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DRAGON, a Bone Morphogenetic Protein Co-receptor *

DRAGON, a Bone Morphogenetic Protein Co-receptor * THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 14, Issue of April 8, pp. 14122–14129, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Received for publication, September 1, 2004, and in revised form, January 21, 2005 Published, JBC Papers in Press, January 25, 2005, DOI 10.1074/jbc.M410034200 ¶  ¶¶ Tarek A. Samad‡§ , Anuradha Rebbapragada§ , Esther Bell**‡‡, Ying Zhang§§, Yisrael Sidis , Sung-Jin Jeong‡, Jason A. Campagna‡, Stephen Perusini , David A. Fabrizio§§, ¶¶ Alan L. Schneyer , Herbert Y. Lin§§, Ali H. Brivanlou**, Liliana Attisano , and Clifford J. Woolf‡ From the ‡Neural Plasticity Research Group, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129, the Department of Biochemistry, University of Toronto, Ontario M5S 1A8, Canada, the **Laboratory of Vertebrate Embryology, The Rockefeller University, New York, ¶¶ New York 10021, the §§Program in Membrane Biology and Renal Unit and Reproductive Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129 plexes, of particular intracellular signaling pathways. The Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF) superfamily of TGF/activin/nodal ligand subfamily contributes to the speci- ligands that regulate many crucial aspects of embryonic fication of endoderm and mesoderm in pregastrula embryos development and organogenesis. Unlike other TGF li- and at gastrula stages, to dorsal mesoderm formation and gands, co-receptors for BMP ligands have not been de- anterior-posterior patterning (3, 4). Later, TGF ligands influ- scribed. Here we show that DRAGON, a glycosylphos- ence the body axis and patterning of the nervous system (5). phatidylinositol-anchored member of the repulsive BMPs, a second major ligand subfamily, contribute to the ven- guidance molecule family, which is expressed early in tralization of germ layers in the early embryo and suppress the the developing nervous system, enhances BMP but not default neural cell fate of the ectoderm (6). BMPs also partici- TGF signaling. DRAGON binds directly to BMP2 and pate later in development in the formation and patterning of BMP4 but not to BMP7 or other TGF ligands. The en- the neural crest, heart, blood, kidney, limb, muscle, and skel- hancing action of DRAGON on BMP signaling is also etal system (7). reduced by administration of Noggin, a soluble BMP Signal transduction in the BMP subfamily is initiated by antagonist, indicating that the action of DRAGON is ligand binding to a receptor complex composed of two type I and ligand-dependent. DRAGON associates directly with two type II receptors. Three different BMP type I receptors BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII (activin receptor-like kinase ALK2, ALK3, and ALK6) and and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In three BMP type II receptors (BMP type II receptor (BMPRII), the Xenopus embryo, DRAGON both reduces the thresh- activin type IIA receptor (ActRIIA), activin type IIB receptor old of the ability of Smad1 to induce mesodermal and (ActRIIB)), each with intracellular serine/threonine kinase do- endodermal markers and alters neuronal and neural mains, have been identified (8). Ligand binding induces phos- crest patterning. The direct interaction of DRAGON phorylation of the type I receptor by the type II receptor, which with BMP ligands and receptors indicates that it is a leads to phosphorylation of cytoplasmic receptor-activated BMP co-receptor that potentiates BMP signaling. Smads. The BMP subfamily signals through one set of receptor- activated Smads (Smad1, Smad5, and Smad8) whereas the TGF subfamily signals via another (Smad2, Smad3). The Transforming growth factor beta (TGF) superfamily li- receptor-activated Smads form heteromeric complexes with a gands that include the TGF, bone morphogenetic protein co-Smad, Smad4, which translocates from the cytoplasm to the (BMP), growth and differentiation factor, and nodal-related nucleus to regulate gene expression. families play a pleiotropic role in vertebrate development by Multiple modulators enhance or reduce TGF and BMP sig- influencing cell specification, differentiation, proliferation, pat- naling. The access of TGF ligands to receptors is inhibited by terning, and migration (1, 2). These functions require the tight the soluble proteins LAP, decorin, and 2-macroglobulin that control of ligand production, ensuring a highly ordered spatio- bind and sequester the ligands (2). Soluble BMP antagonists temporal distribution and specific activation, via receptor com- include Noggin, chordin, chordin-like, the DAN/Cerberus pro- tein family, and sclerostin (2). TGF ligand access to receptors is also controlled by membrane-bound receptors. BAMBI acts * This work was supported in part by the National Institutes of Health (to C. J. W., A. H. B., H. Y. L., and A. L. S.). The costs of publi- as a decoy receptor, competing with the type I receptor (9), cation of this article were defrayed in part by the payment of page -glycan (TGF type III receptor) enhances TGF binding to charges. This article must therefore be hereby marked “advertisement” the type II receptor (10–12), and endoglin enhances TGF in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. binding to ALK1 in endothelial cells (13–15). Cripto, an EGF- § Both authors contributed equally to this work. ¶ To whom correspondence should be addressed. Tel.: 617-724-3621; CFC glycosylphosphatidylinositol (GPI)-anchored membrane Fax: 617-724-3632; E-mail: [email protected]. protein, acts as a co-receptor, increasing the binding of the ‡‡ Supported by a Women in Science Fellowship. TGF ligands nodal, Vg1, and growth and differentiation factor Supported by funds from the Canadian Institute for Health Re- search (CIHR) and is a CIHR Investigator. 1 to activin receptors (16, 17) while blocking activin signaling. The abbreviations used are: TGF, transforming growth factor beta; Only co-receptors acting within the TGF/activin/nodal sig- GPI, glycosylphosphatidylinositol; RGM, repulsive guidance molecule; nal transduction pathway have been identified so far. We now HEK, human embryonic kidney; PBS, phosphate-buffered saline; FBS, find that DRAGON, a 436-amino-acid GPI-anchored protein fetal bovine serum; E, embryonic day; HA, hemagglutinin; DN, domi- nant negative. identified by us and expressed early in the embryonic nervous 14122 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. DRAGON, a Bone Morphogenetic Protein Co-receptor 14123 HepG2, 10 T1/2, or LLC-PK1 cells were transiently transfected with system (18), enhances BMP signaling in cells and developing calcium phosphate/DNA precipitation or Lipofectamine reagent (In- embryos. DRAGON is a member of a gene family comprising two vitrogen). The next day cells were washed with medium containing other GPI-linked proteins, repulsive guidance molecule (RGM) 0.2% FBS and treated overnight with BMP2, BMP4, or TGF as indi- (19) and HFE2 (20) that have diverse roles. DRAGON produces cated. Cell lysates were harvested 48 h post-transfection, and luciferase homophilic and heterophilic cell-cell neuronal adhesion (18), activity was monitored by a plate reader luminometer. Relative light RGM regulates retinotectal projections and neural tube closure units were determined and corrected for transfection efficiency using -galactosidase activity, as all plates were co-transfected with -galac- (19, 21), while mutations in the human HFE2 locus are linked to tosidase expression construct to normalize transfection efficiency. juvenile hemochromatosis (20). Here, we show that DRAGON enhances BMP signaling, binds to specific BMP ligands, associ- Ligands ates with BMP receptors, and potentiates BMP cellular signal- Carrier-free human BMP2, -4, and -7, as well as TGF1, -2, and ing, indicating that DRAGON is a BMP co-receptor. activin were purchased from R&D systems (Minneapolis, MN). Noggin was also purchased from R&D systems. MATERIALS AND METHODS Affinity Labeling, Immunoprecipitation, and Immunoblotting DRAGON Polyclonal Antibody TGF superfamily receptor constructs (28) were transiently trans- The rabbit polyclonal DRAGON antibody was characterized and de- fected into COS-1 cells with Lipofectamine reagent. Cells were affinity- scribed previously (18). Briefly, a rabbit polyclonal antibody was raised labeled with 1.5 nM [ I]-BMP2 or 500 pM TGF for4hat4 °C,andthe against the peptide sequence TAAAHSALEDVEALHPRKC (molecular receptors were cross-linked to ligands with disuccinimidyl suberate weight, 2019.01), which is present in the C terminus of DRAGON up- (Pierce) as described previously (28). Lysates were subjected to immu- stream of its hydrophobic tail and affinity-purified using the same pep- noprecipitation with anti-DRAGON polyclonal or anti-HA monoclonal tide. The antibody binds with high affinity to recombinant DRAGON antibodies (12CA5, Roche Diagnostics) and were separated by SDS- expressed in HEK293T-transfected cells, recognizing a band of 50–55 kDa PAGE, and I-bound ligand was visualized by phosphorimaging. For in Western blots. Western blots of protein extracts from neonatal and co-immunoprecipitation assays, cells lysates were incubated with anti- adult dorsal root ganglion and dorsal root ganglion primary cultures show DRAGON, anti-HA, or anti-FLAG (Sigma) antibodies (1:1000) for 4 h, a similar band with an additional lower band of 35–40 kDa, indicating and immune complexes were collected on protein G-Sepharose. Immu- possible proteolytic cleavage of endogenous DRAGON, similar to that noprecipitates were transferred to nitrocellulose, and proteins were found for chick RGM (19). Preincubation of DRAGON antibody with 1 M detected by immunoblotting with the indicated antibodies. of the above antigenic peptide (4 h at room temperature) results in a loss of both bands by Western blot and staining signals by DRAGON-Fc Preparation and Binding Assay immunohistochemistry. DRAGON-Fc (18) was generated by subcloning DRAGON cDNA Immunohistochemistry without its GPI anchor into the mammalian expression vector pIgplus (R&D Systems, Minneapolis, MN) in-frame with the Fc portion of the To obtain E2.5 embryos, 5-week-old female ICR mice were superovu- human IgG. This allowed us to express a soluble DRAGON-Fc fusion lated by injecting 5 IU of human chorionic gonadotropin (Sigma) 48 h protein. DRAGON-Fc collected in the medium of stably transfected after a prior administration of 5 IU of pregnant mare serum gonado- HEK293T cells was purified using HiTrap protein A affinity columns tropin (Sigma). Treated females were mated with fertile male mice of (Amersham Biosciences) and eluted with 100 mM glycine-HCl, pH 3.0. the same strain and E2.5 embryos were flushed from oviducts. Embryos The elution fraction was neutralized with 0.3 M Tris-HCl, pH 9. Purified were washed with PBS containing 3% FBS (3% FBS-PBS) and fixed in DRAGON-Fc was run on SDS-polyacrylamide gel and immunoblotted 3.7% paraformaldehyde for 10 min at room temperature. They were with anti-Dragon antibody and anti-Fc antibody. permeabilized for 30min with 20 mM HEPES, 300 mM sucrose, 50 mM For ligand binding assays, 2 g of BMP2 ligand/reaction was iodi- NaCl, 3 mM MgCl , and 0.5% Triton X-100, pH7.4. After blocking with nated with I by the modified chloramine-T method as described 3% FBS-PBS for 30min, embryos were incubated with the DRAGON previously (29). Purified soluble DRAGON-Fc (18) was diluted in TBS/ antibody diluted 1:2000 in 3% FBS-PBS overnight at 4 °C, washed, and casein blocking buffer (BioFX, Owings Mills, MD) and incubated over- then incubated with fluorescein isothiocyanate-conjugated anti-rabbit night in the presence or absence of I-labeled radioligands (50,000– IgG (Jackson ImmunoResearch Laboratories) for2hat room tempera- 100,000 counts). For competition binding assays, fixed amounts of ture. After washing with 3% FBS-PBS for 30min at room temperature, radioligands (50,000–100,000 counts) were added to the samples to- embryos were mounted with mounting medium containing propidium gether with increasing amounts (2 pM–500 nM) of non-radioactive TGF iodide (Vector Laboratories, Inc). superfamily ligands or as indicated. Samples were then placed on Whole mount immunohistochemistry in mouse E10.5 embryos was protein A-coated 96-well plates (Pierce) for 90 min, washed 3 times with carried out as described previously (22). Briefly, freshly dissected em- wash buffer (BioFX), and radioactivity was counted using a standard bryos were fixed with 4% paraformaldehyde overnight at 4 °C, washed gamma counter. in saline for 2 h, and then soaked in 80% methanol series. Endogenous peroxidase activity was quenched with 3% H O in 80% methanol and 2 2 Reverse Transcription-PCR Analysis 20% Me SO for 3 h. After washing with Tris-buffered saline containing Reverse transcription-PCR was performed on isolated Xenopus ani- 1% Tween 20 (TBS-T) for 3 h, the embryos were incubated with mal cap explants or embryos, with orthidine decarboxylase as loading DRAGON antibody (1:500) in TBS-T containing 5% skim milk and 5% control (30). Me SO for 2 days at room temperature. The embryos were then washed 3 times and incubated with horseradish peroxidase-coupled anti-rabbit RESULTS IgG (1:200 dilution in TBS-T containing 5% skim milk and 5% Me SO). DRAGON Expression Horseradish peroxidase activity was detected with diaminobenzidine. DRAGON is expressed in mouse E2.5 pre-implantation em- Northern Blot bryos (Fig. 1A), and in postimplantation embryos (E7) (Fig. The premade Northern blot containing 2 g of poly(A) RNA/lane 1B). In E10.5 embryos, DRAGON is found along the neural from mouse embryos at the indicated stages (Clontech, Inc.) was used. tube, in the dorsal root ganglia, and in the tips of the neural The membrane was hybridized with a 300-bp DRAGON cDNA probe as described previously (18). folds and the tail bud (Fig. 1C) as assessed by whole mount immunohistochemistry. Xenopus orthologs of both DRAGON In Situ Hybridization and RGM are expressed maternally and throughout develop- Whole mount in situ hybridizations were carried out as described ment and are detected at high levels by tadpole stages (Fig. previously by Harland (23). Embryos to be sectioned were post-fixed in 1D). Analysis by whole mount in situ hybridization of RGM 4% paraformaldehyde and embedded in 20% gelatin-PBS. Sections expression at stage 12 reveals high levels in the ectoderm by were cut between 50 and 100 m. the blastopore (Fig. 1E, i). More restricted expression is seen by Luciferase Reporter Assays stage 17 and stage 20 in the neural plate, specifically in the dorsal aspect of the neural tube (Fig, 1E, ii and iii). By stage 35, The BMP-inducible BRE-Luc (24), I-BRE-Luc (25), and Msx2-Luc (26) and the TGF-inducible 3TP-lux (27) have been described previously. the expression of RGM is detected in the hindbrain, midbrain, 14124 DRAGON, a Bone Morphogenetic Protein Co-receptor FIG.1. DRAGON is expressed during embryogenesis. A, DRAGON expression in mouse pre-implantation embryos (E2.5) assessed with a DRAGON antibody and visualized by fluorescein isothiocyanate green fluorescence. Nuclei of the embryonic cells are visualized by CY3 red fluorescence (propidium iodide). Pre-adsorption of the DRAGON antibody with the peptide antigen (Pept.) was used as negative control. B, Northern blot showing DRAGON mRNA expression in mouse embryos at embryonic stages E7–17. Cyclophilin mRNA levels were used as loading controls. C, immunohistochemical study of the expression and distribution of DRAGON in mouse E10.5 embryos. D, expression of Xenopus DRAGON (RGMb) and RGMa assessed by reverse transcription (RT)-PCR at the indicated developmental stages. Orthidine decarboxylase (ODC) levels were used as internal control. E, analysis by whole mount in situ hybridization of expression of Xenopus RGMa at stages 12 (i), 17 (ii), 20 (iii), and 35 (iv). Transverse sections through stained stage 12 (top right) and stage 20 (bottom left) embryos are also represented. bp, blastopore; post, posterior; ant, anterior; nc, notochord; rp, roof plate; hb, hindbrain; mb, midbrain; fb, forebrain; ba, branchial arches. and forebrain regions, as well as the branchial arches. In ad- derived reporter construct (24). BMP2 stimulates BRE-Luc dition, expression is seen posteriorly in the somites and tail bud activity in LLC-PK1 cells (Fig. 2). Co-expression of DRAGON (Fig. 1E, iv). with BRE-Luc in the absence of exogenous ligand increases The expression pattern of DRAGON and RGM at different BRE-Luc activity to levels comparable with that achieved by embryonic stages in both the developing mouse and Xenopus is BMP2 stimulation in the absence of DRAGON (Fig. 2A). To comparable with the expression of the BMP type I receptors, assess whether DRAGON regulates the signaling produced by ALK3 and ALK6 and BMP type II receptor (31, 32) (Fig. 1). BMP ligands, LLC-PK1 cells were co-transfected with the This observation prompted us to investigate whether DRAGON BRE-Luc reporter and DRAGON (2 and 20 ng) and incubated contributes to or modulates TGF superfamily signal with BMP2. BMP2 (50 ng/ml) induces a 6-fold increase in transduction. luciferase activity in the absence of DRAGON and a 14-fold increase in DRAGON co-transfected cells (Fig. 2A). DRAGON DRAGON Enhances BMP but Not TGF expression had no effect on TGF signaling, assessed by using Intracellular Signaling a TGF-responsive promoter (TGF-responsive CAGA reporter (12)) in LLC-PK1 cells (data not shown). TGF superfamily ligands are classified as TGF or BMP- like based on the particular Smad-dependent intracellular DRAGON expression activates BMP signaling in the ab- sence of any added ligand (Fig. 2A). To determine whether pathways they activate. We examined whether DRAGON acti- vates these pathways using specific BMP- and TGF-respon- this represents a ligand-independent action or is mediated by endogenous BMP ligands either produced by the cell lines or sive luciferase reporters in LLC-PK1 (kidney epithelial cells), as these cells are highly responsive to many TGF family present in the medium, we investigated whether DRAGON activation of BMP signaling is blocked by administration of members and thereby allow a parallel analysis of any action of DRAGON on TGF superfamily signaling. the soluble BMP antagonist Noggin (2). When added to the To determine whether DRAGON alters BMP signaling we medium Noggin (50–1000 ng/ml) inhibits both the DRAGON- used the BMP-inducible promoter BRE-Luc, an Id1 promoter- and BMP2-induced activation of the BRE-Luc reporter in DRAGON, a Bone Morphogenetic Protein Co-receptor 14125 FIG.2. DRAGON expression enhances BMP signaling and sensitizes cells to BMP. A, LLC-PK1 renal epithelial cells were transiently transfected with BRE-Luciferase construct (BRE-Luc) alone or in combination with 2 or 20 ng of a DRAGON expression construct. Luciferase activity was assessed after incubation in the absence (white bars) or presence (black bars) of BMP2 (1 nM) in the culture medium. Luciferase activity is represented as relative light units (RLU). DRAGON increases and potentiates BMP2-mediated signaling. B, DRAGON-mediated BMP signaling is ligand-dependent. LLC-PK1 cells were transiently transfected with BRE-Luc in the presence or absence of DRAGON and treated with increasing amounts of Noggin as indicated. As a positive control, BRE-Luc-transfected LLC-PK1 cells were treated with BMP2 (1 nM) in the presence of Noggin (50 ng/ml). As a negative control, LLC-PK1 cells transfected with the TGF-responsive reporter construct MLP-Luc were treated with TGF (1 nM) in the presence of Noggin (50 ng/ml). C, 10 T1/2 cells transiently transfected with the BMP-responsive I-BRE-Luc reporter and DRAGON were incubated with increasing doses of BMP2 (25–500 pM). LLC-PK1 cells (Fig. 2B). Noggin has no effect on TGF- through an independent receptor pathway that converges on induced stimulation of the TGF-responsive CAGA reporter the intracellular Smads. We therefore investigated whether (Fig. 2B). DRAGON-mediated activation of BMP signaling is, DRAGON binds to BMP ligands, interacts directly with BMP therefore, ligand-dependent. receptors, and whether a dominant negative BMP receptor or DRAGON significantly enhances BMP signaling and in- Smad mutants reduce the DRAGON-induced enhancement of creases cellular sensitivity to BMP2 in LLC-PK1 cells (Fig. 2, A BMP signaling. and B). To rule out a potential cell-type or reporter-specific DRAGON Binds to BMP but Not TGF Ligands—To deter- effect of DRAGON expression, we examined the generality of mine whether cell surface-localized DRAGON binds directly to this effect, i.e. whether it is restricted to one cell line or to one BMP ligands, COS-1 cells expressing DRAGON were incubated 125 125 BMP-inducible reporter construct. We tested the effect of with [ I]-BMP2 or [ I]-TGF and receptor-bound ligand DRAGON expression in 10 T1/2 cells (mouse mesenchymal cross-linked. HA-tagged BMP type I receptor (ALK6) or TGF stem cells), another BMP-responsive cell line (28), using an- type II receptor (TRII) served as positive controls for BMP2 or other BMP-inducible promoter reporter construct, I-BRE-Luc, TGF binding, respectively. Immunoprecipitation of DRAGON 125 125 which contains a portion of intron 1 of the Smad7 gene (25). from [ I]-BMP2- but not [ I]-TGF-labeled cells revealed DRAGON significantly enhances BMP2-dependent reporter ac- the presence of two [ I]-BMP2-bound proteins of 55–65 kDa tivity in this cell line at BMP2 concentrations of 25–300 pM (Fig. 3A), consistent with the expected size of a BMP-bound (Fig. 2C). This effect is lost at 300 pM or higher, where maximal DRAGON. The presence of two bands suggests that cell sur- signaling is achieved. face-localized DRAGON may be subject to post-translational Similar results were obtained using LLC-PK1, 10 T1/2, and modification. These data, indicating that cell surface-expressed HepG2 cell lines and different BMP-responsive promoters (I- DRAGON can bind BMP2 but not TGF, prompted us to assess BRE-Luc, BRE-Luc, and Msx2-Luc) (Fig. 2, and data not whether DRAGON can directly bind BMP ligands. shown). DRAGON significantly increases cellular sensitivity, We examined whether DRAGON directly binds to BMP li- therefore, to low doses of BMP2 in a manner that is neither gands using a soluble DRAGON-Fc fusion protein in a cell-free reporter- nor cell-specific. These results suggest that the regu- binding system (12). DRAGON-Fc binds [ I]-BMP2 with high latory role of DRAGON in BMP signaling is a generalized affinity and an apparent dissociation rate constant (K )of1.5 phenomenon. This finding prompted us to study the mecha- nM (Fig. 3B). This binding is competed with an excess of unla- nism of the enhancement of BMP signaling by DRAGON and beled BMP2 as well as by BMP4 (4 nM) (Fig. 3, B and C). In where and how it interacts with BMP signaling components. contrast, BMP7 (4 nM), activin A, TGF1, -2, or -3 (4 nM each) do not competitively inhibit the binding of DRAGON-Fc to Mechanism of the Enhancement of BMP BMP2 (Fig. 3C). Pretreatment of cells with DRAGON-Fc de- Signaling by DRAGON creases BMP2- but not BMP7-mediated activation of the BRE- DRAGON could potentially enhance BMP signaling Luc promoter in a dose-dependent manner (60 and 300 ng/ml) through a direct modulation of the BMP signaling complex or (Fig. 3D) but had no effect on TGF-1-dependent activation of 14126 DRAGON, a Bone Morphogenetic Protein Co-receptor FIG.3. DRAGON interacts directly with BMP2 and BMP4. A, DRAGON expressed in COS cells binds to BMP2 but not TGF. COS-1 cells were transiently transfected with DRAGON (DR), HA-tagged ALK6 (A6/HA), or TRII (RII/HA). Cells were affinity-labeled with [ I]-BMP2 (left 125 125 panel)or[ I]-TGF (right panel) and lysates subjected to immunoprecipitation (IP) or immunoblotting with the indicated antibodies. I-Labeled ligand binding to receptors was detected by phosphorimaging. B, DRAGON-Fc binds directly to BMP2. [ I]-BMP2 was incubated with increasing amounts (10–50 ng) of purified DRAGON-Fc then added to protein A-coated plates, and bound radioactivity was measured (left graph). 20 ng of DRAGON-Fc were incubated with [ I]-BMP2 in the presence of increasing amounts of competing unlabeled BMP2 (0–42 nM)(right graph). C, DRAGON-Fc binds directly to BMP2 and BMP4 but not BMP7 or TGF ligands. DRAGON-Fc was incubated with [ I]-BMP2 in the presence of unlabeled BMP2, BMP4, BMP7, Activin A (ActA), TGF1, TGF2, and TGF3, and [ I]-BMP2 binding was measured. D, DRAGON-Fc reduces BMP2 mediated signaling. LLC-PK1 cells were transiently transfected with BRE-Luc construct and treated with BMP2 (150 ng/ml) and DRAGON-Fc (60 and 300 ng/ml). E, DRAGON-Fc (60 ng/ml) reduces BMP2 but not BMP7- or TGF1(1nM each)-mediated signaling. LLC-PK1 cells were transiently transfected with BRE-Luc and treated with BMP2 or BMP7 or transiently transfected with 3TP-Lux construct and treated with TGF1 in the absence or presence of DRAGON-Fc. the CAGA-Luc promoter (Fig. 3E). DRAGON directly interacts immunoprecipitation followed by anti-HA immunoblotting re- with members of the BMP but not the TGF ligand family, with veals that DRAGON associates directly with all BMP type I a preference for BMP2 and BMP4. receptors, (ALK2, ALK3, and ALK6) (Fig. 5A) and with the DRAGON Enhances BMP Signaling Only when Expressed on BMP type II receptors ActRII and ActRIIB (Fig. 5B). the Cell Surface—To assess whether the GPI anchor is required DRAGON Signaling Is Blocked by Dominant Negative ALK3, for the action of DRAGON on BMP signaling, we used the -6, and Smad1—We used dominant negative type I receptors DRAGON-Fc fusion protein where the C-terminal GPI anchor (ALK1-DN, ALK3-DN, and ALK6-DN), which are deficient in is deleted and replaced by human Fc (18). When cotransfected kinase activity and unable to phosphorylate Smads (33), to with the BRE-Luc reporter construct this DRAGON-Fc, which confirm that DRAGON acts through modulation of BMP recep- is secreted into the medium, fails to increase BMP signaling tors and subsequent activation of the downstream Smad1 path- (Fig. 4A). Western blot confirms the expression of both way (Fig. 5, C and D). Co-expression of DRAGON with DRAGON and DRAGON-Fc in the transfected cells (Fig. 4B). ALK3-DN and ALK6-DN in LLC-PK1 cells decreases DRAG- These results demonstrate that the expression of DRAGON on ON-mediated induction of I-BRE luciferase activity to base the cell surface is required for its action on BMP signaling. line, whereas co-expression of ALK1-DN does not affect DRAG- DRAGON Binds to BMP Type I and Type II Receptors—BMP ON-mediated BMP signaling (Fig. 5C). Co-expression of type I and type II receptors are essential for mediating BMP DRAGON in LLC-PK1 cells with a Smad1 dominant negative responses. To test whether DRAGON directly interacts with mutant that lacks the C-terminal phosphoacceptor domain (15, BMP type I and type II receptors, HEK293T cells were tran- 34) also dose dependently reduces DRAGON-induced signal- siently transfected either with DRAGON alone or with HA ing. In contrast, co-expression with wild type Smad1 enhances epitope-tagged type I and type II BMP receptors. Anti-Dragon this signaling (Fig. 5D). These studies demonstrate that DRAGON, a Bone Morphogenetic Protein Co-receptor 14127 DRAGON exerts its effect on BMP signaling via BMP receptors nation with Smad1 mRNA in Xenopus embryos at the two-cell and potentiates Smad1 signaling. stage. We then analyzed the expression levels of mesodermal or endodermal tissue markers as indicators of activation of BMP DRAGON Enhances BMP Signaling in Xenopus Embryos signaling. DRAGON co-injection with Smad1 leads to the in- duction of mRNA for the pan-mesodermal marker Xbra and BMP ligands play pivotal roles in the gastrulation of the two endodermal markers, mix1 and mixer in a dose-dependent embryo (35) and regulate the formation of the mesoderm and manner (Fig. 6A). Injected alone, neither DRAGON nor Smad1 endoderm (36). An analysis of mice lacking genes encoding at the low amounts administered induce the expression of the BMPs, BMP receptors, and their downstream signal transduc- mesodermal and endodermal markers (Fig. 6A). The reduction ers reveal major early and late embryonic developmental alter- in the threshold of the activity of Smad1 by DRAGON indicates ations, with defects in mesoderm and endoderm formation (37). the enhancement of BMP signaling in the embryo and is con- We have examined whether DRAGON expression in Xenopus sistent with the results seen in mammalian cells (Fig. 2). embryos enhances BMP signaling and synergizes with BMP signal transduction components to facilitate BMP signaling in DRAGON Regulates Neural Patterning in vivo. Mouse DRAGON mRNA was injected alone or in combi- Xenopus Embryos Genetic experiments have suggested that various BMPs can promote dorsal neural cell fates (38) and act as morphogens to specify dorsal neural cell types (39). However the contribution of the different BMP signaling components to neural pattern- ing is not fully elucidated. To determine the effect of DRAGON expression on tissue patterning, we analyzed fate changes in Xenopus embryonic explants by reverse transcription-PCR for a variety of molecular markers. DRAGON mRNA was injected at the two-cell stage in the animal caps, and cultured ectoder- mal explants were analyzed for changes in gene expression. DRAGON induces expression of the anterior pan-neural marker Nrp1, the cement gland marker (the most anterior structure in the tadpole) XAG, as well as the early heart FIG.4. DRAGON-mediated BMP signaling requires its GPI an- marker nkx2.5 (Fig. 6B). Up-regulation of both neural and chor. A, DRAGON-Fc (DR-Fc) when expressed by LLC-PK1 cells does heart markers by DRAGON (Fig. 6B) suggests that the induc- not increase BMP signaling. B, Expression of both full-length DRAGON tion of neural tissue by DRAGON is not direct but occurs via (DR) and DRAGON-Fc by the LLC-PK1 cells was confirmed by Western blot analysis. formation of dorsal mesoderm, which induces neural tissue FIG.5. DRAGON interacts directly with BMP type I and type II receptors and signals through the ALK/Smad pathway. HEK293T cells were transiently transfected with HA-tagged type I receptors, ALK2, ALK3, or ALK6 (A), HA-tagged type II receptors, ActRII (RII/HA) or ActRIIB (RIIB/HA), or FLAG-tagged tailless version of BMP type II receptor (B) with or without DRAGON (DR) as indicated. Cell lysates were immunoprecipitated (IP) with anti-Dragon (-DR) antibodies, and associated receptors were visualized by anti-HA or anti-FLAG immunoblotting. Protein expression was confirmed by anti-HA, anti-FLAG, and anti-Dragon immunoblotting of total cell lysates (bottom panels). C, ALK3-DN or ALK6-DN but not ALK1-DN expression reduces DRAGON-induced signaling. LLC-PK1 epithelial cells were transiently transfected with BRE-Luc construct, with or without DRAGON, ALK1-DN, ALK3-DN, or ALK6-DN expression constructs as indicated. D, dominant negative Smad1 reduces DRAGON signaling. LLC-PK1 cells transiently transfected with BRE-Luc and DRAGON constructs, in the absence or presence of increasing amounts (0.1–2 g) of wild type (WT) or dominant negative (DN) Smad1 as indicated. 14128 DRAGON, a Bone Morphogenetic Protein Co-receptor FIG.6. DRAGON potentiates BMP signaling in Xenopus embryos. A, mi- croinjection of mouse DRAGON mRNA in Xenopus laevis embryos at gastrula stages synergizes with Smad1 (mRNA in- jection) to induce mesodermal (xBra and mix1) and endodermal (mixer) markers, as assessed by reverse transcription (RT)- PCR. No induction of EK or Sox2 occurs. B, in animal cap explants DRAGON mRNA injection induces expression of the anterior neural markers nrp1 and Otx2, as well the as cement gland marker (Xag) and nkx2.5, a heart marker. Orthidine decarboxylase (ODC) is the loading con- trol. C, Xenopus embryos were injected in the animal pole of one of two cells at the two-cell stage with DRAGON mRNA and analyzed at late neurula (stage 23), for changes in neural crest patterning, and early tadpole stages (stage 28) for ectopic induction of neural tissue. DRAGON overexpression inhibits twist RNA, a neu- ral crest marker (top panels), and induces ectopic patches of N-tubulin, a neural marker (lower panels). (Fig. 6A). To determine whether DRAGON expression alters cytoplasmic, and nuclear levels and contributes to the forma- neural patterning, we injected mouse DRAGON mRNA into tion and response to gradients of morphogens in development. one cell at the two-cell stage in the animal pole of the Xenopus The role of DRAGON in the embryo may be to differentially embryo and left the injected embryos to develop until early increase or boost the sensitivity of cells to low levels of BMP tadpole stages (Fig. 6C). The uninjected cell serves as control ligand. Such low levels will occur within the spatial BMP for the DRAGON-injected experimental side in the same em- gradients established in the embryo, and DRAGON expression bryo. Overexpression of DRAGON results in an increase in may enable cells to respond earlier or with a greater response N-tubulin (a general neuronal differentiation marker) expres- to a particular level of BMP, than those that do not express sion, as shown by the appearance of ectopic patches of N- DRAGON. Therefore, a differential sensitivity to BMP ligands tubulin-expressing cells (Fig. 6C) and a decrease in neural crest produced by DRAGON expression may contribute to tissue derivatives, as shown by a reduction in twist (an anterior patterning. neural crest marker) expression on the injected side (Fig. 6C) DRAGON interacts directly with specific BMP ligands (40). DRAGON may contribute, therefore, to the patterning of (BMP2 and -4 but not BMP7) in the absence and presence of the developing nervous system by promoting a neuronal phe- BMP type I and type II receptors. How DRAGON enhances notype and inhibiting neural crest differentiation. signaling by the receptor complex needs to be determined. Does it change the affinity of BMP receptor-ligand interactions or DISCUSSION facilitate the formation of heteromeric receptor complexes be- DRAGON was identified by us in a genomic-binding screen tween type I and type II receptors? As a GPI-anchored protein for promoter regions of effector genes regulated by the devel- with no transmembrane domain, DRAGON is unlikely to di- opmentally regulated paired homeodomain transcription fac- rectly alter either the intracellular kinase activity of the BMP tor, DRG11 and is a 436-amino acid member of the RGM family receptors or their coupling to the receptor-activated Smads, but of GPI-anchored proteins (18). We show here that DRAGON it may stabilize the receptor complex. The action of DRAGON, binds to BMP ligands and receptors, and enhances BMP sig- like other GPI-anchored proteins, may depend on localization naling, and conclude that it is a BMP co-receptor. in lipid rafts (41) and in this way contribute to the assembly Given the wide spectrum of responses to the TGF super- and trafficking of the receptor complex in microdomains. family, mechanisms for positive and negative modulation of DRAGON is not necessary for BMP2 and -4 signaling but their signaling exist to exert tight spatiotemporal regulation. rather enhances signaling in response to low concentrations of Positive regulation amplifies signals by reducing activation threshold and potentiating biological activity, whereas nega- these BMP ligands. In this respect, DRAGON differs from nodal, Vg1, and growth and differentiation factor 1, which tive regulation limits the magnitude of signals or terminates signaling. 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DRAGON, a Bone Morphogenetic Protein Co-receptor *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2005 Elsevier Inc.
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0021-9258
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DOI
10.1074/jbc.m410034200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 14, Issue of April 8, pp. 14122–14129, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Received for publication, September 1, 2004, and in revised form, January 21, 2005 Published, JBC Papers in Press, January 25, 2005, DOI 10.1074/jbc.M410034200 ¶  ¶¶ Tarek A. Samad‡§ , Anuradha Rebbapragada§ , Esther Bell**‡‡, Ying Zhang§§, Yisrael Sidis , Sung-Jin Jeong‡, Jason A. Campagna‡, Stephen Perusini , David A. Fabrizio§§, ¶¶ Alan L. Schneyer , Herbert Y. Lin§§, Ali H. Brivanlou**, Liliana Attisano , and Clifford J. Woolf‡ From the ‡Neural Plasticity Research Group, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129, the Department of Biochemistry, University of Toronto, Ontario M5S 1A8, Canada, the **Laboratory of Vertebrate Embryology, The Rockefeller University, New York, ¶¶ New York 10021, the §§Program in Membrane Biology and Renal Unit and Reproductive Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129 plexes, of particular intracellular signaling pathways. The Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF) superfamily of TGF/activin/nodal ligand subfamily contributes to the speci- ligands that regulate many crucial aspects of embryonic fication of endoderm and mesoderm in pregastrula embryos development and organogenesis. Unlike other TGF li- and at gastrula stages, to dorsal mesoderm formation and gands, co-receptors for BMP ligands have not been de- anterior-posterior patterning (3, 4). Later, TGF ligands influ- scribed. Here we show that DRAGON, a glycosylphos- ence the body axis and patterning of the nervous system (5). phatidylinositol-anchored member of the repulsive BMPs, a second major ligand subfamily, contribute to the ven- guidance molecule family, which is expressed early in tralization of germ layers in the early embryo and suppress the the developing nervous system, enhances BMP but not default neural cell fate of the ectoderm (6). BMPs also partici- TGF signaling. DRAGON binds directly to BMP2 and pate later in development in the formation and patterning of BMP4 but not to BMP7 or other TGF ligands. The en- the neural crest, heart, blood, kidney, limb, muscle, and skel- hancing action of DRAGON on BMP signaling is also etal system (7). reduced by administration of Noggin, a soluble BMP Signal transduction in the BMP subfamily is initiated by antagonist, indicating that the action of DRAGON is ligand binding to a receptor complex composed of two type I and ligand-dependent. DRAGON associates directly with two type II receptors. Three different BMP type I receptors BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII (activin receptor-like kinase ALK2, ALK3, and ALK6) and and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In three BMP type II receptors (BMP type II receptor (BMPRII), the Xenopus embryo, DRAGON both reduces the thresh- activin type IIA receptor (ActRIIA), activin type IIB receptor old of the ability of Smad1 to induce mesodermal and (ActRIIB)), each with intracellular serine/threonine kinase do- endodermal markers and alters neuronal and neural mains, have been identified (8). Ligand binding induces phos- crest patterning. The direct interaction of DRAGON phorylation of the type I receptor by the type II receptor, which with BMP ligands and receptors indicates that it is a leads to phosphorylation of cytoplasmic receptor-activated BMP co-receptor that potentiates BMP signaling. Smads. The BMP subfamily signals through one set of receptor- activated Smads (Smad1, Smad5, and Smad8) whereas the TGF subfamily signals via another (Smad2, Smad3). The Transforming growth factor beta (TGF) superfamily li- receptor-activated Smads form heteromeric complexes with a gands that include the TGF, bone morphogenetic protein co-Smad, Smad4, which translocates from the cytoplasm to the (BMP), growth and differentiation factor, and nodal-related nucleus to regulate gene expression. families play a pleiotropic role in vertebrate development by Multiple modulators enhance or reduce TGF and BMP sig- influencing cell specification, differentiation, proliferation, pat- naling. The access of TGF ligands to receptors is inhibited by terning, and migration (1, 2). These functions require the tight the soluble proteins LAP, decorin, and 2-macroglobulin that control of ligand production, ensuring a highly ordered spatio- bind and sequester the ligands (2). Soluble BMP antagonists temporal distribution and specific activation, via receptor com- include Noggin, chordin, chordin-like, the DAN/Cerberus pro- tein family, and sclerostin (2). TGF ligand access to receptors is also controlled by membrane-bound receptors. BAMBI acts * This work was supported in part by the National Institutes of Health (to C. J. W., A. H. B., H. Y. L., and A. L. S.). The costs of publi- as a decoy receptor, competing with the type I receptor (9), cation of this article were defrayed in part by the payment of page -glycan (TGF type III receptor) enhances TGF binding to charges. This article must therefore be hereby marked “advertisement” the type II receptor (10–12), and endoglin enhances TGF in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. binding to ALK1 in endothelial cells (13–15). Cripto, an EGF- § Both authors contributed equally to this work. ¶ To whom correspondence should be addressed. Tel.: 617-724-3621; CFC glycosylphosphatidylinositol (GPI)-anchored membrane Fax: 617-724-3632; E-mail: [email protected]. protein, acts as a co-receptor, increasing the binding of the ‡‡ Supported by a Women in Science Fellowship. TGF ligands nodal, Vg1, and growth and differentiation factor Supported by funds from the Canadian Institute for Health Re- search (CIHR) and is a CIHR Investigator. 1 to activin receptors (16, 17) while blocking activin signaling. The abbreviations used are: TGF, transforming growth factor beta; Only co-receptors acting within the TGF/activin/nodal sig- GPI, glycosylphosphatidylinositol; RGM, repulsive guidance molecule; nal transduction pathway have been identified so far. We now HEK, human embryonic kidney; PBS, phosphate-buffered saline; FBS, find that DRAGON, a 436-amino-acid GPI-anchored protein fetal bovine serum; E, embryonic day; HA, hemagglutinin; DN, domi- nant negative. identified by us and expressed early in the embryonic nervous 14122 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. DRAGON, a Bone Morphogenetic Protein Co-receptor 14123 HepG2, 10 T1/2, or LLC-PK1 cells were transiently transfected with system (18), enhances BMP signaling in cells and developing calcium phosphate/DNA precipitation or Lipofectamine reagent (In- embryos. DRAGON is a member of a gene family comprising two vitrogen). The next day cells were washed with medium containing other GPI-linked proteins, repulsive guidance molecule (RGM) 0.2% FBS and treated overnight with BMP2, BMP4, or TGF as indi- (19) and HFE2 (20) that have diverse roles. DRAGON produces cated. Cell lysates were harvested 48 h post-transfection, and luciferase homophilic and heterophilic cell-cell neuronal adhesion (18), activity was monitored by a plate reader luminometer. Relative light RGM regulates retinotectal projections and neural tube closure units were determined and corrected for transfection efficiency using -galactosidase activity, as all plates were co-transfected with -galac- (19, 21), while mutations in the human HFE2 locus are linked to tosidase expression construct to normalize transfection efficiency. juvenile hemochromatosis (20). Here, we show that DRAGON enhances BMP signaling, binds to specific BMP ligands, associ- Ligands ates with BMP receptors, and potentiates BMP cellular signal- Carrier-free human BMP2, -4, and -7, as well as TGF1, -2, and ing, indicating that DRAGON is a BMP co-receptor. activin were purchased from R&D systems (Minneapolis, MN). Noggin was also purchased from R&D systems. MATERIALS AND METHODS Affinity Labeling, Immunoprecipitation, and Immunoblotting DRAGON Polyclonal Antibody TGF superfamily receptor constructs (28) were transiently trans- The rabbit polyclonal DRAGON antibody was characterized and de- fected into COS-1 cells with Lipofectamine reagent. Cells were affinity- scribed previously (18). Briefly, a rabbit polyclonal antibody was raised labeled with 1.5 nM [ I]-BMP2 or 500 pM TGF for4hat4 °C,andthe against the peptide sequence TAAAHSALEDVEALHPRKC (molecular receptors were cross-linked to ligands with disuccinimidyl suberate weight, 2019.01), which is present in the C terminus of DRAGON up- (Pierce) as described previously (28). Lysates were subjected to immu- stream of its hydrophobic tail and affinity-purified using the same pep- noprecipitation with anti-DRAGON polyclonal or anti-HA monoclonal tide. The antibody binds with high affinity to recombinant DRAGON antibodies (12CA5, Roche Diagnostics) and were separated by SDS- expressed in HEK293T-transfected cells, recognizing a band of 50–55 kDa PAGE, and I-bound ligand was visualized by phosphorimaging. For in Western blots. Western blots of protein extracts from neonatal and co-immunoprecipitation assays, cells lysates were incubated with anti- adult dorsal root ganglion and dorsal root ganglion primary cultures show DRAGON, anti-HA, or anti-FLAG (Sigma) antibodies (1:1000) for 4 h, a similar band with an additional lower band of 35–40 kDa, indicating and immune complexes were collected on protein G-Sepharose. Immu- possible proteolytic cleavage of endogenous DRAGON, similar to that noprecipitates were transferred to nitrocellulose, and proteins were found for chick RGM (19). Preincubation of DRAGON antibody with 1 M detected by immunoblotting with the indicated antibodies. of the above antigenic peptide (4 h at room temperature) results in a loss of both bands by Western blot and staining signals by DRAGON-Fc Preparation and Binding Assay immunohistochemistry. DRAGON-Fc (18) was generated by subcloning DRAGON cDNA Immunohistochemistry without its GPI anchor into the mammalian expression vector pIgplus (R&D Systems, Minneapolis, MN) in-frame with the Fc portion of the To obtain E2.5 embryos, 5-week-old female ICR mice were superovu- human IgG. This allowed us to express a soluble DRAGON-Fc fusion lated by injecting 5 IU of human chorionic gonadotropin (Sigma) 48 h protein. DRAGON-Fc collected in the medium of stably transfected after a prior administration of 5 IU of pregnant mare serum gonado- HEK293T cells was purified using HiTrap protein A affinity columns tropin (Sigma). Treated females were mated with fertile male mice of (Amersham Biosciences) and eluted with 100 mM glycine-HCl, pH 3.0. the same strain and E2.5 embryos were flushed from oviducts. Embryos The elution fraction was neutralized with 0.3 M Tris-HCl, pH 9. Purified were washed with PBS containing 3% FBS (3% FBS-PBS) and fixed in DRAGON-Fc was run on SDS-polyacrylamide gel and immunoblotted 3.7% paraformaldehyde for 10 min at room temperature. They were with anti-Dragon antibody and anti-Fc antibody. permeabilized for 30min with 20 mM HEPES, 300 mM sucrose, 50 mM For ligand binding assays, 2 g of BMP2 ligand/reaction was iodi- NaCl, 3 mM MgCl , and 0.5% Triton X-100, pH7.4. After blocking with nated with I by the modified chloramine-T method as described 3% FBS-PBS for 30min, embryos were incubated with the DRAGON previously (29). Purified soluble DRAGON-Fc (18) was diluted in TBS/ antibody diluted 1:2000 in 3% FBS-PBS overnight at 4 °C, washed, and casein blocking buffer (BioFX, Owings Mills, MD) and incubated over- then incubated with fluorescein isothiocyanate-conjugated anti-rabbit night in the presence or absence of I-labeled radioligands (50,000– IgG (Jackson ImmunoResearch Laboratories) for2hat room tempera- 100,000 counts). For competition binding assays, fixed amounts of ture. After washing with 3% FBS-PBS for 30min at room temperature, radioligands (50,000–100,000 counts) were added to the samples to- embryos were mounted with mounting medium containing propidium gether with increasing amounts (2 pM–500 nM) of non-radioactive TGF iodide (Vector Laboratories, Inc). superfamily ligands or as indicated. Samples were then placed on Whole mount immunohistochemistry in mouse E10.5 embryos was protein A-coated 96-well plates (Pierce) for 90 min, washed 3 times with carried out as described previously (22). Briefly, freshly dissected em- wash buffer (BioFX), and radioactivity was counted using a standard bryos were fixed with 4% paraformaldehyde overnight at 4 °C, washed gamma counter. in saline for 2 h, and then soaked in 80% methanol series. Endogenous peroxidase activity was quenched with 3% H O in 80% methanol and 2 2 Reverse Transcription-PCR Analysis 20% Me SO for 3 h. After washing with Tris-buffered saline containing Reverse transcription-PCR was performed on isolated Xenopus ani- 1% Tween 20 (TBS-T) for 3 h, the embryos were incubated with mal cap explants or embryos, with orthidine decarboxylase as loading DRAGON antibody (1:500) in TBS-T containing 5% skim milk and 5% control (30). Me SO for 2 days at room temperature. The embryos were then washed 3 times and incubated with horseradish peroxidase-coupled anti-rabbit RESULTS IgG (1:200 dilution in TBS-T containing 5% skim milk and 5% Me SO). DRAGON Expression Horseradish peroxidase activity was detected with diaminobenzidine. DRAGON is expressed in mouse E2.5 pre-implantation em- Northern Blot bryos (Fig. 1A), and in postimplantation embryos (E7) (Fig. The premade Northern blot containing 2 g of poly(A) RNA/lane 1B). In E10.5 embryos, DRAGON is found along the neural from mouse embryos at the indicated stages (Clontech, Inc.) was used. tube, in the dorsal root ganglia, and in the tips of the neural The membrane was hybridized with a 300-bp DRAGON cDNA probe as described previously (18). folds and the tail bud (Fig. 1C) as assessed by whole mount immunohistochemistry. Xenopus orthologs of both DRAGON In Situ Hybridization and RGM are expressed maternally and throughout develop- Whole mount in situ hybridizations were carried out as described ment and are detected at high levels by tadpole stages (Fig. previously by Harland (23). Embryos to be sectioned were post-fixed in 1D). Analysis by whole mount in situ hybridization of RGM 4% paraformaldehyde and embedded in 20% gelatin-PBS. Sections expression at stage 12 reveals high levels in the ectoderm by were cut between 50 and 100 m. the blastopore (Fig. 1E, i). More restricted expression is seen by Luciferase Reporter Assays stage 17 and stage 20 in the neural plate, specifically in the dorsal aspect of the neural tube (Fig, 1E, ii and iii). By stage 35, The BMP-inducible BRE-Luc (24), I-BRE-Luc (25), and Msx2-Luc (26) and the TGF-inducible 3TP-lux (27) have been described previously. the expression of RGM is detected in the hindbrain, midbrain, 14124 DRAGON, a Bone Morphogenetic Protein Co-receptor FIG.1. DRAGON is expressed during embryogenesis. A, DRAGON expression in mouse pre-implantation embryos (E2.5) assessed with a DRAGON antibody and visualized by fluorescein isothiocyanate green fluorescence. Nuclei of the embryonic cells are visualized by CY3 red fluorescence (propidium iodide). Pre-adsorption of the DRAGON antibody with the peptide antigen (Pept.) was used as negative control. B, Northern blot showing DRAGON mRNA expression in mouse embryos at embryonic stages E7–17. Cyclophilin mRNA levels were used as loading controls. C, immunohistochemical study of the expression and distribution of DRAGON in mouse E10.5 embryos. D, expression of Xenopus DRAGON (RGMb) and RGMa assessed by reverse transcription (RT)-PCR at the indicated developmental stages. Orthidine decarboxylase (ODC) levels were used as internal control. E, analysis by whole mount in situ hybridization of expression of Xenopus RGMa at stages 12 (i), 17 (ii), 20 (iii), and 35 (iv). Transverse sections through stained stage 12 (top right) and stage 20 (bottom left) embryos are also represented. bp, blastopore; post, posterior; ant, anterior; nc, notochord; rp, roof plate; hb, hindbrain; mb, midbrain; fb, forebrain; ba, branchial arches. and forebrain regions, as well as the branchial arches. In ad- derived reporter construct (24). BMP2 stimulates BRE-Luc dition, expression is seen posteriorly in the somites and tail bud activity in LLC-PK1 cells (Fig. 2). Co-expression of DRAGON (Fig. 1E, iv). with BRE-Luc in the absence of exogenous ligand increases The expression pattern of DRAGON and RGM at different BRE-Luc activity to levels comparable with that achieved by embryonic stages in both the developing mouse and Xenopus is BMP2 stimulation in the absence of DRAGON (Fig. 2A). To comparable with the expression of the BMP type I receptors, assess whether DRAGON regulates the signaling produced by ALK3 and ALK6 and BMP type II receptor (31, 32) (Fig. 1). BMP ligands, LLC-PK1 cells were co-transfected with the This observation prompted us to investigate whether DRAGON BRE-Luc reporter and DRAGON (2 and 20 ng) and incubated contributes to or modulates TGF superfamily signal with BMP2. BMP2 (50 ng/ml) induces a 6-fold increase in transduction. luciferase activity in the absence of DRAGON and a 14-fold increase in DRAGON co-transfected cells (Fig. 2A). DRAGON DRAGON Enhances BMP but Not TGF expression had no effect on TGF signaling, assessed by using Intracellular Signaling a TGF-responsive promoter (TGF-responsive CAGA reporter (12)) in LLC-PK1 cells (data not shown). TGF superfamily ligands are classified as TGF or BMP- like based on the particular Smad-dependent intracellular DRAGON expression activates BMP signaling in the ab- sence of any added ligand (Fig. 2A). To determine whether pathways they activate. We examined whether DRAGON acti- vates these pathways using specific BMP- and TGF-respon- this represents a ligand-independent action or is mediated by endogenous BMP ligands either produced by the cell lines or sive luciferase reporters in LLC-PK1 (kidney epithelial cells), as these cells are highly responsive to many TGF family present in the medium, we investigated whether DRAGON activation of BMP signaling is blocked by administration of members and thereby allow a parallel analysis of any action of DRAGON on TGF superfamily signaling. the soluble BMP antagonist Noggin (2). When added to the To determine whether DRAGON alters BMP signaling we medium Noggin (50–1000 ng/ml) inhibits both the DRAGON- used the BMP-inducible promoter BRE-Luc, an Id1 promoter- and BMP2-induced activation of the BRE-Luc reporter in DRAGON, a Bone Morphogenetic Protein Co-receptor 14125 FIG.2. DRAGON expression enhances BMP signaling and sensitizes cells to BMP. A, LLC-PK1 renal epithelial cells were transiently transfected with BRE-Luciferase construct (BRE-Luc) alone or in combination with 2 or 20 ng of a DRAGON expression construct. Luciferase activity was assessed after incubation in the absence (white bars) or presence (black bars) of BMP2 (1 nM) in the culture medium. Luciferase activity is represented as relative light units (RLU). DRAGON increases and potentiates BMP2-mediated signaling. B, DRAGON-mediated BMP signaling is ligand-dependent. LLC-PK1 cells were transiently transfected with BRE-Luc in the presence or absence of DRAGON and treated with increasing amounts of Noggin as indicated. As a positive control, BRE-Luc-transfected LLC-PK1 cells were treated with BMP2 (1 nM) in the presence of Noggin (50 ng/ml). As a negative control, LLC-PK1 cells transfected with the TGF-responsive reporter construct MLP-Luc were treated with TGF (1 nM) in the presence of Noggin (50 ng/ml). C, 10 T1/2 cells transiently transfected with the BMP-responsive I-BRE-Luc reporter and DRAGON were incubated with increasing doses of BMP2 (25–500 pM). LLC-PK1 cells (Fig. 2B). Noggin has no effect on TGF- through an independent receptor pathway that converges on induced stimulation of the TGF-responsive CAGA reporter the intracellular Smads. We therefore investigated whether (Fig. 2B). DRAGON-mediated activation of BMP signaling is, DRAGON binds to BMP ligands, interacts directly with BMP therefore, ligand-dependent. receptors, and whether a dominant negative BMP receptor or DRAGON significantly enhances BMP signaling and in- Smad mutants reduce the DRAGON-induced enhancement of creases cellular sensitivity to BMP2 in LLC-PK1 cells (Fig. 2, A BMP signaling. and B). To rule out a potential cell-type or reporter-specific DRAGON Binds to BMP but Not TGF Ligands—To deter- effect of DRAGON expression, we examined the generality of mine whether cell surface-localized DRAGON binds directly to this effect, i.e. whether it is restricted to one cell line or to one BMP ligands, COS-1 cells expressing DRAGON were incubated 125 125 BMP-inducible reporter construct. We tested the effect of with [ I]-BMP2 or [ I]-TGF and receptor-bound ligand DRAGON expression in 10 T1/2 cells (mouse mesenchymal cross-linked. HA-tagged BMP type I receptor (ALK6) or TGF stem cells), another BMP-responsive cell line (28), using an- type II receptor (TRII) served as positive controls for BMP2 or other BMP-inducible promoter reporter construct, I-BRE-Luc, TGF binding, respectively. Immunoprecipitation of DRAGON 125 125 which contains a portion of intron 1 of the Smad7 gene (25). from [ I]-BMP2- but not [ I]-TGF-labeled cells revealed DRAGON significantly enhances BMP2-dependent reporter ac- the presence of two [ I]-BMP2-bound proteins of 55–65 kDa tivity in this cell line at BMP2 concentrations of 25–300 pM (Fig. 3A), consistent with the expected size of a BMP-bound (Fig. 2C). This effect is lost at 300 pM or higher, where maximal DRAGON. The presence of two bands suggests that cell sur- signaling is achieved. face-localized DRAGON may be subject to post-translational Similar results were obtained using LLC-PK1, 10 T1/2, and modification. These data, indicating that cell surface-expressed HepG2 cell lines and different BMP-responsive promoters (I- DRAGON can bind BMP2 but not TGF, prompted us to assess BRE-Luc, BRE-Luc, and Msx2-Luc) (Fig. 2, and data not whether DRAGON can directly bind BMP ligands. shown). DRAGON significantly increases cellular sensitivity, We examined whether DRAGON directly binds to BMP li- therefore, to low doses of BMP2 in a manner that is neither gands using a soluble DRAGON-Fc fusion protein in a cell-free reporter- nor cell-specific. These results suggest that the regu- binding system (12). DRAGON-Fc binds [ I]-BMP2 with high latory role of DRAGON in BMP signaling is a generalized affinity and an apparent dissociation rate constant (K )of1.5 phenomenon. This finding prompted us to study the mecha- nM (Fig. 3B). This binding is competed with an excess of unla- nism of the enhancement of BMP signaling by DRAGON and beled BMP2 as well as by BMP4 (4 nM) (Fig. 3, B and C). In where and how it interacts with BMP signaling components. contrast, BMP7 (4 nM), activin A, TGF1, -2, or -3 (4 nM each) do not competitively inhibit the binding of DRAGON-Fc to Mechanism of the Enhancement of BMP BMP2 (Fig. 3C). Pretreatment of cells with DRAGON-Fc de- Signaling by DRAGON creases BMP2- but not BMP7-mediated activation of the BRE- DRAGON could potentially enhance BMP signaling Luc promoter in a dose-dependent manner (60 and 300 ng/ml) through a direct modulation of the BMP signaling complex or (Fig. 3D) but had no effect on TGF-1-dependent activation of 14126 DRAGON, a Bone Morphogenetic Protein Co-receptor FIG.3. DRAGON interacts directly with BMP2 and BMP4. A, DRAGON expressed in COS cells binds to BMP2 but not TGF. COS-1 cells were transiently transfected with DRAGON (DR), HA-tagged ALK6 (A6/HA), or TRII (RII/HA). Cells were affinity-labeled with [ I]-BMP2 (left 125 125 panel)or[ I]-TGF (right panel) and lysates subjected to immunoprecipitation (IP) or immunoblotting with the indicated antibodies. I-Labeled ligand binding to receptors was detected by phosphorimaging. B, DRAGON-Fc binds directly to BMP2. [ I]-BMP2 was incubated with increasing amounts (10–50 ng) of purified DRAGON-Fc then added to protein A-coated plates, and bound radioactivity was measured (left graph). 20 ng of DRAGON-Fc were incubated with [ I]-BMP2 in the presence of increasing amounts of competing unlabeled BMP2 (0–42 nM)(right graph). C, DRAGON-Fc binds directly to BMP2 and BMP4 but not BMP7 or TGF ligands. DRAGON-Fc was incubated with [ I]-BMP2 in the presence of unlabeled BMP2, BMP4, BMP7, Activin A (ActA), TGF1, TGF2, and TGF3, and [ I]-BMP2 binding was measured. D, DRAGON-Fc reduces BMP2 mediated signaling. LLC-PK1 cells were transiently transfected with BRE-Luc construct and treated with BMP2 (150 ng/ml) and DRAGON-Fc (60 and 300 ng/ml). E, DRAGON-Fc (60 ng/ml) reduces BMP2 but not BMP7- or TGF1(1nM each)-mediated signaling. LLC-PK1 cells were transiently transfected with BRE-Luc and treated with BMP2 or BMP7 or transiently transfected with 3TP-Lux construct and treated with TGF1 in the absence or presence of DRAGON-Fc. the CAGA-Luc promoter (Fig. 3E). DRAGON directly interacts immunoprecipitation followed by anti-HA immunoblotting re- with members of the BMP but not the TGF ligand family, with veals that DRAGON associates directly with all BMP type I a preference for BMP2 and BMP4. receptors, (ALK2, ALK3, and ALK6) (Fig. 5A) and with the DRAGON Enhances BMP Signaling Only when Expressed on BMP type II receptors ActRII and ActRIIB (Fig. 5B). the Cell Surface—To assess whether the GPI anchor is required DRAGON Signaling Is Blocked by Dominant Negative ALK3, for the action of DRAGON on BMP signaling, we used the -6, and Smad1—We used dominant negative type I receptors DRAGON-Fc fusion protein where the C-terminal GPI anchor (ALK1-DN, ALK3-DN, and ALK6-DN), which are deficient in is deleted and replaced by human Fc (18). When cotransfected kinase activity and unable to phosphorylate Smads (33), to with the BRE-Luc reporter construct this DRAGON-Fc, which confirm that DRAGON acts through modulation of BMP recep- is secreted into the medium, fails to increase BMP signaling tors and subsequent activation of the downstream Smad1 path- (Fig. 4A). Western blot confirms the expression of both way (Fig. 5, C and D). Co-expression of DRAGON with DRAGON and DRAGON-Fc in the transfected cells (Fig. 4B). ALK3-DN and ALK6-DN in LLC-PK1 cells decreases DRAG- These results demonstrate that the expression of DRAGON on ON-mediated induction of I-BRE luciferase activity to base the cell surface is required for its action on BMP signaling. line, whereas co-expression of ALK1-DN does not affect DRAG- DRAGON Binds to BMP Type I and Type II Receptors—BMP ON-mediated BMP signaling (Fig. 5C). Co-expression of type I and type II receptors are essential for mediating BMP DRAGON in LLC-PK1 cells with a Smad1 dominant negative responses. To test whether DRAGON directly interacts with mutant that lacks the C-terminal phosphoacceptor domain (15, BMP type I and type II receptors, HEK293T cells were tran- 34) also dose dependently reduces DRAGON-induced signal- siently transfected either with DRAGON alone or with HA ing. In contrast, co-expression with wild type Smad1 enhances epitope-tagged type I and type II BMP receptors. Anti-Dragon this signaling (Fig. 5D). These studies demonstrate that DRAGON, a Bone Morphogenetic Protein Co-receptor 14127 DRAGON exerts its effect on BMP signaling via BMP receptors nation with Smad1 mRNA in Xenopus embryos at the two-cell and potentiates Smad1 signaling. stage. We then analyzed the expression levels of mesodermal or endodermal tissue markers as indicators of activation of BMP DRAGON Enhances BMP Signaling in Xenopus Embryos signaling. DRAGON co-injection with Smad1 leads to the in- duction of mRNA for the pan-mesodermal marker Xbra and BMP ligands play pivotal roles in the gastrulation of the two endodermal markers, mix1 and mixer in a dose-dependent embryo (35) and regulate the formation of the mesoderm and manner (Fig. 6A). Injected alone, neither DRAGON nor Smad1 endoderm (36). An analysis of mice lacking genes encoding at the low amounts administered induce the expression of the BMPs, BMP receptors, and their downstream signal transduc- mesodermal and endodermal markers (Fig. 6A). The reduction ers reveal major early and late embryonic developmental alter- in the threshold of the activity of Smad1 by DRAGON indicates ations, with defects in mesoderm and endoderm formation (37). the enhancement of BMP signaling in the embryo and is con- We have examined whether DRAGON expression in Xenopus sistent with the results seen in mammalian cells (Fig. 2). embryos enhances BMP signaling and synergizes with BMP signal transduction components to facilitate BMP signaling in DRAGON Regulates Neural Patterning in vivo. Mouse DRAGON mRNA was injected alone or in combi- Xenopus Embryos Genetic experiments have suggested that various BMPs can promote dorsal neural cell fates (38) and act as morphogens to specify dorsal neural cell types (39). However the contribution of the different BMP signaling components to neural pattern- ing is not fully elucidated. To determine the effect of DRAGON expression on tissue patterning, we analyzed fate changes in Xenopus embryonic explants by reverse transcription-PCR for a variety of molecular markers. DRAGON mRNA was injected at the two-cell stage in the animal caps, and cultured ectoder- mal explants were analyzed for changes in gene expression. DRAGON induces expression of the anterior pan-neural marker Nrp1, the cement gland marker (the most anterior structure in the tadpole) XAG, as well as the early heart FIG.4. DRAGON-mediated BMP signaling requires its GPI an- marker nkx2.5 (Fig. 6B). Up-regulation of both neural and chor. A, DRAGON-Fc (DR-Fc) when expressed by LLC-PK1 cells does heart markers by DRAGON (Fig. 6B) suggests that the induc- not increase BMP signaling. B, Expression of both full-length DRAGON tion of neural tissue by DRAGON is not direct but occurs via (DR) and DRAGON-Fc by the LLC-PK1 cells was confirmed by Western blot analysis. formation of dorsal mesoderm, which induces neural tissue FIG.5. DRAGON interacts directly with BMP type I and type II receptors and signals through the ALK/Smad pathway. HEK293T cells were transiently transfected with HA-tagged type I receptors, ALK2, ALK3, or ALK6 (A), HA-tagged type II receptors, ActRII (RII/HA) or ActRIIB (RIIB/HA), or FLAG-tagged tailless version of BMP type II receptor (B) with or without DRAGON (DR) as indicated. Cell lysates were immunoprecipitated (IP) with anti-Dragon (-DR) antibodies, and associated receptors were visualized by anti-HA or anti-FLAG immunoblotting. Protein expression was confirmed by anti-HA, anti-FLAG, and anti-Dragon immunoblotting of total cell lysates (bottom panels). C, ALK3-DN or ALK6-DN but not ALK1-DN expression reduces DRAGON-induced signaling. LLC-PK1 epithelial cells were transiently transfected with BRE-Luc construct, with or without DRAGON, ALK1-DN, ALK3-DN, or ALK6-DN expression constructs as indicated. D, dominant negative Smad1 reduces DRAGON signaling. LLC-PK1 cells transiently transfected with BRE-Luc and DRAGON constructs, in the absence or presence of increasing amounts (0.1–2 g) of wild type (WT) or dominant negative (DN) Smad1 as indicated. 14128 DRAGON, a Bone Morphogenetic Protein Co-receptor FIG.6. DRAGON potentiates BMP signaling in Xenopus embryos. A, mi- croinjection of mouse DRAGON mRNA in Xenopus laevis embryos at gastrula stages synergizes with Smad1 (mRNA in- jection) to induce mesodermal (xBra and mix1) and endodermal (mixer) markers, as assessed by reverse transcription (RT)- PCR. No induction of EK or Sox2 occurs. B, in animal cap explants DRAGON mRNA injection induces expression of the anterior neural markers nrp1 and Otx2, as well the as cement gland marker (Xag) and nkx2.5, a heart marker. Orthidine decarboxylase (ODC) is the loading con- trol. C, Xenopus embryos were injected in the animal pole of one of two cells at the two-cell stage with DRAGON mRNA and analyzed at late neurula (stage 23), for changes in neural crest patterning, and early tadpole stages (stage 28) for ectopic induction of neural tissue. DRAGON overexpression inhibits twist RNA, a neu- ral crest marker (top panels), and induces ectopic patches of N-tubulin, a neural marker (lower panels). (Fig. 6A). To determine whether DRAGON expression alters cytoplasmic, and nuclear levels and contributes to the forma- neural patterning, we injected mouse DRAGON mRNA into tion and response to gradients of morphogens in development. one cell at the two-cell stage in the animal pole of the Xenopus The role of DRAGON in the embryo may be to differentially embryo and left the injected embryos to develop until early increase or boost the sensitivity of cells to low levels of BMP tadpole stages (Fig. 6C). The uninjected cell serves as control ligand. Such low levels will occur within the spatial BMP for the DRAGON-injected experimental side in the same em- gradients established in the embryo, and DRAGON expression bryo. Overexpression of DRAGON results in an increase in may enable cells to respond earlier or with a greater response N-tubulin (a general neuronal differentiation marker) expres- to a particular level of BMP, than those that do not express sion, as shown by the appearance of ectopic patches of N- DRAGON. Therefore, a differential sensitivity to BMP ligands tubulin-expressing cells (Fig. 6C) and a decrease in neural crest produced by DRAGON expression may contribute to tissue derivatives, as shown by a reduction in twist (an anterior patterning. neural crest marker) expression on the injected side (Fig. 6C) DRAGON interacts directly with specific BMP ligands (40). DRAGON may contribute, therefore, to the patterning of (BMP2 and -4 but not BMP7) in the absence and presence of the developing nervous system by promoting a neuronal phe- BMP type I and type II receptors. How DRAGON enhances notype and inhibiting neural crest differentiation. signaling by the receptor complex needs to be determined. Does it change the affinity of BMP receptor-ligand interactions or DISCUSSION facilitate the formation of heteromeric receptor complexes be- DRAGON was identified by us in a genomic-binding screen tween type I and type II receptors? As a GPI-anchored protein for promoter regions of effector genes regulated by the devel- with no transmembrane domain, DRAGON is unlikely to di- opmentally regulated paired homeodomain transcription fac- rectly alter either the intracellular kinase activity of the BMP tor, DRG11 and is a 436-amino acid member of the RGM family receptors or their coupling to the receptor-activated Smads, but of GPI-anchored proteins (18). We show here that DRAGON it may stabilize the receptor complex. The action of DRAGON, binds to BMP ligands and receptors, and enhances BMP sig- like other GPI-anchored proteins, may depend on localization naling, and conclude that it is a BMP co-receptor. in lipid rafts (41) and in this way contribute to the assembly Given the wide spectrum of responses to the TGF super- and trafficking of the receptor complex in microdomains. family, mechanisms for positive and negative modulation of DRAGON is not necessary for BMP2 and -4 signaling but their signaling exist to exert tight spatiotemporal regulation. rather enhances signaling in response to low concentrations of Positive regulation amplifies signals by reducing activation threshold and potentiating biological activity, whereas nega- these BMP ligands. In this respect, DRAGON differs from nodal, Vg1, and growth and differentiation factor 1, which tive regulation limits the magnitude of signals or terminates signaling. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Apr 8, 2005

References