Journal of Cell Science o:10.1242/jcs.102343 doi: 4026–4035 125, ß Science Cell of Journal 2012 April 10 Accepted iulsse varsi)adtetoohr ( the others to two restricted the is and one humans, (v-arrestin) in system arrestins visual of isoforms three are late cell its deeper to the downregulation. leading the pathway from surface of degradative to the sorting slowly and the lysosomes back to or induces receptor treatment rapidly endosomes, intracellular agonist early Prolonged to recycled endosomes. from directed be surface is to cell receptor compartments the endosomal internalized, the inside Once events signaling of cell. cascade G a trimeric then binding, to and activation leading agonist protein changes by conformational stimulated undergo regulating receptors Once in the etc. vision, role output, neurotransmission, central cardiac as a olfaction, such play functions They physiological 2002). various al., largest et the al., et Takeda (Fredriksson constitute members 2003; thousand a (GPCRs) almost with protein receptors of family coupled G-protein The Introduction words: Key b Summary ( correspondence Franco* for *Author Michel and Luton France Frederic Mole Paleotti, Pharmacologie de Olivia Institut Partisani, Mariagrazia Macia, Eric the of recycling rapid the controls negatively Arf6 4026 rmtn eetritraiain(eiwdi or tal., et Moore in addition, (reviewed In sites 2007). internalization binding clathrin receptor of and AP-2 exposure promoting the that contains the that established domain to terminal well leads of GPCRs been interaction phosphorylated has direct it and the Indeed, complexes the AP-2 molecules. with preventing clathrin binding Second, by direct first proteins. through receptor internalization G the that of of receptor activation desensitization the stimulation phosphorylated induces Agonist (GRK)-dependent of distributed. kinase binding ubiquitously promotes are arrestin2) of accumulation caused mutant Arf6 activated an that or EFA6 report either of we of of degradation activation Here, Expression and ligand-mediated manner. recycling membrane. the dependent the plasma Similarly, regulates activation. the which Arf6 to Arf6, promote of protein back region G C-terminal recycled small the subsequently the that of and activation endosomes, the early promotes into manner dependent inln rvee nDWr ta. 2007). al., et DeWire in (reviewed signaling We Rab4. of upstream acts Arf6 that suggesting Rab4, in of role mutant essential activated an an plays of Arf6 expression which in the model by a rescued propose be could phenotype This deegcreceptor adrenergic h erdto aha,adngtvl otosRb-eedn atrccigt rvn h esniiainof of re-sensitization accumulation the to prevent leads Arf6 to of recycling activation fast The EFA6. Rab4-dependent by controls Arf6 negatively of and activation the pathway, triggers degradation and the membrane, plasma the to arrestin -deegcrcpo ( receptor 2-adrenergic 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. h inln fGCsi imyrgltdb retn.There arrestins. by regulated firmly is GPCRs of signaling The FT rC,cMt rgsre,Infu,Metastasis flux, Ion screen, Drug c-Met, TrpC6, NFAT, b arsiscudas atcpt nreceptor in participate also could -arrestins [email protected] b b A) ebro h PR(-rti ope eetr aiy sitraie nalgn-and ligand- a in internalized is family, receptor) coupled (G-protein GPCR the of member a 2AR), arsist h -rti receptor G-protein the to -arrestins cliee ellie M 25CNRS-Universite 7275 UMR Cellulaire, et ´culaire b arsist iadatvtdand ligand-activated to -arrestins b arsi1itrcsdrcl n iutnosywt r6D n t pcfcecag atrEA,to EFA6, factor exchange specific its and Arf6GDP with simultaneously and directly interacts -arrestin1 b ) arsi otosreceptor controls -arrestin b arsi1and -arrestin1 b arsi C- -arrestin b A eestzto.Telgn-eitdsiuainof stimulation ligand-mediated The desensitization. 2AR b - b ysRAsee oafc h nenlzto fsm GPCRs some of internalization the depletion the affect Arf6 directly including to 2005). seemed al., interacts et siRNA which (Tanabe by chain SMAP1, light clathrin Arf6–GAP the with be the to proposed by also effect was regulated endocytosis The clathrin-mediated the components. on induce clathrin Arf6 coated-pit could of and the Arf6 2007), AP-2 of al., recruitment with et membrane interacting Poupart 2005; directly al., by et (Paleotti and 2003) al., et nAfGPdpnetmne Chne l,20;Sadre al., et Stalder in 2007; membrane al., plasma et the (Cohen at manner an Arf6GTP-dependent recruited ARNO, an is addition, In that GIT1 1998). factor Arf6–GAP al., exchange et Arf1 an Premont of 2000; al., expression et (Claing the following in observed Defects been the 2001). with al., et manner (Claing agonist-dependent the the an of onto in internalization was interacting recruitment the Arf6 2007). regulate AP-2 al., to et and proposed (Poupart clathrin receptor II prevent Angiotensin activated to and 2005) li ta. 06 aaise l,20;Rdarsn and Radhakrishna 2002; PIP al., the et 1995; stimulating Palacios al., By receptor, et 2006; 1997). MHC D’Souza-Schorey transferrin al., Donaldson, 1999; and et al., as et E-cadherin Klein (Altschuler such I receptor, class immunoglobulin cargos polymeric membrane both of in endocytosis in of (reviewed different functions pathways Arf6 crucial -independent and 2006). is clathrin-dependent Chavrier, reorganization and endocytic phagocytosis, structure D’Souza-Schorey transport membrane and as F-actin lipid with coordinating migration such which processes during adhesion, trafficking cellular cytokenesis, many exocytosis, been has in Arf6 transport. involved vesicular intracellular regulate to known A ed otefraino r6T nvv na in vivo in Arf6GTP of formation the to leads 2AR eNc-ohaAtpls 6 ot e uils 66 Valbonne, 06560 Lucioles, des route 660 Antipolis, Nice-Sophia de ´ r6blnst h r aiyo ml rtista are that proteins G small of family Arf the to belongs Arf6 b -deegcrcpo ( receptor 2-adrenergic b A ntedgaainpathway. degradation the in 2AR b b A.W eosrt nvitro in demonstrate We 2AR. A nenlzto aealso have internalization 2AR b A)(onooe al., et (Houndolo 2AR) eerhArticle Research 2 b b 2AR. ytei (Krauss synthesis b A relocates 2AR A ydirectly by 2AR b b b b -arrestins -arrestin- -arrestin- b b -arrestin A in 2AR 2 b - Journal of Cell Science ehv hw htteepeso fEA e othe to led EFA6 K of a TWICK-1, expression as well the as receptor that Indeed, transferrin receptors. of shown membrane redistribution some have of with seems trafficking we EFA6 dynamics the 2008). actin control al., et to cortical (Macia remodeling coordinates membrane plasma it where membrane aha em ob eedn ntecl ye h endocytic the type, cell the on recycling dependent Arf6 be the to However, seems receptors. pathway membrane of role transport a implied endocytosis. observations GPCRs these in bind together, Arf6 Taken for directly 2001). al., to et shown been stimulate has 2011) ic r6D a hw oitrc with interact to shown was Arf6GDP Since membrane with colocalization the of stimulation Ligand-induced of desensitization the to led a Arf6 in activated EFA6 and by The desensitization Arf6 of receptor activation the the of stimulation to Agonist down-modulation. contributing recycling, its the of 1999). activation al., the et and/or Franco internalization 2004; with al., receptor et together membrane (Decressac EFA6 recycling that regulate suggesting could surface Arf6 cell the to channel, 2006; directly interacting al., By et 2007). Li PIP al., 1999; et with al., Sakagami migration 2004; et al., Franco and et 2006; Luton an al., polarization et is (Choi cellular which processes reorganization, EFA6, morphogenesis, actin isoform. in member involved neuronal Arf is each factor, cognate and exchange members specific its Arf6 fifteen activate This least at to 2000). of seems Casanova, up and made is (Jackson family family Arf6- protein these containing all in identified. been activation has molecular Arf6 no pathways tethering now, dependent for Until vesicle the accounting 2003). the al., with activate mechanism et interact (Prigent to to complex and ability exocyst 1998), and al., its (Be et Yang lipid through Caumont 1997; fusogenic producing al., least D1 et neuroendocrine at phospholipase Galas and 1999), 2003; adipocytes Martin, Mueckler, in involved and be (Aikawa exocytosis to shown cells regulated been also the has Arf6 in D’Souza- mechanism described. see molecular been review, no yet a however has (for 2006), cargo Chavrier, the and of Schorey nature the and pathway hte t pcfcecag atrEA ol lobn othis to the bind used also We could adaptor. EFA6 GPCR factor exchange specific its whether and pathway Results endosomes/lysosomes. recycling late the fast to receptor Rab4-dependent the relocalizing the inhibiting nsrmsavdclsadi h bec fiortrnl a isoproterenol, of absence the proteins, in and b the cells examined first serum-starved We in 1998). myc–EFA6, al., Arf6 et of Kallal localization 1998; on the al., stably study et dependent (Gagnon cells to HEK293 used be established extensively to We the 2005). reported expressing al., et been (Houndolo has endocytosis y–F6wr otyfuda h lsammrn Fg 1A, (Fig. membrane plasma the at found mostly were myc–EFA6 A gns –s..A rvosysonfrteendogenous the for shown previously As (–Iso.). agonist 2AR h ciaino rsi civdb h Sec7-domain- the by achieved is Arfs of activation The post-endocytic the in Arf6 implicated also have studies Several eew dniidanwAf-eedn aha htcouples that pathway Arf6-dependent new a identified we Here 2 b b arsi1H a oul whereas soluble was -arrestin1–HA n ci iaet,EA oaie oteplasma the to localizes EFA6 filaments, actin and A nenlzto hnoeepesd(Claing overexpressed when internalization 2AR b A ue oGP hscl iehsbeen has line cell This GFP. to fused 2AR b b -deegcrcpo ihteihbto of inhibition the with receptor 2-adrenergic arsi n F6a h plasma the at EFA6 and -arrestin b b arsi1H n the and -arrestin1–HA A saGC oe eas its because model GPCR a as 2AR b A inln n trafficking and signaling 2AR b arsi-eedn manner. -arrestin-dependent b A ed to leads 2AR b b b arsi easked we -arrestin A rmtdthe promoted 2AR arsisadto and -arrestins ´ b gle A–F and 2AR–GFP ´ ta. 2009; al., et b 2AR–GFP b A by 2AR + –F6(i.1) et hnprfe rtiswr sd we used, were proteins purified VSV- when overexpressing Next, lysate 1B). cells (Fig. BHK G–EFA6 a from and EFA6 the pull-down to manner fused of GST agonist-dependent domain that glutathione C-terminal observed an In we experiments Arf6GDP. pull-down in with (GST) interact exposed to be reported to with known interact physically could EFA6 b whether examined we Next with interacts directly EFA6 epromdGTpl-oneprmnswt he fragments of three C-terminal with the experiments of pull-down GST performed we the map further EFA6 To and Arf6GDP with simultaneously of fragment V319–K357 The demonstrate the with directly experiments regulate interacts these EFA6 conclusion, could that In with S1). domain interact the Fig. PH material longer to the no that accessibility could suggesting domain interaction arrestin1 PH the the for sufficient containing was EFA6 with of portion with C-terminal specifically of and domain directly C-terminus the interacts His–EFA6 that observed hs h iadsiuaino h eetrlast the to leads receptor the found. receptor, are of proteins the stimulation three of the ligand which concentration in the surface cell noticed Thus, We the arrowheads). at see folds +Iso., of 1A, stimulation the (Fig. that surface cell of the translocation at membrane the plasma to a added observed b we was min, (+Iso.) 3 isoproterenol for cells When, shown). not (data r6D rHsEA a bet(i.2,pl down, pull when 2C, to (Fig. compared absent present, was were His–EFA6 higher proteins or as three to Arf6GDP synergistic bound all were be His–EFA6 when to and were Arf6GDP seemed of proteins amounts association both the Arf6GDP When between contrary, binding 2). the of and decrease and no 1 was there lanes and present, the 2C, Arf6GDP both (Fig. constant of to B, and directly a concentrations A bound panel of 2, increasing His–EFA6 Fig. presence pull-down in and observed bind As the GST Arf6GDP EFA6. they end, in of whether this performed concentration determine To were to exclusively. experiments important or was simultaneously it more arrestin1, and constructs New between discriminate possibilities. one. to two needed second these be a lower will experiments bind a quantitative to with or Arf6 molecule to of bound C-terminal the D383) 2B). on to (Fig. region affinity K357 second of (from site binding a binding main arrestin1 the while the also for Arf6GDP, was portion sufficient for same and The 2A). necessary (Fig. was EFA6 K357 to V319 iceeae ftepam membrane. plasma the of area discrete ebaeaeerce nmcEA and myc–EFA6 plasma in the of enriched domains these are numerous of that membrane localization noted be affect should not It did proteins. tags the that confirming –Iso.) oan eefudt eerce nPIP in enriched be to found were domains arsi1admr pcfclywt h -emnldomain C-terminal the with specifically more and -arrestin1 arsi1H n togclclzto ftetreproteins three the of colocalization strong a and -arrestin1–HA sbt r6adEA on otesm einof region same the to bound EFA6 and Arf6 both Arf6 As same the bind to contribute could sub-domain This b b arsi1ee tahg ocnrto fHsEA.In His–EFA6. of concentration high a at even -arrestin1 arsi1 hl ogrCtria oto fEFA6 of portion C-terminal longer a while -arrestin1, r6controls Arf6 b b arsi idn ie o r6adEFA6, and Arf6 for sites binding -arrestin b arsi1 h oto of portion The -arrestin1. b b arsi1 u o S ln,wsal to able was alone, GST not but -arrestin1, A nrae h ubro membrane of number the increased 2AR arsi idn ie(supplementary site binding -arrestin b arsi1(i.1) utemr,the Furthermore, 1C). (Fig. -arrestin1 b arsi1famn V319–K357 fragment -arrestin1 b b arsi1interacts -arrestin1 -arrestin1 b b arsi1adEA within EFA6 and -arrestin1 -arrestin. b A eyln 4027 recycling 2AR 2 b -ci n ezrin and F-actin , A–F.These 2AR–GFP. b arsi1from -arrestin1 S -transferase b -arrestin1 b b b - - - Journal of Cell Science ol etigrdby triggered be the could that hypothesized a in We Arf6 of activation the the of activation Agonist Arf6GDP. of substrate gathering its the and facilitating EFA6 by factor Arf6 exchange of the activation that the concluded and for the allows We of activation, domain 2D). C-terminal (Fig. Arf6 the the activation expected, promoted Arf6 the As EFA6-mediated strongly GDP. of for EFA6 addition Arf6 of of affinity addition the possibly decreasing the Arf6GDP, of by of activation spontaneous presence the facilitated The alone fusion sites. GST binding and EFA6 EFA6 His-tagged of Arf6 absence end, in of exchange this or GDP/GTP To presence of Arf6. the kinetics of in the activation vitro the in facilitate examined to to we platform bind that a factor speculate as to act exchange tempting its was it and simultaneously Arf6GDP both vitro in As ternary Arf6 of a activation catalyzed of form part C-terminal to The EFA6 and Arf6GDP complex. Therefore, 2). to and 1 simultaneously lanes to 3 lane compare 4028 spoeeo a de osrmsavdHK9 el stably cells HEK293 serum-starved to hypothesis, this added test was To internalization. isoproterenol and stimulation GPCR of b arsi1famn 39K5 otiigteAf and Arf6 the containing V319–K357 fragment -arrestin1 ora fCl cec 2 (17) 125 Science Cell of Journal b arsi1famn ute tmltdthe stimulated further fragment -arrestin1 b A ciain as activation, 2AR b arsi1siuae EFA6- stimulates -arrestin1 b b arsi-eedn r6activation Arf6 -arrestin-dependent b deegcrcpo ed to leads receptor adrenergic 2 arsi-eedn manner -arrestin-dependent b arsi1at sapafr that platform a as acts -arrestin1 b arsi sa adaptor an is -arrestin b b arsi1fragment -arrestin1 arsi1cnbind can -arrestin1 b arsi could -arrestin b -arrestin1 r6b F6uo gns tmlto ly oei the in expressing role stably isoproterenol-stimulated cells a HEK293 on internalization. plays expression exogenous stimulation EFA6 of agonist trafficking upon cellular EFA6 the by whether Arf6 asked we Next of recycling relocalizes Rab4-dependent the inhibits EFA6 h diino gns nrae h muto r6T in to Arf6GTP response on of in activation amount Arf6 the that in increased not but agonist control of of addition depleted The cells after agonist-mediated on on level this repeated dependent basal whether were was the determine twofold activation to To (approximately Arf6 returned 3A). min (Fig. and 10 min level) 30 at basal observed, to peaked was Arf6GTP compared activation of the the Upon quantity of detected. higher and absence was agonist, the Arf6GTP of In of addition level down. basal pulled a specifically isoproterenol, was Arf6GTP and expressing el xrsigRPaoeo F–F6 ugsigta Arf6 that suggesting RFP–EFA6, or alone RFP of radio- expressing after rate cells receptor internalization the The of RFP–EFA6 binding. internalization for ligand the encoding monitored plasmid we a and with transfected transiently ciaino r6i vivo. in Arf6 of activation b arsi nrsos oaoitsiuainalw o the for allows stimulation agonist to response in -arrestin oanfsdt S protein. GST to fused domain by His–EFA6 purified of pull-down el yteCtria oanof domain C-terminal the by cells muooaiainwspromda ecie nteMtrasand min. Materials 2 the for in ( isoproterenol described Methods. nM as 100 performed with was (–Iso.) Immunolocalization not or (+Iso.) stimulated ( arrestin expressing stably 1. Fig. hs yc-eriigAfGPadEA,teatvto of activation the EFA6, and Arf6GDP co-recruiting by Thus, b -arrestin. b b arsi1drcl neat ihEFA6. with interacts directly -arrestin1 arH)admcEA eesrmsavdfr5hadthen and h 5 for starved serum were myc–EFA6 and -arr–HA) B b S uldw fVVGtge F6epesdi BHK in expressed EFA6 VSV-G-tagged of pull-down GST ) b A–F n rninl xrsigRFP–EFA6, expressing transiently and 2AR–GFP A olt endosomes/lysosomes late to 2AR b A–F n rninl xrsiga HA-tagged an expressing transiently and 2AR–GFP b arsi-eltdcls(i.3) suggesting 3B), (Fig. cells -arrestin-depleted b A.W is sesdteefc of effect the assessed first We 2AR. b b arsi-eedn ciainof activation -arrestin-dependent arsi ue oGTpoen ( protein. GST to fused -arrestin b arsi ullnt n t C-terminal its and length full -arrestin b b A ciaini dependent is activation 2AR b arsi1ad2b siRNA. by 2 and -arrestin1 arsi,teexperiments the -arrestin, b A a iia in similar was 2AR ( A b E23cells HEK293 ) A–F were 2AR–GFP b A and 2AR C b GST ) 2AR b - Journal of Cell Science -emnli h rsneo nraigcnetaino His–EFA6. of concentration increasing of of domain ( presence V319–K357 the the by in His–EFA6 C-terminal and ARF6GDP of down the of constructs ehd)i h rsneo bec fprfe i–F6(0.5 His–EFA6 purified of of domain absence C-terminal or presence the in Methods) (4 ARF6GDP purified r6D n F6adsiuae r6atvto nvitro. ( in His–EFA6 activation purified Arf6 of stimulates pull-down and EFA6 and Arf6GDP F6ddntafc h lwrccig rsmbyfo more from of presumably overexpression recycling, RFP– min, slow the of 20 early affect following presence not peripheral did the the EFA6 from during in presumably while decreased EFA6, recycling, strongly fast was incubation, of min of endosomes, 10 first rate the during expression the that EFA6 overexpressing observed by EFA6 We affected 4B). in is (Fig. recycling 10% the plasma only that the but indicating to cells min, back 30 recycled after receptors membrane internalized the of for 40% allow cells, to cells the incubated ciaini ihrntrqie rntlmtn for 37 limiting EFA6 at of not min effect 20 the or analyzed then required on We not 4A). (Fig. either internalization is activation the of peptide V319–K357 The 2. Fig. D ieiso [ of Kinetics ) b A eyln.W rae h el ihiortrnlfor isoproterenol with cells the treated We recycling. 2AR ˚ 35 oidc nenlzto,wse n further and washed internalization, induce to C b S]GTP arsi -emnlfsdt S rti.( protein. GST to fused C-terminal -arrestin b m arsi1fsdt S (6 GST to fused -arrestin1 )wsdtrie a ecie nteMtrasand Materials the in described (as determined was M) c odn nAfGP idn f[ of Binding Arf6GDP. on loading S A rprfe r6D ( Arf6GDP purified or ) b arsi -emnlrcut both recruits C-terminal -arrestin b A orcce ncontrol In recycle. to 2AR m M). 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(Fig. the the structures internal of and of EFA6 (+FCS), redistribution present localization and of was strong absence serum the the the of membrane spite when in affect contrast, plasma In 5A). not the (Fig. and did at RFP–EFA6 expression 1, essentially Fig. (–FCS), in starved localized the observed serum previously expressing were as cells stably Arf6 When cells of immunofluorescence. mutants HEK293 internalized activated the in the b or overexpressed of EFA6 end, localization were this intracellular To the receptors. activation, assessed Arf6 upon we unaffected remained rate internalization Ferguson, its and Seachrist in (reviewed 2003). compartments internal i.3 tmlto of Stimulation 3. Fig. A a titydpneto h xrsino F6adon and EFA6 of expression the on dependent strictly was 2AR by determined was localization receptor and 2AR–GFP sterccigrt of rate recycling the As b arsi1ad2adsiuae +s. rnt(Io)wt 0 nM 100 with (–Iso.) not or (+Iso.) stimulated and 2 and -arrestin1 b A–F n rninl xrsigRPEA eeserum were RFP–EFA6 expressing transiently and 2AR–GFP r6controls Arf6 b A yiortrnltigr a triggers isoproterenol by 2AR b A gns,EA xrsinldt a to led expression EFA6 agonist, 2AR b A a togydcesdwhereas decreased strongly was 2AR B E23clssal expressing stably cells HEK293 ) 6 b A eanda h plasma the at remained 2AR tnaddvain(.. fthree of (s.d.) deviation standard b A eyln 4029 recycling 2AR ( A E23clsstably cells HEK293 ) 6 ..o orseparate four of s.d. b -arrestin- b 2AR–GFP b 2AR– Journal of Cell Science eetritraiain hnwse wc ih37 with twice washed then internalization, receptor eyln.Clswr rae ihiortrnl(1 isoproterenol with treated were Cells recycling. ahtm a eemnda ecie nMtosadMtos ( Methods. and Methods in described as determined was time each (1 isoproterenol transfection, after encoding plasmids of sdsrbdi h aeil n Methods. determined and were Materials numbers the receptor in Surface described time. as indicated the for incubated retnwt F6srnl blse h serum-induced the abolished strongly of the form EFA6 of negative serum- redistribution dominant with and the EFA6- of arrestin this co-expression that the of noted Indeed be redistribution should dependent It Arf6. activate the of recycling fast the inhibits EFA6 4. Fig. 4030 xeiet eosrt httesrmtigr h E ciiyof activity GEF the triggers serum the dominant that demonstrate experiments the of redistribution responsible internal the or was the modify Arf6GTP for form of not accumulation wt the did Arf6GTP-induced that Arf6 demonstrating the this of of mutant EFA6, Expression negative to 5C). (Fig. contrast serum in the of and relocalization model with our consistent However, quantification). see of of 5C, one pattern (Fig. modified either distribution strongly expressing a cells exhibited the proteins three of these 80% About +FCS). strong 5A, the (Fig. a of induced redistribution Arf6Q67L–mCherry activated constitutively higher starved significantly serum were 5B). a cells Fig. in the fourfold; steady when resulted (about than the Arf6 At serum activated 5B). of of (Fig. amount presence Arf6 a of to the activation led state, serum robust of very transiently addition but cells The slow HEK293 5B). in (Fig. RFP–EFA6 Arf6GTP to expressing of out formation carried was the Arf6. experiment for pull-down monitor activity a hypothesis, GEF this EFA6 test the To enhances serum the that suggest b h xrsino h atccigAfT5Nmhryadthe and Arf6T157N-mCherry cycling fast the of expression The A nenlzto.HK9 el eetasetyc-rnfce with co-transfected transiently were cells HEK293 internalization. 2AR ora fCl cec 2 (17) 125 Science Cell of Journal m )adteaon of amount the and M) b b A nenlzto a rgee yteadto of addition the by triggered was internalization 2AR A–F n F ln rfsdwt F6t 4h 24 EFA6wt. with fused or alone RFP and 2AR–GFP b b A a needn ntepeec of presence the on independent was 2AR A ntson.Mroe,orresults our Moreover, shown). (not 2AR b 2ARcomparabletothatofEFA6wt b A was 2AR b A Fg C.Atgte these Altogether 5C). (Fig. 2AR b A rsn ttecl ufc at surface cell the at present 2AR b 2AR. b m ˚ E eimand medium HEK C )fr2 i otrigger to min 20 for M) arsi dependent. -arrestin b ( A localization 2AR A iei analysis Kinetic ) B ) b b 2AR 2AR b - lsia oe o PR e oteatvto fAf yEFA6 by Arf6 of activation the a to in led GPCR, for model classical a on togyclclzdwt the with colocalized strongly a found CD63 contrast, lysosomeswas and In endosomes late 6). in located (Fig. mainly endosomes protein tetraspanin early the in of most not that was a indicating receptor proteins, only two showed the protein, between associated overlap endosome weak early an 1), Endosome Antigen (Early EEA1 with receptor the of Co-staining structures. the inhibits structures. turn internal in towards relocalization Arf6 of of activation recycling the fast that and Arf6, on EFA6 aiiae r6atvto yEA.Bohmcladstructural and Biochemical EFA6. of by activation domain Arf6 facilitated to C-terminal led the thus endosomes/lysosomes, signaling downstream of late events. regulation and the the desensitization receptor relocalized to the Arf6 membrane inhibited of but the receptor, activation of the recycling of fast rate Rab4-dependent internalization the affect ebaepoen(i.6.Teepeso ftefs yln Arf6 cycling the fast induced the resident Arf6T157N of lysosomal mutant expression The a 6). Lamp-2 (Fig. protein with membrane co-staining by confirmed was Arf6 the of directs activation EFA6 the that by suggested result This 6). (Fig. structures h erdtv aha.I diini hudb oe that noted be should it addition In pathway. degradative the of accumulation the the induces concluded Arf6 of We activation arrowheads). a the that see that S2, Fig. suggesting structures material endosomal (supplementary early EEA1 through trafficking was Arf6 and of Arf6fraction Arf6T157N partial nor observe could EFA6 between we neither However co-staining that structures. these noted on be found should were It S2). Fig. (supplementarymaterial compartments internal positive Lamp-2 and CD63 uosie l,20)weesRb5i novdi h fast the the If in 2006). involved al., the et is of (Kouranti accumulation receptor Rab35 transferrin whereas the of 2009) recycling al., receptors. et of membrane recycling been Yudowski of fast have recycling the Rab35 fast controls and Rab4 the Rab4 regulate them to among described and traffic membrane lysosomes. to that receptor the suggest of observations sorting the these to response together, in Arf6GTP All of accumulation S3). Fig. material relocalization the the to led of also hours) (3 treatment agonist prolonged bet eceteitaellracmlto of accumulation not intracellular was the Rab35 protein rescue G to small able the of of mutant localization active normal constitutively the affect not b did alone Q67L Rab4Q67L Rab4 while both of presence Arf6T157N, the in and the Indeed, of Rab4. upon mutant protein active G receptor small constitutively be the the should overexpressing of phenotype by recycling rescued this fast activation, of overexpression/Arf6 inhibition EFA6 the by caused nti td,w eosrtdta h tmlto fthe of stimulation the that demonstrated we study, this In Discussion Arf6. of the the activation that the of by suggesting regulated recycling 7), (Fig. fast Arf6T157N Rab4-dependent of overexpression the A nteasneo gns Fg ) ntecnrr,the contrary, the On 7). (Fig. agonist of absence the in 2AR ohEA n r6cudbn ietyadsmlaeul to simultaneously and directly bind could Arf6 and EFA6 Both hnw ogtt eemn h dniyo hs internal these of identity the determine to sought we Then ml rtiso h a aiyaekyrgltr of regulators key are family Rab the of proteins G Small b arsi-eedn anr hsAf ciainddnot did activation Arf6 This manner. -arrestin-dependent b A otedgaaiecmatet (supplementary compartments degradative the to 2AR b b A otepam ebae eutn nits in resulting membrane, plasma the to 2AR A a on ntepam membrane, plasma the on found was 2AR b b A ntelt nooe/yooe was endosomes/lysosomes late the in 2AR A oadtedgaaieptwy This pathway. degradative the toward 2AR b b arsi,adti association this and -arrestin, A eitiuinit h same the into redistribution 2AR b A Sahite l,2000; al., et (Seachrist 2AR b b b A Fg ) Prolonged 8). (Fig. 2AR A rmteplasma the from 2AR b A tmlto ed to leads stimulation 2AR b A a specifically was 2AR A oiieinternal positive 2AR b A nue by induced 2AR b b A in 2AR A,a 2AR, Journal of Cell Science tde aepeiul salse htuo idn othe to binding upon receptor, phosphorylated that and established activated previously have studies eesr o h omto ftetraycmlx( complex ternary the of formation the of tail activation for (reviewed This clathrin C-terminal necessary 2007). and al., AP-2 its et for Moore exposes sites in binding and the contains change which conformational large ed ob ciae n nege ofrainlcags in changes, conformational its undergoes reveal and to EFA6 order activated Therefore, be observation). to EFA6 (unpublished strongneeds of observed domains A C-terminal also interaction. the this was and prevented PH to the domain between bound PH interaction the EFA6 of of addition domain C-terminal the of changes instead conformational EFA6 regulated, on tightly As 1C). be must Fig. C-terminal form; interaction activated this the the (mimicking in and protein the form) of Arf6 domain non-activated the of (mimicking activation arrestin subsequent the stimulation. receptor and to EFA6), response and Arf6 hc suulymse.Wa ol eteatvtro F6 In EFA6? of activator the be would What masked. usually is which ncnrs oAf,EA neatdwt ohtefull-length the both with interacted EFA6 Arf6, to contrast In b arsi idn iea h -emnldomain, C-terminal the at site binding -arrestin b arsi a erqie.Cnitnl,while Consistently, required. be may -arrestin b b arsi ol lobe also could -arrestin arsi nege a undergoes -arrestin b arsi strongly, -arrestin b -arrestin, b - retnCtria einadt stimulate to and region C-terminal arrestin and cells the activation. to EFA6–GEF the addition for serum looking couples now that are mechanism We molecular 2008). al., et has (Morishige factor reported exchange been receptor, the EGF of phosphorylation phosphorylated EGF-dependent the no but with interact directly to been has shown GEP100/Brag2 activity. exchange the seems affect and negatively 1999) to al., et Arno/cytohesin (Santy described previously Arf1–GEF been has family the of phosphorylation (ThePKC-induced ubiquitylation EFA6 or phosphorylation of serum-activated as modifications such post-traductional Specific trigger protein. would the or receptors agonist of by the change affected structural As a conformational be from result Arf6. to could activity exchange on seem the exposure, not activity serum does EFA6–GEF EFA6 reveals of that localization the stimulus trigger not a did of serum of addition the the of internalization that noticed be Arf6 should to It led EFA6 expressing cells and in activation serum of addition study, this ro nte rGF a ensont neatwt the with interact to shown been has ArfGEF, another Arno, b A eitiuinit nrclua compartments. intracellular into redistribution 2AR r6controls Arf6 b A ntson.Ti stefrtdescription first the is This shown). (not 2AR ersnaieo he eaaeexperiments. separate are three shown of results representative The Methods. the and of in Materials described amount as was the determined (FCS) was and Arf6 serum time activated the indicated Then the h. for 5 added for (+) not serum-starved or and EFA6 RFP-tagged plasmids encoding with transfected transiently were noigRPtge F6o F6E4K(A) EFA6(E242K or EFA6 plasmids RFP-tagged with encoding transfected transiently were GFP h ro asrpeettesadr deviations. standard the ( represent bars error experiments. The independent three triplicate from in samples combination plasmid each for internal showing b cells 10 HEK293 bars: of Scale percentage h. 5 serum-staved for or (–FCS) and (+FCS) (C) serum mutants with or incubated wt Arf6 mCherry-tagged or ( compartments. internal towards the induces of Arf6 redistribution of Activation 5. Fig. B A A–F-aee tutrswsdetermined was structures 2AR–GFP-labeled E23clssal expressing stably cells HEK293 ) , C E23clssal expressing stably cells HEK293 ) b A eyln 4031 recycling 2AR b A rmtecl surface cell the from 2AR b ´ A internalization 2AR r ta. 00.A 2010). al., et ard m .The m. b 2AR–GFP b 2AR– b - Journal of Cell Science acd novn r6adAf.Hwvrarl o r1in Arf1 for The role a effector. However Arf6 Arf1. an and b as Arf6 involving Arno, that cascade Arf6 one, the suggests an second of as this EFA6, activation a one, Thus, first and 2011). a nucleotide ArfGEF, al., activator DiNitto two the et 2007; recruit al., could Stalder control arrestin et 2007; (Cohen could Arf1 al., for Arf6 et now Arno clear of that appears activity studies exchange It 2001). several al., from et (Claing overexpression after Scale antibodies. and lysosomes) endosomes for late marker 10 both a bars: for (as marker anti-Lamp2 a and marker a (as lysosomes) (as anti-CD63 anti-EEA1 FCS- endosomes), with in early stained h and for 24 fixed were After cells EFA6. medium, RFP-tagged containing encoding plasmid with transfected of accumulation the pathway. induces EFA6 6. Fig. 4032 yokltnrognzto n neihla elpolarity cell epithelial in and reorganization cytoskeleton inhibition from release recycling. the removal fast to agonist Rab4-dependent leading that of Arf6 propose of that activation in we in the Thus, demonstrating is stops increase 2009). study al., This rapid et elegant a Arf6. (Yudowski an produced of removal recent activation agonist a the with by agreement regulated of was accumulation intracellular pathway this rescue of could b active Arf6, Rab4 accumulation constitutively of of mutant The mutants intracellular compartments. receptor active the endosomal/lysosomal of or to steps EFA6 leading overexpressed later of for of presence recycling required the receptor be the Indeed, minutes, trafficking. could the 10 around Arf6 As peaked Arf6 activated Arf6GTP addition the limited. and agonist of minutes, upon not few amount activation a within is the usually occurs that internalization internalization suggesting for study, required this in EFA6 the of of Arf6 endocytosis of internalization the depletion affected Consistently, siRNA 2003). by al., et (Krauss membrane ta. 07 n yicesn h PI(4,5)P the increasing Poupart 2005; by al., and et 2007) directly (Paleotti al., by clathrin et assembly, and coat AP-2 clathrin with interacting in participate may Arf6GTP A inln n/rtafcigrmist euncovered. be to remains trafficking and/or signaling 2AR A,sgetn htteRb-eedn atrecycling fast Rab4-dependent the that suggesting 2AR, ehv rvosysonta F6i novdi actin in involved is EFA6 that shown previously have We hti h ucino h gns-nue ciaino Arf6? of activation agonist-induced the of function the is What m m. E23clssal expressing stably cells HEK293 ora fCl cec 2 (17) 125 Science Cell of Journal b b A ol ea h edo molecular a of head the at be would 2AR A a o fetdb vrxrsinof overexpression by affected not was 2AR b b A–F eetransiently were 2AR–GFP b A a niie nthe in inhibited was 2AR A ntedegradative the in 2AR b 2 A.Hwvr the However, 2AR. ee tteplasma the at level b A recycling 2AR b A nthe in 2AR b - nomto nterl fEA/r6i hsclathrin- this the any in provide as redistribution intracellular not EFA6/Arf6 However, an induces does of EFA6/Arf6 internalization. of study role expression constitutive Our the independent ligand. on in internalization of detectable information any absence the the undergo as than not the peculiar slower does be times receptor to M2 four seems but (about internalization) slow agonist-induced 2009). is Donaldson, internalization and This (Scarselli pathway the clathrin-independent agonist, of absence regulation. actin for for required not only and is binding domain arrestin C-terminal EFA6 we the the contrast, stimulation induce In to competent 2004). fully al., are b et mutants Luton Arf6GTP function the 2008; that full al., show the et to (Klein reproduce al., ability EFA6 cannot et of alone its (Macia Arf6GTP organization through while F-actin 2008), remodel presumably and required interact directly strictly is domain hs he xeiet r plotted. means are The experiments experiments. three independent these three in samples duplicate on b of 10 redistribution bars: Scale combination. in ( plasmids myc-tagged both or or Arf6(T157N) Rab4(Q67L) mCherry-tagged encoding plasmid with i.7 r6T csusra a4i the in Rab4 upstream ( acts Arf6GTP 7. Fig. Drine l,20;Fac ta. 99 uo ta. 2004; al., et Luton 1999; al., et Franco 2002; The al., et (Derrien B A A–F-aee tutrswsdtrie o ahpamdcombination plasmid each for determined was structures 2AR–GFP-labeled A nrclua eitiuinsgetn htudragonist under that suggesting redistribution intracellular 2AR uniiaino h feto a4adRb5o h Arf6-induced the on Rab35 and Rab4 of effect the of Quantification ) E23clssal expressing stably cells HEK293 ) crel n oado aercnl eosrtdta nthe in that demonstrated recently have Donaldson and Scarselli ´ r ta. 00.I hs w rcse,teEA C-terminal EFA6 the processes, two these In 2010). al., et ard b A.Tepretg fHK9 el hwn internal showing cells HEK293 of percentage The 2AR. b A osiuieyitraie na in internalizes constitutively 2AR b A–F eetasetytransfected transiently were 2AR–GFP b A eyln pathway. recycling 2AR m m. 6 ..of s.d. b - Journal of Cell Science ai recycling. rapid 8. Fig. orsodn oDlec’ oiidEgesmdu otiig1%FCS, 10% containing 100 medium Eagle’s penicillin, modified U/ml 100 Dulbecco’s penicillin, to U/ml corresponding 100 (Gibco- broth, medium phosphate BHK-21 tryptose in 10% grown FCS, 100 were 5% (BHK-21) containing cells BRL), kidney hamster antibodies Baby and reagents culture, Cell Methods and Materials to interesting be transferrin will plays the also It in JIP3/4 of role of 2011). the recruitment a recycling Arf6-dependent al., the once fast if et determine the (i.e. (Montagnac for auxilin receptor required of of recruitment was Arf6-dependent burst this JIP3/4 the that the to and associated occurred), also after uncoating became They an JIP3/4 vesicles 2011). effectors in that endocytic Arf6 al., pits the demonstrated et that clathrin-coated showed (Montagnac with have fashion associated colleagues AP-2-dependent be could and observed. Arf6GTP was occurs, Chavrier Arf6GTP activation of Arf6 Recently staining by where positive recycling membrane EEA1 plasma intracellular regulate mainly the is at EFA6 would whereas located Consistently, it to Rab4–GAP. transported a where stimulating is structures vesicles the AP-2/clathrin that endosomal speculate to to and associated we tempting is Arf6GTP constitutive It stimulation, pathway. recycling both dependent agonist control after could recycling recycling. Arf6 agonist-dependent that fast hypothesize the inhibits the of opeetdwt 200 with complemented b ftafcigt eit eetrdsniiainuo prolonged agonists. upon of desensitization stages stimulation receptor later at mediate required to effectors trafficking Arf6 of of would recruitment accumulation the regulated for endocytic timely allow same and the spatially in This subsequently vesicles. micro-domains and membrane same plasma the the within of molecules both of accumulation (here of activation a in EFA6 -Rfsdt F (HEK293- GFP to fused 2-AR ua mroi inycls(E23 eegoni E media HEK in grown were (HEK293) cells kidney embryonic Human usin eano o r6atvto slne oRab4- to linked is activation Arf6 how on remain Questions ncnlso,orrslsidct htAf satvtdby activated is Arf6 that indicate results our conclusion, In m /lsrpoyi n ML-glutamine. mM 2 and streptomycin g/ml b ceai oe fterl fAf ntecnrlof control the in Arf6 of role the of model Schematic b A)adteatvtdAf ol nueaspecific a ensure could Arf6 activated the and 2AR) A nteasneo gns n htterexpression their that and agonist of absence the in 2AR b A atrecycling. fast 2AR b b arsi-eedn anruo agonist-mediated upon manner -arrestin-dependent A.Tepyia oncinbtentecargoes the between connection physical The 2AR. m /lG418. g/ml m /lsrpoyi.HK9 tbyepesn the expressing stably HEK293 streptomycin. g/ml b A)wr anandi E medium HEK in maintained were 2AR) b 2AR lneh.Alcntut eevrfe ydul-taddDAsequencing. DNA p(mCherry-C1; double-stranded into inserted by and verified PCR were Q67L, by T157N, constructs in-frame generated and All for Arf6wt were Healthcare) mCherry-tagged Clontech). mutants (GE N-terminus. (full Arf6 pGEX-3X the into EFA6A 1–645 T27N at cloned human GST residues of and with encoding PCR (PH-Cter) fusion in-frame by 351–645 Sequences for obtained (Cter); N-terminus. to 490–645 were the sites (Sec7); cloning 125–347 at HindIII/SacII length); Macia by RFP using obtained 1999; with were pRFP-C1 al., mutant into fusion et E242K and cloned Franco wt and 2002; EFA6A PCR al., encoding Sequences N-terminus. et 2001). (Derrien the al., et at elsewhere His–Arf6 GST described GFP–EFA6A, with been EFA6A, have fusion (Thomas VSV-G-tagged in-frame His–EFA6A, Benovic for encoding Plasmids J. Healthcare) (GE and pGEX2T 319– bovine Moore into 319–418; of C. residues 383–418 encoding 357–383, by 357, Sequences provided Philadelphia). were University, Jefferson plasmids C-ter) named rne n a3Q7 a bandfo .Ehr Isiu atu,Paris, Nice, Pasteur, (Institut (I3M, Echard Cormont A. M. from by obtained was provided France). Rab35Q67L was and Rab4Q67L France) myc-tagged pcDNA3 eesprtdb 0 D-AEadtaserdt ircluoemembranes. nitrocellulose to transferred and SDS-PAGE 10% by separated were ojgtdatbde eefo ako muoeerh(neci,France). Cy5- (Interchim, and ImmunoResearch Texas-Red- Jackson FITC, from (France). were Biosciences antibodies BD conjugated from anti- were anti-EEA1 antibodies anti-CD63 and monoclonal and Mouse Heidelberg). C19) Cruz, (Santa and anti- Biotechnology goat Sainte- AC17), (K16 Bourgoin, (clone S. antibody by arrestin1 Mannheim, anti-Lamp2 provided monoclonal anti-His 8A6-2 GmbH, Mouse (clone monoclonal Canada). Arf6 Foy, Diagnostics mouse against (Clontech), mAb Roche GFP mouse (Sigma), P5D4; against antiserum (clone rabbit epitope Germany), VSV-G MO). against Louis, (St Chemical Sigma from were nucleotides, pcDNA3- constructs DNA H-1cls(2 cells TCS-SP5 BHK-21 Leica a Co-immunoprecipitation with out carried and was paraformaldehyde, Microsystems). analysis 0.3% (Leica al., et in microscope as microscopy (Franco fixed washed previously Confocal reagents then described were as transfection, 1998). cells transfection analysis after immunofluorescence the h (Roche) for stated, 24 processed otherwise 6 PBS Unless in Fugene manufacturers. twice the or by (Polyplus France) described Jet-PEI the transiently Illkirch, using were coverslips constructs glass transfection, pEGFP-N1/C1 round or mm pcDNA3 11 with on transfected plated previously cells HEK293 or as BHK-21 microscopy prepared immunofluorescence Confocal was recombinant EFA6A Prigent his-tagged 1995; and 2008). al., al., Recombinant et Arf6GDP et Franco (Macia 2003). described 2001; recombinant prepared Franco, al., were and assays, tag et (Chavrier hexa-histidine elsewhere binding C-terminal described a as vitro with wild-type in Arf6 myristoylated the proteins recombinant For of purification and Expression Fujifilm the were using of software. lysates intensity revealed AIDA using The and cell quantified system. then procedure blotting imaging total western ECL fluorescence of by LAS3000 the manufacturer’s analyzed aliquots with and anti- the chemiluminescence measure treatment, experiment siRNA to To down with GST-pull h according France). before 72 reagent removed Illkirch, after transfection, Interferin depletion (Polyplus using instruction nM) (20 siRNA (Belgium). Eurogentec from purchased were siSiah and (5 b duplex siRNA A interference RNA uentn eekp o ute nlss h eann uentn a mixed was supernatant remaining 4 The analysis. 5 further with for kept 13,000 were at supernatant centrifugation by clarified were Lysates rnfcin n05m flssbfe 2 MHps H74 %N-0 0 mM 100 NP-40, 1% 7.4, pH Hepes, after mM MgCl h [20 mM 48 buffer 1 lysis lysed of NaCl, were ml cells 0.5 alone. in pEGFP-C1 transfection, or constructs EGFP-EFA6A various GGUCUGCUCCUCAGAAGCU-3 n [ and ˚ arsi1and -arrestin1 .Tebaswr hnwse nlssbfe n muorcpttdproteins immunoprecipitated and buffer lysis in washed then were beads The C. hshlpd eefo vniPlrLpd Briga,A) [ AL), (Birmingham, Lipids Polar Avanti from were Phospholipids h olwn nioiswr sd os oolnlatbde (mAb) antibodies monoclonal mouse used: were antibodies following The E23clswr ltda 0 ofunyadtasetdwt h indicated the with transfected and confluency 40% at plated were cells HEK293 3 ]G117wr rmPri le.Aoetn spoeeo,unlabeled isoproterenol, Azolectin, Elmer. Perkin from were H]CGP12177 m fat-F nioyad20 and antibody anti-GFP of g b A-F,pcDNA3- 2AR-GFP, b arsi niois on nioiswr eetdby detected were antibodies Bound antibodies. -arrestin 6 10 b 2 arsi2(et-amre l,20) iN (5 siRNA A 2006). al., et (Gesty-Palmer -arrestin2 .5m MFadatbe fpoes niios(Roche)]. inhibitors protease of tablet a and PMSF mM 0.25 , 9 6 -ACCTGCGCCTTCCGCTATG-3 el n10m isecluedse)wr rnfce with transfected were dishes) culture tissue mm 100 in cells r6controls Arf6 b arsi1adpcDNA3- and -arrestin1 9 b a sda oto.Bt si Both control. a as used was ) arsi1wr bandb C n cloned and PCR by obtained were -arrestin1 b arsi2(1)wr rmSnaCruz Santa from were (C18) -arrestin2 m rti-–ehrs LBfr4hat h 4 for CL4B protein-A–Sepharose l b A eyln 4033 recycling 2AR g o 0mn n lqoso the of aliquots and min, 10 for 9 httresbt human both targets that ) b b arsi inl was signals -arrestin arsi1(319–418; -arrestin1 b -arrestin1/2 35 b S]GTP -arrestin c b 9 S - - Journal of Cell Science ainl el ehrh AR0-LN06-2t .. and Scientifique. M.F.] Recherche to la de [ANR-08-BLAN-0060-02 National Recherche Centre la de Nationale noguahoeSpaoebasi idn ufr(0m rsHl H7.5, pH Tris-HCl, mM (50 MgCl mM buffer 2 binding NaCl, mM in 100 beads was glutathione–Sepharose eluate of onto fragments the various containing in antibody. proteins EFA6 monoclonal fusion anti-VSV-G of using presence analysis The blot western SDS-PAGE. by on detected separated and buffer upeetr aeilaalbeoln at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.102343/-/DC1 online available material Supplementary Recherche la pour Association the by Cance Cancer; supported le contre was work the This of results. reading our Funding critical on A. comments for helpful thanked Echard, for are Montagnac A. We G. acquisition. Singer reagents. and image Karen Cormont, manuscript with providing and help M. for his Yu for Vitale May Benovic, Brau N. N. F. L. acknowledge and and gratefully Lent Chavrier J. A. P. Moore, Mallard, Benmerah, We H. experiments. C. pull-down Marchal, GST thank with C. work thank preliminary for to Clement like would We Acknowledgements (10 cells HEK293 b as transfection essentially after h assayed 24 were Briefly, HEK293- 2003). otherwise), proteins al., indicated Arf6 et (unless (Niedergang endogenous previously of described levels Arf6GTP Activation for assay down Pull (4 Arf6GDP [ MgCl mM in 10 lysed NaCl, were mM cells 100 h, 8.0, 24 pH After Tris (Roche). reagent mM VSV- 6 50 encoding Fugene plasmids using with EFA6A transfected G-tagged or mock-transfected were cells BHK-21 experiments pull-down GST 4034 lcrl MDTwt rtaeihbtr) yae eecaiidby clarified were 4 (Amersham at Lysates min beads 40 glutathione–Sepharose for inhibitors). Biotech.) to Pharmacia protease bound GST–Arf-binding-domain with of 13,000 DTT at centrifugation mM 2 glycerol, rtnX10 . MET,admaue o aiatvt sn scintillation a using radioactivity for measured and analyzer. EDTA, liquid mM 2.5 X-100, Triton 0m MgCl mM 10 ihGPbudAf n/rHstge F6(2 EFA6 His-tagged and/or Arf6 GDP-bound with ufradbudpoen ltdi 30 in eluted proteins bound and buffer iho ihu a niae ntefgr eed i-agdEA (0.5 EFA6 His-tagged legend) figure the in indicated (as without or with el eetaserdt 4 to transferred were cells ebae el eeicbtdwt 0n [ of nM 10 concentration with the incubated were measure cells To membrane, events. trafficking ottaseto.Frteitraiainasy elwr ahdwt 37 with washed were cell assay, internalization the For post-transfection. ye t4 at lysed 93 o t4 at h 5 for 1983) eyln sa,clswr nuae ih1 37 with with incubated twice were cells 1 assay, with recycling incubated and medium a and (DTT) dithiothreitol 15,000 mM at 2 centrifuged and SDS, (Roche) 4 0.005% inhibitors protease cholate, of cocktail sodium 0.05% 100, ttd n nuae o t25 at h 1 for incubated and stated) mgn ytm h muto r6T inl a uniiduigteAIDA the using (ECL quantified was fluorescence chemiluminescence signals LAS3000 Arf6GTP by Fujifilm of software. amount the antibodies anti-Arf6 the using an system, the bound revelation using imaging on blot and of loaded western procedure) by were detection Amersham Arf6GTP lysates of After starting presence the the antibody. for from assessed Arf6 and total gel of amounts Normalized GST- h rsneo .%BA hnguahoeSpaoebaswr de o h 1 for added were beads glutathione–Sepharose 25 Then BSA. at 0.5% of presence the ml n5 MTi/C p .) 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Cronin, T., M. Lee, Gabe A., Delprato, P., J. DiNitto, eie .M,An . ekwt,R .adSeo,S K. S. Shenoy, and J. R. Lefkowitz, S., Ahn, M., S. DeWire, ere,V,Culal,C,Fac,M,Mrieu . otorir . Houlgatte, P., Montcourrier, S., Martineau, M., Franco, C., Couillault, J., V., Derrien, Barhanin, S., Knauer, R., Warth, S., Bendahhou, M., Franco, S., Decressac, ’oz-coe,C,L,G,Clmo .I n th,P D. P. Stahl, and I. M. Colombo, G., Li, C., D’Souza-Schorey, li,S,Fac,M,Cadn .adLtn F. Luton, and P. Chardin, M., L. Franco, S., J. Klein, Benovic, and B. R. Penn, W., A. Gagnon, L., Kallal, oe,L . od,A,Vra,P,Bon .D,Bla .adDnlsn .G. J. P. Donaldson, Chavrier, and and C. T. D’Souza-Schorey, Balla, D., F. Brown, P., Varnai, A., Honda, A., L. Cohen, lig . hn . ilr .E,Vtl,N,Ms,J,Peot .T and T. R. Premont, J., Moss, N., Vitale, E., W. Miller, W., Chen, A., Claing, ako,C .adCsnv,J E. J. Casanova, and L. C. A. Jackson, Claing, and L. P. Boulay, T., Houndolo, lig . er,S . ciioi,M,Wle,J . laei .P,Lefkowitz, P., J. 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