Bone Morphogenetic Proteins Signal through the Transforming Growth Factor-β Type III Receptor
2008; Elsevier BV; Volume: 283; Issue: 12 Linguagem: Inglês
10.1074/jbc.m704883200
ISSN1083-351X
AutoresKellye C. Kirkbride, Todd A. Townsend, Monique W. Bruinsma, Joey V. Barnett, Gerard C. Blobe,
Tópico(s)Bone Metabolism and Diseases
ResumoThe bone morphogenetic protein (BMP) family, the largest subfamily of the structurally conserved transforming growth factor-β (TGF-β) superfamily of growth factors, are multifunctional regulators of development, proliferation, and differentiation. The TGF-β type III receptor (TβRIII or betaglycan) is an abundant cell surface proteoglycan that has been well characterized as a TGF-β and inhibin receptor. Here we demonstrate that TβRIII functions as a BMP cell surface receptor. TβRIII directly and specifically binds to multiple members of the BMP subfamily, including BMP-2, BMP-4, BMP-7, and GDF-5, with similar kinetics and ligand binding domains as previously identified for TGF-β. TβRIII also enhances ligand binding to the BMP type I receptors, whereas short hairpin RNA-mediated silencing of endogenous TβRIII attenuates BMP-mediated Smad1 phosphorylation. Using a biologically relevant model for TβRIII function, we demonstrate that BMP-2 specifically stimulates TβRIII-mediated epithelial to mesenchymal cell transformation. The ability of TβRIII to serve as a cell surface receptor and mediate BMP, inhibin, and TGF-β signaling suggests a broader role for TβRIII in orchestrating TGF-β superfamily signaling. The bone morphogenetic protein (BMP) family, the largest subfamily of the structurally conserved transforming growth factor-β (TGF-β) superfamily of growth factors, are multifunctional regulators of development, proliferation, and differentiation. The TGF-β type III receptor (TβRIII or betaglycan) is an abundant cell surface proteoglycan that has been well characterized as a TGF-β and inhibin receptor. Here we demonstrate that TβRIII functions as a BMP cell surface receptor. TβRIII directly and specifically binds to multiple members of the BMP subfamily, including BMP-2, BMP-4, BMP-7, and GDF-5, with similar kinetics and ligand binding domains as previously identified for TGF-β. TβRIII also enhances ligand binding to the BMP type I receptors, whereas short hairpin RNA-mediated silencing of endogenous TβRIII attenuates BMP-mediated Smad1 phosphorylation. Using a biologically relevant model for TβRIII function, we demonstrate that BMP-2 specifically stimulates TβRIII-mediated epithelial to mesenchymal cell transformation. The ability of TβRIII to serve as a cell surface receptor and mediate BMP, inhibin, and TGF-β signaling suggests a broader role for TβRIII in orchestrating TGF-β superfamily signaling. Members of the transforming growth factor-β (TGF-β) 2The abbreviations used are: TGF-βtransforming growth factor-βALKactivin receptor-like kinaseAVatrioventricularBMPbone morphogenetic proteinEMTepithelial to mesenchymal transitionGDFgrowth/differentiation factorTβRIIITGF-β type III receptorsTβRIIIsoluble TβRIIITβRIIIΔgagTβRIII minus glycosaminoglycan chainsshRNAshort hairpin RNAGFPgreen fluorescent protein. 2The abbreviations used are: TGF-βtransforming growth factor-βALKactivin receptor-like kinaseAVatrioventricularBMPbone morphogenetic proteinEMTepithelial to mesenchymal transitionGDFgrowth/differentiation factorTβRIIITGF-β type III receptorsTβRIIIsoluble TβRIIITβRIIIΔgagTβRIII minus glycosaminoglycan chainsshRNAshort hairpin RNAGFPgreen fluorescent protein. superfamily (including the TGF-β, the activin/inhibin, and the bone morphogenetic protein (BMP)/growth differentiation factor (GDF) subfamilies) are involved in many cellular processes including growth regulation, migration, apoptosis, and differentiation (1Miyazono K. Maeda S. Imamura T. Cytokine Growth Factor Rev. 2005; 16: 251-263Crossref PubMed Scopus (706) Google Scholar, 2Shi Y. Massague J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4737) Google Scholar, 3ten Dijke P. Korchynskyi O. Valdimarsdottir G. Goumans M.J. Mol. Cell. Endocrinol. 2003; 211: 105-113Crossref PubMed Scopus (172) Google Scholar, 4Balemans W. Van Hul W. Dev. Biol. 2002; 250: 231-250Crossref PubMed Google Scholar). The BMP subfamily, with 20 members, is the largest and has essential roles in development and well established roles in bone formation (1Miyazono K. Maeda S. Imamura T. Cytokine Growth Factor Rev. 2005; 16: 251-263Crossref PubMed Scopus (706) Google Scholar, 5Zhao G.Q. Genesis. 2003; 35: 43-56Crossref PubMed Scopus (301) Google Scholar).BMP initiates signaling upon ligand binding to the high affinity type I BMP signaling receptors, activin-like receptor kinase-1 (ALK1) (6Brown M.A. Zhao Q. Baker K.A. Naik C. Chen C. Pukac L. Singh M. Tsareva T. Parice Y. Mahoney A. Roschke V. Sanyal I. Choe S. J. Biol. Chem. 2005; 280: 25111-25118Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar), ALK2, ALK3, or ALK6 (7Miyazono K. Kusanagi K. Inoue H. J. Cell Physiol. 2001; 187: 265-276Crossref PubMed Scopus (452) Google Scholar). The serine/threonine kinase activity of the type I receptor is activated upon recruitment and phosphorylation by a type II receptor, either the BMP type II receptor (BMPRII), or one of the activin type II receptors (ActRII or ActRIIB) (8Miyazawa K. Shinozaki M. Hara T. Furuya T. Miyazono K. Genes Cells. 2002; 7: 1191-1204Crossref PubMed Scopus (577) Google Scholar). Upon activation the type I receptor phosphorylates the intracellular effector proteins, Smad1/5/8 transcription factors, which complex with the common Smad, Smad4, and enter the nucleus to induce BMP-mediated target gene transcription (1Miyazono K. Maeda S. Imamura T. Cytokine Growth Factor Rev. 2005; 16: 251-263Crossref PubMed Scopus (706) Google Scholar). Whereas most BMPs are able to elicit distinct cellular effects, the mechanism by which a limited number of cell surface receptors mediate these divergent effects remains to be established.Co-receptors are important components of many signaling pathways (9Kirkbride K.C. Ray B.N. Blobe G.C. Trends Biochem. Sci. 2005; 30: 611-621Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The TGF-β type III receptor (TβRIII or betaglycan), endoglin (10Cheifetz S. Bellon T. Cales C. Vera S. Bernabeu C. Massague J. Letarte M. J. Biol. Chem. 1992; 267: 19027-19030Abstract Full Text PDF PubMed Google Scholar), and members of the repulsive guidance molecule family, DRAGON, RGMa, and hemojuvelin (11Samad T.A. Rebbapragada A. Bell E. Zhang Y. Sidis Y. Jeong S.J. Campagna J.A. Perusini S. Fabrizio D.A. Schneyer A.L. Lin H.Y. Brivanlou A.H. Attisano L. Woolf C.J. J. Biol. Chem. 2005; 280: 14122-14129Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 12Babitt J.L. Zhang Y. Samad T.A. Xia Y. Tang J. Campagna J.A. Schneyer A.L. Woolf C.J. Lin H.Y. J. Biol. Chem. 2005; 280: 29820-29827Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 13Babitt J.L. Huang F.W. Wrighting D.M. Xia Y. Sidis Y. Samad T.A. Campagna J.A. Chung R.T. Schneyer A.L. Woolf C.J. Andrews N.C. Lin H.Y. Nat. Genet. 2006; 38: 531-539Crossref PubMed Scopus (824) Google Scholar), have been characterized as TGF-β superfamily co-receptors. TβRIII is an abundantly and ubiquitously expressed cell surface receptor that enhances binding of all three isoforms of TGF-β to the TGF-β signaling receptor complex (14Andres J.L. Ronnstrand L. Cheifetz S. Massague J. J. Biol. Chem. 1991; 266: 23282-23287Abstract Full Text PDF PubMed Google Scholar), and is required for high affinity cell surface binding of TGF-β2. TβRIII also binds inhibin, another TGF-β superfamily member (15Lewis K.A. Gray P.C. Blount A.L. MacConell L.A. Wiater E. Bilezikjian L.M. Vale W. Nature. 2000; 404: 411-414Crossref PubMed Scopus (492) Google Scholar). In addition to directly regulating ligand availability, TβRIII also alters the subcellular localization of the signaling receptor complex through interactions with the PDZ domain containing protein, GIPC (16Blobe G.C. Liu X. Fang S.J. How T. Lodish H.F. J. Biol. Chem. 2001; 276: 39608-39617Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), and β-arrestin2 (17Chen W. Kirkbride K.C. How T. Nelson C.D. Mo J. Frederick J.P. Wang X.F. Lefkowitz R.J. Blobe G.C. Science. 2003; 301: 1394-1397Crossref PubMed Scopus (207) Google Scholar). The demonstration that TβRIII is required for TGF-β2-stimulated epithelial to mesenchymal transformation (EMT) in vitro (18Brown C.B. Boyer A.S. Runyan R.B. Barnett J.V. Science. 1999; 283: 2080-2082Crossref PubMed Scopus (329) Google Scholar) and the embryonic lethality of the TβRIII knock-out mouse (19Stenvers K.L. Tursky M.L. Harder K.W. Kountouri N. Amatayakul-Chantler S. Grail D. Small C. Weinberg R.A. Sizeland A.M. Zhu H.J. Mol. Cell. Biol. 2003; 23: 4371-4385Crossref PubMed Scopus (198) Google Scholar, 20Compton L.A. Potash D.A. Brown C.B. Barnett J.V. Circ. Res. 2007; 101: 784-791Crossref PubMed Scopus (103) Google Scholar) has fostered consideration of a unique and non-redundant role for TβRIII that is independent of ligand presentation to the kinase receptor complexes.Several observations suggest that TβRIII may serve as a cell surface receptor for BMP. First, BMP shares structural similarity with ligands known to bind TβRIII (21Griffith D.L. Keck P.C. Sampath T.K. Rueger D.C. Carlson W.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 878-883Crossref PubMed Scopus (244) Google Scholar). Second, TβRIII shares extracellular domain homology with endoglin (22Lopez-Casillas F. Cheifetz S. Doody J. Andres J.L. Lane W.S. Massague J. Cell. 1991; 67: 785-795Abstract Full Text PDF PubMed Scopus (544) Google Scholar, 23Wang X.F. Lin H.Y. Ng-Eaton E. Downward J. Lodish H.F. Weinberg R.A. Cell. 1991; 67: 797-805Abstract Full Text PDF PubMed Scopus (539) Google Scholar), which binds BMP-2 and BMP-7 in the presence of their respective type II receptors (24Barbara N.P. Wrana J.L. Letarte M. J. Biol. Chem. 1999; 274: 584-594Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). Finally, TβRIII is a heparan sulfate proteoglycan (25Cheifetz S. Andres J.L. Massague J. J. Biol. Chem. 1988; 263: 16984-16991Abstract Full Text PDF PubMed Google Scholar, 26Segarini P.R. Seyedin S.M. J. Biol. Chem. 1988; 263: 8366-8370Abstract Full Text PDF PubMed Google Scholar) and these glycosaminoglycan modifications have been shown to mediate basic fibroblast growth factor binding to TβRIII (27Andres J.L. DeFalcis D. Noda M. Massague J. J. Biol. Chem. 1992; 267: 5927-5930Abstract Full Text PDF PubMed Google Scholar). As BMP has a strong affinity for heparan sulfate (28Irie A. Habuchi H. Kimata K. Sanai Y. Biochem. Biophys. Res. Commun. 2003; 308: 858-865Crossref PubMed Scopus (105) Google Scholar), these modifications may confer the ability of TβRIII to bind BMP as well. Here we investigate whether TβRIII functions as a cell surface receptor for BMP.EXPERIMENTAL PROCEDURESCell Culture, Plasmids, Antibodies, and Growth Factors—COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum (Invitrogen). NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. PC-3 cells were maintained in F12 Kaighn's (Invitrogen) supplemented with 10% fetal bovine serum.Human TβRIIIΔgag was generated using XL-Site directed mutagenesis (Stratagene) to mutate serine 532 to alanine (forward: 5′-CCCTTGGGGACAGTGCTGGTTGGCCAGA and reverse 5′-CATCTGGCCAACCAGCACTGTCCCCAAG), followed by mutating serine 543 to alanine (forward: 5′-GGAAATCCATTATCACCTGCCTCCAGAT and reverse 5′-GGTTATGAAGATCTGGAGGCAGGTGATA) to make the double mutant. Plasmids were generous gifts from Kohei Miyazono (ALK3 and ALK6), Petra Knaus (BMPRII), and Fernando Lopez-Casillas (myc-TβRIII extracellular domain deletions) (29Esparza-Lopez J. Montiel J.L. Vilchis-Landeros M.M. Okadome T. Miyazono K. Lopez-Casillas F. J. Biol. Chem. 2001; 276: 14588-14596Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar).Adenoviruses for EMT assays were generated using the pAdEasy system (30He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3229) Google Scholar). All concentrated viruses were titered by performing serial dilutions of the concentrated virus and counting the number of GFP-expressing 293 cells after 18-24 h.Adenoviruses containing sequences for human TβRIII and non-targeting control short hairpin RNA were generated by Dharmacon and inserted into a vector co-expressing the DS-Red fluorophore using the Adeno-X™ Expression System (Clontech). Specificity for TβRIII has previously been demonstrated (31You H.J. Bruinsma M.W. How T. Ostrander J.H. Blobe G.C. Carcinogenesis. 2007; 28: 2491-2500Crossref PubMed Scopus (52) Google Scholar, 32Gordon K.J. Dong M. Chislock E.M. Fields T.A. Blobe G.C. Carcinogenesis. 2008; (in press)Google Scholar). Recombinant human BMP-2, BMP-4, BMP-7, GDF-5, and soluble TβRIII were purchased from R&D Systems.BMP receptors were detected with an anti-hemagglutinin antibody (Roche). A polyclonal antibody against the cytoplasmic tail of TβRIII was generated by our laboratory and characterized previously (33Dong M. How T. Kirkbride K.C. Gordon K.J. Lee J.D. Hempel N. Kelly P. Moeller B.J. Marks J.R. Blobe G.C. J. Clin. Investig. 2007; 117: 206-217Crossref PubMed Scopus (196) Google Scholar). The β-actin antibody was purchased from (Sigma). Both anti-mouse and anti-rabbit antibodies were purchased from Amersham Biosciences.Iodination of BMP Family Members—125I-BMP were generated using the chloramine T method as previously described (34Cheifetz S. Hernandez H. Laiho M. ten Dijke P. Iwata K.K. Massague J. J. Biol. Chem. 1990; 265: 20533-20538Abstract Full Text PDF PubMed Google Scholar). Ten micrograms of carrier-free recombinant human BMP-2, BMP-7, BMP-4, and GDF-5 were used for each labeling. 125I-TGF-β1 was purchased from Amersham Biosciences.Cross-linking and Immunoprecipitation of Receptors—Binding assays were performed as previously described (35Lopez-Casillas F. Wrana J.L. Massague J. Cell. 1993; 73: 1435-1444Abstract Full Text PDF PubMed Scopus (770) Google Scholar). COS-7 cells (150,000 cells/well in 6-well plates) were transiently transfected with 2 μg (or otherwise indicated) of plasmid DNA using FuGENE 6 (Roche) 18 h after plating. Transiently transfected cells were incubated for 3 h at 4 °C with 10 nm BMP (150 pm TGF-β1), unless indicated otherwise. Endogenous TβRIII studies were incubated overnight at 4 °C. Competition studies were carried out similarly, except that the indicated concentrations of cold BMP-7 (×0.1, 1, 10, and 100) were added alongside the hot ligand (2 nm) for 3 h at 4 °C. Cell surface complexes were cross-linked with disuccinimidyl suberate and quenched with 1 m glycine. The cells were then lysed with RIPA buffer containing protease inhibitors and immunoprecipitated at 4 °C. Before immunoprecipitation lysate was removed for control gel analysis. The immunoprecipitated proteins were resolved using SDS-PAGE. These gels were subsequently dried and exposed to an audioradiograph.Surface Plasmon Resonance—BMP-2 (1600 response units) was immobilized on a CM5 sensor chip using amine coupling (sodium acetate pH 4.5). Soluble TβRIII (R & D Systems) was diluted in running buffer (10 mm Hepes, pH 7, 0.15 m NaCl, 3 mm EDTA, and 0.005% Surfactant P20) and flowed at concentrations ranging from 7.81 to 250 nm for 5 min at a flow rate of 50 μl/min. TGF-β1 (900 response units) was immobilized in sodium acetate, pH 5.5. In the TGF-β studies, sTβRIII (concentrations ranging from 8.75 to 560 nm) was flowed at a rate of 50 μl/min for 3 min. The resulting sensograms were then fit using nonlinear least squares analysis and numerical integration of differential rate equations and the fits were then analyzed by considering the distribution of the residuals.Smad1 Phosphorylation—PC-3 cells were plated at 125,000 cells/well and infected with adenovirus (50 multiplicity of infection) containing either non-targeting control short hairpin RNA (shRNA) or shRNA directed against human TβRIII 24 h after plating. The cells were then incubated for 96 h, serum-starved for 5 h, and treated with the indicated concentrations of rhBMP-2 for 10 min followed by direct lysis. Smad1 phosphorylation was assayed by Western blot with phospho-Smad1 antibody (Cell Signaling), with total Smad1 antibody as a loading control (Cell Signaling).Viral Injections and Collagen Gel Assays—Injections and assays were performed as previously described by Desgrosellier et al. (36Desgrosellier J.S. Mundell N.A. McDonnell M.A. Moses H.L. Barnett J.V. Dev. Biol. 2005; 280: 201-210Crossref PubMed Scopus (68) Google Scholar) with the exception of the addition of vehicle (bovine serum albumin/HCl), 200 pm TGF-β2, or 5 nm BMP-2, BMP-4, BMP-7, or GDF-5 12 h after placement of the explant on collagen. Each GFP-expressing cell was scored as epithelial, activated, or transformed as described (36Desgrosellier J.S. Mundell N.A. McDonnell M.A. Moses H.L. Barnett J.V. Dev. Biol. 2005; 280: 201-210Crossref PubMed Scopus (68) Google Scholar). For the total number of explants and cells counted, refer to supplemental Tables 3 and 4.RESULTSTβRIII Is a Cell Surface Receptor for BMP-2—To determine whether TβRIII functions as a BMP receptor, we expressed TβRIII, along with the BMP receptors, ALK3, ALK6, or BMPRII, in COS-7 cells, which express low endogenous levels of these cell surface receptors and assessed BMP-2 binding by chemically cross-linking 125I-BMP-2 to binding partners on the cell surface. As expected, 125I-BMP-2 bound to ALK3 and ALK6, but not to BMPRII, which cannot bind ligand on its own (Fig. 1A). 125I-BMP-2 was also detected bound to TβRIII in the presence of ALK3, ALK6, and BMPRII (Fig. 1A, lanes 3, 5, and 7) suggesting that TβRIII binds BMP-2. TβRIII expression also modestly increased BMP-2 binding to ALK3 and ALK6, but did not confer ligand binding to BMPRII (Fig. 1A).BMPRII binds BMP ligands only when in complex with either ALK3 or ALK6 (8Miyazawa K. Shinozaki M. Hara T. Furuya T. Miyazono K. Genes Cells. 2002; 7: 1191-1204Crossref PubMed Scopus (577) Google Scholar). To determine whether TβRIII affects the ability of BMPRII to bind BMP in the presence of ALK3 or ALK6, we expressed TβRIII with these traditional BMP signaling complexes. As expected, BMPRII could be detected bound to 125I-BMP-2 when co-expressed with ALK3 or ALK6 (Fig. 1B, lanes 1 and 3), and TβRIII did not significantly alter BMP-2 binding to BMPRII (Fig. 1B, lanes 2 and 4).To determine whether BMP receptor expression was necessary for the ability of TβRIII to bind BMP-2, we expressed TβRIII alone. In the absence of ALK3 and ALK6, 125I-BMP-2 formed a cross-linked complex with fully processed TβRIII in a dose-dependent fashion (Fig. 1C, left) establishing that BMP-2 is able to bind TβRIII independent of other ligand binding receptors.BMPs bind heparan sulfate with high affinity (28Irie A. Habuchi H. Kimata K. Sanai Y. Biochem. Biophys. Res. Commun. 2003; 308: 858-865Crossref PubMed Scopus (105) Google Scholar, 37Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) and TβRIII is a heparan sulfate and chondroitin sulfate proteoglycan (38Lopez-Casillas F. Payne H.M. Andres J.L. Massague J. J. Cell Biol. 1994; 124: 557-568Crossref PubMed Scopus (345) Google Scholar). These glycosaminoglycan modifications are important for basic fibroblast growth factor binding to TβRIII (27Andres J.L. DeFalcis D. Noda M. Massague J. J. Biol. Chem. 1992; 267: 5927-5930Abstract Full Text PDF PubMed Google Scholar), but not for TGF-β (38Lopez-Casillas F. Payne H.M. Andres J.L. Massague J. J. Cell Biol. 1994; 124: 557-568Crossref PubMed Scopus (345) Google Scholar) or inhibin binding (39Wiater E. Harrison C.A. Lewis K.A. Gray P.C. Vale W.W. J. Biol. Chem. 2006; 281: 17011-17022Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). To determine whether these glycosaminoglycan modifications were important for BMP binding to TβRIII, we used a mutant of TβRIII in which the serines (Ser-535 and Ser-546) necessary for glycosaminoglycan chain attachment are converted to alanines preventing this modification (TβRIIIΔgag) (38Lopez-Casillas F. Payne H.M. Andres J.L. Massague J. J. Cell Biol. 1994; 124: 557-568Crossref PubMed Scopus (345) Google Scholar). In these studies, the core protein of TβRIIIΔgag was affinity labeled with 125I-BMP-2 in a dose-dependent fashion (Fig. 1C, right) indicating that the heparan sulfate modifications were not necessary for BMP-2 binding to TβRIII.TβRIII exists in two forms, a membrane bound form and a soluble form, sTβRIII, derived from ectodomain shedding (38Lopez-Casillas F. Payne H.M. Andres J.L. Massague J. J. Cell Biol. 1994; 124: 557-568Crossref PubMed Scopus (345) Google Scholar). sTβRIII consists of the extracellular domain of TβRIII and is able to bind TGF-β, sequester ligand from the cell surface receptors, and antagonize TGF-β signaling (38Lopez-Casillas F. Payne H.M. Andres J.L. Massague J. J. Cell Biol. 1994; 124: 557-568Crossref PubMed Scopus (345) Google Scholar, 40Velasco-Loyden G. Arribas J. Lopez-Casillas F. J. Biol. Chem. 2004; 279: 7721-7733Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). To determine whether sTβRIII is able to bind BMP-2, we exposed recombinant, purified sTβRIII to 125I-BMP-2. As with membrane-bound TβRIII, sTβRIII was affinity labeled with 125I-BMP-2 in a dose-dependent fashion (Fig. 1D, lanes 2-4), with a BMP-2 binding pattern similar to that of the well characterized TβRIII ligand, TGF-β1 (Fig. 1D, lane 1). These data demonstrate that sTβRIII is able to bind BMP-2 and confirm that the binding of BMP-2 to the extracellular domain of TβRIII is direct.Kinetics and Affinity of BMP Binding to TβRIII—To characterize the interaction between TβRIII and BMP-2 we used surface plasmon resonance (also known as BIAcore), a sensitive method to measure protein-protein interactions (41Fivash M. Towler E.M. Fisher R.J. Curr. Opin. Biotechnol. 1998; 9: 97-101Crossref PubMed Scopus (170) Google Scholar, 42Karlsson R. J. Mol. Recognit. 2004; 17: 151-161Crossref PubMed Scopus (373) Google Scholar). BIAcore has been used to define the interactions of multiple TGF-β superfamily ligands with their receptors (6Brown M.A. Zhao Q. Baker K.A. Naik C. Chen C. Pukac L. Singh M. Tsareva T. Parice Y. Mahoney A. Roschke V. Sanyal I. Choe S. J. Biol. Chem. 2005; 280: 25111-25118Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 43Hatta T. Konishi H. Katoh E. Natsume T. Ueno N. Kobayashi Y. Yamazaki T. Biopolymers. 2000; 55: 399-406Crossref PubMed Scopus (49) Google Scholar, 44De Crescenzo G. Grothe S. Zwaagstra J. Tsang M. O'Connor-McCourt M.D. J. Biol. Chem. 2001; 276: 29632-29643Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 45Natsume T. Tomita S. Iemura S. Kinto N. Yamaguchi A. Ueno N. J. Biol. Chem. 1997; 272: 11535-11540Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Both BMP-2 and TGF-β1 were immobilized to a dextran sensor chip and purified sTβRIII was the analyte. Upon mathematically fitting the response curves, the model that best fit the binding of TβRIII to BMP-2 was the bivalent analyte (or avidity) model (Fig. 2 and supplemental Table S1). This model also provided the best fit for TGF-β1 binding to TβRIII, based on previous BIAcore studies (44De Crescenzo G. Grothe S. Zwaagstra J. Tsang M. O'Connor-McCourt M.D. J. Biol. Chem. 2001; 276: 29632-29643Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and confirmed here (supplemental Fig. 1). The fit to the bivalent analyte model suggested two ligand binding sites on TβRIII for both TGF-β1 and BMP-2 (supplemental Table S2). Using the bivalent model, we established kinetic and thermodynamic constants for BMP-2 interacting with the ligand binding site on sTβRIII, with data from three independent experiments establishing a dissociation constant of 10 μm (±3.66) for BMP-2 and 5 μm (±1.71) for TGF-β1. These results are comparable with previously published reports for TβRIII and TGF-β1 (44De Crescenzo G. Grothe S. Zwaagstra J. Tsang M. O'Connor-McCourt M.D. J. Biol. Chem. 2001; 276: 29632-29643Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). These studies indicate that the affinity of TβRIII for BMP-2 is on the same order of magnitude as the affinity of TβRIII for TGF-β1.FIGURE 2The bivalent analyte model best fits the kinetics for BMP-2 binding to TβRIII using surface plasmon resonance (BIAcore). A, global fitting analysis was carried out on the response units of TβRIII (the analyte) binding to rhBMP-2 (1600 response units) immobilized using amine coupling to a CM5 dextran sensor chip after subtracting out bovine serum albumin binding as background. TβRIII was flowed over BMP-2 at a rate of 50 ml/min for 5 min at concentrations ranging from 7.81 to 250 nm. The data were then fit to kinetic models. Represented is the best fit of the data, the bivalent analyte (also known as the avidity model). This data are representative of three independent experiments. B, graphical representation of the residuals of the data fit to the bivalent analyte model.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Multiple Members of the BMP Subfamily Bind to TβRIII—To determine whether TβRIII could bind other BMP subfamily members, we radiolabeled representative members of the three distinct BMP subfamilies, BMP-4, BMP-7, and GDF-5 (Fig. 3A) (1Miyazono K. Maeda S. Imamura T. Cytokine Growth Factor Rev. 2005; 16: 251-263Crossref PubMed Scopus (706) Google Scholar). 125I-BMP-2, 125I-BMP-4, 125I-BMP-7, and 125I-GDF-5 each formed a cross-linked complex with both the fully processed form of TβRIII, along with TβRIIIΔgag (Fig. 3B), suggesting that a broad range of BMP family members can bind to the core protein of TβRIII. Intriguingly, there were subtle differences in the binding patterns of certain BMP subfamily members to TβRIII, particularly to the core protein.FIGURE 3Multiple members of the BMP family specifically bind to the core protein of TβRIII. A, evolutionary tree diagram generated by the MacVector program from NCBI sequence alignment of known TβRIII ligands and the BMP members used in this study. B, COS-7 cells expressing wild type (wt) TβRIII or TβRIIIΔgag were exposed to 125I-BMP-2, 125I-BMP-4, 125I-BMP-7, 125I-GDF-5, or 125I-TGF-β1 as indicated and chemically cross-linked followed by immunoprecipitation with an antibody to the cytoplasmic tail of TβRIII. Total cellular TβRIII is shown as an expression control (bottom panel). *, indicates a nonspecific band. C and D, COS-7 cells expressing wild type TβRIII (C) or TβRIIIΔgag (D) were simultaneously exposed to 2 nm 125I-BMP-7 in the presence of increasing amounts of cold BMP-7 (0.2, 2, 20, and 200 nm) as indicated, followed by chemical cross-linking and immunoprecipitation. E, NIH3T3 cells were exposed to 125I-BMP-2, 125I-BMP-7, and 125I-TGF-β1. All lysates were immunoprecipitated with either preimmune serum or a TβRIII antibody, separated by SDS-PAGE, and detected by phosphorimaging. The data are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To define the specificity of the interaction of BMP with TβRIII, we performed competition experiments with iodinated ligand in the presence of increasing concentrations of unlabeled ligand. Consistent with a previous report (46Koenig B.B. Cook J.S. Wolsing D.H. Ting J. Tiesman J.P. Correa P.E. Olson C.A. Pecquet A.L. Ventura F. Grant R.A. Chen G.X. Wrana J.L. Massague J. Rosenbaum J.S. Mol. Cell Biol. 1994; 14: 5961-5974Crossref PubMed Scopus (311) Google Scholar), we were unable to specifically compete off BMP-2 binding with excess cold ligand (data not shown). The nonspecific binding of 125I-BMP-2 is likely due to the presence of basic residues at the amino terminus of BMP-2, as previously reported (46Koenig B.B. Cook J.S. Wolsing D.H. Ting J. Tiesman J.P. Correa P.E. Olson C.A. Pecquet A.L. Ventura F. Grant R.A. Chen G.X. Wrana J.L. Massague J. Rosenbaum J.S. Mol. Cell Biol. 1994; 14: 5961-5974Crossref PubMed Scopus (311) Google Scholar). Accordingly, we investigated specificity using BMP-7, which also binds TβRIII, and for which these amino-terminal basic residues are not present. Here, 100-fold excess cold ligand successfully competed off 125I-BMP-7 from wild-type TβRIII (Fig. 3C) with ∼40% nonspecific binding remaining (Fig. 3C and supplemental Fig. S2). Unlabeled BMP-7 also competed with 125I-BMP-7 for binding to TβRIIIΔgag (Fig. 3D). Taken together, these data support the ability of TβRIII to specifically bind a broad range of BMP subfamily members.To establish the physiological relevance of BMP binding to TβRIII, we investigated whether BMP family members bound to endogenous TβRIII. For these studies, we used NIH3T3 cells, which abundantly express TβRIII. Both 125I-BMP-2 and 125I-BMP-7 bound to high molecular weight complexes corresponding to the fully processed endogenous form of TβRIII, which were specifically immunoprecipitated by a TβRIII antibody, and not by preimmune serum, in a pattern similar to that of 125I-TGF-β1 (Fig. 3E). These data confirm that both BMP-2 and BMP-7 are able to bind to endogenous TβRIII.BMP Binds to Both TβRIII Ligand Binding Domains—Our BIAcore data suggested two ligand binding sites for BMP-2 and TGF-β1 on the core protein of TβRIII. Consistent with this, previous studies have established two TGF-β binding regions on TβRIII, with one in the membrane-distal half (Binding Reg
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