Báo cáo khoa học: Differential membrane compartmentalization of Ret by PTB-adaptor engagement pdf

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Differential membrane compartmentalization of Retby PTB-adaptor engagementT. K. Lundgren, Moritz Luebke, Anna Stenqvist and Patrik ErnforsDivision of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, SwedenThe Ret receptor tyrosine kinase (RTK) has many dif-ferent functions during development and in adult life.Ret is activated upon engagement of its ligands glialcell line-derived neurotrophic factor (GDNF), neurtu-rin, persephin, and artemin, and the coreceptorsGDNF family receptors (GFRs) a1–4. DysregulatedRet is implicated in diseases such as the multiple endo-crine neoplasia (MEN) syndromes (MEN2a andMEN2b), as well as in agangliosis of the colon, one ofthe most common developmental defects in young chil-dren [1]. In the developing nervous system, neural crestcell migration depends on Ret ligands produced in thesurrounding tissue, which guide the migrating cellsto a correct position within the embryo [2]. In theKeywordsfractionation; Frs2; lipid rafts; PTB adaptors;RetCorrespondenceP. Ernfors, Division of MolecularNeurobiology, Department of MedicalBiochemistry and Biophysics, KarolinskaInstitute, 171 77 Stockholm, SwedenFax: +468 341960Tel: +468 52487659E-mail: patrik.ernfors@ki.se(Received 14 December 2007, revised 23February 2008, accepted 26 February 2008)doi:10.1111/j.1742-4658.2008.06360.xGlial cell line-derived neurotrophic factor family ligands act through thereceptor tyrosine kinase Ret, which plays important roles during embryonicdevelopment for cell differentiation, survival, and migration. Ret signalingis markedly affected by compartmentalization of receptor complexes intomembrane subdomains. Ret can propagate biochemical signaling fromwithin concentrates in cholesterol-rich membrane microdomains or lipidrafts, or outside such regions, but the mechanisms for, and consequencesof, Ret translocation between these membrane compartments remain lar-gely unclear. Here we investigate the interaction of Shc and Frs2 phos-photyrosine-binding domain-containing adaptor molecules with Ret andtheir function in redistributing Ret to specialized membrane compartments.We found that engagement of Ret with the Frs2 adaptor results in anenrichment of Ret in lipid rafts and that signal transduction pathways andchemotaxis responses depend on the integrity of such rafts. The competingShc adaptor did not promote Ret translocation to equivalent domains, andShc-mediated effects were less affected by disruption of lipid rafts. How-ever, by expressing a chimeric Shc protein that localizes to lipid rafts, weshowed that biochemical signaling downstream of Ret resembled that ofRet signaling via Frs2. We have identified a previously unknown mecha-nism in which phosphotyrosine-binding domain-containing adaptors, bymeans of relocating Ret receptor complexes to lipid rafts, segregate diversesignaling and cellular functions mediated by Ret. These results reveal theexistence of a novel mechanism that could, by subcellular relocation ofRet, work to amplify ligand gradients during chemotaxis.AbbreviationsCO, cholesterol oxidase; CTB, cholera toxin B; DRG, dorsal root ganglia; DRM, detergent-resistant membrane; E, embryonic day; eGFP,enhanced green fluorescent protein; ERK, extracellular signal-related kinase; GFR, glial cell line-derived neurotrophic factor family receptor;GNDF, glial cell line-derived neurotrophic factor; HA, hemagglutinin; MAPK, mitogen-activated protein kinase; MCD, methyl- b -cyclodextrin;MEN, multiple endocrine neoplasia; MLS, membrane localization signal; PI3K, phosphoinositide-3-kinase; PTB adaptor, phosphotyrosine-binding domain-containing adaptor; PVDF, poly(vinylidene difluoride); RTK, receptor tyrosine kinase; SUP, detergent-soluble supernatant;TfR, transferrin receptor.FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2055developing and adult mouse, Ret is also instrumentalin promoting survival of neurons, including parasym-pathetic neurons and dopaminergic neurons of the sub-stantia nigra pars compacta [3–5].Many of the functions regulated by Ret activationdepend on an intact Tyr1062 in the intracellulardomain [6,7]. Upon Ret phosphorylation, this tyrosinemediates biochemical signal transduction via interac-tion with adaptor proteins. Several phosphotyrosine-binding domain-containing adaptors (PTB adaptors)compete for interaction with Tyr1062, but only oneadaptor may interact at any given time [8]. The dis-tinct functional outcome of Ret activation is corre-lated with the different PTB adaptors interacting withRet [9,10]. We have shown previously that expressionof Ret mutants, selective for binding to either theadaptor protein Shc or Frs2 to Tyr1062, results indistinctly different patterns of plasma membranelocalization of Ret. Frs2 recruitment resulted in aconcentration of Ret into membrane foci [4,11]. TheRet receptor has recently been shown to signal fromwithin different cellular compartments. The oncogenicprecursor of Ret (Men2B) can be activated andinduce downstream signaling from within the endo-plasmic reticulum [12]. Ret can also be recruited tospecialized lipid raft domains in the plasma mem-brane, where it can be phosphorylated, interact withadaptor proteins and induce downstream signaling[12–14].Lipid rafts are membrane microdomains, rich insphingolipids and cholesterol, that form lateral assem-blies in the plasma membrane [15]. Lipid rafts seques-ter a number of different proteins that arepalmitoylated or contain a number of other lipidanchors, which may be regulated by the selectiveinteraction with these domains. In this way, therecruitment of Ret to lipid rafts can lead to the acti-vation of distinct signaling pathways, due to the com-partmentalized cell signaling events. However, themechanism for directing Ret into lipid rafts remainslargely unknown. We report here that the PTB adap-tor Frs2 functions to translocate Ret to membranesubdomains of the lipid raft type. Interactions of Retwith the Shc adaptor, which, in contrast to Frs2,lacks a palmitoylation tail that confers attachment tolipid rafts, did not result in redistribution of Ret tolipid rafts. Targeting the Shc adaptor to lipid rafts byengineering the adaptor with a raft targeting tail ledto altered biochemical signaling resembling, in severalrespects, signal transduction downstream of Frs2. Weshow that the distinct biological outcomes of Ret acti-vation largely depends on the targeting to, and signal-ing from within, lipid rafts.ResultsA PTB-adaptor-dependent membrane relocationof Ret receptorsTo investigate whether Tyr1062 is important for Rettranslocation into lipid rafts, we performed crudefractionations of neuronal SK-N-MC cells transfectedwith the MEN2a version of Ret (2aRet). The MEN2amutation C634R is found in more than 85% ofpatients with MEN syndrome, and renders Ret con-stitutively active, thus omitting the need for ligandfor its activation. Lipid rafts and nonraft membraneswere isolated according to their resistance to and sol-ubilization by detergent. Cells expressing2aRet or a2aRetY1062Fmutant that is incapable of adaptor inter-action with the phosphorylated Tyr1062 were har-vested in detergent, and the detergent-resistantmembrane (DRM) fraction was separated from thedetergent-soluble supernatant (SUP) fraction by cen-trifugation. Each fraction was subjected to PAGEand transferred to poly(vinylidene difluoride) (PVDF)membranes. Immunoblotting against Ret revealed thatthe majority of Ret partitioned in the DRM fraction.This was in contrast to the2aRetY1062Fmutant, wherethe majority of Ret was found in the SUP fraction(Fig. 1A). To test whether Ret distribution wasaffected by interaction with either the Frs2 or the Shcadaptor, we overexpressed Shc or Frs2 constructstogether with2aRet in SK-N-MC cells. Lysates fromcells overexpressing Shc or Frs2 were divided intoequal halves, and each half was immunoprecipitatedfor Shc or Frs2 and immunoblotted against Ret.Nearly all Ret precipitated with the overexpressedadaptor, showing that overexpression of one adaptorleads to outcompetition of the other with respect toRet interaction (Fig. 1B). Next, we fractionatedlysates of cells expressing2aRet or2aRetY1062Ftogether with overexpressed Frs2 or Shc into DRMand SUP fractions. Adaptor overexpression led tohigh amounts of Ret in the DRM fraction in bothFrs2 and Shc conditions, whereas2aRetY1062F,asbefore, displayed a much lower partitioning intoDRMs (Fig. 1C). Ret localization to the supernatantfraction was largely unaffected by the Y1062 muta-tion in the presence of Frs2 or Shc. To confirm therelative purity of raft and nonraft membranes, DRMand SUP fractions were immunoblotted towards thelipid raft marker flotillin-1 and the transferrin recep-tor (TfR), the latter of which is excluded from raft-like domains. As expected, flottillin-1 was foundnearly exclusively in the DRM fraction, whereas TfRwas mainly found in the SUP farction (Fig. 1C).Ret PTB-adaptor translocation to rafts T. K. Lundgren et al.2056 FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBSFurthermore, the DRM and SUP fractions were im-munoblotted towards the adaptor proteins themselves.Both Shc and Frs2 were found predominantly in theDRM fractions (Fig. 1D). These results suggest thatRet localization to nonraft membrane regions occursindependently of adaptor engagement, whereas itslocalization to lipid rafts depends on Shc or Frs2interactions with Ret Tyr1062.To examine whether Ret and adaptor partitioningis affected by disruption of lipid-ordered regions, weexposed cells to methyl-b-cyclodextrin (MCD), anagent that is commonly used to extract cholesterolfrom cell membranes in order to disrupt lipid-orderedregions in the plasma membrane. Cells were treatedfor 30 min with 8 mm MCD or vehicle prior to frac-tionation and subsequent immunoblotting towardsRet. The amount of Ret present in the DRM fractionwas significantly reduced, with a correspondingincrease of Ret in the SUP fraction, when cells weretreated with MCD (Fig. 1D). This indicates that theintegrity of cholesterol-rich regions is necessary forRet partitioning into DRMs, agreeing with previousdata [14].The insoluble material recovered in the DRM frac-tion represents a collection of many discrete lipid-ordered structures, and is not exclusively composed oflipid rafts [16]. Previous studies have found that Retpartitions into DRM fractions upon ligand induction[14], and that interaction of Ret with the Frs2 adap-tor, but not the Shc adaptor, occurs in such fractions[13]. To further investigate whether the previouslydetermined localization of Ret to membrane foci,depending on adaptor engagement [11], had any rela-tion to lipid rafts, we expressed Ret tagged withenhanced green fluorescent protein (eGFP) (ReteGFP)in cells. Fusion of eGFP to Ret allows direct visuali-zation of the subcellular distribution of Ret. ReteGFPwas expressed with Shc or Frs2 in SK-N-MC neuro-nal cells that were stained for visualization of themembrane lipid rafts using a fluorescently conjugatedcholera toxin B (CTB) subunit that binds to the pen-tasaccharide chain of plasma membrane gangliosideGM1, which selectively partitions into lipid rafts [17].Using confocal microscopy, we found that there wasa consistent punctuate pattern of lipid rafts with highabundance in neurites after 30 min of ligand stimulus.Interestingly, extensive localization of ReteGFPto lipidrafts was seen only in Frs2-expressing cells, and notin cells expressing Shc (Fig. 2A,C). The distinct recep-tor localization was also correlated with morphologi-cal differences between Shc- and Frs2-expressing cells;Frs2-expressing cells often contained markedly morecell processes and neurites than Shc-expressing cells.Frs2 is exclusively localized to lipid rafts [18], whereasShc has been shown to inducibly localize to lipid-ordered regions in some instances, e.g. in T-cell recep-tor signaling [19]. To examine whether lipid rafttargeting of Shc could mediate translocation of Retto a subcellular localization similar to that mediatedby Frs2, we expressed an Shc construct containingthe Ras membrane localization signal (MLS)(ShcMLS), which permanently localizes Shc to lipidrafts [20]. This Shc construct was shown in previouswork to activate the mitogen-activated protein kinase(MAPK) pathway constitutively [20]. Intriguingly,sustained activation of MAPK is one prominent dis-tinguishing feature of Ret signaling by Frs2 recruit-ment, in contrast to Ret signaling via Shc [4]. WhenShcMLSwas overexpressed in cells along withReteGFP, the membrane localization of Ret was lar-gely confined to lipid raft regions, similar to that ofReteGFP- and Frs2-expressing cells (Fig. 2B), indicat-ing that lipid raft targeting of Shc leads to a redistri-bution and enrichment of Ret in lipid rafts.ACDBFig. 1. Tyr1062 is necessary for Ret partitioning into lipid rafts. (A)Mutation of Tyr1062 results in a loss of2aRet partitioning to theDRM fraction.2aRetWTor2aRetY1062Fwas expressed in SK-N-MCcells. Cells were harvested in 1% Triton buffer, separated intoDRM or SUP fractions, separated on polyacrylamide gels, andtransferred to PVDF membranes, with subsequent blotting for Ret(n = 3 with similar results). (B) Overexpression of Shc or Frs2 adap-tors forces2aRet to interact with either adaptor at the expense ofthe other. Lysates of SK-N-MC cells were separated, and each halfwas immunoprecipitated for Shc or Frs2 and immunoblottedagainst Ret (n = 2). (C) Overexpression of Shc or Frs2 adaptorsresults in2aRetWTbut not2aRetY1062Fpartitioning into DRM frac-tions. SK-N-MC cells were treated and harvested as in (A), and blot-ted for detection of Ret (n = 3). (D) Shc and Frs2 partitioning of Retto the DRM fraction depends on intact lipid rafts. SK-N-MC cellsexpressing2aRetWTand2aShc or Frs2 were treated with MCD orvehicle, harvested, and separated into DRM and SUP fractions.Immunoblot towards Ret and Frs2 or Shc as indicated (n = 3).T. K. Lundgren et al. Ret PTB-adaptor translocation to raftsFEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2057A critical role of Ret membrane localizationin downstream signaling events in cell linesand primary cellsWe next examined the intracellular signaling down-stream of Ret in cells expressing the Shc, ShcMLSorFrs2 adaptors. Phosphorylated (active) Akt or p42,44extracellular signal-related kinase (ERK) MAPK pro-tein levels were examined by immunoblotting at differ-ent time points after Ret ligand stimuli. ShcMLSexpression together with Ret resulted in signal activa-tion resembling the Ret ⁄ Frs2 coexpression characteris-tics. Specifically, a higher and sustained ERK p42,44MAPK level was also observed at 12 h in cells express-ing ShcMLSas compared to Shc (Fig. 3A). Further-more, Ret signaling via ShcMLSdid not lead to asrobust activation ⁄ phosphorylation of Akt as did Shc,but levels were higher than for signaling via Frs2(Fig. 3C). To investigate the importance of rafts fordownstream ERK p42,44 MAPK activation, weapplied cholesterol oxidase (CO) to cells during ligandstimuli. CO was chosen because cells cannot withstandMCD treatment for long time periods, and CO hasbeen shown to disrupt the biochemical effects of mem-brane-localized Ret as well as other receptor complexesin a fashion similar to what is accomplished by usingMCD [11,21,22]. In the presence of CO, ERK p42,44MAPK activation was markedly reduced both in cellsexpressing Ret ⁄ ShcMLSas well as in cells expressingRet ⁄ Frs2 at all time points, whereas activation ofERK p42,44 MAPK downstream of Shc was much lessaffected (Fig. 3A). The specificity of cholesterol speciesfor phosphorylated ERK p42,44 MAPK was alsoimportant in MEN2a versions of Ret when expressedwith Shc or Frs2. In cells expressing2aRet, phosphory-lated ERK p42,44 MAPK levels were high when Shcand Frs2 were coexpressed. CO treatment attenuatedphosphorylated ERK p42,44 MAPK levels down-AA'BB'CC'Fig. 2. Labeling of plasma membrane gan-glioside GM1 lipid rafts shows extensivecolocalization of Frs2-associated but notShc-associated Ret to lipid rafts. (A–C)SK-N-MC cells expressing ReteGFP(green)together with Shc (A,A¢), ShcMLS(B,B¢)orFrs2 (C, C¢). Actin was stained with Alexa-648 phalloidin and Alexa-546 (red) conju-gated to CTB to mark lipid rafts (n = 5).Arrows indicate colocalization of ReteGFPwith lipid rafts. Scale bar = 25 lm.Ret PTB-adaptor translocation to rafts T. K. Lundgren et al.2058 FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBSstream of Shc and led to an almost complete absenceof phosphorylated ERK p42,44 MAPK downstream ofFrs2 (Fig. 3B).Most studies on receptor signaling from within raftshave been performed on immortalized cell lines in cul-ture or by using artificial membranes. We attempted tosee whether the PTB adaptors determine Ret subcellu-lar distribution and signaling in primary neurons of amore complex system. To this end, we electroporatedReteGFPand either adaptor into the neural tube ofchicken embryos. DNA was injected in ovo into theneural tube of embryos at Hamburger–Hamiltonstage 11 [approximately embryonic day (E)2], and elec-troporation was performed by placing electrodes alongthe rostral–caudal axis of the neural tube. The egg wasthen closed and placed in an incubator until theembryos had grown to stage 24 (E4.5). At this stage,the expression of transfected ReteGFPwas found in thedeveloping spinal cord and in dorsal root ganglia(DRG), and the transfected tissue was dissected underfluorescent light (supplementary Fig. S1). eGFP-posi-tive spinal cord segments were pooled and weighed,and equal amounts of tissue were trypsinized into sin-gle cells and immediately incubated with Ret ligandsfor 30 min. After ligand stimulation, the cells werefractionated into DRM and SUP fractions. In accor-dance with the results obtained with neuronal SK-N-MC cells, immunoblotting against Ret revealed that itwas predominantly located in DRM fractions, regard-less of which adaptor (Shc, ShcMLS, or Frs2) was con-comitantly overexpressed (Fig. 3D). Immunoblottingof DRM and SUP fractions against the adaptor pro-teins showed that ShcMLSand Frs2 were nearly exclu-sive to the DRM fraction, whereas Shc was presentalso in the SUP fractions (Fig. 3E).We investigated the activation of MAPK and Aktpathways in the developing chick embryo. Previousstudies have found that the ERK MAPK pathway [andto a lesser extent, also the phosphoinositide-3-kinase(PI3K) ⁄ Akt pathway] can be temporally and quantita-tively affected by activation within or outside DRMfractions [23]. To examine the role of the PTB adaptorsin the activation of the MAPK and Akt pathways,spinal cords electroporated with Ret together witheither Shc, ShcMLSor Frs2 were separated into DRMand SUP fractions, as above. Phosphorylated Akt lev-els were generally low for all PTB adaptors in theDRM fraction, showing that phosphorylated AktACDFGEBFig. 3. The raft-targeting adaptors Frs2 and ShcMLSshow enhanced ERK phophorylation, which is dependent on the integrity of cholesterol-rich domains. (A) SK-N-MC cells expressing Ret and either Shc, ShcMLSor Frs2 adaptors were lysed after Ret ligand stimulation for indicatedtimes and subsequently immunoblotted towards phosphorylated ERK and all ERK. CO was applied to cells as indicated. (B) Experiments asin (A) with MEN2a versions of Ret. (C) Experiments as in (A) with immunoblotting towards phosphorylated Akt and Akt. (D) E2 chickenembryos were electroporated in ovo with Ret and adaptor constructs as indicated, and allowed to develop to E4.5. At E4.5, positively trans-fected spinal cord segments were dissected out and pooled to equal amounts. After dissociation into single cells and stimulation with Retligands for 30 min cells, were lysed and fractionated into DRM and SUP fractions. Fractions were immunoblotted for detection of Ret. (E)Experiments as in (C) with immunoblotting for the hemagglutinin (HA)-tag of Shc and ShcMLSor for Frs2. (F, G) Experiments as in (C) withimmunoblots towards phosphoryated Akt or phosphorylated ERK.T. K. Lundgren et al. Ret PTB-adaptor translocation to raftsFEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2059signaling predominantly takes place in cellular struc-tures partitioning into the SUP fraction (Fig. 3G).Phosphorylated Akt levels were greater in the SUPfraction when Shc or ShcMLSwas expressed with Retas compared to Frs2, where levels were overall lowerand more similar between the DRM and SUP fractions(Fig. 3G). Phosphorylated ERK p42,44 MAPK immu-noblotting revealed the strongest signal when Frs2 wasexpressed with Ret in both the DRM and the SUPfractions (Fig. 3F). ShcMLSresulted in activation ofERK p42,44 MAPK in the DRM. Shc, on the otherhand, was very poor in mediating ERK p42,44 MAPKactivation in the DRM fraction (Fig. 3G). These resultstherefore agree with the results obtained with SK-N-MC cells, and further support the idea that phos-phory lated ERK p42,44 MAPK is often localized tocomplexes of PTB adaptors with Ret in lipid rafts andthat phosphorylated Akt is mostly found outside rafts.To characterize the Ret distribution in greater detailthan is permitted by DRM ⁄ SUP fractionation, we useda recently published fractionation protocol that omitsthe need for detergent and is highly specific withregard to molecular distribution within and outsidelipid raft fractions [24]. Dissected spinal cords fromchick embryos were lysed in detergent-free bufferand fractionated by ultracentrifugation in OptiPrepgradients. Seven fractions were aspirated fromcolumns, and the protein content within each fractionwas concentrated by precipitation, loaded on poly-acrylamide gels, and transferred to PVDF membranes.The separation of raft fractions from nonraft fractionswas confirmed by immunoblotting against flotillin-1and TfR (Fig. 4). As determined by immunoblottingagainst Ret, this more sensitive method revealed thatthe fractional distribution of Ret varied with the adap-tor expressed in the embryos. Frs2 overexpression ledto Ret being directed towards lower-density fractions(corresponding to lipid rafts), peaking in fraction 2.Shc overexpression resulted in significantly less Retpartitioning into the fractions of lowest density, withlittle or no Ret in fraction 2 and peak levels in frac-tion 3 (Fig. 4). Overexpression of the raft-localizingShcMLSconstruct resulted in a significant portion ofRet being partitioned into both fraction 2 and frac-tion 3. Immunoblotting against the adaptors them-selves showed that whereas both Shc and ShcMLSwerepresent in both low-density and high density fractions,Frs2 was nearly exclusive to the same fraction as Ret(fraction 2) (Fig. 4).Functional consequences of signaling from raftand nonraft membrane compartmentsAssociation of Ret with the different adaptors mayresult in distinct cellular responses to Ret ligand stimu-lation [4,9]. In functional terms, the chemotactic prop-erties of Ret signaling via Frs2 are much greater thanthose of Ret signaling via Shc [11]. Ret signaling viarecruitment of Shc, on the other hand, is necessary forRet-mediated cell survival when neuronal cells are pre-sented to toxic agents, and also for neurite formationin certain cell types [4,9]. We investigated how disrup-tion of lipid-ordered domains affected these functionalaspects, and whether the ShcMLSadaptor is similar toFrs2 or Shc in terms of cellular response to Ret stimu-lation. Chemotactic migration towards Ret ligands wasexamined by seeding neuronal SK-N-MC cells express-ing Ret receptors that are selective for binding of onlyFrs2 (RetFrs+) or Shc (RetShc+) to the region ofTyr1062 [4]. Ret mutants binding to Frs2 showed anearly three-fold higher migrational capacity than didRetShc+, and this effect was ligand-dependent, as allconditions displayed a similar low level of random cellmigration without ligand being supplied to the lowerculture compartment (Fig. 5A). Cells expressingRetShc+⁄ Shc did not show any statistically significantligand-induced migration. Cells expressing ShcMLStogether with RetShc+displayed a two-fold increase ofmigration towards Ret ligand (Fig. 5A). The sameexperiment was then performed in the presence of CO.AFig. 4. Density fractionation of the membrane localizes Frs2 andShc partly to different membrane compartments. (A) E2 chickenembryos were electroporated in ovo and allowed to develop to E5.At E5, positively transfected spinal cord segments were dissectedout and pooled to equal amounts of input. After dissociation intosingle cells and stimulation with Ret ligands for 30 min, cells wereharvested in detergent-free buffer and subjected to ultracentrifuga-tion in OptiPrep density gradients. Fractions were taken out aftercentrifugation, and concentrated protein from each fraction wasimmunoblotted against Ret or HA-tag for Shc and ShcMLSoragainst Frs2, as indicated. Fraction 1 is the less dense fraction, andfraction 7 is the fraction with the highest density. The bottom panelshows immunoblotting for the lipid raft marker flotillin-1 and thenonraft marker TfR.Ret PTB-adaptor translocation to rafts T. K. Lundgren et al.2060 FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBSCO treatment of cells led to a large decrease in thenumber of migrating cells, almost reaching baselinelevels in all conditions (Fig. 5A), indicating that theintegrity of cholesterol-rich membrane domains is nec-essary for the occurrence of directional migrationtowards Ret ligand.The ability of Ret selective mutants to promote cellsurvival was next examined. SK-N-MC cells treatedwith anisomycin to induce apoptosis were rescued bysupplementing the medium with Ret ligands. To detectthe apoptotic response to anisomycin with high sensi-tivity, single cells were examined in the comet assay. Inthis system, fragmented DNA moves out of the cellsoma in the shape of comet tails when cells are embed-ded in agarose and subjected to an electric field. Afterstaining of DNA with SYBR-green, images were cap-tured using fluorescence microscopy. The comet-tailmoment was determined by measuring pixel intensityin the comet head and tail, and calculating themomentum of the comet tails [25]. In accordance withwhat was previously found [4], Ret ligands resulted ina much greater cell survival effect for cells expressingRetShc+than for those expressing RetFrs+(Fig. 5B,C).When ShcMLSwas coexpressed with RetShc+toenforce Shc signaling in lipid rafts, an intermediatesurvival-promoting effect was seen (Fig. 5B,C). Dis-ruption of cholesterol-rich membrane compartmentswith CO had no effects on cell survival (Fig. 5B,C).Furthermore, CO alone without anisomycin did notresult in any detectable cell injury (Fig. 5B,C), suggest-ing that CO at the concentrations used does not havean effect on cell survival. Thus, these data suggest thatRet-mediated cell migration, but not cell survival,requires intact lipid rafts.DiscussionThe present study was conducted to determine whetherPTB adaptors determine Ret localization to differentmembrane compartments, and whether pathway-spe-cific signaling and functional outcomes are affected bysignaling from within and outside rafts. It has previ-ously been shown that Ret localizes to lipid rafts ina glycophosphatidylinositol-bound GFRa1 coreceptor-dependent fashion [26]. However, more recent resultshave demonstrated that both soluble and glycophos-ABCFig. 5. Frs2- dependent chemotaxis but not Shc-dependent cellsurvival depends on raft integrity. (A) SK-N-MC cells expressing Retmutants were analyzed in transwell chemotaxis assays after 14 h,with Ret ligands supplied below the membrane and CO suppliedabove and below the membrane. Two-wayANOVA compared to themaximally migrating RetFrs+⁄ Frs2 condition, ***P < 0.001 (n = 3).(B) SK-N-MC cells were treated with anisomycin and CO as indi-cated, and subjected to the comet assay. (C) Quantification of (B).The cell-rescuing effect of Ret ligands against the apoptosis-induc-ing agent anisomycin was measured by quantifying apoptosis ⁄ DNAdamage expressed as comet-tail momentum. Two-wayANOVAcompared to the maximally rescuing RetShc+⁄ Shc condition,**P < 0.01, *P < 0.05 (n = 3). Scale bar in (B) = 50 lm.T. K. Lundgren et al. Ret PTB-adaptor translocation to raftsFEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2061phatidylinositol-anchored GFRa1 increase Ret distri-bution to rafts [13]. In the same study, Frs2 and Shcadaptor engagement with Tyr1062 was found to occurpredominantly within and outside rafts, respectively.We report here that PTB adaptor engagement is criti-cal for Ret to localize to lipid rafts, suggesting thatupon ligand engagement, the PTB adaptor associationresults in a movement of Ret from nonraft to raftmembrane regions. Consistently, phosphorylated ERKp42,44 MAPK signaling by a MEN2a form of Ret,which signals even in the absence of ligands andGFRa1, was more sensitive to disruption of choles-terol-rich membranes by CO upon Frs2 as comparedto Shc recruitment. Our data also suggest that recruit-ment of Ret to rafts by PTB adaptors results in dis-tinct signal transduction patterns. The biochemicalintegrity of Ret signaling depends on undisruptedlipid-ordered domains when Ret assembles togetherwith adaptors localizing to rafts, but less so when thecomplex is localized outside rafts. Furthermore, amodified version of the Shc adaptor that localizes tolipid rafts in a similar way to Frs2 results in signalingresembling Frs2 recruitment by Ret.Our results show that Ret resides largely in DRMfractions both in cell lines and in vivo in the chickspinal cord. This localization was critically dependenton Tyr1062 and its interaction with Frs2 and Shc,because eliminating interactions at this site resulted ina clear loss of Ret in the DRM fraction and anincrease in the SUP fraction. Cyclodextrins such asMCD effectively remove cholesterol from the plasmamembrane [27]. This property has led to the extensiveuse of MCD to study the function of lipid rafts, whichare membrane microdomains whose integrity dependson the presence of cholesterol. In this article, we showthat cholesterol depletion by MCD results in a loss ofboth Frs2- and Shc-induced increases of Ret in theDRM fraction.Analysis of the DRM and SUP fractions suggestedthat both Shc and Frs2 reside largely in the DRMfraction, and that signaling from Ret by means of Shcand Frs2 might be initiated from within lipid raftmembrane compartments. However, with the use of amore sensitive density-dependent fractionation, it wasclear that Ret associated with Frs2 and Shc resides indifferent membrane compartments, with Ret interac-tions with Frs2 being more strongly associated withthe flottilin-containing fractions, which are believed toinclude lipid rafts [28]. These experiments also showedthat, unlike Shc, which is present in very low amountsin the Frs2-associated Ret fractions, ShcMLSis locatedin both raft and nonraft fractions. Details of the lipiddistribution of the sphingolipid- and cholesterol-richlipid rafts are not well characterized. Clearly, there is agreat spatial and functional heterogeneity of thesemembrane domains. Recently, it was found thatplasma membrane sphingomyelin-rich domains arespatially distinct from ganglioside GM1-rich mem-brane domains in Jurkat T cells, and may form distinctand unique signaling platforms [29]. Distinct cellularlocalization of Shc and Frs2 with Ret ⁄ Frs2 but notRet ⁄ Shc localized to GM1-rich lipid rafts was alsoconfirmed using CTB labeling. In this experiment, aclear colocalization of ReteGFPto lipid rafts in thepresence of Frs2 but not Shc was evident.We noticed a high baseline activity without ligand inFrs2 conditions throughout our study. This is consis-tent with previous results on ERK MAPK and onother downstream effector proteins activated via Frs2,and is most likely due to the sustained interaction ofFrs2 with Ret and also other RTKs [4,30]. In a recentstudy, we have shown that selective interaction of Retwith Shc results in the activation of AKT to a muchgreater extent than when Ret signals by recruitment ofFrs2, and conversely that, unlike Shc signaling, Frs2signaling leads to ERK p42,44 MAPK activation athigh levels. Interestingly, the difference in signaling wasreflected not only by more robust activation, but alsoby a significantly sustained activation from 5 min to atleast 12 h. Signaling by Ret via Shc activates ERK to alesser degree, and peaks at about 30 min [4]. Severallines of evidence suggest that the sustained activationof ERK is dependent on the raft context rather than itbeing PTB adaptor-specific. Signaling via Frs2 afterdisruption of the rafts by CO resulted in a markedattenuation ERK MAPK signaling, with a duration ofminutes instead of hours. Furthermore, recruitment ofShc to lipid rafts by introduction of the Ras membranelocalization signal resulted in elevated and sustainedERK p42,44 MAPK activation, similar to that seen forFrs2. Whereas activation of ERK p42,44 MAPK bythe normal Shc adaptor was less attenuated by lipidraft disruption, ERK p42,44 MAPK activation byShcMLSwas dependent on intact rafts, and when therafts were disrupted, ERK p42,44 MAPK activationwas almost completely absent. On the basis of theseresults, we conclude that sustained ERK p42,44MAPK signaling by Ret depends on intact lipid rafts.It is interesting to note from our in vivo data result-ing from the chick experiments that Shc activates Aktalmost exclusively outside of rafts, with there beinglittle ERK p42,44 MAPK activation either within oroutside of rafts, as seen by its partitioning into theDRM fraction. In contrast, whereas ShcMLSactivatesAkt mostly outside of rafts, its activation of ERKp42,44 MAPK was almost exclusively within theRet PTB-adaptor translocation to rafts T. K. Lundgren et al.2062 FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBSDRM raft fraction. Frs2 activation of Akt was overallvery weak, whereas ERK p42,44 MAPK activationwas strong in both the DRM fraction and in the non-raft SUP fraction. Because our density-dependentfractionation and colabeling of Ret with lipid rafts incellular staining both suggest that Ret associated withFrs2 and Shc resides in different membrane compart-ments, our results suggest that Akt activation mayresult largely from nonraft activation of Shc by Ret.Unlike Akt, ERK p42,44 MAPK, which is activateddownstream of Frs2, appeared to localize both to raftmembranes and to nonraft membrane fractions.Because Ret association with Frs2 is almost exclusiveto the lipid rafts, this suggests that ERK MAPK par-titioning into nonraft fractions is presumably initiatedfrom within the raft. It is interesting that ERK p42,44MAPK signaling resulting from ShcMLSassociationwith Ret takes place exclusively in the DRM fraction,suggesting that, unlike signaling downstream of Frs2,components of the ERK MAPK signaling pathwaystay associated with the raft-localized ShcMLSalsoafter activation.Both Shc and Frs2 have been implicated in cellmigration, and we cannot exclude a role also forPI3K ⁄ Akt signaling from Ret. This pathway has previ-ously been implicated in such cellular functionsmediated by Ret in, for example, kidney epithelialMadin–Darby canine kidney cells [31]. However, ourfindings suggest that PI3K⁄ Akt signaling is not suffi-cient for cell migration by itself in the absence ofMAPK ERK signaling via Frs2. Directional signalingonto RTKs at the leading edge appears to be criticalfor chemotaxis, and defines the direction of actin poly-merization and subsequent cell migration. It is notclear how the cells measure gradients of RTK ligandsresulting in gradients of receptor activation along thesurface that are translated into polarization of thecytoskeleton, extension of cell processes, and eventu-ally translocation of the cell body. Our results showthat Ret-mediated chemotaxis is critically dependenton lipid rafts, whereas cell survival signaling via Shc isnot significantly affected by CO. This is consistent withthe conclusion that Shc activation by Ret may takeplace in nonraft membrane regions, similar to whathappens with many other tyrosine kinase receptors[13,32], whereas Frs2, which is necessary for Ret-elic-ited directional migration, is activated in raft-like foci[11,18]. Our results do not allow us to distinguishwhether the effects of CO on migration result directlyfrom the loss of ERK signaling or from the physicallocalization of Ret to the growing axons, as seen bythe Ret colocalization with CTB. As for other RTKs,Ret-stimulated migration is dependent on ERK signal-ing, as blocking of this pathway prevents Ret-inducedmigration [11,33]. However, this does not exclude thepossibility that that a raft-dependent localization ofRet to filopodia ⁄ lamellipodia may also be importantfor directional migration and axonal extension. Consis-tent with this hypothesis are data showing that localstimulation of cells expressing the epidermal growth fac-tor receptor with a bead soaked in epidermal growthfactor leads to ERK activation spreading throughoutthe cell, whereas actin polymerization remains local [34].Our results open the possibility that Frs2-dependentrecruitment of Ret receptors to lipid rafts upon ligandengagement may participate in increasing receptor levelsin the direction of increasing ligand concentrations. Thismay provide a molecular mechanism for cellular amplifi-cation of ligand gradients that could play importantroles in directed cell migration.Experimental proceduresCell culture, DNA constructs, and mutagenesisAll Ret mutants were harbored and expressed in PJ7W plas-mids and subcloned into peGFP vectors (Clonetech Inc.,Mountain View, CA, USA) to make fluorescent constructs,as described previously [11]. SK-N-MC cells were main-tained in DMEM supplemented with 10% fetal bovineserum, 2% horse serum and 1 mm glutamine. Starvationswere done in DMEM containing 0.5% total serum. Allligand stimulations were performed for 30 min unless statedotherwise, using 50 ngÆmL)1recombinant human GDNFand 100 ngÆmL)1recombinant human GFRa1 ⁄ FC chimera(R&D Systems, Minneapolis, MN, USA). CO (Sigma,Munich, Germany) was used at 8 mm, as previouslydescribed [21]; specifically, cultured cells were incubated withCO at 1.8 UÆmL)1for 1 h prior to and during ligand stimu-lation. Anisomycin (Sigma) was applied to cells 30 minbefore ligand application at a final concentration of12 lgÆmL)1, and cells were incubated for another 2 h beforeexamination. Transfections were performed using Lipofecta-mine LTX (Invitrogen, Karlsruhe, Germany), according tothe manufacturer’s instructions. Growth medium wasreplaced approximately 7 h after transfection. Transfectionefficiency was continuously monitored by eGFP fluores-cence.Antibodies and reagentsAntibodies against Ret (Ret H-300), and phosphotyrosine(PY99) were obtained from Santa Cruz Biotechnology(Santa Cruz, CA, USA). Antibodies against HA tags werefrom BD Biosciences (CA, USA). Antibodies against Frs2were from Sigma. Antibodies against flotillin-1 were fromTransduction Labs (Lexington, KY, USA). AntibodiesT. K. Lundgren et al. Ret PTB-adaptor translocation to raftsFEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2063against TfR were from Zymed (San Francisco, CA, USA).Antibodies against Akt, phosphorylated Akt Ser473,p44 ⁄ 42 MAPK and phosphorylated p44 ⁄ 42 MAPK werefrom Cell Signalling (Hitchin, UK). Alexa-546-conjugatedGM1-CTB was from Molecular Probes (Leiden, the Neth-erlands), and was used according to the manufacturer’sinstructions. After staining with GM1-CTB, cells werefixed in 4% paraformaldehyde and examined onZeiss LSM 5 exciter microscopes with axiovision software(Zeiss, Karlsruhe, Germany).Immunoblotting and SDS ⁄ PAGECells were lysed in Laemmli buffer for phospho-proteinblots. Precipitated proteins from fractionations were elutedby boiling in Laemmli buffer. Proteins were fractionated onpolyacrylamide gels and immobilized on PVDF membranes(GE Healthcare, Uppsala, Sweden). Western blot detectionwas carried out by the enhanced chemiluminescence method(GE Healthcare), according to standard darkroom proce-dures. Quantifications were done using imagej software(http://rsb.info.nih.gov/ij).Preparation of DRMs and lipid raft fractionationDRM and SUP fractions were prepared by harvesting cellsin 1% Triton X-100 buffer for SK-N-MC cells and 0.9%Triton X-100 buffer for chick spinal cord cells, as previouslydescribed [14]. Detergent-free fractionation of chick spinalcord was done by electroporation of E2 (approximatelystage 11) chicken embryos in ovo. Electroporator settingswere as described previously [35]. The embryos were incu-bated until E5. At E5, positively transfected spinal cord andDRG segments were dissected out and pooled to equalamounts of input (eight embryos were routinely needed perexperiment and condition). After dissociation into singlecells and stimuli with Ret ligands for 30 min, cells were har-vested in detergent-free buffer and subjected to ultracentrif-ugation in 0–20% OptiPrep density gradients as previouslydescribed [24]. Seven fractions of 0.68 mL each were takenfrom columns after centrifugation, and concentrated proteinfrom each fraction was immunoblotted against proteins asindicated in the figure legends.Vertical cell migration assaysCell migration response towards Ret ligands was assessedusing transwell cell culture inserts (Falcon, Europe) with12 lm pores. Transfected cells were seeded (1–5 · 105cells)in the chambers and allowed to migrate towards ligands, asindicated in the figures. Quantification was done under amicroscope by counting cells in three visual fields afterremoval of stationary cells on the upper side of membranesusing a cotton-tip.Comet assaySK-N-MC cells were treated as indicated in the figures.Cells were spun down in ice-cold NaCl ⁄ Piand immediatelycombined with low-melt agarose to a final concentration of0.9% agarose. The samples were immobilized on precoatedagarose slides (Trevigen, Gaithersburg, MD, USA) andallowed to settle for 20 min at 4 °C. The slides wereimmersed in lysis solution (Trevigen) for 55 min at 4 °C.Slides were submersed in alkaline solution (NaOH ⁄EDTA ⁄ H2O) with a pH > 13 for 50 min at room tempera-ture. After equilibration in 1 · TBE buffer three times for5 min each, the slides were placed in a horizontal electro-phoresis chamber with a voltage of 0.9 VÆcm)1for 14 min.DNA was fixed in MeOH and EtOH and dried at roomtemperature. Examination was performed by staining slideswith SYBR-green, and images were captured at 200· mag-nification. Quantification was done on digital images of 25random cells per condition using tritek comet scoringsoftware bridged to Mac OSX software via a Parallel Win-dows emulator.AcknowledgementsThis work was supported by the Swedish Cancer Soci-ety, the Swedish Foundation for Child Cancer, theSwedish Medical Research Council and the SwedishFoundation for Strategic Research (CEDB andDBRM grants) for P. Ernfors, the Karolinska InstituteMD-PhD programme for T. K. Lundgren and theLERU Graduate Programme for A. Stenqvist.References1 Arighi E, Borrello MG & Sariola H (2005) RET tyro-sine kinase signaling in development and cancer. Cyto-kine Growth Factor Rev 16, 441–467.2 Newgreen D & Young HM (2002) Enteric nervous sys-tem: development and developmental disturbances –part 2. 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