The Mouse Soluble Gfra4 Receptor Activates RET Independently of Its Ligandpersephin

Oncogene (2007) 26, 3892–3898 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc SHORT COMMUNICATION The mouse soluble GFRa4 receptor activates RET independently of its ligandpersephin

J Yang, P Runeberg-Roos, V-M Leppa¨ nen and M Saarma

Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Helsinki, Finland

Glial cell line-derived neurotrophic factor (GDNF) family There are four different ligands, all of which belong to ligands (GFLs) all signal through the transmembrane the glial cell line-derived neurotrophic factor (GDNF) receptor tyrosine kinase RET. The signalling complex family (GDNF, neurturin, artemin, persephin), and consists of GFLs, GPI-anchoredligandbinding GDNF correspondingly four different GPI-anchored co-recep- family receptor alphas (GFRas) andRET. Signalling via tors, which are named GDNF family receptor alpha RET is requiredfor the development of the nervous system (GFRa) 1–4 (Airaksinen and Saarma, 2002). The andthe kidney,as well as for spermatogenesis. However, persephin binding co-receptor GFRa4 is of special constitutive activation of RET is implicatedas a cause in interest, as its restricted expression pattern suggests that several diseases. Mutations of the RET proto-oncogene it may play a role in the MEN 2 syndrome (Lindahl cause the inherited cancer syndrome multiple endocrine et al., 2000, 2001). Several splice variants of GFRa4 neoplasia type 2 (MEN 2). Recently, it has been suggested have been found both in the human and in the mouse that mutations in the persephin binding GFRa4 receptor (Lindahl et al., 2000, 2001). The variants have been may have a potentially modifying role in MEN 2. Several postulated to encode putative GPI-anchored, transmem- naturally occurring, different splice variants of the brane or soluble receptors. A schematic picture of the mammalian GFRa4 have been reported. A 7 bp inser- mouse GFRa4 splicing variants is shown in Figure 1d. tion–mutation in the human GFRa4 gene causes a shift of Recently, a mutation, which may contribute to a more reading frame and thereby changes the balance between aggressive disease course of MEN 2, was identified in the transcripts encoding GPI-anchored and soluble human GFRa4 (Vanhorne et al., 2005). The mutation GFRa4 receptors. We report here that the mammalian consists of a 7 bp insertion that causes a frameshift in all soluble GFRa4 can activate RET independently of its of the human GFRa4 splice variants. This mutation preferential ligand, persephin. Our data show that soluble affects the C-terminal end of the different forms of the GFRa4 can associate with, andinduce, phosphorylation receptor, and thereby seems to alter the balance between of RET. In addition, our data show that this isoform of GPI-anchored and soluble forms of human GFRa4. GFRa4 can induce downstream signalling, as well as Both the human and mouse GPI-anchored variants bind neuronal survival anddifferentiation, in the absence of persephin (PSPN) and mediate RET-activation (Lindahl persephin. These results suggest that, in line with the et al., 2001; Yang et al., 2004), but none of the soluble previous report, GFRa4 may be a candidate gene for, or GFRa4 receptors have ever been characterized. modifier of, the MEN 2 diseases. Generally, the GFRa receptors consist of three Oncogene (2007) 26, 3892–3898. doi:10.1038/sj.onc.1210161; homologous cysteine-rich domains (D1, D2, D3), with published online 8 January 2007 the exception of the mammalian GPI-anchored GFRa4 receptors that lack the first N-terminal D1. Crystal Keywords: GFRa4; RET; PSPN; neurite outgrowth; structure analysis of the D3, as well as functional testing survival of the GFRa1 model, has located the ligand binding residues to D2 (Scott and Iba´ n˜ ez, 2001; Leppa¨ nen et al., 2004). Recently, the artemin/GFRa3 complex structure revealed a conserved binding interface in domain 2, and Inherited multiple endocrine neoplasia type 2 (MEN 2) a convergent ligand recognition was suggested (Wang is a cancer syndrome which, in most patients, is caused et al., 2006). We found (Figure 1a–d) that the mouse and by mutations in the proto-oncogene RET (Kodama human wild-type soluble GFRa4, as well as the two et al., 2005). RET is a receptor tyrosine kinase which, human mutated forms of soluble GFRa4, have in under normal conditions, is activated by a complex common a preserved D2 with five cysteine bridges and consisting of a dimeric ligand and a dimeric co-receptor. six a-helices. However, all the soluble forms of GFRa4 have a non-conserved D3 domain with a significant Correspondence: Professor M Saarma, Institute of Biotechnology, homology only up to the first a-helix, where only one of Viikki Biocenter, University of Helsinki, Viikinkaari 9, PO Box 56, the cysteine bridges seems to be preserved (Figure 1a). FIN-00014 Helsinki, Finland. E-mail: mart.saarma@helsinki.fi In order to characterize soluble GFRa4, we Received 25 May 2006; revised 3 October 2006; accepted 23 October established a stable cell line (mouse neuroblastoma 2006; published online 8 January 2007 Neuro 2a) expressing an N-terminally FLAG–tagged Mouse soluble GFRa4 receptor activates RET J Yang et al. 3893 mouse soluble GFRa4, which is secreted into the reticulum signal sequence, see Figure 1e). As shown in medium (Figure 1e). The molecular weight of the Figure 1a, the non-homologous part of the D3 domain expressed protein corresponds to the calculated 21 kDa of mouse soluble GFRa4 harbors two to three new (including a double FLAG-tag (ASDYKDDDDKAS cysteine residues. These new cysteine residues could be DYKDDDDKAS) and excluding the endoplasmic involved in the formation of intermolecular cysteine

bc NN

d e

D2 D2 D2 D2

1 2 3 4 5 6 D3 D3 D3 ←

50 kDa ← ← dimer

37 kDa

25 kDa ← ← ←monomer PM 20 kDa

GFRα1-GPI mGFRα 4-GPI mGFRα 4-TM mGFRα 4-sol

Transmembrane domain Hinge region GPI-linking region Lipid rafts GPI anchor (For caption see next page)

Oncogene Mouse soluble GFRa4 receptor activates RET J Yang et al. 3894 bridges, and could thereby contribute to a spontaneous GPI transfected Neuro 2a cell lines (Yang et al., 2004). dimerization of the receptor. Therefore, we analysed the With the aid of Amicon Ultra-15 (Millipore, 10 kDa dimerization of the mouse soluble GFRa4 under cut-off), we concentrated soluble GFRa4 from the reducing and non-reducing conditions, and found that serum-free RPMI 1640 medium (80 Â ) of Neuro 2a a portion of the receptor indeed forms dimers cells expressing the N-terminally FLAG-tagged mouse (Figure 1e). As GFRa1 D2 has been postulated to soluble GFRa4. This 80 Â concentrate, which is here- interact with RET (Leppa¨ nen et al., 2004), a dimeric after called sGFRa4-concentrate, was used with or GFRa4 D2 could have the potential to recruit two RET without PSPN to investigate if the mouse soluble tyrosine kinase receptors and, thereby, induce its GFRa4 has the capacity to interact with, and activate, activation even in the absence of PSPN. In order to RET. The sGFRa4-concentrate was added to naive analyse if PSPN binds to mouse soluble GFRa4, we Neuro 2a cells that endogenously express high levels of collected the secreted mouse soluble GFRa4 into a RET, but no GFRa4 receptors (Yang et al., 2004). After HEPES-based medium. [125I]-PSPN was added to this lysis, RET was immunoprecipitated from the samples medium in the absence or presence of increasing and the precipitates were analysed by Western blotting amounts of unlabelled PSPN. After rotation at þ 41C with FLAG antibodies. To ensure equal precipitation of for 4 h, the receptor was immunoprecipitated with anti- RET in all samples, the precipitates were also analysed FLAG antibodies. The amount of co-precipitated [125I]- with RET antibodies (Figure 2c). Contradictory results PSPN was measured in each sample, and an IC50 of have previously been reported on whether the GFRa1 1.6 nM was determined (Figure 2a), using the nonlinear can associate with RET in the absence of the ligand regression analysis program Prism 3.02 (GraphPad (Eketja¨ ll et al., 1999), or only in the presence of the Prism Software, San Diego, CA, USA). From these ligand (Tansey et al., 2000). Our co-immunoprecipita- experiments, we conclude that mouse soluble GFRa4 tion assays show that the soluble GFRa4 receptor has the capacity to bind PSPN with a binding constant interacts with RET already in the absence of its ligand comparable to that of mouse GPI-anchored GFRa4 PSPN. In order to assay if the soluble GFRa4 also has (2 nM) (Figure 2a). This result is in line with previously the capacity to activate RET, we added the sGFRa4- published data which predict that D2 is central to the concentrate to naive Neuro 2a cells and immunopreci- binding of GDNF to the GFRa1 receptor and of pitated RET from the lysates of these samples. The artemin to GFRa3 receptor (Scott and Iba´ n˜ ez, 2001; myelin basic protein has previously been reported to Leppa¨ nen et al., 2004; Wang et al., 2006). It is also function as an exogenous substrate in in vitro kinase supported by our recent finding that the purified D2 assays with kinase activated RET (Asai et al., 1995; from GFRa1 has the capacity to bind GDNF (data not Kato et al., 2000). The result of our in vitro kinase assay shown). (Figure 2d) is in line with our co-immunoprecipitation Translation of full-length PSPN has previously been results, and shows that soluble GFRa4 activates RET shown to occur only from the spliced variant of its both in the absence and presence of PSPN. mRNA (Milbrandt et al., 1998). Therefore, we first Signalling through RET has been reported to verified by RT–PCR that the stable cell line did not promote neuronal survival through the PI-3 kinase/ express the spliced mRNA encoding full-length PSPN AKT signalling pathway (Kodama et al., 2005), and we (Figure 2b). Furthermore, the absence of PSPN expres- therefore set out to determine if the mouse soluble sion in Neuro 2a cells is supported by previously GFRa4 without PSPN could activate the PI-3 kinase/ published data showing that exogenously added PSPN AKT pathway and promote cell survival. The biochemi- induces a robust phosphorylation of RET in GFRa4- cal detection of activated downstream signalling was

Figure 1 Molecular characterization of soluble GFRa4. (a) A sequence alignment of D2 and D3 domains of rat GFRa1, mouse GPI- anchored GFRa4 (mGFRa4-GPI), mouse soluble GFRa4 (mGFRa4-sol), the putative human soluble GFRa4 (hGFRa4-sol), as well as the two putative soluble mutant forms of human GFRa4 (hSCa and hSCb) (Vanhorne et al., 2005). The conserved cysteines are highlighted in yellow, and the putative disulfide bridges are numbered as in the homologous rat GFRa1 D3 (Leppa¨ nen et al., 2004) and in human GFRa3 D2 (Wang et al., 2006) shown in (c). The cysteines not following the conserved disulfide pattern are highlighted in magenta. The conserved amino-acid triplet RRR reported to be important for GFRa1/GDNF binding (Scott and Iba´ n˜ ez, 2001; Leppa¨ nen et al., 2004) and GFRa3/artemin binding (Wang et al., 2006) is highlighted in red. The alpha helices are indicated for domain 2 as shown in (c) and for domain 3 as in Leppa¨ nen et al. (2004). The sequence alignment was generated with CLUSTALW (Thompson et al., 1994). (b) A ribbon diagram model of the mouse GFRa4 D2 with a-helices shown as coils. The Ca-atoms corresponding to the GFRa1 RRR triplet are highlighted in blue. The cysteines involved in the putative disulfide bridges shown as a ball-and-stick representation, and the disulfide bridges are highlighted with yellow lines. N- and C-termini are also shown. (c) The template used for modelling, a ribbon diagram of the crystal structure of GFRa3 D2 (Wang et al., 2006) is shown in (b). The alpha helices are labelled from a1toa6 from N- to C-terminus. Disulfide bridges are labelled similarly from 1 to 5 and highlighted with yellow lines. The mouse GFRa4 D2 model was prepared using the automated protein homology-modelling server Swiss-Model (Schwede et al., 2003). The figures were prepared with MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Bacon, 1997). (d) A schematic presentation of the different splicing variants in mouse. (e) Secretion of FLAG-tagged mouse soluble GFRa4 by stably transfected Neuro 2a cells. Medium was collected from parental Neuro 2a cells (lanes 1, 3 and 5) or transfected Neuro 2a cells (lanes 2, 4 and 6). In lanes 1 and 2, the samples were mixed with a reducing Laemmli buffer and boiled for 5 min; in lanes 3 and 4, the samples were mixed with a non-reducing sample buffer and boiled for 2 min; in lanes 5 and 6, the samples were mixed with the same non-reducing sample buffer without boiling. The samples were analysed by 15% SDS-PAGE and subsequent Western blotting with antibodies to the FLAG-epitope. (For figure see previous page)

Oncogene Mouse soluble GFRa4 receptor activates RET J Yang et al. 3895 carried out in Neuro 2a cells. Western blot analysis domain, which shows that signalling is dependent both shows that soluble GFRa4 can activate the PI-3 kinase/ on sGFRa4 as well as RET (Figure 3b). The survival AKT pathway both in the presence or absence of PSPN assays (Figure 3c) were performed in transiently (Figure 3a). Furthermore, the ability of the FLAG- transfected rat 7 days postnatal cerebellar granule tagged sGFRa4 to activate PI-3 kinase/AKT can be neurons (CGNs), which previously have been used to blocked by FLAG antibodies and RET extracellular characterize the functionality of different GFRa receptors (Tansey et al., 2000). CGNs do not endogenously express RET, GFRa4 or PSPN (Yang et al., 2004). Our a data from the survival assay are consistent with the mGFRα4-sol 10000 activation of both RET and the PI-3 kinase/AKT mGFRα4-GPI pathway and show that mouse soluble GFRa4 can signal through RET even in the absence of PSPN. This was determined with three different types of controls. In 5000 the ﬁrst control, we asked if the sGFRa4-concentrate cpm could promote a survival response in non-transfected CGNs (which do not express RET), and found that the response is fully RET-dependent. In the second control, we attempted to determine whether the activity of the 0 FLAG-tagged soluble GFRa4 could be blocked by anti- −15.0 −12.5 −10.0 −7.5 −5.0 FLAG antibodies. Here, we found that both an addition log M (PSPN) of FLAG-antibodies directly to the medium, as well as a

b 2a A H O MW Adrenal Neuro 2 cDN 700 bp → cDNA Figure 2 The mouse soluble GFRa4 interacts with and activates RET. (a) Binding assay between FLAG-tagged mouse soluble 600 bp → ← unspliced PSPN GFRa4 and [125I]-PSPN (&). Cells secreting FLAG-tagged mouse 500 bp → ← spliced PSPN soluble GFRa4 were incubated in 5 mM glucose, 10 mM HEPES (pH 7.4), 1 mM CaCl , 0.5 mM MgCl and 0.2% BSA. After 4 h, the → 2 2 400 bp medium was collected and incubated with [125I]-PSPN (1 nM), þ /À cold PSPN (1, 5, 10, 100 and 200 nM, PeproTech EC LtdAQ) for c l 4 h. The receptor was subsequently immunoprecipitated with anti- -sol FLAG antibodies and Sepharose beads. The beads were washed, 4 4-so ated α α and the amount of bound [125I]-PSPN was measured by a Liquid ive N2a 125 untre na GFR GFR Scintillation Counter (LAB, WallacAQ). The binding of [ I]- + PSPN PSPN to Sepharose beads, in the absence of unlabelled PSPN, was 25 kDa → 20 kDa→ ← soluble GFRα4 used as control and is indicated in the ﬁgure (). The results from binding assays with mouse GPI-anchored GFRa4 (mGFRa4-GPI) is shown in parallel (m). Neuro 2a cells were transiently transfected 1234 with mGFRa4–GPI and subsequently (after 24 h) seeded on pre- IP: RET, IB: FLAG coated 24-well plates. After an additional overnight incubation, the cells were washed with ice-cold DMEM medium and incubated 170 kDa → with 1 nM [125I]-PSPN in DMEM containing 0.2% bovine serum 150 kDa → albumin, 15 mM HEPES (pH 7.5) for 4 h on ice, þ /À cold PSPN (0.1, 0.5, 1.0, 2.0, 20 and 100 nM). Cells were washed four times and IP: RET, IB: RET lysed in 1 M NaOH. The amount of [125I]-PSPN was measured as above. (b) RT–PCR analysis of transcripts encoding PSPN in d ) Neuro 2a cells, according to Lindfors et al. (2006). No spliced PSPN transcript was detected in Neuro 2a cells. cDNA from the adrenal gland was used as a positive control. (c) Co-immunopre- l ol (60o min d -s cipitation of FLAG-tagged mouse soluble GFRa4 and RET. The 4 ate α4-GPIα4-solα (30 αmin)4-s sGFRa4-concentrate (GFRa4-sol) was added þ /À PSPN (100 ng/ ive N2aR a FR ml) to non-transfected Neuro2a cells that were lysed. Untreated untren GF G GFRGFR PSPN (60 min) 123456 + cells, as well as cells treated with medium from naive Neuro 2a cells (naive N2a), were included as controls. RET was immunoprecipitated with RET-antibodies, and the precipitates were analysed with

antibodies to the FLAG-epitope and RET. (d) In vitro kinase assay.

250 kDa ← The sGFRa4-concentrate (GFRa4-sol) was added in the absence or ← ← pRET presence of PSPN (100 ng/ml) to naive, starved Neuro 2a cells, and

150 kDa incubated for 30 or 60 min. Untreated cells and cells treated with 75 kDa ← medium from naive Neuro 2a cells (naive N2a), or medium from Neuro 2a cells transfected with the GPI-anchored splice variant of

mouse (GFRa4-GPI), were included as controls. RET was ← immunoprecipitated from each lysate and the precipitate was re- 37 kDa

suspended in the kinase buffer with 2 mg myelin basic protein

25 kDa ← (MBP) and 2 mCi [g-32P]ATP. The kinase reaction was carried out at ← ← 20 kDa pMBP 301C for 20 min, and terminated by adding 2 Â Laemmli sample buffer. The samples were loaded on a 4–20% gradient SDS–PAGE, which was dried and analysed by autoradiography.

Oncogene Mouse soluble GFRa4 receptor activates RET J Yang et al. 3896 withdrawal of the FLAG-tagged soluble GFRa4 protein Supplementary Figure 1. Our results with soluble from the medium by immunoprecipitation, reduced the GFRa4 show that the soluble receptor can induce survival response dramatically. In the third control, we neurite outgrowth even in the absence of PSPN. asked whether concentrated media from either naive It is generally assumed that the dimeric GFLs induce Neuro 2a, or Neuro 2a cells which overexpress GPI- the dimerization of the GFRa receptors, which in turn anchored GFRa4, could promote a survival response. trigger the dimerization and activation of RET. In the We found that the media in both these cases were case of the mouse soluble GFRa4 receptor, it is tempting inactive. to speculate that its ability to activate RET directly Recently, we have reported that PSPN mediates could be owing to the non-conserved cysteine residues in differentiation in PC6-3 cells transfected with GPI- D3 domain. These residues may be involved in the anchored GFRa4 (Yang et al., 2004). The PC6-3 cell line formation of intermolecular cysteine bridges, thereby is a clone of rat PC12 cells that expresses low levels of giving rise to dimeric GFRa4 receptor complexes in the endogenous RET. Here, we show that soluble GFRa4 absence of PSPN (Figure 1e). Recently, a mutation that also has the capacity to induce neurite outgrowth in gives rise to two putative forms of soluble GFRa4 these cells (Figure 4). We and others have previously receptors was reported to be involved in a more shown that soluble GFRa1 (both full-length and D1- aggressive disease course of MEN 2 (Vanhorne et al., truncated forms) can induce an activation of RET only 2005). As these mutated forms of the GFRa4 receptor in the presence of GDNF (Paratcha et al., 2001; also harbor non-conserved cysteine residues in D3 Virtanen et al., 2005). A representative RET-phos- (Figure 1a), it remains to be determined if the described phorylation assay with soluble GFRa1 is shown in phenotype could be caused by the formation of covalently linked dimeric GFRa4 receptor complexes. Our ﬁndings also offer an insight into MEN 2 cases a where RET is not mutated. sol 4-GPI 4- 4-sol α α α R untreatednaive N2aGF GFR GFR µ + PSPN LY 294002 ( 20 M ) - - - - - + - +

DMSO - - - + - - - - 75 kDa ←

IB: pAKT 50 kDa ← Figure 3 Mouse soluble GFRa4 promotes PI-3 kinase/AKT 1 2 3 4 5 6 7 8 activation and neuronal survival. (a) Activation of the PI-3 kinase/AKT pathway in Neuro 2a cells. Naive Neuro 2a cells were starved for 7–8 h in serum-free RPMI 1640 medium. The sGFRa4- IB: AKT concentrate (GFRa4-sol) was added with or without PSPN (100 ng/ml) for 30 min, whereafter the samples were washed with ice-cold PBS and lysed. The same controls as in Figure 2d were b included. The selective inhibitor of PI-3 kinase, LY 294002, was -sol 4-sol 4-sol 4-sol 4 ECD also included as a control. In this control, the cells were pre-treated α α α e N2a α with LY 294002 at 20 mM for 1 h before adding the sGFRa4- iv ET R concentrate in the presence of the inhibitor (30 min). DMSO is untreated na GFR GFR GFR +FLAG GFRab + PSPN + a solvent for the inhibitor, and was therefore used as a control.

75 kDa ← The extracts were analysed by Western blotting with antibodies IB: pAKT to phospho-AKT (pAKT). Equal loading of the samples was

50 kDa ← ensured by re-probing the membrane with antibodies to AKT. 1 2 3 4 5 6 (b) Inhibition of the PI-3 kinase/AKT-activation in Neuro 2a cells. The sGFRa4-concentrate was pre-incubated with either FLAG- antibodies (FLAG ab) at 3.5 mg/ml (Sigma AQ), or RET IB: AKT extracellular domain protein (RET ECD; R&D SystemsAQ) at 7 mg/ml before the concentrate was added to the Neuro 2a cells.

The assay was performed as in (a). (c) Cell survival assay in CGNs. P

c % neuronal survival On day 5 of culture, the neurons were co-transfected with EGFP

ET R 0 102030405060708090 EGF and RET as indicated in the ﬁgure. After a 24-h transfection, the initial number of EGFP-positive cells was counted in designated - + + ﬁelds (around 200 cells). Where indicated, the cultures were mock N2a + + subsequently switched from high potassium (25 mM) with serum (K25 þ S) to low potassium (5 mM) medium without serum naive N2a + + (K5ÀS). Concentrated media from mock transfected Neuro 2a cells (mock N2a), naive Neuro 2a cells (naive N2a), the GFRa4- GFRα4-GPI + + GPI stable cell line (GFRa4-GPI) and the GFRa4-soluble stable K5-S GFRα4-sol + - cell line (GFRa4-sol) were exchanged for K5ÀS with the aid of * Amicon Ultra-15 columns and added to the CGNs. PSPN (100 ng/ GFRα4-sol + + ml) or antibodies to the FLAG-epitope (FLAG ab) were added GFRα4-sol + PSPN + + (1 mg/ml) as indicated. As an additional control, the FLAG-tagged * GFRa4-sol was immunoprecipitated (IP) from the media before GFRα4-sol + FLAG ab + + adding it to the CGNs. The side effects of FLAG antibodies were excluded by adding the antibodies to CGNs grown in K25 þ S GFRα4-sol + IP + + medium. EGFP-positive CGNs were re-counted after 3.5 days. For K25+S FLAG ab + - each treatment, two parallel samples were counted and the assay was repeated three times.

Oncogene Mouse soluble GFRa4 receptor activates RET J Yang et al. 3897 Acknowledgements

We thank Maria Lindahl for providing us with the cDNA from the adrenal gland and the primers for the RT–PCR reaction. We also thank Pa¨ ivi Linholm for providing us with the cDNA from Neuro 2a cells, Matthew Phillips for correcting the language, and Kerstin Krieglstein for comment- ing on the manuscript. This work was supported by grants from the Academy of Finland and Sigrid Juse´ lius Foundation (to MS). – Naive N2a Mock N2a References

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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