autoregulatory circuit regulating basolateral cargo export ... · 26/05/2020 · 1 autoregulatory...

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Autoregulatorycircuit regulatingbasolateralcargoexport fromtheTGN:roleof theorphanreceptorGPRC5AinPKDsignalingandcellpolarityRosariaDiMartino1,AnitaCapalbo1,LuciaSticco1,AlessandraVaravallo2,VidyaKunnathully1,ValentinaDeLuca1,NamrataRaviIyengar1,MatteoLoMonte1,PetraHenklein2,JorgeCancino3andAlbertoLuini1*Affiliations:1 InstituteofBiochemistryandCellBiology,NationalResearchCouncil,ViaPietroCastellino111,80131Naples,Italy2TelethonInstituteforGeneticsandMedicine(TIGEM),Pozzuoli,Italy.3InstitutfurBiochemie,ChariteUniversitätsmedizin,Berlin,Germany.4 Centro de Biología Celular y Biomedicina (CEBICEM), Facultad de Medicina y Ciencia,UniversidadSanSebastián,Lota2465,Santiago7510157,Chile.*Correspondence:[email protected]

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 27, 2020. . https://doi.org/10.1101/2020.05.26.114710doi: bioRxiv preprint

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AbstractThe membrane transport apparatus comprises a series of separate membrane boundcompartments, or transport stations, that are responsible for the synthesis, processing,transport,sortinganddeliverytotheirfinalcellulardestinationsofmosttransmembraneandsoluble lumenal proteins. Over the last decades themembrane transport system has beenshowntobeextensivelyregulatedbothbyenvironmentalinputsandbyinternalhomeostaticsignallingsystems,orcontrolsystems,thatoperatetomaintainthehomeostasisandoptimalfunctionalityofthemaintransportstations,suchastheendoplasmicreticulumandtheGolgi,inthefaceof internalandexternalperturbations.Thetrans-Golginetwork(TGN)isamajortransportandprocessingstationandthemainsortingcompartmentofthetransportapparatus.However, the mechanisms that control cargo export and sorting at the TGN have so farremainedelusive.Herewe focuson thesortingofbasolateralcargoproteinsandshowthattheseproteinsbindtotheTGNlocalizedorphanreceptorGPRC5A.Thecargo-GPRC5Acomplextriggers the activation of a signaling pathway that involves the Gβγ subunits dependentactivationofthephospholipaseCbeta3(PLCβ3),whichinturninducesdiacylglycerol(DAG)production.DAGrecruitsandactivatesproteinkinaseD(PKD)andthephosphorylationofitssubstrates. This step results in the formation of basolateral carriers for delivery of thesecargoes to the basolateral plasma membrane domain. We term this mechanism “ARTG”(AutoRegulationofTGNexport).Remarkably,theimpairmentofARTGpathwaycomponents,andinparticularofGPRC5A,causesdefectsinthepolarizedorganizationofepithelialcells.Keywords:Apico-basal polarity, autoregulatory systems, basolateral cargo, GPRC5A, PLCβ3, PKD,signaling,TGNexport.


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IntroductionThebiosyntheticmembranetransportsystemisaseriesofmembrane-boundcompartmentsarranged in a linear sequence that is responsible for the synthesis, processing, transport,sorting anddelivery to their final cellulardestinationsofmost transmembraneand solublelumenalproteins(henceforthcalledcargoes),andofmostlipids.Itincludestheendoplasmicreticulum (ER), where the initial synthesis and processing of cargoes occurs, the ER-Golgiintermediatecompartment(ERGIC),theGolgicomplex,andthetrans-Golginetwork(TGN).TheTGNisconsideredtobethemainsortingstationfromwherecargoproteinsaretransportedtotheir final destinations, such as lysosomes, theplasmamembrane (PM)or the extracellularmilieu(DeMatteisandLuini,2008).Throughout their journey, cargo proteins are processed by specialized reactions (folding,followedbypost-translationalmodificationslikeglycosylation,andthensorting)insuccessivestations,eachofwhichresutsinthegenerationofintermediateproducts,suchasunfoldedorpartiallymodifiedproteins,whicharefurtherprocessedandtransportedacrossthesuccessivesecretorystations(MellmanandWarren,2000).Over the last several years, a growing body of evidence has led to the recognition thatmembranetransportissupervisedbymultipleregulatorymechanisms,amongwhichwecanrecognize two different types. One class of regulatory devices acts in response to externalsignals,andintegratesthetransportsystemtoavarietyofcomplexcellularresponsesinitiatedby PM receptors, such as such growth,migration or differentiation responses (Farhan andRabouille2011;Farhanetal.,2017).Asecondtypeofmechanism,calledinternalhomeostaticregulatorydevices,operatetomonitortheoptimalworkingofthetransportcompartmentsandfuction toA)maintain their homeostasis and efficiency in the face of internal and externalperturbations, and B) ensure that the compartments act in a coordinated fashion amongstthemselves andwith other functionalmodules in cells, such asmetabolism or cytoskeletalmotility(Luiniet.al.,2014;LuiniandParashuraman,2016)A well characterized homeostatic device is the unfolded protein response (UPR), whichregulates the levels of unfolded proteins in the ER through multiple signaling andtranscriptional cascades (Walter and Ron, 2011). Other examples are the autoregulatorysystemsthatoperateintheER(Subramanianetal.,2019;Centonzeetal.,2019)andtheGolgi(Pulvirentietal.,2008;Giannottaetal.,2012;Cancinoetal.,2014;Solisetal.,2017;Tapiaetal.,2019) that ensure the correct trafficking through these stations. So far, the organization,moleculardeterminantsandfunctionofapotentialautoregulatorycircuitattheTGNhasnotbeenexamined.Whencargoproteinsreach the lumenof theTGNtheyaresortedandshipped toeither thebasolateralorapicalPMdomains,ortothelysosomes.However,intheabsenceofahomeostaticmechanism, they may accumulate in the TGN lumen, creating the risk of ectopic andfunctionallyaberrant signaling (MellmanandNelson,2008).Moreover, theaccumulationofcargointheTGNmightresultintheirmis-sorting.Indeed,itisknownthatinasortingorganelle


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if thepreferredexport route fora certain cargo, forexampleabasolateral cargoprotein, isblocked, the cargo proteinswill end up leaving the organelle bymeans of apical transportcarriers,withpotentiallyharmfulfunctionalconsequences(StoopsandCaplan,2014;Wilson,2011).Basedontheseconsiderations,wepredictthatanautoregulatorydeviceisexpectedtooperateattheTGNtoregulatecargosortingandexport.To reveal the presence of such a device, we challenged the transport apparatus with anoverloadofbasolateralcargointheTGNandanalyzedthecellularresponse.First,weusedatargetedphosphoproteomicsapproachtodetectthemodulationofsignalingpathways,usinganassaydesignedtodetecttheactivationofalargenumberofkinasesandotherregulatorymolecules. Second, to complement these findings, we undertook an unbiased proteomicsapproachtodeterminetheinteractingpartnersofbasoloteralcargoaccumulatedattheTGNbyimmuno-precipitationandmassspectrometry(seeMaterialsandMethods).Wereportherethatbasolateralcargoproteins,uponarrivingattheTGN,activateanorphanTGNresidentG-ProteinCoupledReceptorGPRC5AthatisincomplexwithheterotrimericG-proteinsubunitsGαi3andGβγ.GPRC5AactsasasensorofcargoattheTGN,andactivatesanetworkofsignalingpathways,thusformingacentralcomponentoftheautoregulatorydevice.Amongtheactivatedcascades,GPRC5AsimulatesaGβγ-PLCβ3-PKDsignalingpathwaythathasbeencharacterizedpreviouslytoregulateexportfromtheTGN(Jamoraetal.,1999;Liljedahletal.,2001;BaronandMalhotra,2002,DiazAñelandMalhotra2005).ActivatedGβγsubunitsrecruitandactivatetheenzymephospholipaseCbeta3(PLCβ3),whichproducesthediacylglycerol(DAG)requiredforproteinkinaseD(PKD)recruitmentandactivation.PKDactivationresultsinphosphorylationofmanysubstratesandintheformationofbasolateralcargocarriersthatexportcargooutoftheGolgi,attenuatingbasolateralproteincargoloadintheTGNlumenandenhancingthefidelityofsorting.TheseseriesofeventsresultinanegativefeedbackactionontheconcentrationofcargointheTGN,thusreducingtheriskofaberrantcargoaccumulationandmis-sorting.We call this circuitARTG (AutoRegulationofTGNexport). Importantly, theARTGsignalingpathwaysupportsthemaintenanceofcellpolarity,andmaycontrol thedevelopmentof theepithelial-mesenchymaltransition(EMT).ResultsAcargooverloadintheTGNtriggerssignalingresponsesthatregulatecargoexportTochallenge theautoregulatorydeviceexpected tooperateat theTGNwithanappropriateperturbation,we set up conditions to generate a basolateral cargo protein overload in thiscompartmentusingdifferenttransportsynchronizationmethods.Suchmethodswerebasedontheuseofretentionusingselectivehooks(RUSH)(Boncompainetal.,2012)andFM(Riveraet al., 2000; Rollins et al., 2000) constructs of basolateral cargo proteins, as well as thetemperature-sensitivevariantofthevesicularstomatitisvirus(ts045)Gprotein(forbrevity,VSVG), an extensively characterized basolateral cargo protein. These proteins can be


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accumulated in controlledamounts in theERbyblocking theirERexport (seeMethods forexperimentaldetails),andcanthenbeallowed,byremovingtheexportblock,toexittheERandmoveasasynchronous‘wave’throughthesuccessivestationsofthesecretorypathway,including the TGN. A well-characterized variant of this protocol is based on an additionaltemperature block at 20°C for 2 hours after release from the ER. At 20°C, cargo proteinsaccumulateintheTGN(MatlinandSimons,1983).Uponshiftingthetemperatureagainto37°C(or32°CincaseofVSVG)theyleavetheTGNforthePM.Wecallthesetwoprotocolsthe“wave”andthe“20°Caccumulation”,respectively.TheexportofVSVGfromtheTGNhasbeenextensivelycharacterizedbefore(Polishchuketal.,2003;Luinietal.,2007)andhasbeenshowntotakeplacevialargepleiomorphiccarriersthatformanddelivercargotothePMatratesroughlyproportionaltotheloadofcargointheTGNindicatingthattherateofcarrierformationreflectstheTGNcargoload(DeMatteisandLuini,2008;Polishchuketal.,2009).Touncoverthemechanismresponsibleforcouplingtherateofexporttotheamountofcargoload,wesoughttoidentifytheexpectedsignalingresponsesusinga targeted phosphoproteomic approach based on a phospho-antibodymicroarray.We firstgeneratedaVSVGtraffic“wave”usingpreviouslycharacterizedconditionsi.e.byaccumulatingthis protein at 40°C in the ER for 3 hours and then releasing the block by shifting thetemperature to 32°C (Mironov et al., 2003). Cycloheximide (CHX)was added from 30minbeforereleasingtheblockuntiltheendoftheexperimenttoexcludeeffectsfromendogenouscargoes. VSVG exited the ER synchronously and reached its peak concentration in theTGNbetween20and25minutesandthenreachedthePMbetween30-40minutesafterthereleaseof theERblock.Thephospho-antibodymicroarraywasused to assess thephosphorylationstateofseveralhundredkinasesandregulatoryproteinsatthetimeofaccumulatingVSVGintheTGN(i.e.,20minutesat32°CafterthereleaseoftheERblock)incomparisontowhencargoaccumulation in the ER at 40°C (Figure 1A). The TGN cargo peak induced the hyper-phosphorylation or de-phosphorylation of approximately 90 proteins on specific residues(Figure1B).Analysisofthisdata-setusingbioinformatictoolsandmanualcuration,revealedtheactivationofvarioussignalingpathways(Figure1C).ThemostenrichedweretheERK/MEKsignaling,Calcium/DAG/PKC/PKDsignaling,JAK/STAT,andmTORpathways.Inaddition,wealso noted an elevated phosphorylation of eIF2α, a mechanism linked to the inhibition ofproteinsynthesis(Pakos-Zebrucka2016;Costa-MattioliandWalter,2020).Itisplausiblethattheabovesignalsmightbegeneratedbycargotraversingearlierstationsofthesecretorypathway (Cancinoetal.,2014;Subramanianetal.,2019).Toaddress this,weperformedtheprotocolbasedoncargoaccumulationspecificallyattheTGNbyincubatingcellsat20°C.Wethenvalidatedtheactivationofkeysignalingproteins intheabovecascadesbyimmuno-blotting. PKD, ERK1/2 and eIF2α were robustly phosphorylated during cargoaccumulationattheTGN(Figure1Dand1E),whileSTATwasdephosphorylated(Figure1D).Asa further control, the same temperature shift in absence of cargo (cells treated withcycloheximide,Figure1E)didnotelicitanyoftheabove-mentionedresponses,indicatingthattheobservedsignalingresponseisexclusivelydependentonthecargoaccumulationsanditisnotabyproductofthetemperaturechangesorothermanipulations.


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Wenextaskediftheactivationoftheabovepathwaysmightberesponsibleforcargoexportfrom the TGN to the PM. We depleted or inhibited the relevant kinases/molecules andmonitoredthePMarrivalofbasoltarealcargoproteinVSVG.Strikingly,wefoundthatonlytheinhibitionofPKDcausedablockofVSVGexitfromtheTGN(datanotshown).ThisisinconcordancewithearlierstudiesthatindicatedtherequirementofPKDactivityfortheformationofbasolateralcargocarriersandtheirtraffictothePM(Yeamanetal.,2004).PKDisactivateddownstreamofasignalingpathwaythatcomprisesGβγ,PLCβ3,DAG,andPKC(DiazAñelA.M.2007;Lauetal.2013;DiazAñelandMalhotra,2005).Weconfirmedthateitherthedepletion of PLCβ3 or the inhibition of Gβγ significantly arrested cargo at the TGN(SupplementaryFigure1Aand1C).TheseresultsinsumsuggestthatcargoaccumulationattheTGNactivatesmultiplesignalingevents.Specifically,thecargodependentactivationofPKDinturnregulatescargoexporttothebasolateralPM.BasolateralcargooverloadintheTGNlumentriggerstheactivationandrecruitmentofPLCβ3andPKDontheTGNmembraneThetargetedphosphoproteomicsanalyses indicatedanactivationofPLC-PKDsignalingasaconsequenceofcargoarrivalattheTGN.WehencefocusedonexaminingtherecruitmentofthesesignalingcomponentstoTGNmembranesinresponsetoTGNcargoaccumulation.First,wedeterminedwhichspecificcargoesattheTGNelicitedPKDactivation.PKDismainlycytosolicatsteadystateandisrecruitedtomembranesbyDAGviathekinase’sC1adomain(Maeda et al., 2001). Its activation canbedetectedby assessing its autophosphorylation atposition ser916, a signal diagnostic of the kinase activity (Matthews et al., 1999). We co-transfectedHeLacellswithGST-taggedPKD1andwithvarious synchronizable cargoes thattraffictoeitherthebasolateralPM(E-cadherin,TNFαandhGH)ortheapicalPM(GPI-GFP),andexaminedtheeffectofelevatedTGNcargoloadsontherecruitmentandactivationofPKDatthe Golgi (Figure 2D and Supplementary Figure 2 A-B-C). The cargo proteins were firstaccumulatedintheERandthenallowedtoexitandaccumulateintheTGNcompartmentat20°Cfor2hours.ReleaseofthesecargoesfromtheTGNwastheninducedbyshiftingthecellstothepermissivetemperatures.Strikingly,theaccumulationofonlythebasolateralcargoesintheTGNlumenresultedintheactivationandrecruitmentofPKDtotheTGN(Figure2D).TheapicalcargoGPI-GFPhadnoeffect(SupplementaryFigure2C). Anessential step forPKDactivation is the localDAGproduction thathasadual functionofrecruitingandactivatingPKDviaPKCdependentphosphorylation(Maedaetal.,2001;DiazAñelandMalhotra,2005).AcommonapproachtomonitorDAGproductionincellsistousetheC1a-(PKD)-GSTplasmidderivedfromtheDAGbindingdomainofPKD,whichisrecruitedtocellmembranesenrichedwithDAG.ToassayforDAGproductionontheTGNmembranes,HeLacellsweretransfectedwiththeC1a-(PKD)-GSTplasmidandaTGNcargopulsewascarriedout.C1a-(PKD)-GSTwasrecruitedtotheTGNmembranesduringTGNaccumulationofVSVGat20°C(Figure2C).


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ThegenerationofDAGonTGNmembranesduringbasolateralcargosecretionispossiblyduetotheactionofthePLCβ3(DiazAñel,2007;Sicartetal,2015;Aniteietal,.2017).PLCβ3isapredominantly cytosolic protein that can be recruited to membranes by activation of Gβγsubunits(Foggetal.,2001).PLCβ3canalsolocalizeattheGolgiandisnecessaryfortheTGNexportof basolateralcargoes(DiazAñel,2007;Aniteietal,.2017).WetestedtheeffectsofelevatedTGNcargolevelsonthedistributionofPLCβ3.Atsteadystate,PLCβ3localizedmostlyinthecytosolandontheTGN(Figure2A).DuringTGNaccumulationofthecargoat20°C,PLCβ3markedlyshiftedfromthecytosoltotheGolgi.Then,ascargolefttheTGNforthePM,uponreleaseof theTGN20°Ctemperatureblock, it redistributedbackto thecytosol (Figure2B).SincePLCβ3isactivatedbytheGβγsubunit,wetestedtheeffectofaninhibitorypeptideoftheGβγ subunits (Blüml et al., 1997). Gβγ inhibition blocked both the cargo dependent PKDactivation(Figure2F)andthearrivalofVSVGonthePM(SupplementaryFigure1C).ThesedataoverallindicatethatthePLCβ3-PKDpathwayactivatedbycargoattheTGN,inaccordancewiththe activation of a pathway previously reported to be necessary for the TGN export ofbasolateralcargo(MalhotraandCampelo,2011).Altogether,thesedatasuggestthepresenceofanautoregulatorymechanismthatsensestheamountofcargointheTGNlumenandtriggersaGβγ-PLCβ3-PKDpathwaythatactivatescargoexportoutoftheTGN.WenamedthisautoregulatorysystemasARTG(autoregolatorysystemforTGNexport).BasolateralcargoexportfromtheTGNrequirestheorphanreceptorGPRC5A Thecoreofanautoregulatorycircuitisthepresenceofasensorthatdetectsthecriticalvariabletobecontrolled,whichinthiscaseistheloadofbasolateralcargoattheTGN.FortheARTGsystem,wepredictedthatasensormustbesituatedontheGolgimembrane.ToidentifytheputativesensorofTGNcargoload,weresortedtoanunbiased‘interactomics’approachbasedon the assumption that the sensor might be directly or indirectly associated with theaccumulatingcargo.WeimmunoprecipitatedVSVGatthreedistincttimepointsduringacargotransportpulse:i)at40°CwhenVSVGisintheER;ii)at20°CwhenVSVGaccumulatesattheTGN;and iii)30minutesafter release from theTGNat32°CwhenVSVGactively traffics incarriers destined toward the basolateral PM. Under each of these conditions, the cargointeractingpartnersweredetectedbyiterativeroundsofmassspectrometry(Figure3A).Strictfiltering based on a ranked score (see Figure 3B) resulted in a list of approximately 100interacting proteins that were subsequently analyzed. We focused on cargo interactingpartnersthatwerepresentonlyduringTGNaccumulationandaftercargoreleasefromtheTGN,and excluded those proteins that interacted with cargo arrested in the ER (Figure 3B).Remarkably,ourproteomicsscreenhighlightedaninteractionbetweencargoandamemberofthe classCofGPCRs, namedGPRC5A (ChengandLotan, 1998;Ye et al., 2009). Co-immunoprecipitation and immuno-blotting confirmed that GPRC5A is a bona fide interactor ofbasolteralcargoesVSVGandhGHattheTGN(Figure3Cand3D),suggestingthatthisorphanreceptormightinitiatethesignalingeventsthatactivatetheGβγ-PKDpathway.GPRC5A localizes to the PM, Golgi and post Golgi vesicles (Zhou and Rigoutsos, 2014).Weconfirmed that the endogenous protein localizes to the Golgi and perinuclear vesicles in


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multiplecelltypes(SupplementaryFigure4A).WetheninvestigatedthepotentialroleofthisGPCR in the TGN export of cargoes. GPRC5A depletion resulted in a significant block ofbasolateral cargoes VSVG, E-cadherin, TNFα, and hGH exiting the TGN (Figure 4A,Supplementary Figure 4C-E). An siRNA resistant tagged-mutant GPRC5A when expressedrescuedcargotransporttothePM(Figure4D).Importantly,GPRC5AdepletiondidnotaffecttheexportofapicalcargoesGPI-GFPandGP135-GFPandlysosomalcargoesLAMP1,CathepsinDandheparanase(SupplementaryFigure4B-D-Fanddatanotshown).These results strongly indicate a specific role for GPRC5A in the transport of basolateralcargoes,andhencetheregulationofsortingstringencyattheTGN.TheARTG-PKDpathwayisregulatedbyGPRC5AOur results so far suggest that GPRC5A senses basolateral cargo arrival at the TGN andsubsequently activates downstream heterotrimeric G proteins, thus initiating the signalingcascade that culminateswith PKD activation. To test thismodel,we depleted GPRC5A andassayedforPKDactivation.Indeed,weobservedadecreaseintheactivatedformofthePKD(Ser916)byimmuno-blotting(Figure4C).Moreover,GPRC5AdepletedcellsexhibitedapoorrecruitmentofPKDser916andPLCβ3totheTGNandapredominantcytosoliclocalization,evenunderconditionsofsubstantialTGNcargoaccumulation(Figure4Band4E).Atsteadystate(i.e.)intheabsenceofcargooverloads,PLCβ3waspoorlyrecruitedtothemembranefractionincellsdepletedofGPRC5A(Figure4F).As statedpreviously, bothPLCβ3 andPKD activation requiresGβγ subunits (Diaz-Añel andMalhotra, 2005). Classically, G-protein coupled receptors (GPCR) remain in complex withheterotrimericGαandGβγsubunitsinaninactivestate(Hilgeretal.,2018).GPCRactivationresults inaGDPtoGTPexchangeontheGα subunitandasubsequentreleaseofactiveGβγsubunitstoinitiatesubsequentdownstreamsignalingevents(Hilgeretal.,2018).ToidentifytheGαprotein(s)activatedbytheGPRC5AduringTGNcargoaccumulation,weperformedasecondunbiased‘interactome’screenbypullingdownGPRC5Aandanalyzingtheinteractingpartner proteins by mass spectrometry (Figure 5A). We identified the heterotrimeric G-proteins Gαi3, Gβ2 and Gγ12 in complexwith GPRC5A and determined Gαi3 as abonafideinteractorofGPRC5Abyimmunoblotting(Figure5B).WetheninvestigatedifGαi3isactivatedduringcargoaccumulationattheTGN.WemeasuredtheamountofactivatedGαi3(Gαi3-GTP)bytakingadvantageofacharacterizedantibodythatrecognizesandimmunoprecipitatestheGTP-bound(seeMethodsfordetails).ImmunoblottinganalysesofimmunoprecipitatedGαi3showedasignificantincreaseintheactiveGTPboundformofGαi3onlywhencargoaccumulatedattheTGN(Figure5C).DepletingGPRC5Ainsteadystate HeLa cells resulted in a decrease of active Gαi3 compared to controls (Figure 5D).Significantly,GPRC5AandactiveGαi3were important forcargo inducedPKDactivationat theTGN as their absence resulted in a complete loss of PKD phosphorylation (Figure 5E),suggestingthatthetwoproteinsactinthesamesignalingpathway.


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Finally,wealsotestedifGPRC5AsignalingregulatedtheintensitytheeIF2αphosphorylation.Strkingkly,eIF2αphosphorylationwasmarkedlyincreasedinGPRC5A-KDcellsbothatsteadystateandduringcargooverloadattheTGN(datanotshown),presumablyasaconsequenceofaberrant TGN cargo accumulation. The signaling events that result in this markedphosphorylationremaintobeunderstood.Altogether these results suggest that the ARTG signaling pathway on TGN membranes isinitiatedbybasolateralcargointeractionswithGPRC5A.Thecargo-GPRC5AcomplexactivatesGαi3andthereleaseofGβγsubunits,thatinturnactivatethePLCβ3-nPKC-PKDpathway.PKDactivation induces the export of basolateral cargoes from theTGN.Notably,ARTGdoesnotcontroltheexportofapicalorlysosomalcargoesfromtheTGN,highlightingthepossibilitythatthissignalingcircuitmaintainsoptimalsortingandpolarityincells.MechanismofARTGactivationbybasolateralcargothroughGPRC5AOurdatasuggestthatbasolateralcargoesintheTGNlumeninteractwithGPRC5A,orwithaproteincomplexcontainingGPRC5A.TheanalysisofthisinteractioninthelumenoftheTGNiscomplicatedbyanabundanceoffactors(pH,ionicconcentration,othertypesofproteinsandcomplex carbohydrate and lipid composition) (Paroutis et al., 2004; Boutté Y., 2018;Kellokumpu,2019).Tocircumventtheseissues,wesoughttostudytheinteractionbetweenbasolateralcargoesandGPRC5Aunder simplified conditions by expressing the receptor on the cell surface. This ispossiblesinceoverexpressionofGPRC5AresultsinextensivePMlocalization,inadditiontoitsGolgiandPGClocalization(Figure6F).Similarresultsarealsoachievedbytreatingcellswithretinoicacid(RA)(Figure6B),whichisknowntoincreasetheexpressionofthereceptor(Chengand Lotan,1998; Ye et al, 2009). Under these conditions, which results in a homogenouslocalizationofthereceptoratthePM,cellswerefirstwashedwithbufferandthenincubatedwith soluble basolateral cargoes, such asTNFα and albumin, or the apical cargo, lysozyme.Soluble cargo proteins were added in serum-free media for 15 minutes to the cells pre-incubatedat20°Cfor3hourstopreventfurthertransportofGPRC5Atoorfromtheplasmamembrane.Thesolublebasolateralcargoes(albuminorTNFα)inducedamarkedactivationofPKD(Figure6Cand6D)andrecruitmentoftheactivatedkinasetothePM(Figure6F),whilethesolubleapicalcargolysozymehadnoeffect(Figure6A).ThisdiffersfromtheeffectsofcargoonGPRC5A,whichresultsinPKDrecruitmentattheTGN.IncontrolcellsthatdonotexpressGPRC5AatthePM,theadditionofalbuminorTNFαtothecellmediumhadnoeffectonPKD.Aschematicmodeloftheresults fromtheseexperiments isdescribedinFigure6H.Thedose-responsecurves for thesecargoes identifyanEC50of0.28mMforAlbuminand1.3nMforTNFα(Figure6G).ThesevaluesarecompatiblewiththehighsecretedlevelsofalbuminandthelowlevelsforTNFαconsideringthattheaffinityoftheseproteinsfortheirownreceptorsisintheorderof100pMforTNFαR(DavidJ.MacEwan,2002)andintheorderof6-600nMfordifferenttypesAlbuminreceptors(Bernetal.,2015).Notably,theadditionofcargototheabove-describedsystemat37°CdidnotactivatePKD.Fromaphysiologicalpointofview, theabsenceof receptoractivationat the temperatureof37°C


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could mean that under basal conditions this signaling machinery cannot be erroneouslyactivatedbythesolublebasolateralcargoesthatarenormallypresentintheextracellularspace.ReceptorsoftheGPRC5familyhaveapartiallyredundantfunctionindifferentcelllinesWeexpecttheARTGtobeabasallyactiveinallcells,eventhoughthespecificmembersoftheGPRC5 family might differ in expression levels. GPRC5 family members are differentiallyexpressedamonghealthyhumantissues(ZhouandRigoutsos,2014).Weaskedifanyoftheother GPRC5 paralogues replace GPRC5A function, thus implying redundant roles in TGNexport.TheGPRC5familymembersinmammalsareGPRC5A,GPRC5B,GPRC5C,andGPRC5D;withallmembersbeingorphanreceptors(Bjarnadottiretal,2005).GPRC5Aisexpressedathigh levels in the lung (Taoetal.,2004;Xuetal.,2005),whereas theexpressionpatternofGPRC5B ismostly restricted to neuronswith limited expression in other tissues (Brauner-Osborneetal.,2000;Robbinsetal.,2000;Robbinsetal.,2002;Kimetal.,2018).Theexpressionof GPRC5C is more widespread than GPRC5A/B and is prevalent in the kidney and liver(Robbinsetal.,2000).GPRC5Disassociatedwithhard-keratinizedstructures,likecorticalcellsof the hair shaft (Inoue et al., 2004).We ectopically expressedmyc tagged versions of theisoformsinGPRC5AdepletedHeLacellsandassayedforcargotransporttothePM.AsshowninFigure4D,onlyGPRC5CwasabletorescueVSVGtraffickingoutoftheTGN,whiletheothertwoGPCRshadnoeffect(datanotshown).GPRC5AandGPRC5Csharearound30%aminoacididentity, but localize to similar compartments in HeLa cells (Supplementary Figure 4B),supportingthehypothesisthatGPRC5CcouldhaveredundantfunctionstoGPRC5Ainothercelltypes,suchashepatocytes,whereitismajorlyexpressed.RoleoftheARTGpathwayinpolarizedsecretionandcellpolarityWe finally explored the functions of the ARTG in regulating cell polarity with focusedexperimentsinpolarizedHepG2hepatocytes.Thiscelllinehasbeenextensivelyusedtostudymembrane polarity (AnneMüsch, 2014) and expresses high levels of GPRC5C. In addition,hepatocytesareprofessionalsecretorycellspresumablyhighburdensofcargoproteins(suchas albumin) traversing the secretory pathway.Normal polarizedHepG2 showdifferent cellmembrane domains where the asymmetry is achieved through the establishment of tightjunctionsthatmarkthebordersbetweenapicalandbasolateralmembranessuchthatapicaldomainsofadjacenthepatocytesformthebilecanaliculiwhosefunctionistoexcretebilesaltsfrom the cell (Gissen and Arias, 2015).Bile canaliculi formation is an important feature ofpolarizedhepatocytes and canbeobservedas channels that are clearlymarkedbyadensebundleofactinmicrofilamentsandaring-likestructureofthetightjunctionzonulaoccludens1(ZO-1),asshowninFigure7D.We examined the effects of GPRC5C depletion on the trafficking of endogenous basolateralproteins, such as ZO-1, E-cadherin, and albumin in HepG2 cells (Figure 7A). Bothimmunoblotting(Figure7A)andmicroscopy(Figure7C)analysesrevealedgrossalterationsinthepolaritymarkerZO-1.Moreover,GPRC5C-KDcellsdidnotformclusterswithanapparentloss of a polarized PM domain architecture. Interestingly, the levels of E-cadherin wereincreased,aphenotypelinkedwithadisruptedhepaticcellpolarity(Konopkaetal.,2007).


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We next investigated the effects of GPRC5C depletion on bile canaliculi formation. Usually,around30%to40%ofHepG2cells(culturedinamonolayerforatleast72hrs)polarizewithbile canaliculi-like structures (Van IJzendoorn et al., 2004). GPRC5Cdepletion resulted in adramaticreductioninthepercentageofcellswithbilecanaliculi(~7%;Figure7E).Moreover,intracellularalbumin(Figure7A)andalpha1anti-trypsin(Figure7D)levelswereelevatedinGPRC5Cdepletedcells,suggestingdefectsinthenormalsecretionoftheseproteins.Indeed,thesecretionofalbuminintotheextracellularmediumwasmarkedlydelayedincellsdepletedofGPRC5C(Figure7B).These results indicate a role for ARTG in the sorting and secretion of endogenous solublebasolateralcargoesandintheoptimalpolarizationoftheHepG2cells.DiscussionInthisstudy,wereportthattheTGNisequippedwithanautoregulatorymoleculardevice,theARTG complex, that is activated by the binding of basolateral cargo to an orphan receptor,GPRC5A (also known as RAI3) in the TGN and then activates the TGN basolateral exportmachinery.ARTGcanthusbemodeledasacontroldeviceoperatingtokeepthebasolateralcargolevelsatlowlevelstoavoidthepotentiallyharmfuleffectsofcargooverloads.GPRC5Aactsasacargosensorandsignaltransducerand,uponbinding(directlyorindirectly)withbasolateral cargoproteins, induces the recruitmentandactivationofa setof signalingcomponentsthatincludetheGproteinGαi3andGβγsubunits,PLCβ3,theproductionofDAG,andthephosphorylationofPKD.TheactivationofPKDresultsintheformationofapost-Golgicarriertargetedtothebasolateralmembrane.Severalcomponentsofthispathway,fromtheGβγ subunits to PKD had been identified previously. Their activation was proposed to beinitiatedatthePMbythestimulationofaGPCR-GproteinscomplexandtheconsequentreleaseandtranslocationtotheTGNoftheGβγsubunits(DiazAñel,2007).Adifferentbutanalogousmechanism of activation of PKD at the TGN by PM receptors has been proposed by otherauthors(Eisleretal.,2018).AlsoGαi3hasbeenpreviouslyreportedtolocalizeintheGolgiindifferent cell types (Stow et al., 1991a;Wilson et al., 1994) and to play role in intra-Golgitransportofheparansulfateproteoglycan(Stowetal.,1991b).Moreover,recently,ithashasbeenshowntobeactivatedbyanon-GPCRdependentmechanismattheGolgi(Loetal.,2015).Gαi3 is also activated on other subcellular membranes, like LC3-positive autophagosomes(Garcia-Marcosetal.,2011),melanosomes(Youngetal.,2011)andattheplasmamembrane(Thompsonetal.,2007;Kuwanoetal.,2016).Thus,thesamesignalingmoleculescanoperateindifferentways indifferentregulatorynetworks fordifferentpurposes.ThecurrentstudyshowsthatthePKDpathwaycanbeactivatedbycargobindingtoGPRC5A-Gαi-3attheTGN,andthatit isacomponentofaregulatorydevicethatsupportsoptimalTGNfunctionalityaswellascellpolarity.A mechanistic question raised by these findings concerns the recognition by GPRC5A of amultitudeofbasolateralcargoes,whosestructuralcharacteristicsvaryconsiderably.Sequencehomologies and/or structural motifs common to all basolateral cargoes that would bindGPRC5Ahavenotsofarbeenidentifiedbyusorothers.Weproposeamodelinwhichdifferent


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classesofbasolateralcargoesactivatethetheGPRC5Areceptorthroughoneormoreadaptorsendowedwithmultiple binding sites, that form a complexwith the GPRC5a receptor. ThismechanismwouldbeanalogoustothatproposedfortheexportofthelargenumberofdifferentproteinspeciesfromtheERthroughmultiplerecognitiondomainsindifferentSec24adaptors(Milleretal.,2003;Wendeleretal.,2007;Zanettietal.,2012).RelatedtotheabveistheproblemofthepreciseroleoftheGPRC5AcomplexinthesortingmechanismatTGN.TheactivationofGPRC5AbybasolateralcargoproteinsisessentialfortheselectivetransportoftheseproteinstothebasolateraldomainofthePM.Onemayhoweveraskif the function of the GPRC5A complex is only due to the activation of the formation ofbasolateral carriers in which cargo proteins are then recruited by traditional sortingmechanisms,forinstancebymeansoftheclathrinadaptor(AP)complexes(Rodriguez-Boulanetal.,2005;Rodriguez-BoulanandMusch,2005),oriftheGPRC5Acomplex,whichrecognizesa lumenal region of the cargo to be activated, may also function as an adaptor for therecruitmentofcargointhebasolateralcarrier.Thefactthatonlyafractionofthebasolateralcargoesexhibitmotifsthatcanbelinkedtoknownsortingmechanism(DiMartinoetal.,2019)makesadirectroleoftheGPRC5Acomplexanattractivehypothesis,andingeneraltheroleofthiscomplexinTGNsortingafascinatingterritorytoexploreinthisarea.Not all human tissues express GPRC5A. There are fourmammalian genes belonging to theGPRC5family,eachwithaspecificexpressionprofileacrossdifferenthumantissues.TheothergenesGPRC5genesareGPRC5B,GPRC5C,andGPRC5D.Atleastsomeofthemareinvolvedinregulationofofbasolateralcargoexportindifferentcelltypes.GPRC5Cmanagestocompensatefor the lack of GPRC5A in restoring transport of basolateral cargo molecules (Figure 4D).Furthermore,GPRC5C,whichisexpressedathighlevelsinvarioustissuesincludingtheliver,regulates the secretion in HepG2 cells of endogenous basolateral cargomolecules, such asalbumin,a1AT,andE-cadherin,andtheconsequentformationofcelljunctionsthatdeterminethecorrectpolarizationofthisparticularcelltype(Figure7).ManyobservationshavehighlightedastrongconnectionbetweenalterationsinGPRC5Aandseveral human cancers (Jiang et al., 2018), and in particular with the presence of moreaggressive and metastatic phenotypes through the induction of the EMT (Liu et al., 2016;Sawadaetal.,2020;Liuetal.,2017).EMT takes place physiologically during embryonic development, tissue regeneration, organfibrosis,andwoundhealing.Throughthisprocess,epithelialcellscanswitchtoamesenchymalphenotype.Thisphenomenonisalsoinvolvedintumorprogressionwithmetastaticexpansionandinthegenerationofcancercellswithstemcellproperties,whichplayanimportantroleinresistancetocancertreatment(Nietoetal.2016,Lambertetal.2017).E-cadherinisoneofthemainregulatorsoftheEMTprocess.Itisthoughttoactbymaintainingcell-celladhesion,withaglobalanti-proliferative,anti-metastatic,andanti-invasioneffect.Epithelialpolaritylossisacomponent of the EMT process, and plays a crucial role in the correct localization of keybasolateralmolecules,suchasE-cadherin.GPRC5Adepletioninmouseepithelialtrachealcellshas been shown to cause a strongEMT induction following exposure to silica (Wang et al.,


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2015),suggestingaroleforGPRC5AinmaintainingepithelialidentityespeciallyinthecontextoflungtumorsinwhichGPRC5Ahasbeenshowntoactasatumorsuppressor(Taoetal.,2007).ThecurrentstudyindicatesthattheroleofGPRC5Aincancercouldbeexplainedthroughthepromotion and regulation of themechanisms that support the activation of themachinerynecessaryfortheexportofbasolateralmoleculesinparticularbyactingonPKD.InMDCKcells,apolarizedcellsystem(Yeamanetal.2004),ithasbeenfoundthatbothPKD1andPKD2playacriticalroleintraffickingofendogenousproteinstothebasolateralmembrane,suchasE-cadherinandβ1-integrin.ByregulatingtheproductionofTGNcarriersdestinedforthebasolateralmembrane,PKD1andPKD2canplayan important role in thegenerationofepithelial polarity. In addition, PKD regulation has also been linked to cancer invasivenessthrough EMT. Evidence of reduced E-cadherin and PKD1 in advanced prostate, breast, andstomachcancersfurthersupportsthistypeofroleforbothproteins(Durandetal.,2016;Royetal,2017).Therelationshipbetweenthetwoproteinsishighlightedbyseveralobservations:PKD1 is essential to maintain the transcription of the E-cadherin gene and repress theexpressionofmesenchymalproteinsthroughthephosphorylationofthetranscriptionfactorSNAIL(Duetal.,2010);activatedPKD1phosphorylatesthecytoplasmictailofE-cadherin,thusstabilizingitsassociationwithβ-cateninandtheactincytoskeleton(Jaggietal.,2005),whichstrengthens adherent junctions with consequent inhibition of cell motility. Therefore, thedownregulationofPKD1couldfacilitatetheEMTphenomenonbycompromisingbothE-cadfunctionandexpressionandpromotingtheexpressionofgenesinvolvedinthemesenchymalphenotype.ThecomponentsoftheARTGmachineryarealsoinvolvedingeneticdiseasesandpathologicalconditionsthatcanberelatedtodefectsinthecorrectproteinsecretionorcellpolarization.Gαi-3isassociatedwithararegeneticdisease,theauriculocondylarsyndrome(OMIM602483and614669),characterizedbyseverecraniofacialmalformationsanddefectsinearfunction(Riederetal.,2012;Gordonetal.,2013;RomanelliTavaresetal.,2015).StudiesconductedonGαi-3knock-outmicehaverevealeddefectsintheimmaturepre-hearingorgan(Beer-Hammeretal.,2018)whichdependontheessentialroleplayedbyGαi-3inthecontrolofasymmetriccelldivisionandofthepolarityandshapingofthebasalcochleahaircellbundleintheinnerear(Ezanetal.,2013;Mauriacetal.,2017).The PLCβ3 mutation or depletion also gives rise to several important phenotypes: ahomozygousmutationwith lossofPLCβ3 functionhasbeen linkedtospondylometaphysealdysplasiawithcornealdystrophy,anautosomal recessivediseasecharacterizedbymultipledefectsinbonegrowthandcornealopacity,whosepatients'fibroblastsshowedanalteredactincytoskeleton due to increased PIP2 levels (Ben-Salem et al., 2018). In PLCβ3-KOmice, thehyperproliferationofhematopoieticstemcellsleadstomyeloproliferativeneoplasiacausedbythelackofsuppressionofSTAT5signalingmediatedbyPLCβ3throughtheproteinphosphataseShp1(Xiaoetal.,2009).ηPKCknock-outmiceshowgreatersusceptibilitytotumorformationintwo-stageskincarcinogenesisandshowadelayedanddefectiveskinre-epithelializationduringcutaneouswoundhealing,suggestingaroleforηPKCinthemaintenanceofepithelialtissuearchitecture


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Figure1:Thephospho-antibodymicroarrayrevealsthesignalsactivatedbythearrivalofcargointheTGN.A)Theglobalsignalingresponsegeneratedbythearrivalofcargo(VSVG)in the TGNwas analyzed through the phospho-antibodymicroarraymade available by thecompanyKinexus.Thetwosamplesanalyzedwerethe“CargointheER”versus“CargointheTGN”.Briefly,afterinfectingthecells,theywerecollectedattheirrespectivetimepointsandtheircellpelletsweresenttotheKinexusCompanyforanalysis.B)Shortenedlistofresultsobtainedfromtheanalysisofthephospho-antibodyarray.TheZ-ratiovaluesgreaterthan1andlessthan-1wereconsideredsignificant(ingreenthevaluesthatindicateanincreaseinphosphorylation and in red those that indicate a decrease). The cells in the protein namecolumnarecoloredbasedontheirbelongingtospecificsignalingpathways:blueforthePI3K-mTORpathway,greenfortheMitogenicpathway,orangefortheCalcium/DAGpathwayandfinallypurplefortheJak-STATsignaling.C)TheproteinsobtainedfromtheshortenedlisthavebeenanalyzedbyGeneOntologyandmanualcurationinordertohighlightparticularsignalingpathwaysactivatedorinactivedbythearrivalofthecargointheTGN.ThisanalysishighlightedtheactivationoftheMAPK(Mitogenic),PI3K-MTORandCalcium/DAGpathways,whiletheJaK-STATpathwayisinhibited.Intherepresentationofthepathway,thefollowinggraphiccodeshavebeenassigned:themoleculeswritteninboldbelongdirectlytotheshortenedlist,whiletheregularoneshavebeenaddedtocomplete thepathway; themoleculeswrittenwith thecolorthatidentifiesthepathway(bothboldandregular)havebeenvalidatedbywesternblotanalysis.D)WesternblotvalidationofselectedsignalingmoleculesfromtheabovementionedsignalingpathwayduringVSVGtrafficpulsefromtheTGNtothePM.E)WesternblotanalysisofthedependenceonthepresenceofthecargointheTGNfortheactivationofthesignalingduring the temperature shift at 20°C in the presence or absence of Cycloheximide (CHX,50ug/ml). The Cycloheximidewas added at 37°C half an hour before the shift to 20°C andmaintaineduntil theendof theassaytocompletelyemptytheTGNandavoidthearrivalofnewlysynthesizedcargo.


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Figure2:Basolateral cargoaccumulation in theTGN inducesa local recruitmentandactivation of thePLCb3-DAG-PKD signalingpathway.A) Conventional signaling cascadethat leads to PKD activation on TGN membranes. B) IF analysis of the localization of theendogenousproteinPLCb3 inHeLacellsduring theVSVGtrafficpulseat the indicated timepoints.ThegraphrepresentsthePLCb3levelsinGolgicomparedtothetotal.C)LocalizationoftheDAG-reporter(C1a-PKD-GST)duringVSVGaccumulationintheTGN.Thegraphshowstheco-localizationprofilebetweenVSVGandtheC1a-PKD-GST.D)IFanalysisofthelocalizationofthe active form of the PKD protein by labeling the PKD1-GST overexpressedwith the antipPKDser916 antibody (auto-phosphorylation residue) during the traffic pulse of VSVG. ThegraphhighlightsthelevelsofpPKDser916intheGolgiattheindicatedtimepoints.E)Westernblot analysis of endogenous pPKDser916 levels during VSVG traffic pulse.F)Western blotanalysisofendogenouspPKDser916levelsduringVSVGtrafficpulseat40°C,andatthetimepointof the20°Cblock in thepresenceof thebeta-gammasubunit inhibitor (R8-phosducinaa215-233,50uM,1h,lane3)orPKD(Gö697610uM,1h,lane4),orvehicle(DMSO,lane2).


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Figure 3: Proteomic analysis of VSVG interactome in the different stations of thesecretorypathwayrevealsGPRC5AasnovelcargointeractorintheTGNcompartment.A)ExperimentaldesignoftheVSVGbindingproteinsanalysisacrossthesecretorypathway.B)VenndiagramofVSVGputativeinteractorsinthedifferentsamples.Weappliedsomefilterstoexcludeun-specificallyboundproteinsusingproteinscorebetween500and120(assignedbythemassspecfacilityasthesumofthesinglepeptidescores)asarbitrarycut-off.Fromthisstepofanalysis,weexcludedtheproteinsincommontoallthesamples,andthenwefocusedontheproteins foundtoco-immunoprecipitatewithVSVGboth in theTGNand in thePGCs(samples20°Cand30minutesat32°C).Thoseproteinsarelistedinthetablealongwiththeirsubcellularlocalization.C)WestenblotanalysisconfirmsthatGPRC5Aco-immunoprecipitateswith VSVG during the 20C block.D) Co-IP analysis of the interaction between the solublebasolateralcargohGHandGPRC5AduringhGHtrafficpulse.


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Figure 4: GPRC5A regulates through the pathway that leads to the activation andrecruitmentoftheproteinKinaseD.A)IFanalysisofVSVGproteintransportat30mintimeat32°C(afterblockat20°Cfor2hours)inHeLacells.ThequantificationinthegraphshowstheaccumulationofVSVGintheTGNintheGPRC5A-KDcellscomparedtothecontrolcells.B)IFanalysisofthelocalizationoftheactiveformoftheproteinkinaseD(overexpressedPKD1-GSTandlabeledwiththeantipPKDser916antibody)incontrolcellsandGPRC5A-KDduringthetrafficpulsewithVSVGintheblocktimepointat20°C.Thequantificationshowsthat intheGPRC5A-KDcellstheactiveformofPKDisnotrecruitedtotheTGNwithrespecttothecontrolcells.C)RescueexperimentofthetransportofVSVGinGPRC5A-KDcellsbyexpressionoftheGPRC5AsiRNAresistantconstruct(si-rGPRC5A-Myc)oroftheGPRC5(B-C-D)-Mycconstructs.The IF analysis of VSVG transport shows that both si-rGPRC5A-Myc and GPRC5C-MycconstructsareabletorecoverVSVGtrafficdefectsinducedbythedepletionofGPRC5A,whilethe overexpression of GPRC5B or GPRC5Ddoes not recover (data not shown), indicating apotentialpartialredundancyoffunctionoftheproteinsGPRC5AandGPRC5C.D)WesternblotanalysisofpPKDser916levelsduringVSVGtrafficpulseincontrolandGPRC5A-KDcells.E)IFanalysisofendogenousPLCb3localizationincontrolandGPRC5A-KDHeLacells.QuantificationinthegraphhighlightsareducedGolgilocalizationofPLCb3inGPRC5A-KDcellscomparedtocontrolcells(scalebars10nm).F)WesternblotanalysisofcytosolicandmembranefractionsofcontrolandGPRC5A-KDsteadystateHeLacells.ThequantificationinthegraphshowstheratioofthemembranefractionofPLCb3tothetotalusingCalnexin(membranefraction)andGAPDH(cytosolicfraction)asnormalizers.


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Figure5:Galphai-3iscoupledtoGPRC5AanditisactivatedduringcargoaccumulationintheTGN.A)ListofGproteinsidentifiedinGPRC5AIPsbyLC-MS/MSanalysis.B)Westernblot(WB)validationofGαi-3co-immunoprecipitationwithGPRC5AinsteadystateHeLacells.C)WBanalysisofGαi-3activeform(Gαi-GTP)immunoprecitiationduringVSVGtrafficpulse.ThequantificationhighlightstheincreasedamountofactiveGαi-3duringVSVGaccumulationintheTGN.D)WBanalysisGαi-GTPinmmunoprecipitatedlevelsincontrol,GPRC5A-KDandGαi-3-KDsteadystateHeLacells.ThequantificationshowsthedecreasedlevelsofactiveGαi-3inabsenceofGPRC5A,Gαi-3-KDcellsareusedasnegativecontroloftheassay.E)WBanalysisofPKDser916levels incontrol,GPRC5A-KDandGαi-3-KDduringVSVGtrafficpulseinHeLacells. The graph clearly shows that in eitherGPRC5A-KDorGαi-3-KD condition there is noactivationofPKDinresponsetoVSVGaccumulationintheTGNcompartment.


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Figure6:GPRC5AonplasmamembranecanactivatePKDinresponsetotheadditionofsolublecargointheextracellularmedium.A)RT-PCRanalysisofGPCR5AtranscriptlevelsuponDMSOorretinoicacid(RA)treatment(2uM,16hrsinserum-freemedia)incontrolandGPRC5A-KDHeLa cells.B)Westernblot analysis of endogenouspPKDser916 levels in cellstreated with RA (or DMSO) and two different soluble cargoes, TNFα (15nM, basolaterallysecreted)andLysozyme(15mM,apicallysecreted),orvehicle.AfterRA(orDMSO)treatmentallconditionshavebeenincubatedat20°Cfor3hrsinserum-freemedia,thensolublecargoeshavebeenaddedfor15minutes.C-D)WesternblotanalysisofendogenouspPKDser916levelsincontrol(lanes1to4)andGPRC5A-KD(lanes5to8)cellstreatedwithRA(2uM,16hrsinserum-freemedia,lanes2,4,6and8)andalbumin(750uM,15min,panelClanes3,4,7and8)orrecombinant TNFα (15nM, 15min, panel D lanes 3,4,7 and 8). All conditions have beenperformedasabovementionedforpanelB.E)QuantificationofpPKDser916levelsfromCandDexperiments.F)IFanalysisofpPKDser916(overexpressedPKD1-GST)localizationincellsoverexpressingGPRC5AtreatedwithrecombinantTNFαorvehicle(scalebar10nm).G)Dose-responsecurvesforpPKDser916activationwithdifferentconcentrationofTNFαandalbumin.H)Graphicalsummaryandmodeloftheexperimentsdescribedinthisfigure.


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Figure7:GPRC5CregulatespolarizationandsecretionofbasolateralsolubleproteinsinHepG2cells.A)IFanalysisofthelocalizationoftheendogenousalpha1anti-trypsinprotein(A1AT)incontrolandGPRC5C-KDHepG2cellsat20°Cfor2hrs(time0)and15minutesaftertheshiftoftemperatureto37°C.B)WesternblotanalysisofA1ATsecretedinthemediumofHepG2controlandGPRC5A-KDcellsatdifferenttimepointsat37°Cafterblockingat20°Cfor2 hours (time 0). C) IF analysis of Phalloidin-488 staining to visualize the bile-canaliculi,visualizedasacircularanddensestructureofvariablediameter, insteadystatecontrolandGPRC5A-KD HepG2cells. The graph represents the percentage (%) of canaliculi in bothconditions.D)IFanalysisofthelocalizationofthejunctionproteinZonulaoccludens1(ZO-1),incontrolandGPRC5C-KDcells.Co-stainingwithPhalloidinshowsthatincontrolcellstheZO-1proteinformsaringaroundtheactinthatformsthecanaliculus,whileinGPRC5C-KDthisdoesnothappenandZO-1remainslocalizedinsidethecellortothecellperiphery(scalebars10nm).E)WesternblotanalysisofdifferentpolarityandcelljunctionmarkersincontrolandGPRC5C-KDHepG2cells,withtheirrelativequantification.F)IFanalysisofGPRC5CincontrolandGPRC5C-KDHepG2cells.


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Figure8:CargodependentARTGsignalingactivationmodel.BasolateralcargoesarrivalintheTGN,fromwhichtheymustbesubsequentlysorted,activatetheorphanreceptorGPRC5A.The mechanism that directly associates the cargo to the receptor is still being analyzed,however,theassociatedGβγsubunitsstartasignalingcascadethatflowsontotheactivationandrecruitmentofPKD.Previousliteraturehad,infact,identifiedtheGβγsubunits,PLCβ3andηPKCasmoleculesupstreamoftheactivationofPKDontheTGN(Jamoraetal.,1999;BaronandMalhotra,2002;DiazAñelandMalhotra,2005;DiazAñel,2007).The finaleffectof thissignaling is thephosphorylationbyPKDofsubstraterrequired for theexportofbasolateralcargoes.WecalledthiscircuitARTG,forAutoregulationofTGNexport.


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