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

Differential membrane compartmentalization of Ret by PTB-adaptor engagement T. K. Lundgren, Moritz Luebke, Anna Stenqvist and Patrik Ernfors Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden The Ret receptor tyrosine kinase (RTK) has many dif- ferent functions during development and in adult life. Ret is activated upon engagement of its ligands glial cell line-derived neurotrophic factor (GDNF), neurtu- rin, persephin, and artemin, and the coreceptors GDNF family receptors (GFRs) a1–4. Dysregulated Ret is implicated in diseases such as the multiple endo- crine neoplasia (MEN) syndromes (MEN2a and MEN2b), as well as in agangliosis of the colon, one of the most common developmental defects in young chil- dren [1]. In the developing nervous system, neural crest cell migration depends on Ret ligands produced in the surrounding tissue, which guide the migrating cells to a correct position within the embryo [2]. In the Keywords fractionation; Frs2; lipid rafts; PTB adaptors; Ret Correspondence P. Ernfors, Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden Fax: +468 341960 Tel: +468 52487659 E-mail: patrik.ernfors@ki.se (Received 14 December 2007, revised 23 February 2008, accepted 26 February 2008) doi:10.1111/j.1742-4658.2008.06360.x Glial cell line-derived neurotrophic factor family ligands act through the receptor tyrosine kinase Ret, which plays important roles during embryonic development for cell differentiation, survival, and migration. Ret signaling is markedly affected by compartmentalization of receptor complexes into membrane subdomains. Ret can propagate biochemical signaling from within concentrates in cholesterol-rich membrane microdomains or lipid rafts, or outside such regions, but the mechanisms for, and consequences of, 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 and their function in redistributing Ret to specialized membrane compartments. We found that engagement of Ret with the Frs2 adaptor results in an enrichment of Ret in lipid rafts and that signal transduction pathways and chemotaxis responses depend on the integrity of such rafts. The competing Shc adaptor did not promote Ret translocation to equivalent domains, and Shc-mediated effects were less affected by disruption of lipid rafts. How- ever, by expressing a chimeric Shc protein that localizes to lipid rafts, we showed that biochemical signaling downstream of Ret resembled that of Ret signaling via Frs2. We have identified a previously unknown mecha- nism in which phosphotyrosine-binding domain-containing adaptors, by means of relocating Ret receptor complexes to lipid rafts, segregate diverse signaling and cellular functions mediated by Ret. These results reveal the existence of a novel mechanism that could, by subcellular relocation of Ret, work to amplify ligand gradients during chemotaxis. Abbreviations CO, 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 2055 developing and adult mouse, Ret is also instrumental in 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 activation depend on an intact Tyr1062 in the intracellular domain [6,7]. Upon Ret phosphorylation, this tyrosine mediates biochemical signal transduction via interac- tion with adaptor proteins. Several phosphotyrosine- binding domain-containing adaptors (PTB adaptors) compete for interaction with Tyr1062, but only one adaptor may interact at any given time [8]. The dis- tinct functional outcome of Ret activation is corre- lated with the different PTB adaptors interacting with Ret [9,10]. We have shown previously that expression of Ret mutants, selective for binding to either the adaptor protein Shc or Frs2 to Tyr1062, results in distinctly different patterns of plasma membrane localization of Ret. Frs2 recruitment resulted in a concentration of Ret into membrane foci [4,11]. The Ret receptor has recently been shown to signal from within different cellular compartments. The oncogenic precursor of Ret (Men2B) can be activated and induce downstream signaling from within the endo- plasmic reticulum [12]. Ret can also be recruited to specialized lipid raft domains in the plasma mem- brane, where it can be phosphorylated, interact with adaptor proteins and induce downstream signaling [12–14]. Lipid rafts are membrane microdomains, rich in sphingolipids and cholesterol, that form lateral assem- blies in the plasma membrane [15]. Lipid rafts seques- ter a number of different proteins that are palmitoylated or contain a number of other lipid anchors, which may be regulated by the selective interaction with these domains. In this way, the recruitment of Ret to lipid rafts can lead to the acti- vation of distinct signaling pathways, due to the com- partmentalized cell signaling events. However, the mechanism for directing Ret into lipid rafts remains largely unknown. We report here that the PTB adap- tor Frs2 functions to translocate Ret to membrane subdomains of the lipid raft type. Interactions of Ret with the Shc adaptor, which, in contrast to Frs2, lacks a palmitoylation tail that confers attachment to lipid rafts, did not result in redistribution of Ret to lipid rafts. Targeting the Shc adaptor to lipid rafts by engineering the adaptor with a raft targeting tail led to altered biochemical signaling resembling, in several respects, signal transduction downstream of Frs2. We show that the distinct biological outcomes of Ret acti- vation largely depends on the targeting to, and signal- ing from within, lipid rafts. Results A PTB-adaptor-dependent membrane relocation of Ret receptors To investigate whether Tyr1062 is important for Ret translocation into lipid rafts, we performed crude fractionations of neuronal SK-N-MC cells transfected with the MEN2a version of Ret ( 2a Ret). The MEN2a mutation C634R is found in more than 85% of patients with MEN syndrome, and renders Ret con- stitutively active, thus omitting the need for ligand for its activation. Lipid rafts and nonraft membranes were isolated according to their resistance to and sol- ubilization by detergent. Cells expressing 2a Ret or a 2a Ret Y1062F mutant that is incapable of adaptor inter- action with the phosphorylated Tyr1062 were har- vested in detergent, and the detergent-resistant membrane (DRM) fraction was separated from the detergent-soluble supernatant (SUP) fraction by cen- trifugation. Each fraction was subjected to PAGE and transferred to poly(vinylidene difluoride) (PVDF) membranes. Immunoblotting against Ret revealed that the majority of Ret partitioned in the DRM fraction. This was in contrast to the 2a Ret Y1062F mutant, where the majority of Ret was found in the SUP fraction (Fig. 1A). To test whether Ret distribution was affected by interaction with either the Frs2 or the Shc adaptor, we overexpressed Shc or Frs2 constructs together with 2a Ret in SK-N-MC cells. Lysates from cells overexpressing Shc or Frs2 were divided into equal halves, and each half was immunoprecipitated for Shc or Frs2 and immunoblotted against Ret. Nearly all Ret precipitated with the overexpressed adaptor, showing that overexpression of one adaptor leads to outcompetition of the other with respect to Ret interaction (Fig. 1B). Next, we fractionated lysates of cells expressing 2a Ret or 2a Ret Y1062F together with overexpressed Frs2 or Shc into DRM and SUP fractions. Adaptor overexpression led to high amounts of Ret in the DRM fraction in both Frs2 and Shc conditions, whereas 2a Ret Y1062F ,as before, displayed a much lower partitioning into DRMs (Fig. 1C). Ret localization to the supernatant fraction was largely unaffected by the Y1062 muta- tion in the presence of Frs2 or Shc. To confirm the relative purity of raft and nonraft membranes, DRM and SUP fractions were immunoblotted towards the lipid 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 found nearly exclusively in the DRM fraction, whereas TfR was 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 FEBS Furthermore, the DRM and SUP fractions were im- munoblotted towards the adaptor proteins themselves. Both Shc and Frs2 were found predominantly in the DRM fractions (Fig. 1D). These results suggest that Ret localization to nonraft membrane regions occurs independently of adaptor engagement, whereas its localization to lipid rafts depends on Shc or Frs2 interactions with Ret Tyr1062. To examine whether Ret and adaptor partitioning is affected by disruption of lipid-ordered regions, we exposed cells to methyl-b-cyclodextrin (MCD), an agent that is commonly used to extract cholesterol from cell membranes in order to disrupt lipid-ordered regions in the plasma membrane. Cells were treated for 30 min with 8 mm MCD or vehicle prior to frac- tionation and subsequent immunoblotting towards Ret. The amount of Ret present in the DRM fraction was significantly reduced, with a corresponding increase of Ret in the SUP fraction, when cells were treated with MCD (Fig. 1D). This indicates that the integrity of cholesterol-rich regions is necessary for Ret partitioning into DRMs, agreeing with previous data [14]. The insoluble material recovered in the DRM frac- tion represents a collection of many discrete lipid- ordered structures, and is not exclusively composed of lipid rafts [16]. Previous studies have found that Ret partitions 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 previously determined localization of Ret to membrane foci, depending on adaptor engagement [11], had any rela- tion to lipid rafts, we expressed Ret tagged with enhanced green fluorescent protein (eGFP) (Ret eGFP ) in cells. Fusion of eGFP to Ret allows direct visuali- zation of the subcellular distribution of Ret. Ret eGFP was expressed with Shc or Frs2 in SK-N-MC neuro- nal cells that were stained for visualization of the membrane lipid rafts using a fluorescently conjugated cholera toxin B (CTB) subunit that binds to the pen- tasaccharide chain of plasma membrane ganglioside GM1, which selectively partitions into lipid rafts [17]. Using confocal microscopy, we found that there was a consistent punctuate pattern of lipid rafts with high abundance in neurites after 30 min of ligand stimulus. Interestingly, extensive localization of Ret eGFP to lipid rafts was seen only in Frs2-expressing cells, and not in 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 more cell processes and neurites than Shc-expressing cells. Frs2 is exclusively localized to lipid rafts [18], whereas Shc 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 raft targeting of Shc could mediate translocation of Ret to a subcellular localization similar to that mediated by Frs2, we expressed an Shc construct containing the Ras membrane localization signal (MLS) (Shc MLS ), which permanently localizes Shc to lipid rafts [20]. This Shc construct was shown in previous work 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]. When Shc MLS was overexpressed in cells along with Ret eGFP , the membrane localization of Ret was lar- gely confined to lipid raft regions, similar to that of Ret eGFP - 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. A C D B Fig. 1. Tyr1062 is necessary for Ret partitioning into lipid rafts. (A) Mutation of Tyr1062 results in a loss of 2a Ret partitioning to the DRM fraction. 2a Ret WT or 2a Ret Y1062F was expressed in SK-N-MC cells. Cells were harvested in 1% Triton buffer, separated into DRM or SUP fractions, separated on polyacrylamide gels, and transferred to PVDF membranes, with subsequent blotting for Ret (n = 3 with similar results). (B) Overexpression of Shc or Frs2 adap- tors forces 2a Ret to interact with either adaptor at the expense of the other. Lysates of SK-N-MC cells were separated, and each half was immunoprecipitated for Shc or Frs2 and immunoblotted against Ret (n = 2). (C) Overexpression of Shc or Frs2 adaptors results in 2a Ret WT but not 2a Ret Y1062F partitioning 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 Ret to the DRM fraction depends on intact lipid rafts. SK-N-MC cells expressing 2a Ret WT and 2a Shc or Frs2 were treated with MCD or vehicle, 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 rafts FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2057 A critical role of Ret membrane localization in downstream signaling events in cell lines and primary cells We next examined the intracellular signaling down- stream of Ret in cells expressing the Shc, Shc MLS or Frs2 adaptors. Phosphorylated (active) Akt or p42,44 extracellular signal-related kinase (ERK) MAPK pro- tein levels were examined by immunoblotting at differ- ent time points after Ret ligand stimuli. Shc MLS expression together with Ret resulted in signal activa- tion resembling the Ret ⁄ Frs2 coexpression characteris- tics. Specifically, a higher and sustained ERK p42,44 MAPK level was also observed at 12 h in cells express- ing Shc MLS as compared to Shc (Fig. 3A). Further- more, Ret signaling via Shc MLS did not lead to as robust activation ⁄ phosphorylation of Akt as did Shc, but levels were higher than for signaling via Frs2 (Fig. 3C). To investigate the importance of rafts for downstream ERK p42,44 MAPK activation, we applied cholesterol oxidase (CO) to cells during ligand stimuli. CO was chosen because cells cannot withstand MCD treatment for long time periods, and CO has been shown to disrupt the biochemical effects of mem- brane-localized Ret as well as other receptor complexes in a fashion similar to what is accomplished by using MCD [11,21,22]. In the presence of CO, ERK p42,44 MAPK activation was markedly reduced both in cells expressing Ret ⁄ Shc MLS as well as in cells expressing Ret ⁄ Frs2 at all time points, whereas activation of ERK p42,44 MAPK downstream of Shc was much less affected (Fig. 3A). The specificity of cholesterol species for phosphorylated ERK p42,44 MAPK was also important in MEN2a versions of Ret when expressed with Shc or Frs2. In cells expressing 2a Ret, phosphory- lated ERK p42,44 MAPK levels were high when Shc and Frs2 were coexpressed. CO treatment attenuated phosphorylated ERK p42,44 MAPK levels down- AA' BB' CC' Fig. 2. Labeling of plasma membrane gan- glioside GM1 lipid rafts shows extensive colocalization of Frs2-associated but not Shc-associated Ret to lipid rafts. (A–C) SK-N-MC cells expressing Ret eGFP (green) together with Shc (A,A¢), Shc MLS (B,B¢)or Frs2 (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 Ret eGFP with 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 FEBS stream of Shc and led to an almost complete absence of phosphorylated ERK p42,44 MAPK downstream of Frs2 (Fig. 3B). Most studies on receptor signaling from within rafts have been performed on immortalized cell lines in cul- ture or by using artificial membranes. We attempted to see whether the PTB adaptors determine Ret subcellu- lar distribution and signaling in primary neurons of a more complex system. To this end, we electroporated Ret eGFP and either adaptor into the neural tube of chicken embryos. DNA was injected in ovo into the neural tube of embryos at Hamburger–Hamilton stage 11 [approximately embryonic day (E)2], and elec- troporation was performed by placing electrodes along the rostral–caudal axis of the neural tube. The egg was then closed and placed in an incubator until the embryos had grown to stage 24 (E4.5). At this stage, the expression of transfected Ret eGFP was found in the developing spinal cord and in dorsal root ganglia (DRG), and the transfected tissue was dissected under fluorescent 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 ligands for 30 min. After ligand stimulation, the cells were fractionated into DRM and SUP fractions. In accor- dance with the results obtained with neuronal SK-N- MC cells, immunoblotting against Ret revealed that it was predominantly located in DRM fractions, regard- less of which adaptor (Shc, Shc MLS , or Frs2) was con- comitantly overexpressed (Fig. 3D). Immunoblotting of DRM and SUP fractions against the adaptor pro- teins showed that Shc MLS and Frs2 were nearly exclu- sive to the DRM fraction, whereas Shc was present also in the SUP fractions (Fig. 3E). We investigated the activation of MAPK and Akt pathways in the developing chick embryo. Previous studies have found that the ERK MAPK pathway [and to a lesser extent, also the phosphoinositide-3-kinase (PI3K) ⁄ Akt pathway] can be temporally and quantita- tively affected by activation within or outside DRM fractions [23]. To examine the role of the PTB adaptors in the activation of the MAPK and Akt pathways, spinal cords electroporated with Ret together with either Shc, Shc MLS or Frs2 were separated into DRM and SUP fractions, as above. Phosphorylated Akt lev- els were generally low for all PTB adaptors in the DRM fraction, showing that phosphorylated Akt A C DF G E B Fig. 3. The raft-targeting adaptors Frs2 and Shc MLS show enhanced ERK phophorylation, which is dependent on the integrity of cholesterol- rich domains. (A) SK-N-MC cells expressing Ret and either Shc, Shc MLS or Frs2 adaptors were lysed after Ret ligand stimulation for indicated times and subsequently immunoblotted towards phosphorylated ERK and all ERK. CO was applied to cells as indicated. (B) Experiments as in (A) with MEN2a versions of Ret. (C) Experiments as in (A) with immunoblotting towards phosphorylated Akt and Akt. (D) E2 chicken embryos 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 Ret ligands 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 Shc MLS or for Frs2. (F, G) Experiments as in (C) with immunoblots towards phosphoryated Akt or phosphorylated ERK. T. K. Lundgren et al. Ret PTB-adaptor translocation to rafts FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2059 signaling predominantly takes place in cellular struc- tures partitioning into the SUP fraction (Fig. 3G). Phosphorylated Akt levels were greater in the SUP fraction when Shc or Shc MLS was expressed with Ret as compared to Frs2, where levels were overall lower and more similar between the DRM and SUP fractions (Fig. 3G). Phosphorylated ERK p42,44 MAPK immu- noblotting revealed the strongest signal when Frs2 was expressed with Ret in both the DRM and the SUP fractions (Fig. 3F). Shc MLS resulted in activation of ERK p42,44 MAPK in the DRM. Shc, on the other hand, was very poor in mediating ERK p42,44 MAPK activation in the DRM fraction (Fig. 3G). These results therefore 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 to complexes of PTB adaptors with Ret in lipid rafts and that phosphorylated Akt is mostly found outside rafts. To characterize the Ret distribution in greater detail than is permitted by DRM ⁄ SUP fractionation, we used a recently published fractionation protocol that omits the need for detergent and is highly specific with regard to molecular distribution within and outside lipid raft fractions [24]. Dissected spinal cords from chick embryos were lysed in detergent-free buffer and fractionated by ultracentrifugation in OptiPrep gradients. Seven fractions were aspirated from columns, and the protein content within each fraction was concentrated by precipitation, loaded on poly- acrylamide gels, and transferred to PVDF membranes. The separation of raft fractions from nonraft fractions was confirmed by immunoblotting against flotillin-1 and TfR (Fig. 4). As determined by immunoblotting against Ret, this more sensitive method revealed that the fractional distribution of Ret varied with the adap- tor expressed in the embryos. Frs2 overexpression led to Ret being directed towards lower-density fractions (corresponding to lipid rafts), peaking in fraction 2. Shc overexpression resulted in significantly less Ret partitioning into the fractions of lowest density, with little or no Ret in fraction 2 and peak levels in frac- tion 3 (Fig. 4). Overexpression of the raft-localizing Shc MLS construct resulted in a significant portion of Ret being partitioned into both fraction 2 and frac- tion 3. Immunoblotting against the adaptors them- selves showed that whereas both Shc and Shc MLS were present 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 raft and nonraft membrane compartments Association of Ret with the different adaptors may result 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 than those of Ret signaling via Shc [11]. Ret signaling via recruitment of Shc, on the other hand, is necessary for Ret-mediated cell survival when neuronal cells are pre- sented to toxic agents, and also for neurite formation in certain cell types [4,9]. We investigated how disrup- tion of lipid-ordered domains affected these functional aspects, and whether the Shc MLS adaptor is similar to Frs2 or Shc in terms of cellular response to Ret stimu- lation. Chemotactic migration towards Ret ligands was examined by seeding neuronal SK-N-MC cells express- ing Ret receptors that are selective for binding of only Frs2 (Ret Frs+ ) or Shc (Ret Shc+ ) to the region of Tyr1062 [4]. Ret mutants binding to Frs2 showed a nearly three-fold higher migrational capacity than did Ret Shc+ , and this effect was ligand-dependent, as all conditions displayed a similar low level of random cell migration without ligand being supplied to the lower culture compartment (Fig. 5A). Cells expressing Ret Shc+ ⁄ Shc did not show any statistically significant ligand-induced migration. Cells expressing Shc MLS together with Ret Shc+ displayed a two-fold increase of migration towards Ret ligand (Fig. 5A). The same experiment was then performed in the presence of CO. A Fig. 4. Density fractionation of the membrane localizes Frs2 and Shc partly to different membrane compartments. (A) E2 chicken embryos were electroporated in ovo and allowed to develop to E5. At E5, positively transfected spinal cord segments were dissected out and pooled to equal amounts of input. After dissociation into single cells and stimulation with Ret ligands for 30 min, cells were harvested in detergent-free buffer and subjected to ultracentrifuga- tion in OptiPrep density gradients. Fractions were taken out after centrifugation, and concentrated protein from each fraction was immunoblotted against Ret or HA-tag for Shc and Shc MLS or against Frs2, as indicated. Fraction 1 is the less dense fraction, and fraction 7 is the fraction with the highest density. The bottom panel shows immunoblotting for the lipid raft marker flotillin-1 and the nonraft 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 FEBS CO treatment of cells led to a large decrease in the number of migrating cells, almost reaching baseline levels in all conditions (Fig. 5A), indicating that the integrity of cholesterol-rich membrane domains is nec- essary for the occurrence of directional migration towards Ret ligand. The ability of Ret selective mutants to promote cell survival was next examined. SK-N-MC cells treated with anisomycin to induce apoptosis were rescued by supplementing the medium with Ret ligands. To detect the apoptotic response to anisomycin with high sensi- tivity, single cells were examined in the comet assay. In this system, fragmented DNA moves out of the cell soma in the shape of comet tails when cells are embed- ded in agarose and subjected to an electric field. After staining of DNA with SYBR-green, images were cap- tured using fluorescence microscopy. The comet-tail moment was determined by measuring pixel intensity in the comet head and tail, and calculating the momentum of the comet tails [25]. In accordance with what was previously found [4], Ret ligands resulted in a much greater cell survival effect for cells expressing Ret Shc+ than for those expressing Ret Frs+ (Fig. 5B,C). When Shc MLS was coexpressed with Ret Shc+ to enforce Shc signaling in lipid rafts, an intermediate survival-promoting effect was seen (Fig. 5B,C). Dis- ruption of cholesterol-rich membrane compartments with CO had no effects on cell survival (Fig. 5B,C). Furthermore, CO alone without anisomycin did not result in any detectable cell injury (Fig. 5B,C), suggest- ing that CO at the concentrations used does not have an effect on cell survival. Thus, these data suggest that Ret-mediated cell migration, but not cell survival, requires intact lipid rafts. Discussion The present study was conducted to determine whether PTB adaptors determine Ret localization to different membrane compartments, and whether pathway-spe- cific signaling and functional outcomes are affected by signaling from within and outside rafts. It has previ- ously been shown that Ret localizes to lipid rafts in a glycophosphatidylinositol-bound GFRa1 coreceptor- dependent fashion [26]. However, more recent results have demonstrated that both soluble and glycophos- A B C Fig. 5. Frs2- dependent chemotaxis but not Shc-dependent cell survival depends on raft integrity. (A) SK-N-MC cells expressing Ret mutants were analyzed in transwell chemotaxis assays after 14 h, with Ret ligands supplied below the membrane and CO supplied above and below the membrane. Two-way ANOVA compared to the maximally migrating Ret Frs+ ⁄ 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 ⁄ DNA damage expressed as comet-tail momentum. Two-way ANOVA compared to the maximally rescuing Ret Shc+ ⁄ 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 rafts FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2061 phatidylinositol-anchored GFRa1 increase Ret distri- bution to rafts [13]. In the same study, Frs2 and Shc adaptor engagement with Tyr1062 was found to occur predominantly within and outside rafts, respectively. We report here that PTB adaptor engagement is criti- cal for Ret to localize to lipid rafts, suggesting that upon ligand engagement, the PTB adaptor association results in a movement of Ret from nonraft to raft membrane regions. Consistently, phosphorylated ERK p42,44 MAPK signaling by a MEN2a form of Ret, which signals even in the absence of ligands and GFRa1, was more sensitive to disruption of choles- terol-rich membranes by CO upon Frs2 as compared to Shc recruitment. Our data also suggest that recruit- ment of Ret to rafts by PTB adaptors results in dis- tinct signal transduction patterns. The biochemical integrity of Ret signaling depends on undisrupted lipid-ordered domains when Ret assembles together with adaptors localizing to rafts, but less so when the complex is localized outside rafts. Furthermore, a modified version of the Shc adaptor that localizes to lipid rafts in a similar way to Frs2 results in signaling resembling Frs2 recruitment by Ret. Our results show that Ret resides largely in DRM fractions both in cell lines and in vivo in the chick spinal cord. This localization was critically dependent on Tyr1062 and its interaction with Frs2 and Shc, because eliminating interactions at this site resulted in a clear loss of Ret in the DRM fraction and an increase in the SUP fraction. Cyclodextrins such as MCD effectively remove cholesterol from the plasma membrane [27]. This property has led to the extensive use of MCD to study the function of lipid rafts, which are membrane microdomains whose integrity depends on the presence of cholesterol. In this article, we show that cholesterol depletion by MCD results in a loss of both Frs2- and Shc-induced increases of Ret in the DRM fraction. Analysis of the DRM and SUP fractions suggested that both Shc and Frs2 reside largely in the DRM fraction, and that signaling from Ret by means of Shc and Frs2 might be initiated from within lipid raft membrane compartments. However, with the use of a more sensitive density-dependent fractionation, it was clear that Ret associated with Frs2 and Shc resides in different membrane compartments, with Ret interac- tions with Frs2 being more strongly associated with the flottilin-containing fractions, which are believed to include lipid rafts [28]. These experiments also showed that, unlike Shc, which is present in very low amounts in the Frs2-associated Ret fractions, Shc MLS is located in both raft and nonraft fractions. Details of the lipid distribution of the sphingolipid- and cholesterol-rich lipid rafts are not well characterized. Clearly, there is a great spatial and functional heterogeneity of these membrane domains. Recently, it was found that plasma membrane sphingomyelin-rich domains are spatially distinct from ganglioside GM1-rich mem- brane domains in Jurkat T cells, and may form distinct and unique signaling platforms [29]. Distinct cellular localization of Shc and Frs2 with Ret ⁄ Frs2 but not Ret ⁄ Shc localized to GM1-rich lipid rafts was also confirmed using CTB labeling. In this experiment, a clear colocalization of Ret eGFP to lipid rafts in the presence of Frs2 but not Shc was evident. We noticed a high baseline activity without ligand in Frs2 conditions throughout our study. This is consis- tent with previous results on ERK MAPK and on other downstream effector proteins activated via Frs2, and is most likely due to the sustained interaction of Frs2 with Ret and also other RTKs [4,30]. In a recent study, we have shown that selective interaction of Ret with Shc results in the activation of AKT to a much greater extent than when Ret signals by recruitment of Frs2, and conversely that, unlike Shc signaling, Frs2 signaling leads to ERK p42,44 MAPK activation at high levels. Interestingly, the difference in signaling was reflected not only by more robust activation, but also by a significantly sustained activation from 5 min to at least 12 h. Signaling by Ret via Shc activates ERK to a lesser degree, and peaks at about 30 min [4]. Several lines of evidence suggest that the sustained activation of ERK is dependent on the raft context rather than it being PTB adaptor-specific. Signaling via Frs2 after disruption of the rafts by CO resulted in a marked attenuation ERK MAPK signaling, with a duration of minutes instead of hours. Furthermore, recruitment of Shc to lipid rafts by introduction of the Ras membrane localization signal resulted in elevated and sustained ERK p42,44 MAPK activation, similar to that seen for Frs2. Whereas activation of ERK p42,44 MAPK by the normal Shc adaptor was less attenuated by lipid raft disruption, ERK p42,44 MAPK activation by Shc MLS was dependent on intact rafts, and when the rafts were disrupted, ERK p42,44 MAPK activation was almost completely absent. On the basis of these results, we conclude that sustained ERK p42,44 MAPK 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 Akt almost exclusively outside of rafts, with there being little ERK p42,44 MAPK activation either within or outside of rafts, as seen by its partitioning into the DRM fraction. In contrast, whereas Shc MLS activates Akt mostly outside of rafts, its activation of ERK p42,44 MAPK was almost exclusively within the Ret 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 FEBS DRM raft fraction. Frs2 activation of Akt was overall very weak, whereas ERK p42,44 MAPK activation was strong in both the DRM fraction and in the non- raft SUP fraction. Because our density-dependent fractionation and colabeling of Ret with lipid rafts in cellular staining both suggest that Ret associated with Frs2 and Shc resides in different membrane compart- ments, our results suggest that Akt activation may result largely from nonraft activation of Shc by Ret. Unlike Akt, ERK p42,44 MAPK, which is activated downstream of Frs2, appeared to localize both to raft membranes and to nonraft membrane fractions. Because Ret association with Frs2 is almost exclusive to the lipid rafts, this suggests that ERK MAPK par- titioning into nonraft fractions is presumably initiated from within the raft. It is interesting that ERK p42,44 MAPK signaling resulting from Shc MLS association with Ret takes place exclusively in the DRM fraction, suggesting that, unlike signaling downstream of Frs2, components of the ERK MAPK signaling pathway stay associated with the raft-localized Shc MLS also after activation. Both Shc and Frs2 have been implicated in cell migration, and we cannot exclude a role also for PI3K ⁄ Akt signaling from Ret. This pathway has previ- ously been implicated in such cellular functions mediated by Ret in, for example, kidney epithelial Madin–Darby canine kidney cells [31]. However, our findings suggest that PI3K⁄ Akt signaling is not suffi- cient for cell migration by itself in the absence of MAPK ERK signaling via Frs2. Directional signaling onto RTKs at the leading edge appears to be critical for chemotaxis, and defines the direction of actin poly- merization and subsequent cell migration. It is not clear how the cells measure gradients of RTK ligands resulting in gradients of receptor activation along the surface that are translated into polarization of the cytoskeleton, extension of cell processes, and eventu- ally translocation of the cell body. Our results show that Ret-mediated chemotaxis is critically dependent on lipid rafts, whereas cell survival signaling via Shc is not significantly affected by CO. This is consistent with the conclusion that Shc activation by Ret may take place in nonraft membrane regions, similar to what happens 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 distinguish whether the effects of CO on migration result directly from the loss of ERK signaling or from the physical localization of Ret to the growing axons, as seen by the Ret colocalization with CTB. As for other RTKs, Ret-stimulated migration is dependent on ERK signal- ing, as blocking of this pathway prevents Ret-induced migration [11,33]. However, this does not exclude the possibility that that a raft-dependent localization of Ret to filopodia ⁄ lamellipodia may also be important for directional migration and axonal extension. Consis- tent with this hypothesis are data showing that local stimulation of cells expressing the epidermal growth fac- tor receptor with a bead soaked in epidermal growth factor leads to ERK activation spreading throughout the cell, whereas actin polymerization remains local [34]. Our results open the possibility that Frs2-dependent recruitment of Ret receptors to lipid rafts upon ligand engagement may participate in increasing receptor levels in the direction of increasing ligand concentrations. This may provide a molecular mechanism for cellular amplifi- cation of ligand gradients that could play important roles in directed cell migration. Experimental procedures Cell culture, DNA constructs, and mutagenesis All 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 bovine serum, 2% horse serum and 1 mm glutamine. Starvations were done in DMEM containing 0.5% total serum. All ligand stimulations were performed for 30 min unless stated otherwise, using 50 ngÆmL )1 recombinant human GDNF and 100 ngÆmL )1 recombinant human GFRa1 ⁄ FC chimera (R&D Systems, Minneapolis, MN, USA). CO (Sigma, Munich, Germany) was used at 8 mm, as previously described [21]; specifically, cultured cells were incubated with CO at 1.8 UÆmL )1 for 1 h prior to and during ligand stimu- lation. Anisomycin (Sigma) was applied to cells 30 min before ligand application at a final concentration of 12 lgÆmL )1 , and cells were incubated for another 2 h before examination. Transfections were performed using Lipofecta- mine LTX (Invitrogen, Karlsruhe, Germany), according to the manufacturer’s instructions. Growth medium was replaced approximately 7 h after transfection. Transfection efficiency was continuously monitored by eGFP fluores- cence. Antibodies and reagents Antibodies against Ret (Ret H-300), and phosphotyrosine (PY99) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against HA tags were from BD Biosciences (CA, USA). Antibodies against Frs2 were from Sigma. Antibodies against flotillin-1 were from Transduction Labs (Lexington, KY, USA). Antibodies T. K. Lundgren et al. Ret PTB-adaptor translocation to rafts FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2063 against TfR were from Zymed (San Francisco, CA, USA). Antibodies against Akt, phosphorylated Akt Ser473, p44 ⁄ 42 MAPK and phosphorylated p44 ⁄ 42 MAPK were from Cell Signalling (Hitchin, UK). Alexa-546-conjugated GM1-CTB was from Molecular Probes (Leiden, the Neth- erlands), and was used according to the manufacturer’s instructions. After staining with GM1-CTB, cells were fixed in 4% paraformaldehyde and examined on Zeiss LSM 5 exciter microscopes with axiovision software (Zeiss, Karlsruhe, Germany). Immunoblotting and SDS ⁄ PAGE Cells were lysed in Laemmli buffer for phospho-protein blots. Precipitated proteins from fractionations were eluted by boiling in Laemmli buffer. Proteins were fractionated on polyacrylamide gels and immobilized on PVDF membranes (GE Healthcare, Uppsala, Sweden). Western blot detection was 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 fractionation DRM and SUP fractions were prepared by harvesting cells in 1% Triton X-100 buffer for SK-N-MC cells and 0.9% Triton X-100 buffer for chick spinal cord cells, as previously described [14]. Detergent-free fractionation of chick spinal cord was done by electroporation of E2 (approximately stage 11) chicken embryos in ovo. Electroporator settings were as described previously [35]. The embryos were incu- bated until E5. At E5, positively transfected spinal cord and DRG segments were dissected out and pooled to equal amounts of input (eight embryos were routinely needed per experiment and condition). After dissociation into single cells 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 previously described [24]. Seven fractions of 0.68 mL each were taken from columns after centrifugation, and concentrated protein from each fraction was immunoblotted against proteins as indicated in the figure legends. Vertical cell migration assays Cell migration response towards Ret ligands was assessed using transwell cell culture inserts (Falcon, Europe) with 12 lm pores. Transfected cells were seeded (1–5 · 10 5 cells) in the chambers and allowed to migrate towards ligands, as indicated in the figures. Quantification was done under a microscope by counting cells in three visual fields after removal of stationary cells on the upper side of membranes using a cotton-tip. Comet assay SK-N-MC cells were treated as indicated in the figures. Cells were spun down in ice-cold NaCl ⁄ P i and immediately combined with low-melt agarose to a final concentration of 0.9% agarose. The samples were immobilized on precoated agarose slides (Trevigen, Gaithersburg, MD, USA) and allowed to settle for 20 min at 4 °C. The slides were immersed in lysis solution (Trevigen) for 55 min at 4 °C. Slides were submersed in alkaline solution (NaOH ⁄ EDTA ⁄ H 2 O) with a pH > 13 for 50 min at room tempera- ture. After equilibration in 1 · TBE buffer three times for 5 min each, the slides were placed in a horizontal electro- phoresis chamber with a voltage of 0.9 VÆcm )1 for 14 min. DNA was fixed in MeOH and EtOH and dried at room temperature. Examination was performed by staining slides with SYBR-green, and images were captured at 200· mag- nification. Quantification was done on digital images of 25 random cells per condition using tritek comet scoring software bridged to Mac OSX software via a Parallel Win- dows emulator. 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