Samad TA et al. (2005), DRAGON, a bone morphogenetic protein co-receptor.

XB-ART-2423

J Biol Chem 2005 Apr 08;28014:14122-9. doi: 10.1074/jbc.M410034200.

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DRAGON, a bone morphogenetic protein co-receptor.

Samad TA , Rebbapragada A , Bell E , Zhang Y , Sidis Y , Jeong SJ , Campagna JA , Perusini S , Fabrizio DA , Schneyer AL , Lin HY , Brivanlou AH , Attisano L , Woolf CJ .

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Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)beta superfamily of ligands that regulate many crucial aspects of embryonic development and organogenesis. Unlike other TGFbeta ligands, co-receptors for BMP ligands have not been described. Here we show that DRAGON, a glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, which is expressed early in the developing nervous system, enhances BMP but not TGFbeta signaling. DRAGON binds directly to BMP2 and BMP4 but not to BMP7 or other TGFbeta ligands. The enhancing action of DRAGON on BMP signaling is also reduced by administration of Noggin, a soluble BMP antagonist, indicating that the action of DRAGON is ligand-dependent. DRAGON associates directly with BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In the Xenopus embryo, DRAGON both reduces the threshold of the ability of Smad1 to induce mesodermal and endodermal markers and alters neuronal and neural crest patterning. The direct interaction of DRAGON with BMP ligands and receptors indicates that it is a BMP co-receptor that potentiates BMP signaling.

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Species referenced: Xenopus laevis
Genes referenced: acta1 acvr1 acvr2a acvr2b acvrl1 alk babam2 bmp2 bmp4 bmp7.1 bmp7.2 bmpr1a bmpr1b csrp3 gnpda1 inhba nog nr2e1 nrp1 otx2 pklr rgma rgmb smad1 sox2 tgfb1 tubb2b twist1

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	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.
	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).
	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 [125I]-BMP2 (left panel) or [125I]-TGF (right panel) and lysates subjected to immunoprecipitation (IP) or immunoblotting with the indicated antibodies. 125I-Labeled ligand binding to receptors was detected by phosphorimaging. B, DRAGON-Fc binds directly to BMP2. [125I]-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 [125I]-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 [125I]-BMP2 in the presence of unlabeled BMP2, BMP4, BMP7, Activin A (ActA), TGF1, TGF2, and TGF3, and [125I]-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 (1 nM 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.
	FIG. 4. DRAGON-mediated BMP signaling requires its GPI anchor. A, DRAGON-Fc (DR-Fc) when expressed by LLC-PK1 cells does not increase BMP signaling. B, Expression of both full-length DRAGON (DR) and DRAGON-Fc by the LLC-PK1 cells was confirmed by Western blot analysis.
	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.
	FIG. 6. DRAGON potentiates BMP signaling in Xenopus embryos. A, microinjection of mouse DRAGON mRNA in Xenopus laevis embryos at gastrula stages synergizes with Smad1 (mRNA injection) 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 control. 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 neural crest marker (top panels), and induces ectopic patches of N-tubulin, a neural marker (lower panels).
	rgmb (repulsive guidance molecule family member b) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, lateral view, dorsal right.
	rgmb (repulsive guidance molecule family member b) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal left
	rgmb (repulsive guidance molecule family member b) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35, lateral view, anterior right, dorsal up.