The Mouse Soluble Gfra4 Receptor Activates RET Independently of Its Ligandpersephin
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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 a bc NN d e D1 D2 D2 D2 D2 D3 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).