelicesofproteinsof thisfamily,investigationsinthisfieldcanleadtogreaterunderstandingofallR4 proteins.PreviousdatahasshownthathumanmutationsexistwithintheNTD amphipathichelixofRGS2andthatthesemayhaveimportantfunctionalconsequences. Thisprojectsetouttodeterminewhetherspecificmodulationofbasicaminoacid chargesintheamphipathichelixdomainwassufficienttoalterthemembraneaffinityof theRGSaminoterminaldomain.

29

1.gHypotheses Hypothesis.RGS5signalingregulatesVSMCfunctionandvasculartone. Hypothesis2.Theamphipathichelixiscriticalforlipidbilayerinteractionandfunctionof R4subfamilyRGSproteinsasinhibitorsofGqvasoconstrictorsignaling. 30

Chapter2:MaterialsandMethods 2.aRGS5ReporterKnockoutMouse Deltagenlaboratoriesconductedgeneknockoutprocedures.ALacZneomycin cassettewasinsertedintothesecondexonoftheRGS5geneinmouseembryonicstem cells,whichwerethenimplantedintoC57BL/6mice.Asaresult,Invivoexpressionof theRGS5LacZgeneinsertwasdrivenbythewildtypeRGS5promoter.(weblink; http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=markerDetail&key=338 91) 2.a.iBreedingandgenotypingofRGS5KOmouse AllexperimentsconformedtotheGuidefortheCareandUseofLaboratory AnimalspublishedbytheUSNationalInstitutesofHealth(NIHPublicationNo.8523, revised1996),InstitutionalGuidelinesandtheCanadianCouncilonAnimalCare. GenotypingwasconductedasdescribedbyDeltagenlaboratories (www.informatics.jax.org/external/ko/deltagen/459.html).Lossofgeneexpressionwas verifiedinknockoutanimalsbyRTPCRasdescribedinCifelli,etal(2008).Animalsused inexperimentswereRGS5KOandwildtypelittermatecontrolsfromRGS5 heterozygousheterozygousmatings. 31

2.bPressureMyography Micewereanesthetizedusingisofluoraneandsacrificedbycervicaldislocation. Intactlargeandsmallintestineswereisolatedfromthelowergastrointestinaltractand placedincold(4°C)physiologicalMOPS(3(Nmorpholino)propanesulfonicacid)buffer. Allsubsequentstepsoccurat4°Cuntilstatedotherwise.Intactvesselsareisolatedusing acombinationofbluntandsharpdissectionmethods,atwhichpointtheyarestoredin freshbufferuntilcannulationandperfusion. Intactarterieswereplacedinsmallvesselpressuremyographchambers(Living Systems,Virginia,USA)containing5mlMOPSbuffer.Thevesselswerecannulatedand securedtoglassmicropipettesusingsurgicalsuture.Vesselsareinitiallypressurizedto 40mmHgandthenslowlyincreasedto120mmHg.Theresultingbowedvesselwas separateduntilstraightenedbetweenthemicropipettes,atwhichpointpressurewas returnedto40mmHg.Thevesselbathwasthenwarmedto37°Candallowedto equilibratewithinthechamberfor30minutes. Phenylepherine(PE),Acetylcholine(ACh)andSphingosine1phosphate(S1P) doseresponseswereconductedat40mmHgpressureand37°C.Forexperiments examiningmyogenicresponse,pressurewasincreasedusingaServopump(Living Systems,Virginia,USA)routedthroughapressuremonitorexamininginflowand outflowpressure(LivingSystems,Virginia,USA). VesseldiameterwasvisualizedusingaNikonTMSmicroscopeconnectedtoa PanasonicCCTVcameraanddisplayedonaconventionalblackandwhitemonitor.The 32

imagewasroutedthroughavideodimensionanalyzer(LivingSystems,Virginia,USA) anddiameterdatawasrecordedusingAcqknowledgesoftwareonalaboratory computer.Diameterdatawassavedfromthelaboratorycomputerandanalyzedusing MicrosoftExcel.Theformulaeusedtoanalyzerawdiameterdataweredesignedto presentcontractileordilatoryresponsesofvesselsinreferencetothebaselinediameter priortocommencingeachexperiment.Assuch,forexperimentsusingvasoconstrictor agonists,baselinediameterwastakenasthediameterofthevesselat40mmHg,prior totheadditionofanyvasoconstrictor.Forvasodilatorystudies,measurementswere analyzedinordertodisplaytheamountofAChmediatedvasodilationoccurringinthe presenceof1mMPEconcentrationwithintheorganbath.Themyogenicvasoconstrictor responsewasassessedbycomparingthediamatersofeachvesselinthepresenceand absenceofCalciumateachpressurestep. 2.b.iMyogenicresponse:Oncearteriesareheatedandequilibratedto40mmHg, pressureisreducedto20mmHgandincreasedinstepwiseincrementsof20mmHgup to120mmHg.Thevesselsaremaintainedateachpressurestepfor5min(20,40,60 mmHg)and7min(80,100,120mmHg).Thediameterdataforactivemyogenictone wascombinedwiththepassivevesseltonedatabelowasfollows: [(Passivediameteratpressurestep)­(Activediameteratpressurestep)]/(Passive diameteratpressurestep)

33

Passivemyogenictone:ArteriesarewashedwithCa2+freeMOPSbuffer(37°C)4times at810minuteintervals.Pressureisthenreducedto20mmHgandincreasedin20 mmHgstepwiseincrementsto120mmHg.Thevesselwasallowedtoremainateach pressurestepfor1min. 2.b.iiPEdoseresponse:Foreachdose,1mlofMOPSbufferwasremovedfromthe organbathandreplacedwithPEdosespreparedin1mlofMOPSbuffer.The concentrationswereasfollows:1,3,10,30,100,300,1000,3000,10000,30000(nM). PEdoseswereheatedto37°Candaddedin5minintervals.Diameterdatawasanalyzed usingthefollowingformula: [(BaselineDiameter)(StablereadingofdiameteratPEdose)]/(BaselineDiameter) 2.b.iiiAChdoseresponse:BeforeAChaddition,arteriesareinitiallyconstrictedto1mM PEtoestablishbackgroundvasculartone.EachdoseofAChispreparedcontaining1 mMPEtomaintainconstantPEconcentrationwithintheorganbath.ACh concentrationswereincreasedasfollows:1,3,10,30,100,300,1000,3000,10000, 30000(no).Diameterdatawasanalyzedusingthefollowingformula: [(StablereadingofdiameteratAChdose)(Diameterchangeinresponseto PE)]/(DiameterchangeinresponsetoPE)

34

2.b.ivS1Pdoseresponse:S1Pdoseswerepreparedfollowingestablishedprotocolsfrom theresearchlaboratoryofSteffenBolz.Briefly,S1Pwasdissolvedin4%BSAtoa concentrationof1X104µM.Thiswasusedasthepeakconcentrationwhilethe remaining4doseswerepreparedusing1in10dilution.Diameterdatawasanalyzed usingthefollowingformula: [(BaselineDiameter)(DiameteratS1Pdose)]/(BaselineDiameter) 2.cAorticSmoothMuscleCellCultureExperiments Alltissuecultureexperimentswereconductedonprimaryisolatedaorticsmooth musclecellsfromwildtypeandRGS5knockoutmice.Briefly,aortasfromaorticrootto renalbranchwereisolatedfromwildtypeandknockoutanimalsandprocessedusing elastase,collagenaseandmechanicaldegradationuntilsurroundingconnectivetissue wasremoved.Unlessotherwisenoted,cellsaregrowninCompleteMedia(DMEM/F12, 10%FBS,P/G/Santibody,PDGF).Cellsweregrowninaheracell150incubatorand experimentsandprocedureswereconductedinaclassIIabiologicalsafetycabinet. 2.c.iERKSignalingAssay PrimaryculturedwildtypeandRGS5knockoutaorticsmoothmusclecellswere platedin6wellplates.Cellswereserumstarvedfor24hourspriortotreatmentand thentreatedwithAngII,ET1,S1P,PDGFandSNP,orvehiclecontrol.Cellswere incubatedfor10minutesandharvestedoniceusinganSDSglycerollysisbuffer. 35

2.c.iiVASPSignalingAssay Primaryculturedsmoothmusclecellswereplatedon6wellplatesandserum starvedfor24hourspriortotreatmentwith1.5mMSNPorvehiclecontrol.Once treated,treatedandcontrolwildtypeandknockoutcellswereharvestedusinganSDS glycerollysisbufferat10,45,and90minuteintervals. 2.c.iiiCellProliferationAssay Primaryculturedsmoothmusclecellswereplatedin6wellplatesata concentrationof1X105cellsperwell.Sufficientwellswereplatedtoallow2wellsper genotype,perdayforatotalof14wellspergenotype.Allcellswereplated simultaneouslyandallowedtogrowinparallel.Twowellsfromeachgenotypewere harvestedeachdayandcountedusingahemocytometer.Theresultantnumberswere averagedbetweenwellsforeachgenotype.Shownistheaveragecellcountateach timepoint(n=3)witherrorbarsdepictingstandarderrorfromthemean. 2.dRGS2Nterminaldomainhelixsecondarystructureandpeptidelipidassociation RGS2peptidelipidinteractionsandsecondarystructureassayswereconducted usingsynthesizedNTDpeptidesfromtheHospitalforSickChildren:AdvancedProtein TechnologyCentre(Toronto,ON). 2.d.iTryptophanFluorimetry TryptophanfluorescencespectraofRGS2WT,R44H,andL45DNterminal

domainpeptidesweremeasuredusinganAVIVratiospectrofluorometerATF105 36

(Lakewood,NJ).TheNTDPeptidesampleswerethenresuspendedinRBStoafinal concentrationof0.2µM.Extrudedunilaminarliposomes(EncapsulaNanoSciences, Nashville,TN)weremadefrombovinebrainlipids(AvantiPolarLipids)andwerediluted inPBS.Liposomeswereaddedtopeptidesolutionfor5minbeforemeasurementto allowconsistentlipidassociation.Foreachlipidconcentration,fluorescenceemission spectraafter295nmexcitationwererecordedat2nmstepsfrom310to400nm. SimilarlygeneratedliposomeandPBSalonecontrolemissionspectraweresubtracted frompeptidespectratoaccountfornonpeptide,backgroundfluorescenceemission.In experimentsinvolvingtrifluoroethanol(TFE),solutionswithpeptideswerethoroughly mixedandincubatedfor5minbeforemeasurement. 2.d.iiCircularDichroism PeptidesecondarystructurewasassessedusinganAVIVCircularDichroism

Spectrometermodel202.WildtypeRGS2,L45D,andR44Hmutantpeptideswere analyzedwithorwithoutliposomes.UnilaminarliposomesforCDstudiesweremadeas describedpreviously(Bernsteinetal.,2000).Inbrief,a3:2solutionof dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol(AvantiPolarLipids)in chloroformwasdriedundernitrogenandresuspendedinPBS.Lipidsinsolutionwere sonicatedfor5minwith20spulsesandchilledonice.Liposomesweremadefreshfor eachexperiment.Peptides(721µM)weredilutedinPBSwithandwithoutlipidsorTFE, andspectraweremeasuredfrom190to260nmin1nmincrementsaveragedover4s aftera5minincubationperiod.ThespectraoflipidsandPBSaloneweresubtracted

37

fromsamplemeasurementstoaccountfornonpeptide,backgroundfluorescence emission. 2.eStatisticalmethods Unlessotherwisestateddataarepresentedasmean+/standarderrorofthe mean(SEM).Oneway,twowayandrepeatedmeasuresANOVAwereusedasindicated intheFigurelegends.Inordertodeterminethesignificanceofcellsignalingassays, student'sttestswereconducted,withsignificancebeingassignedtoresultswithp valuesoflessthan0.05. 38

Chapter3:Results 3.aPressureMyography LossofRGS5doesnotaltermyogenicresponsiveness MyogenicResponse(MR):Thebaylisseffect,ormyogenicresponse,isamechanism inherenttoVSMCs,whichcausescontractioninresponsetoincreasesintransmural pressureandcellularstretching.InordertoexaminewhetherRGS5playsarolein mediatingthemyogenicresponse,isolatedarterioleswereexposedtoincreasing pressurefrom20mmHgto120mmHg,instepwiseincrementsof20mmHg.These experimentswererepeatedincalciumfreephysiologicalMOPSbufferandtheresultant ratiowasdeterminedtobetheactivemyogenictone.Themeasurementstakenin calciumfreebufferallowfortheexaminationofcomplianceindependentofthe contractileactivityoftheVSMCs.IfRGS5isplayingaroleinthemediationofthe myogenicresponse,weexpecttoobserveoneorbothofthefollowing:initiationof contractionatlowerpressurestepsorincreasedoverallcontractionatanyorall pressuresteps.TheresultsshowaslightlyincreasedmyogenicresponseintheRGS5 knockoutascomparedtowildtype,manifestinginincreasedsensitivity(lowersetpoint) topressurewhilemaintainingsimilaroverallconstrictionatpeakpressure(120mmHg) (Figure5).Passivevesseltonewasanalyzedseparatelyinordertoinvestigatewhether thematrixcompositionofRGS5knockoutvesselsvariesascomparedtowildtype vessels.TherewasnosignificantdifferenceinpassivetonebetweenwildtypeandRGS5 knockoutmesentericvessels(Figure6).Significanceofmyogenicresponsecurvesand 39

passivetonecurveswereassessedusingtwowayANOVAwithrepeatedmeasures. Vesseldiameterwasalsoanalyzedinordertoensurethereisnovariationbetween vesselsusedforpressuremyographyexperiments,andnosignificantdifferencewas observed(Figure7).Forvesseldiameter,student'sttestwasusedtoassesswhether therewasasignificantdifferenceinvesseldiameterbetweenwildtypeandknockout vessels. 40

FIGURE5

FIGURE5:NosignificantdifferenceinmyogenicresponseofwildtypeandRGS5 knockoutmesentericarterioles.Cannulatedmesentericresistancearterioleswere maintainedinMOPSphysiologicalbufferat37°C.Intramuralpressurewasincreasedin 20mmHgstepwiseincrementsfor5min(20,40,60mmHg)or7min(80,100,120 mmHg)periods,andstablereadingofvesseldiameterwasrecorded.Significancewas assessedbytwowayANOVAwithrepeatedmeasures.Errorbarsshownarestandard errorfromthemean.Datadescribediscalculatedusingthefollowingformula: [(Passivediameteratpressurestep)­(Activediameteratpressurestep)]/(Passive diameteratpressurestep)

41

FIGURE6

FIGURE6:Nosignificantdifferenceinvascularwallcompliancebetweenwildtypeand RGS5KOmesentericresistancearterioles.Following(3X10min)washingofmesenteric resistancearterioleswithCalciumfreeMOPSphysiologicalbuffer,cannulatedvessels areexposedtostepwiseincreasesinintralumenalpressure.Resultantcurvesdisplaythe complianceofwildtypeandRGS5KOvessels.Errorbarsshownarestandarderrorfrom themean.SignificancewasassessedbytwowayANOVAwithrepeatedmeasuresThe datashowniscalculatedasfollows: [(Diameteratpressurestep)­(Diameterat20mmHg)]/(Diameteratpressurestep mmHg)

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FIGURE7

FIGURE7:NosignificantdifferenceinvesseldiameterbetweenwildtypeandRGS5KO vesselsusedinpressuremyography.Diametersofvessels(at40mmHg)usedfor pressuremyographywererecordedthroughoutandcompiled.Theerrorbarsshown representthestandarderrorfromthemean.Significancewasascertainedusinga student'sttest,withsignificanceassignedtopvaluesoflessthan0.05.(n=8KO,n=12 WT)

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Shingosine1phosphate(S1P):S1P,asphingolipidandbioactivecellsignalingmediator, bindstotwosubtypesofGPCRsonVSMCs:S1P2andS1P3(Waeber,Blondeauetal. 2004).ThesereceptorsareinturncoupledtobothGiandGqboundheterotrimers. Assuch,thevarietyofdownstreamsignalingeffectsS1Pinducesareplentifulandof these,vasoconstrictionisacloselyexaminedone.Furthermore,S1Psignalinghasbeen showntobeessentialforthemyogenicresponse(Bolz,Vogeletal.2003).Importantly, RGS5hasbeenshowntospecificallytargetS1PsignalinginVSMCs(Cho,Harrisonetal. 2003).Takentogether,thesepointssuggestthatRGS5maybeanimportantregulator oftheS1Pmediatedcomponentofthemyogenicresponse.Ourstudiesinresistance arteriolesfromRGS5knockoutmicewereaimedattestingthispossibility.Toexamine whetherRGS5isactingontheS1PreceptorcoupledGqsubunit,isolatedresistance arteriolesweretreatedwithincreasingdosesofS1P,avasoconstrictorandkeyregulator ofthemyogenicresponse.IfRGS5displaysspecificityfortheS1Preceptor,weexpectto seeoneoforbothofthefollowing:increasedsensitivityofthevesseltoexogenously appliedS1PandincreasedoverallconstrictioninresponsetoS1P.RGS5knockout vesselsdemonstratednosignificantdifferenceincontractileresponsecomparedtowild typevessels(Figure8).SignificancewasdeterminedusingatwowayANOVAwith repeatedmeasures.

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FIGURE8

FIGURE8:NosignificantdifferenceinresponsetoS1PinwildtypeandRGS5KO mesentericresistancearterioles.Cannulatedmesentericresistancearterieswere maintainedat40mmHgand37°C,andtreatedwithincreasingconcentrationsof sphingosine1phosphate.SignificancewasdeterminedusingatwowayANOVAwith repeatedmeasures.Theerrorbarsshownrepresentthestandarderrorsfromthemean. Thedatashownwascalculatedasfollows: [[(BaselineDiameter)(DiameteratS1Pdose)]/(BaselineDiameter)]*100 45

LossofRGS5doesnotaltervasoconstrictororvasodilatorresponsesinresistance arterioles Phenylepherine(PE)DoseResponse:Phenylepherineiswidelyusedinstudiesof adrenergicsignaling.Briefly,VSMCscontainanumberofcatecholaminereceptorsthat areessentialinmediatingsympatheticsignalinginmammalianorganisms.Thecellular effectsofthesecatecholamines,whichincludeepinephrine,norepinepherineand dopamine,aredependantuponthetypeofcellsurfacereceptor(GPCRswithvaryingG subunits)theybind.Ofthese,alphaadrenergicreceptorsareGqcoupledandassuch inducePLCactivationandasubsequentincreaseinintracellularcalcium.This mechanism,aspreviouslydescribed,isessentialfortheinitiationofcalciumdependant SMCcontraction,andinvessels,calciumdependantvasoconstriction.Consequently, nearlyallknownpotentvasoconstrictorsactthroughGqcoupledreceptors.Inorderto examinewhetherRGS5isactinginageneralGqvasoconstrictorpathway,isolated resistancearteriolesweretreatedwithincreasingdosesofPE,apotentvasoconstrictor andalphaadrenergicagonist.adrenergicreceptorsfollowtheclassicalGqmediated pathwaydescribedabovewhichculminatesinphosphorylationofMLCbyMLCK. Changesinvesseldiameterwererecordedandanalyzedtoassessvasoconstrictor response.IfRGS5attenuatedGqmediatedsignaling,wewouldexpecttosee enhancedcontractileactivityintheknockoutvesselsascomparedtowildtypecontrols. TheresultsshownosignificantdifferenceinvasoconstrictiontoPEbetweenwildtype andRGS5knockoutvessels(Figure9).Significancewasdeterminedusingtwoway ANOVAwithrepeatedmeasures. 46

FIGURE9

FIGURE9:Nodifferenceinphenylepherinemediatedcontractionbetweenwildtype andRGS5KOmesentericresistancearterioles.Cannulatedarteriolesweremaintained at40mmHgand37°C,whiletheyweretreatedwithincreasingconcentrationsof phenylepherine.SignificancewasdeterminedusingatwowayANOVAwithrepeated measures.Errorbarsshownrepresentthestandarderrorfromthemean.Thedata shownwascalculatedasfollows: [[(BaselineDiameter)(StablereadingofdiameteratPEdose)]/(Baseline Diameter)]*100 47

Acetylcholine(ACh)DoseResponse:InordertoexaminewhetherRGS5isactingatsome pointinsmoothmusclecellrelaxation,isolatedresistancearteriolesweretreatedwith ACh,inordertoinduceNOdependantvasodilation.Ifthecurrentreportsarecorrectwe mayhaveexpectedtoobserveenhancedvasodilatoryactivityinRGS5deficient arterioles.However,therewasnosignificantdifferenceinvascularresponsetoACh betweenwildtypeandRGS5knockoutvessels(Figure10)suggestingthatRGS5didnot playasignificantroleinendothelialdependentvasodilation.Significancewascalculated usingtwowayANOVAwithrepeatedmeasures.

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FIGURE10

FIGURE10:NosignificantdifferenceinAChmediatedrelaxationofPEinduced constrictionbetweenwildtypeandRGS5KOmesentericresistancearterioles. Cannulatedarterioleswereconstrictedwithphenylepherine(1µM)andmaintainedat 40mmHgand37°C.Vesselswerethentreatedwithincreasingconcentrationsof acetylcholine.SignificancewasdeterminedusingatwowayANOVAwithrepeated measures.Errorbarsshownarestandarderrorfromthemean.Thedatashownwas calculatedasfollows: [[(StablereadingofdiameteratAChdose)(Diameterchangeinresponseto PE)]/(DiameterchangeinresponsetoPE)]*100

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3.bWesternimmunoblots DownstreamsignalingpathwaysarealteredinRGS5deficientprimaryculturedsmooth musclecells ERKsignalingassay:Extracellularregulatedkinases1and2areubiquitouslyutilized proteinsforassessingvasoactivesignalingduetotheirroleinthepropagationof extracellularsignalingthroughGPCRs(Leroy,Missottenetal.2007).Specifically,this signalinghasbeenconnectedtoGqandGisignaling,andasdescribedearlier,results inanumberofintracellulareffectsrangingfrommigrationandadhesionto proliferation.CurrentliteraturepresentsincreasedERK1/2phosphorylationinvehicle treatedculturedRGS5knockoutVSMCs.WedefinetheseERKphosphorylationresults fromvehicletreatedgroupsasbasalERKphosphorylation,astheyarethebasallevelsof phosphorylationwithouttheadditionofagonists(stimulated).Weundertook experimentstoexamineERK1/2signalinginprimaryculturedVSMCsinorderto understandthediscrepancybetweenourwholeanimaldataandthepublishedresults. Weexpectedtoseeeitherincreasedbasaland/orstimulatedERK1/2levelsinRGS5KO primaryculturedcellswhencomparedtocontrols.CellsweretreatedwithS1P,AngII, ET1,SNPandPDGFandresultingimmunoblotswereanalyzedforphosphoERK/totalERK ratio.(S1P,AngII,ET1andSNPresultsareshown;Figure11)Resultsdemonstrateno significantdifferenceinstimulatedERK1/2phosphorylationbetweenRGS5KOandwild typecells,howeverbasalERK1/2phosphorylationinRGS5KOcellsappearedhigherif notequaltothelevelsobservedinwildtypecells.Assuch,vehicletreatedlaneswere

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pooledandsignificantlyhigherlevelsofERK1/2phosphorylationwasobservedinthe RGS5knockoutsmoothmusclecellsintheabsenceofagonists,ascomparedtowild typecontrols(Figure12).

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FIGURE11

FIGURE11:NosignificantdifferenceinERK1/2phosphorylationwhenstimulatedby vasoactiveagonists.PrimaryculturedwildtypeandRGS5KOaorticsmoothmusclecells weretreatedwithS1P,AngII,ET1,andSNP,incubatedfor10min,andharvested. LysateswereanalyzedbywesternimmunoblotforERK1/2phosphorylation.Results shownarephosphorylatedERKbands,normalizedtototalERKandanalyzedusing ImageJ.Errorbarsdepictthestandarderrorfromthemean.Significancewasassessed usingastudent'sttest.(n=4,foreachtreatmentgroup) 52

FIGURE12

FIGURE12:RGS5KOprimaryculturedVSMCsdisplayincreasedbasallevelsofERK phosphorylation.AlthoughnodifferencewasobservedinstimulatedERK1/2 phosphorylationlevels,basalERK1/2phosphorylationinRGS5KOcellsappearedtobe thesameasorhigherthanwildtypelevels.VehicletreatedbandsfromallERK1/2 signalingassaytreatmentgroups(showninFigure11)werecompiledandanalyzed usingImageJandsignificancewasdeterminedusingastudent'sttest.Errorbarsshown arestandarderrorfromthemean.

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VASPsignalingassay:VASP,asdescribedearlier,isawidelyutilizedproteininthe measurementofPKAandPKGactivity.Furthermore,asitsnameimplies,VASPis phosphorylatedinresponsetovasodilatorstimulus.Previousstudieshave demonstratedincreasedVASPphosphorylationinresponsetoSNPaddition,in conjunctionwithobservedhypotension,inRGS5knockoutmice.Theseresults comprisedthebulkoftherationaleforthecurrentproposedroleforRGS5asa regulatorofvasodilatorsignaling.Inordertoassesswhetherourprimarycultured VSMCsweredisplayingsimilarcharacteristicstothoseintheliterature,weconducted westernimmunoblotsexaminingVASPphosphorylationinresponsetoSNPtreatment. CellsweretreatedwithSNP(4mM)for10,45and90mintodeterminewhetherRGS5 wasaffectingNOinduced(vasodilatory)signaling.WeexpectedincreasedVASP phosphorylationintheRGS5knockoutcellsinresponsetoSNPtreatment,asthe literaturereports.OurresultsshowincreasedVASPphosphorylationinRGS5knockout VSMCsascomparedtowildtypecontrolsafter45and90minuteincubationperiods. (Figure13)

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FIGURE13

FIGURE13:RGS5KOculturedVSMCsdisplayhigherlevelsofVASPphosphorylation thanwildtypecontrols.PrimaryculturedVSMCsweretreatedwith1.5mMSNPfor10, 45and90minuteincubationperiodsandharvested.Lysateswereanalyzedbywestern immunoblotingforVASPphosphorylation.DatashownisphosphorylatedVASPbands normalizedtototalVASPandthenanalyzedbyImageJsoftware.Lanescorrespondingto vehicletreatedcellsarelabeledC(Control)andlanescorrespondingtoSNPtreatedcells arelabeledT(Treatment),whileWTandKOrefertowildtypeandRGS5KOcells, respectively.ThenumericalvaluescorrespondtoSNPincubationtimepriortoaddition oflysisbuffer.Errorbarsrepresentstandarderrorfromthemean,andsignificancewas determinedusingastudent'sttest.(n=4)

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3.cCellproliferationassay RGS5knockoutprimaryculturedsmoothmusclecellsdonotdisplayanydifferencein cellularproliferationwhencomparedtowildtypecontrols InordertoassesswhetherRGS5affectsoverallsmoothmusclecellERK

signaling,weassessedproliferationofculturedarterialsmoothmusclecells.Gprotein pathways,aspreviouslydescribed,arenotlimitedtocontractilesignaling,andcan activateproliferativesignalingthroughtheMAPkinasesignalingpathway.Thisassay providedtwopotentialinsightsintothefunctionofRGS5:wewereabletoexamine whetherRGS5attenuatesgrowthfactorsignalingleadingtoproliferation,aswellas assesswhethertheincreaseinbasalERK1/2phosphorylationobservedinwestern immunoblotsalsoaffectsalternateMAPkinasesignalingpathways.Ourexpectedresults wereenhancedproliferativeactivityinRGS5KOcellscomparedtowildtype,specifically intherateofcellgrowth.However,resultsshowednosignificantdifferencein proliferationratebetweenwildtypeandRGS5knockoutcells(Figure14).Therewasa slightdiscrepancyinpeakcellconcentrationhowever,consistentinallexperimental trials. 56

FIGURE14

FIGURE14:Thereisnosignificantdifferenceingrowthratebetweenwildtypeand RGS5KOprimaryculturedSMCs.1.0X105cellswereplatedin14wellsforeach genotype(28totalwells).2wellspergenotypewerecountedeachdayusinga hemocytometerandanaveragewastaken.Errorbarsdepictstandarderrorfromthe mean,andsignificancewasassessedatspecifictimepointsusingastudent'sttest.

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3.dR44HMutationDoesNotDisrupttheHelixFormingCapabilityoftheRGS2NTDHelix. TheasparticacidinthepreviouslypublishedmutantL45Disaknownhelixbreakerand thustheL45DmutantdoesnotassociatewiththePMbecauseofitsinabilitytoforma helix.TotestwhethertheR44Hmutant'sinabilitytoassociatewiththePMisduetothe samemechanism,wecomparedthesecondarystructureofRGS2,R44H,andL45D peptidesinthepresenceandabsenceofanionicliposomesusingcirculardichroism.As expected,intheabsenceoflipids,allofthepeptidesshowadisorderedrandomcoilCD signature.Inthepresenceofanioniclipids,however,bothRGS2andR44Hmutant peptidesshowcharacteristichelixformationwithamolarellipticityminimaat222nm (Figure15),consistentwiththenonhelixbreakingnatureofhistidine.Incontrast,CD spectraofL45Dshowarandomcoilspectrumeveninthepresenceofliposomes (Heximeretal.,2001).Together,thesedataindicatethattheR44HNTD,despite retainingtheabilitytoformaproperhelix,wasunabletoformastableinteraction withthelipidbilayer.

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FIGURE15

FIGURE15:R44Hmutantpeptidesformhelicesinthepresenceoflipids.R44H mutationshowsnormalhelixformingpotentialinthepresenceofnegativelycharged phospholipids.Theindicatedmutantandwildtypepeptidescorrespondingtoresidues 34to57inPBSwereincubatedeitherwithorwithoutlipidsandCDspectrawere 59

collectedasdescribedunderMaterialsandMethods.Blackcirclesrepresentcontrol spectrumofpeptidesinPBSsolution;Whitecirclesrepresentspectrumofpeptide solutioncontaining1.4mMlipidliposomes.Shownisarepresentativeexperimentfrom threeindependentexperiments.(FigurefromGu,Tirgarietal,2008,Molecular Pharmacology) 60

3.eTheAmphipathicHelixDomainoftheR44HMutantDoesNotStablyAssociatewith PurifiedLiposomes TheNTDamphipathichelixisrequiredforinteractionofRGS2withnegativelycharged phospholipidsontheinnerleafletoftheplasmamembrane.Mutationsthatdisrupt amphipathicityarepredictedtodisruptitslocalizationandfunction.BecausetheR44H mutationspecificallychangesanarginineresidueadjacenttothehydrophobicfaceof theRGS2helixtohistidine,weproposedthatthismutationwouldresultinareduction inRGS2lipidbilayeraffinity.Tryptophanspectroscopyhasbeenusedtostudythe interactionoftryptophancontainingamphipathichelicalpeptideswithlipidbilayers (Burstein,Vedenkinaetal.1973).Thisassaymeasureschangesinthefluorescence emissionspectrumofatryptophanresidueonthehydrophobicfaceofanamphipathic helix.Asthelocalenvironmentofthetryptophanchangesfrompolar(solution)to hydrophobic(lipids),thereisablueshiftinthespectrummaxima(Burstein,Vedenkina etal.1973).Spectrawerecorrectedforbackgroundasdescribedpreviously.Corrected spectrafromwildtypeRGS2showsthemaximashiftwithincreasingconcentrationsof lipids,whereastheR44Hspectraisunaffectedbythepresenceoflipids.Likewise,L45D, amutantpeptidepreviouslyshownnottointeractwiththePMorformanhelixinthe presenceoflipids(Heximeretal.,2001)showsnochangeinitstryptophanpropertiesin thisassay.Onepossibleexplanationforthedifferencesintryptophanspectroscopyof theR44Hmutantisthatthelonghydrophobicsidechainofarginine44couldcontribute toanincreasedlocalhydrophobicenvironmentoftryptophanwhenthepeptideisina helicalformation.Toaddressthispossibility,wetestedwhetherthetryptophan 61

spectrumwasalteredbyTFE,ahelixpromotingsolvent,andintheabsenceof liposomes.Asshowninfigure17,theadditionofTFEresultsinamarkedincreasein helixformationofthewildtypeRGS2peptidewithoutinducingablueshiftinthe tryptophanspectra.AlthoughwecannotexcludethepossibilitythattheArg44residue contributestothehydrophobicenvironmentofthenearbyTrp41,suchaninteraction cannotexplaintheprofoundblueshiftobservedinthepresenceofliposomes. Accordingly,thesedatasuggestthatthespectralchangesoftryptophanobservedinthe presenceofliposomesareduetoitsinsertionintothehydrophobiccoreofthelipid bilayer.(Figure16)Theseresults,inconjunctionwiththosefromcirculardichroism analysisleadtotheproposed"snorkeling"mechanismforNTDamphipathichelixlipid association.(Figure18)

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FIGURE16

FIGURE16:R44Hmutantpeptidesdonotinteractwithlipidbilayers.R44Hmutation resultsindecreasedpenetrationofthehydrophobicfaceoftheRGS2helixintothelipid bilayercore.RGS2helixdomainpeptidescorrespondingtoresidues34to57fromWT, 63

R44H,andL45Dwereincubatedwithincreasingamountsofpurifiedunilaminar liposomesfrombrainlipidextracts.TryptophanfluorescenceemissionspectraofTrp41 onthehydrophobicfaceoftheRGS2amphipathichelixwerecollectedwithlipid concentrationsrangingfrom0to0.4mg/ml.Shownisarepresentativeexperimentfrom threeindependentexperiments.(FigurefromGu,Tirgarietal,2008,Molecular Pharmacology) 64

FIGURE17

FIGURE17:TFEadditiontoR44Hpeptidescauseshelixformationwithoutany associatedblueshiftinthetryptophanspectra.TryptophanFluorimetryandCircular DichroismstudieswereconductedontheR44Hmutantpeptideinthepresenceof trifluoroethanolandtheabsenceoflipidsinordertoassesswhetherthelocal environmentofthetryptophanresiduecontributingtotheblueshiftassociatedwith lipidinteraction.Theresultsdemonstratenoblueshiftinthetryptophanemission 65

spectra,inspiteofprominenthelixformation.Datashownisonerepresentative experimentofthreeindependantexperiments.(FigureisfromGu,Tirgarietal,2008, MolecularPharmacology)

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FIGURE18

FIGURE18:A)PeptideconservationbetweenR4familyNTDs.B)Proposed "snorkeling"mechanismbywhichNTDsstablyinteractwithlipidbilayers.Figure18A describesthehighdegreeofsequencehomologyaswellastheheavilyconserved highlightedbasicresidues.TheseresiduesarebelievedtoberequiredforstableRGS2 NTDhelixinteractionwithalipidbilayer,asdescribedinfigure18B.Weproposethat theR44HmutationhindersNTDhelixinteractionwiththelipidbilayerduetoits shorterandstericallyunfavourablesidechain,makingitincapableof"snorkeling". (FigurefromGu,Tirgarietal,2008,MolecularPharmacology) 67

Chapter4:Discussion Generaldiscussion Bloodpressureregulationisessentialformaintenanceofnormal

cardiovascularfunctioninallmammalianorganisms.Ithasalreadybeenestablished thatGproteinsareintegralinthecontrolofbloodpressureandflowatthelevelofthe heart,thekidneysandthevasculature,andfurthermore,thatthefinetuningoftheseG proteinsignalsbyRGSproteinsisessentialfornormalfunction.Whenthesignaling requiredforproperfunctionbecomescompromised,theresultsincludeanumberof vasculopathiesandCVDsincludinghypertension,anestablishedriskfactorfor numerousseriousclinicalcomplicationssuchasmyocardialinfarction,andstroke.The currentliteraturehasfirmlyestablishedaroleforRGS2asapotentregulatorofVSMC contractileactivitythroughitsabilitytoinhibitGqsignaling.Existingliterature proposesaroleforRGS5asaregulatorofVSMCactivity,butasamediatorof vasodilatorysignaling,andnotasamediatorofGqvasoconstrictorsignaling.Thedata describedhereinprovidesbothfurthersupportandnewinsightsintothestudyofRGS proteinsandtheirpotentialbiologicrolesasregulatorsofVSMCfunction. 4.aRGS5discussion Interestingly,theresultsdescribedinthisthesisdonotsupportadirectrolefor

RGS5asakeyregulatorofvasculartoneorsystemicpressure.BothPEandAChdose responsecurves,aswellasmyogenicresponseandS1Pdoseresponsecurves,showno significantdifferencebetweenwildtypeandRGS5knockoutvessels.Furthermore, 68

wholeanimalstudiespreviouslyconductedinourlabsupporttheseobservations(Zhang andHeximer,unpublished).Thoughtheseobservationsarecontrarytotheexperimental hypothesis,aswellastheearlierliteratureinsupportoftherationaleforthisstudy, theseresultsmaybeindicativeofamorecomplexroleinpreviouslyunderexamined mechanisms,suchasSMCmigration/interactionandmyoendothelialcoupling.Our currentresultsallsupporttheconclusionthattheabsenceofRGS5haslittleornoeffect intheregulationofVSMCcontractionanddilationinresponsetotheagoniststested. Ofcourse,thisdoesnotruleoutthepossibilitythatRGS5specificallyregulatesanother GPCRmediatedpathwaythatwasnottestedinthiswork.Interestingly,theliterature alsodescribesaleanbodyhabitusinRGS5knockoutmicecomparedtowildtype controls,whichisalsonotevidentinourfindings.Astheseknockoutsweremadewith differenttargetingconstructsthismayindicatedifferencesbetweendifferentgenetic RGS5KOmodels,wherethesiteofgeneticmanipulationishasdifferenteffectsonthe expressionofgenesnearbyonthesamechromosome.Alternatively,thegenetic backgroundsofthedifferentstrainsmaynotbeidenticalallofouranimalswere backcrossed>8generationsintoaC57Bl/6background. Althoughwedidnotobservethehypotensivephenotypereportedinthe

literature,anumberofobservationsdidmatchtheexistingdataandarelikelytobe specificforthelossofRGS5.ParticularlytheobservationofincreasedbasalERK1/2 phosphorylation,aswellasanincreaseinVASPphosphorylationinRGS5knockoutcells ascomparedtowildtype(Cho,Parketal.2008).Asdescribedearlier,bothofthese intracellularsignalingproteinsareinvolvedincytoskeletalmodulation,whileERKhas 69

beenspecificallyshowntobeinvolvedintheactivationofthemyogeniccontractile response(Lidington,Peteretal.2009).Thiscombinationofalteredcellsignalingis intriguingasitmaydescribeacompensatorymechanismbywhichincreasedmyogenic responseand/oragonistmediatedcontractionisbeingtemperedbyincreasedVASP signaling.Consistentwiththispossibility,nophenotypewasobservedinthewhole animalorisolatedarteriolarpreparations,despiteourobservationofalteredsignalingin culturedprimaryRGS5KOVSMCs. 4.a.iMyogenicResponseinRGS5knockoutresistancevessels InourattemptstoelucidatetheroleofRGS5,wefocusedourattentionona

VSMCspecificmechanism­themyogenicresponse.Althoughthefindingsfromthe pressuremyographystudiesshowednosignificantdifferencebetweengenotypes,there wasaslightlyhigher,albeitnonsignificantincreaseinmyogenicresponseintheRGS5 knockoutvessels.Thesepreliminaryresults,aswellasdatasupportingapotentialrole forRGS5inmediatingS1Psignaling(andthusthemyogenicresponse)(Cho,Harrisonet al.2003;Peter,Lidingtonetal.2008),weretherationaleforthedoseresponse experimentsexaminingvascularapplicationofS1P.Theresultantdoseresponsecurves, however,werenotsignificantlydifferent.Nevertheless,wemustconsiderthatthese studieswerecarriedoutinmesentericresistancearteries,whichshowarelativelyweak responsetoS1Pcomparedtoothervascularbedsandthusitispossiblethat investigatingmyogenicandS1Presponsesinothervascularbedsmayrevealmore intriguingfindingsthanthoseobservedinmesentericvessels.Wealsoconsidered

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literaturethatestablishedaconnectionbetweenmyogenicresponsesandconstriction inducedbyvasoactiveagonists.Thisdata,fromdeWitetal,describesanincreasein norepinepherinemediatedconstrictionduetotheactionofthemyogenicresponse(de Wit,Bolzetal.1998).Thus,sinceRGS5mayactonbothGproteinmediatedpathways (S1PandNE)wepostulatedaleftwardshift(greatersensitivity)inRGS5KOduringthePE doseresponseascomparedtowildtype,correspondingwiththediscrepancyobserved betweenRGS5KOandwildtypemyogenicresponsecurves.Thistooisnotevidentfrom ourfindingsasthePEdoseresponsesshownodifferencebetweenexperimentalgroups. Therefore,theseresults,despiteexistingliteraturetothecontrary,suggesttheabsence ofRGS5doesnotsignificantlyalterthemyogenicresponseorconstrictoractivityin responsetoS1P.However,thereareanumberofstudiesthathaveexaminedalternate pathwaysformodulationofvasculartone,andmorespecifically,thetriggeringand modulationofthemyogenicresponse(DavisandHill1999)Theseincludepathways implicatingstretchactivatedcationchannelsthatallowcalciumentry,ormechanisms implicatingthetraffickingofglobularactinandit'sdynamicpolymerizationinSMCs, whilenumerousmechanismsareproposedsupportingtheRho/ROK/MLCPsignaling thatresultsinsustainedcontraction(Cipolla,Gokinaetal.2002;Fanchaouy,Bychkovet al.2007;Schubert,Lidingtonetal.2008).Manyofthesesamesignalingpathwaysact throughGproteincoupledreceptorsandanumberofthoseincludeGiandGq coupledheterotrimericGproteins.AsaresultthereispotentialforRGS5toplayarole inmanycellularmechanisms.Intheexperimentspresentedherein,S1PandAngII receptoractivitywasassessedastheyhavebeenidentifiedasbothtargetsofR4family 71

RGSproteinsaswellaspotentialmediatorsofthemyogenicresponse(Wang,Liuetal. 2002;MederosySchnitzler,Storchetal.2008;SharifNaeini,Folgeringetal.2009). Althoughwedonotobserveanysignificantdifferenceinmyogenicresponsebetween wildtypeandknockoutvessels,therecouldpotentiallybeacompensatorymechanism throughwhichtheconstrictorresponseisbeingdulled.ThehigherlevelsofVASP phosphorylationinourknockoutcellsshowaneffectoftheabsenceofRGS5atanend pointofthevasodilatorysignalingpathway,namelyPKGactivity.Thus,usingincreased VASPphosphorylationasamarkerforincreasedPKGactivity,ourresultssupporta potentialincreasedvasodilatorymechanismactiveinthesevessels. Asdescribedearlier,themyogenicresponsehasbeendemonstratedtobe

closelyregulatedbytheactivityofS1PthroughtheS1Preceptor,aGPCR,andCa2+ independentvasoconstriction(Schubert,Lidingtonetal.2008).Asaresult,the observationofanincreased,althoughnonsignificant,initialconstrictionatalower intravascularpressure(lowersetpoint)causedustoconsidertestingS1P responsivenessinthesesamevessels.Ourhypothesispredictedaleftwardshiftinthe S1PdoseresponsecurvefromtheRGS5knockoutvesselsascomparedtowildtype controls,indicatinganincreaseinVSMCsensitivityattheleveloftheGPCR.Surprisingly, theS1Pdoseresponsesfortheexperimentalgroupswerenotsignificantlydifferent.As such,thecurrentevidencesupportsnoroleforRGS5inregulatingS1Pvasoconstrictor signalingthroughtheS1P2orS1P3receptor.

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4.a.iiCellproliferationandmigratorysignalinginRGS5KOVSMCs Aspreviouslyillustrated,ratesofproliferationofculturedVSMCsfromwild

typeandRGS5KOmicewereassessed.Theresultsfromtheproliferationassaysare intriguingastheypresentapotentialforalternatepathwaysinwhichRGS5maybe acting.Priortodiscussingthispossibility,itisimportanttonotethatAngIIandS1P signaling,bothofwhichhavebeenshowntobetargetedbyRGS5,arenotonly responsibleforvasoconstrictoractivitybutalsohavebeenimplicatedincellmotilityand proliferativeactivity(Hobson,Rosenfeldtetal.2001;Watterson2004;CottonandClaing 2009).Morespecifically,recentfindingsconcerningS1PR5haveshownapotentiallink betweenthefunctionofcentrosomesinmitoticproliferationandS1P(Gillies,Leeetal. 2009).Assuch,theabsenceofanydifferenceinS1Pmediatedvasoconstrictiondoesnot necessarilyremovethepotentialforRGS5tobemediatingS1PRsignals,andmayinfact pointtohighlyspecializedS1PsignalingpathwaysinVSMCsthatareentirelyisolated fromcontractilesignals. InordertoaddresswhetherornotRGS5affectsmitoticactivity,cell

proliferationassayswereconducted.Theresultsoftheseexperimentsdemonstratedno roleoreffectofRGS5intermsofgrowthrate,howeveraninterestingobservationwas thefluctuationinpeakcellconcentrations.Althoughnostatisticalsignificancewas foundbetweenpointsofcurves,thismodulationattheupperplateauofthegrowth curvecouldbearesultofmodifiedcellsizeorcellcellcontactcharacteristicsand affectedSMCcontactinhibition.Theresultsdescribedhereindonotsupportarolefor

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RGS5inmediatingS1Pdependantvasoconstriction,butdohoweverdemonstratea changeinS1Pmediatedsignaling,suggestingaroleforRGS5inoneofthemany processesthismoleculeaffects.AnotherinterestingfindingisthatofChitaleyand colleagues(2003)whichidentifiesVASPasasubstrateforPKC(Chitaley,Chenetal. 2004).PKC,whichisadownstreamsignalingpartnertoGPCRsthroughDAGactivity,is anactiveplayerintheRho/ROKsignalingpathway.Assuch,aroleforRGS5inregulating migratoryoradhesionsignalingmightbeapparentinchangesinRhoactivityorthe activityofagonistsaffectingitsfunctions.TheresultsfromERK1/2phosphorylation assaysfurthersupportthisconclusionasbasalERK1/2phosphorylationisheightenedin knockoutVSMCs.IndeedithasbeenestablishedthatS1Pactsinanautocrineand paracrinemanner,supportingthehypothesisthatincreasedbasalERK1/2maybea resultofchangesinautocrine/paracrineS1Psignalinginculturedcells(RosenandGoetzl 2005).TheaboveresultsshowingincreasedVASPphosphorylationserveasafurther indicatorofaffectedcellcellinteractionsignaling.Theinitialrationalederivedfrom existingliteraturehypothesizedthatRGS5knockoutbloodvesselswoulddemonstrate prolongedvasodilatorsignalingorheightenedresponsetovasodilatordrugs.The myographyresultshoweverarecontrarytothis,withwildtypeandRGS5knockout vesselsbehavingsimilarlyinacetylcholinedoseresponseexperiments.Vasodilator stimulatedproteinisbelievedtobeinvolvedinanumberofcellularmechanisms,with establishedconnectionstocellmatrixmodificationandmigration(Bear,Loureiroetal. 2000;Machesky2000).Theseobservations,madeinfibroblasts,describeattenuated cellmigratoryactivityinVASPknockoutcells.Assuch,ifRGS5playsaroleinVASP

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phosphorylation,furtherexperimentsexaminingthemigratoryactivityofRGS5 knockoutVSMCswouldbeprudenttohelpelucidateitsinvivorole. 4.a.iiiLimitationsofRGS5study AlthoughtheLacZreporterknockoutmodeliseffectiveinidentifyingtissues

whereRGS5promoteractivityishigh,itcannotbeusedtospecificallydeterminethe halflife,cellularlocalization,orbindingcharacteristicsoftheRGS5protein,sincethe additionoftheLacZinsertmaycompromiseallofthesefactors.Furthermore,LacZ expressionandsubsequentcolorationinresponsetoXGalstaininghasbeenassociated withathresholdeffect,whichmayservetoconfoundobservedexpressionlocation. QuantitativeRealTimePCRstudiesestablishthepresenceofthisthresholdeffect.For example,carotidvessels,whichdisplaynoXGalstaining,containsixthousand moleculesofRGS5mRNApertwonanogramsofRNA,comparedtodescendingaorta RGS5levelswhichcontain170thousandmoleculesper2nanogramsofRNA.Thus,the presenceofRGS5mRNAwiththeassociatedabsenceofXGalstaining,demonstratesa thresholdeffect. AnadditionallimitationconcerningtheverityoftheRGS5KOatthegenetic

levelconcernsthecellularresponsetotheabsenceofRGS5.Aswithanygenetically manipulatedbiologicalmodel,thepossibilityexistsforcompensatoryactivitybysimilar genesorgenesinrelatedpathways.Whilewecannotruleoutthepossibilityofparallel pathwaycompensatorymechanisms,ourRTPCRanalysisofRGS5deficientvessels showednoupregulationofanyoftheotherRGSfamilymRNAlevels. 75

Anadditionallimitationconcernstheuseofmesentericarteriolesforthe

pressuremyographyassays.Themesentericvasculatureisusefulinitsrelativeeaseof isolationandanalysishoweverinwholeanimalphysiology,thesevesselsdonotprovide asignificantcontributiontotheestablishmentofperipheralresistanceandmaintenance ofsystemicpressure.Duetothetechnicalchallengesassociatedwiththeisolationand measurementofthesevesselshowever,mesentericvesselswerechosen.Finally,ourX GalstainingassaysshowednotableRGS5promoteractivityinthemesentericarterioles, howevertheselevelswererelativelylowerthanthestrongeststaining,whichwe observedintheaorta,iliacandrenalarteries.Ultimately,furtheranalysisusingmore pertinentvesselstobloodpressureregulationmayrevealamoreprominenteffectofa lackofRGS5invascularfunction.Anadditionallimitation,asdescribedearlier,concerns changesincellsize,whichmayberesponsibleforthevariationinpeakcellnumber observedinproliferationassays.Furtherexperimentsexaminingvariationsincellsize andshapemayelucidatethisquestion. 4.a.ivFuturestudiesforRGS5 Theresultsfromthesestudieshavebeenquitehelpfulinansweringthe

question:DoesRGS5playsaroleinmediatingVSMCcontractilityorrelaxationinorder tocontrolvascularpressure?Incompletingthesestudies,wehaveeffectivelyremoved thispathwayamongthelistofpotentialrolesforRGS5.Unfortunately,thebiologicrole

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ofRGS5inarteriolesstillremainsunclear,howeverthereareanumberofstudiesand currentlyunexaminedcellularmechanismsthatawaitattention. Onesuchareaisthatoftheinteractionandsignalingthatoccursbetween

VSMCsandendothelialcells.Recentstudieshavedemonstratedanimportantrolefor myoendothelialjunctionsinthisinteraction.Myoendothelialcouplingoccursthrough transinternalelasticlamina(transIEL)endothelialcellprotrusions,whichmaybe anotherareawhereingproteinsignalingisplayingarole.Briefly,myoendothelial couplingappearstoincreaseasthevasculaturemovesfromconduittoresistance arteries.Furthermore,Ca2+signaling("sparks"and"pulsars"),whichisinfluencedby changesintransmuralpressure,hasbeenlocalizedtoregionscloselyassociatedwith theseprotrusions(Ledoux,Tayloretal.2008).Intriguingly,transIELprotrusionnumber appearstobeinverselyrelatedtothepatternofRGS5expressionwehaveobservedin ourLacZstainingstudies(Siegl,Koeppenetal.2005).Briefly,theregionsofhighestLacZ stainingareinkeyconduitarteriesandregionsexperiencinghighlevelsofintravascular pressureorturbulentflow,namelytheaorticarch,descendingaorta,andrenaland femoralarteries.Expressioniscompletelyabsenthoweverinthecarotidartery,withthe exceptionofthecarotidbulb.Thesepointsofexpression,combinedwithresults showingabrogationofleakytumourvasculatureinRGS5knockoutmicesuggesta structural/adhesionmediatingroleforRGS5.Morespecifically,RGS5maybeinvolvedin mediatingtheinteractionofVSMCswithneighboringVSMCsand/orendothelialcells. FurtherevidencetosupportthislineofresearchisevidentintheworkofLaddsand colleagues.Briefly,studiesbyLaddsandcolleagues(2009)examinedRGStranscript 77

levelsinthehumanmyometrium,andtheshiftsintheirlevelsthroughpregnancy.This isintriguingastheprocessofpregnancyandbirthingismadepossiblethroughashiftin SMCphenotypeandfunction,fromproliferativetocontractileatterm,anditisatthis timepointthatitappearsRGS5levelsfluctuate(Ladds,Zervouetal.2009).Thisareaof researchisparticularlystrikingasitallowsfurtherconsiderationofRGS5asamediator ofvasculardevelopment,andaregulatorofSMCdifferentiationandinteraction. Furthermore,itisestablishedthatinordertoshiftfromaproliferative(during pregnancy)tocontractile(duringlabour)phenotype,SMCsmustmodifytheirECMand intercellularadhesionqualities,allowingthemyometriumtocontractasasyncitium.As such,ashiftinRGSlevels,asdescribedbyLaddsetal,couldbeinvolvedinfinetuning theexacttimingorspatialarrangementofthisSMCcontractilesyncitium.These potentialmechanismsfromstudiesinmyometrialSMCscanberelatedtonewliterature discussingthevascularroleofRGS5.Asdiscussedearlier,currentliteraturefrom Hamzahandcolleagues(2008)describesnormalizedtumourvasculatureinRGS5 knockoutmousemodels.TheauthorsproceedtofurtherreviewthepotentialforRGS5 asamediatorofvascularremodelingandcorrelateit'scardiovascularrolein conjunctionwithRGS2andRGS4,theotherknowncardiovascularRGSproteins(Manzur andGanss2009).Thenormalizationoftumourvasculaturecouldbearesultofa numberoffactorsincludingvariationsinmatrixproteincomposition,modifiedSMC migrationandadhesioncharacteristics,aswellaschangesinmyoendothelialcoupling. Inclosing,althoughthecurrentdatadoesnotdefineaconclusiveroleforRGS5

inthevasculature,itisclearthatRGS5doesnotplayaroleinregulatingarterialtonein 78

responsetovasoactiveagonists.Asdescribedearlier,theremaybeaneffectatthelevel ofmyogenicresponsethatisbeingmaskedbyanincreasedvasodilatoryresponsein knockoutarteries.Thisnegativedataaddstoourunderstandingofthephenotypeofthe RGS5KOmodel.Withthedefinitionthatnoclearvascularphenotypewasidentified, futureresearchcanbeconductedinadditionalareasofvascularresearchasoutlined. Thus,ournegativeoutcomeshavecontributedtoimprovedunderstandingofRGS proteinsinvascularbiology. 4.bRGSNTDAmphipathicHelixsecondarystructurediscussion,andfuturedirections RGS2haslongbeenidentifiedasapotentmediatorofvasoconstrictorsignalingthrough itsattenuationofGqactivity.ItisuniqueintheRGSsuperfamilyforthisGq specificity.Morerecentdata,asdescribedearlier,haslinkedmutationsinRGS2tocases ofhypertensioninhumans.Weexaminedonemutation,R44H,whichoccurredwithin theNTDamphipathicalphahelixofRGS2,inordertodetermineapotentialmechanism whereinRGS2isinvolvedinthedevelopmentofhypertension.Studiesconductedinour labinculturedcellsshowedreducedRGS2mutantGqsignalingattenuation, demonstratedbyincreasedCalciuminfluxinmutanttransfectedcells.Theseresults confirmedafunctionaldetrimentresultingfromtheR44Hmutation.Inorderto determinethemechanismforthis,spectroscopicanalysisofthesecondarystructure andlipidinteractionoftheNTDalphahelixwasconducted.Interestingly,thepoint mutationinthehydrophobiccoreoftheamphipathichelixdidnotabolishalphahelix 79

formation,asisevidentbythecirculardichroismdata.However,analysisoftryptophan fluorimetrydatarevealedreducedalphahelixlipidbilayerinteraction,leadingtothe developmentofasnorkelingmodelofRGS2NTDinteractionwiththeplasma membrane.ThearginineresidueinwildtypeRGS2NTDamphipathichelicesare essentialforlipidinteractionasthelongnonpolarsidechainsstablyinteractwiththe nonpolarcoreofthelipidbilayerwhileallowingtheconjugatedpolarheadgroupsto interactwiththepolarregionsofthephospholipidbilayerandcytosol.Themutationof argininetoalargerandstericallynonidealhistidineresiduehinderstheabilityofthe helixtointeractandassuchdisruptsoverallplasmamembraneassociationwithout disruptinghelixformation.Itisinterestingtonotethatpropermembraneassociationis essentialtonormalRGS2function,promotingfurtherstudiesintoNTDcharacteristicsof otherR4subfamilymembers.

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