In the Absence of Type III Receptor, the Transforming Growth Factor (TGF)-β Type II-B Receptor Requires the Type I Receptor to Bind TGF-β2
2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês
10.1074/jbc.m401350200
ISSN1083-351X
AutoresElisabetta C. del Re, Jodie L. Babitt, Alnoor Pirani, Alan L. Schneyer, Herbert Y. Lin,
Tópico(s)Genetic factors in colorectal cancer
ResumoTransforming growth factor β (TGF-β) ligands exert their biological effects through type II (TβRII) and type I receptors (TβRI). Unlike TGF-β1 and -β3, TGF-β2 appears to require the co-receptor betaglycan (type III receptor, TβRIII) for high affinity binding and signaling. Recently, the TβRIII null mouse was generated and revealed significant non-overlapping phenotypes with the TGF-β2 null mouse, implying the existence of TβRIII independent mechanisms for TGF-β2 signaling. Because a variant of the type II receptor, the type II-B receptor (TβRII-B), has been suggested to mediate TGF-β2 signaling in the absence of TβRIII, we directly tested the ability of TβRII-B to bind TGF-β2. Here we show that the soluble extracellular domain of the type II-B receptor (sTβRII-B.Fc) bound TGF-β1 and TGF-β3 with high affinity (Kd values = 31.7 ± 22.8 and 74.6 ± 15.8 pm, respectively), but TGF-β2 binding was undetectable at corresponding doses. Similar results were obtained for the soluble type II receptor (sTβRII.Fc). However, sTβRII.Fc or sTβRII-B.Fc in combination with soluble type I receptor (sTβRI.Fc) formed a high affinity complex that bound TGF-β2, and this complex inhibited TGF-β2 in a biological inhibition assay. These results show that TGF-β2 has the potential to signal in the absence of TβRIII when sufficient TGF-β2, TβRI, and TβRII or TβRII-B are present. Our data also support a cooperative model for receptor-ligand interactions, as has been suggested by crystallization studies of TGF-β receptors and ligands. Our cell-free binding assay system will allow for testing of models of receptor-ligand complexes prior to actual solution of crystal structures. Transforming growth factor β (TGF-β) ligands exert their biological effects through type II (TβRII) and type I receptors (TβRI). Unlike TGF-β1 and -β3, TGF-β2 appears to require the co-receptor betaglycan (type III receptor, TβRIII) for high affinity binding and signaling. Recently, the TβRIII null mouse was generated and revealed significant non-overlapping phenotypes with the TGF-β2 null mouse, implying the existence of TβRIII independent mechanisms for TGF-β2 signaling. Because a variant of the type II receptor, the type II-B receptor (TβRII-B), has been suggested to mediate TGF-β2 signaling in the absence of TβRIII, we directly tested the ability of TβRII-B to bind TGF-β2. Here we show that the soluble extracellular domain of the type II-B receptor (sTβRII-B.Fc) bound TGF-β1 and TGF-β3 with high affinity (Kd values = 31.7 ± 22.8 and 74.6 ± 15.8 pm, respectively), but TGF-β2 binding was undetectable at corresponding doses. Similar results were obtained for the soluble type II receptor (sTβRII.Fc). However, sTβRII.Fc or sTβRII-B.Fc in combination with soluble type I receptor (sTβRI.Fc) formed a high affinity complex that bound TGF-β2, and this complex inhibited TGF-β2 in a biological inhibition assay. These results show that TGF-β2 has the potential to signal in the absence of TβRIII when sufficient TGF-β2, TβRI, and TβRII or TβRII-B are present. Our data also support a cooperative model for receptor-ligand interactions, as has been suggested by crystallization studies of TGF-β receptors and ligands. Our cell-free binding assay system will allow for testing of models of receptor-ligand complexes prior to actual solution of crystal structures. Transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; BMP, bone morphogenetic protein; TβRI, TGF-β type I receptor; TβRII, TGF-β type II receptor; TβRIII, TGF-β type III receptor; TβRII-β type II-B receptor; ActRII, activin type II receptor; sTβRII.Fc, soluble human TβRII fused to the Fc region of human immunoglobulin; sTβRIIB.Fc, soluble human TβRII fused to the Fc region of human immunoglobulin; sTβRI.Fc, soluble mouse TβRI fused to the Fc region of human immunoglobulin; Mv1Lu cells, mink lung epithelial cells; ECD, extracellular domain. 1The abbreviations used are: TGF-β, transforming growth factor-β; BMP, bone morphogenetic protein; TβRI, TGF-β type I receptor; TβRII, TGF-β type II receptor; TβRIII, TGF-β type III receptor; TβRII-β type II-B receptor; ActRII, activin type II receptor; sTβRII.Fc, soluble human TβRII fused to the Fc region of human immunoglobulin; sTβRIIB.Fc, soluble human TβRII fused to the Fc region of human immunoglobulin; sTβRI.Fc, soluble mouse TβRI fused to the Fc region of human immunoglobulin; Mv1Lu cells, mink lung epithelial cells; ECD, extracellular domain. represents a large superfamily of dimeric growth factors that include the TGF-βs, inhibins, activins, Mullerian inhibiting substance, growth and differentiation factors, and bone morphogenetic proteins (BMPs) in mammals (1Massague J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (2996) Google Scholar, 2Shi Y. Massague J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4740) Google Scholar). These cytokines play important roles in an array of processes such as growth, differentiation, and development (3Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4201) Google Scholar). There are three TGF-β isoforms that share a high degree of homology and overlapping biological activities (4Roberts A.B. Sporn M.B. Peptide Growth Factors and Their Receptors. Springer-Verlag, New York1990Google Scholar). However, distinct expression patterns and unique, isoform-specific phenotypes of the corresponding knockout mice demonstrate significant non-redundancy of TGF-β function (5Shull M.M. Ormsby I. Kier A.B. Pawlowski S. Diebold R.J. Yin M. Allen R. Sidman C. Proetzel G. Calvin D. Annunziata N. Doetschman T. Nature. 1992; 359: 693-699Crossref PubMed Scopus (2604) Google Scholar, 6Kulkarni A.B. Huh C.G. Becker D. Geiser A. Lyght M. Flanders K.C. Roberts A.B. Sporn M.B. Ward J.M. Karlsson S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 770-774Crossref PubMed Scopus (1629) Google Scholar, 7Kaartinen V. Voncken J.W. Shuler C. Warburton D. Bu D. Heisterkamp N. Groffen J. Nat. Genet. 1995; 11: 415-421Crossref PubMed Scopus (870) Google Scholar, 8Sanford L.P. Ormsby I. de Gittenberger G.A. Sariola H. Frieman R. Boivin G.P. Cardell E.L. Doetschman T. Development. 1997; 124: 2659-2670Crossref PubMed Google Scholar, 9Piek E. Heldin C.H. Ten Dijke P. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (737) Google Scholar).TGF-βs exert their biological effects through three cell surface receptors designated as type I, II, and III (TβRI, TβRII, and TβRIII) (2Shi Y. Massague J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4740) Google Scholar), all of which have been cloned (10Lopez-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, 11Wang 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, 12Lin H.Y. Wang X.F. Mg-Eaton E. Weinberg R.A. Lodish H.F. Cell. 1992; 68: 775-785Abstract Full Text PDF PubMed Scopus (966) Google Scholar, 13ten Dijke P. Ichijo H. Franzen P. Schulz P. Saras J. Toyoshima H. Heldin C.H. Miyazono K. Oncogene. 1993; 8: 2879-2887PubMed Google Scholar). In addition, the type II-B receptor (TβRII-B), an alternatively spliced isoform of TβRII containing an insert of 26 amino acids replacing Val51, has also been identified (14Suzuki A. Shioda N. Maeda T. Tada M. Ueno N. FEBS Lett. 1994; 335: 19-22Crossref Scopus (36) Google Scholar, 15Hirai R. Fijita T. Exp. Cell Res. 1996; 223: 135-141Crossref PubMed Scopus (27) Google Scholar, 16Rotzer D. Roth M. Lutz M. Lindemann D. Sebald W. Knaus P. EMBO J. 2001; 20: 480-490Crossref PubMed Scopus (78) Google Scholar). Type I and type II receptors have intracellular serine/threonine kinase domains, whereas the type III receptor has only a short intracellular domain. On binding of TGF-β ligands, constitutively active type II receptors recruit and phosphorylate type I receptors; the activated type I receptor kinase then interacts with and phosphorylates downstream signaling molecules, the RSmads (13ten Dijke P. Ichijo H. Franzen P. Schulz P. Saras J. Toyoshima H. Heldin C.H. Miyazono K. Oncogene. 1993; 8: 2879-2887PubMed Google Scholar, 17Wrana J.L. Attisano L. Wieser R. Ventura F. Massague J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2094) Google Scholar, 18Feng X.H. Derynck R. EMBO J. 1997; 16: 3912-3923Crossref PubMed Scopus (161) Google Scholar). The exact stoichiometry of the active receptor signaling complex is not known. Type II and type I receptors have been shown to form homodimers in the absence of ligand (19Henis Y.I. Moustakas A. Lin H.Y. Lodish H.F. J. Cell Biol. 1994; 126: 139-154Crossref PubMed Scopus (168) Google Scholar, 20Gilboa L. Wells R.G. Lodish H.F. Henis Y.I. Cell Biol. 1998; 140: 767-777Crossref PubMed Scopus (122) Google Scholar), thus raising the possibility that the active receptor signaling complex could be a large multimeric complex consisting of a minimum of two type II receptors, two type I receptors, and the TGF-β ligand homodimer. The crystal structures of TGF-β2 (21Daopin S. Piez K.A. Ogawa Y. Davies D.R. Science. 1992; 257: 369-373Crossref PubMed Scopus (374) Google Scholar, 22Schlunegger M.P. Grutter M.G. Nature. 1992; 358: 430-444Crossref PubMed Scopus (284) Google Scholar), and TGF-β3 (23Mittl P.R. Priestle J.P. Cox D.A. McMaster G. Cerletti N. Grutter M.G. Protein Sci. 1996; 5: 1261-1271Crossref PubMed Scopus (127) Google Scholar) ligands have been solved. In addition, the extracellular domain of TβRII alone (24Boesen C.C. Radaev S. Motyka S.A. Patamawenu A. Sun P.D. Structure. 2002; 10: 913-919Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and in complex with TGF-β3 (25Hart P.J. Deep S. Taylor A.B. Shu Z. Hinck C.S. Hinck A.P. Nat. Struct. Biol. 2002; 9: 203-208PubMed Google Scholar), have also been crystallized. These structures yield qualitatively different information than the crystal structure of the BMP type IA receptor (BMPR1A)· BMP-2 complex (26Kirsch T. Sebald W. Dreyer M.K. Nat. Struct. Biol. 2000; 7: 492-496Crossref PubMed Scopus (269) Google Scholar) and the activin type II receptor (ActRII)· BMP-7 complex (27Greenwald J. Groppe J. Gray P. Wiater E. Kwiatkowski W. Vale W. Choe S. Mol. Cell. 2003; 11: 605-617Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). In aggregate, these studies suggest that the TGF-β receptor and ligand interactions may involve a cooperative model of binding in which the extracellular domains of the type II and type I receptors make physical contact, whereas the BMP/ActRII and BMP ligands may utilize an allosteric model of binding in which the extracellular domains of the type II and type I receptors do not interact.Of the three TGF-β isoforms, TGF-β2 and TGF-β3 have been less well investigated than TGF-β1. TGF-β3 appears to bind receptors and signal in a manner that is similar to TGF-β1. In contrast, TGF-β2 has much lower affinity for TβRII than TGF-β1 and -β3. It has been demonstrated that an accessory receptor, TβRIII, is necessary for efficient binding and cross-linking of TGF-β2 and subsequent signaling (28Lopez-Casillas F. Wrana J.L. Massague J. Cell. 1993; 73: 1435-1444Abstract Full Text PDF PubMed Scopus (770) Google Scholar). In this model, TGF-β2 binds to TβRIII, which then recruits TβRII and TβRI resulting in phosphorylation of TβRI and downstream signaling (28Lopez-Casillas F. Wrana J.L. Massague J. Cell. 1993; 73: 1435-1444Abstract Full Text PDF PubMed Scopus (770) Google Scholar). Interestingly, the recently published phenotype of the TβRIII null mouse (29Stenvers 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. Mol. Cell. Biol. 2003; 23: 4371-4385Crossref PubMed Scopus (198) Google Scholar) is not completely overlapping with the phenotype of the TGF-β2-deficient mouse (8Sanford L.P. Ormsby I. de Gittenberger G.A. Sariola H. Frieman R. Boivin G.P. Cardell E.L. Doetschman T. Development. 1997; 124: 2659-2670Crossref PubMed Google Scholar), suggesting the existence of alternative methods for TGF-β2 binding and signaling that do not involve TβRIII. Cross-linking studies of cell-surface TGF-β receptors with 125I-TGF-β2 in transfected COS cells have suggested that high affinity TGF-β2 binding and downstream signaling in these cells can occur via complexes of type I and type II receptors (30Rodriguez C. Chen F. Weinberg R.A. Lodish H.F. J. Biol. Chem. 1995; 270: 15919-15922Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Alternatively, Rotzer et al. (16Rotzer D. Roth M. Lutz M. Lindemann D. Sebald W. Knaus P. EMBO J. 2001; 20: 480-490Crossref PubMed Scopus (78) Google Scholar) has proposed that TβRII-B binds TGF-β2 with ensuing signaling in the absence of TβRIII. Of note, an earlier report showing the inability of unlabeled TGF-β2 to compete for 125I-TGF-β1 binding to TβRII-B inferred that TβRII-B resembles TβRII in its inability to bind TGF-β2 (14Suzuki A. Shioda N. Maeda T. Tada M. Ueno N. FEBS Lett. 1994; 335: 19-22Crossref Scopus (36) Google Scholar). In general, these studies of TGF-β receptor binding to ligand were performed on receptors expressed at the cell surface where binding can only be measured indirectly via cross-linking to radioligand followed by autoradiography. These studies are therefore limited by an inability to directly quantify binding and thus obtain an accurate measurement of receptor-ligand affinities. In addition, the presence of other extracellular cell surface-associated proteins that may act as accessories to binding cannot be ruled out.To overcome the limitations of cell surface expression studies and to specifically assess the ability of TβRII-B to bind TGF-β2, we have developed a cell-free system using soluble TGF-β receptors. We demonstrate that in comparison with TGF-β1 and -β3, TGF-β2 bound poorly to soluble TGF-β type II (sTβRII.Fc) or TGF-β type II-B receptors (sTβRII-B.Fc) alone. However, TGF-β2 did bind sTβRII.Fc or sTβRII-B.Fc in complex with the soluble type I receptor (sTβRI.Fc) in solution, and cell-surface TβRII or TβRII-B together with TβRI could mediate TGF-β2 signaling in the absence of TβRIII. Our heteromeric receptor binding assay system provides supporting evidence for a cooperative model of type II and type I receptor interactions with TGF-β2 ligand, and provides a rapid and straightforward way to measure the binding of receptor and/or ligand mutants that arise from structure-function studies.EXPERIMENTAL PROCEDUREScDNA Subcloning—The cDNA encoding the extracellular domain of human TβRII was amplified by PCR from human TβRII cDNA (12Lin H.Y. Wang X.F. Mg-Eaton E. Weinberg R.A. Lodish H.F. Cell. 1992; 68: 775-785Abstract Full Text PDF PubMed Scopus (966) Google Scholar). The PCR product was digested and ligated in-frame into the restriction sites BamHI (5′) and HindIII (3′) of the vector pIg-Tail (31Komesli S. Vivien D. Dutartre P. Eur. J. Biochem. 1998; 254: 505-513Crossref PubMed Scopus (54) Google Scholar) to generate the sTβRII.Fc mammalian expression construct. The primers used were 5′-CCCAAGCTTATGCCGCTGCTGCTACTGCTG-3′ (forward) and 3′-ATATTGTGGTCGTTAGGACTGCGCCTAGGG-5′ (reverse). The cDNA was sequenced on both strands to confirm the fidelity of the construct.To generate cDNA for the extracellular domain of human TβRII-B, the 26-amino acid insert was generated by an overlapping primer strategy using PCR. The N-terminal half of the insert was generated by PCR using the following primers: 5′-CCCAAGCTTGCCGCCACCATGGGTCGGGGGCTGCTCAGG-3′ (forward) and 3′-CTGGGGCAGATGTTCTGGGCCTCCATTTCCACATCCGACTTCTGAACGTGCGGT-5′ (reverse). The C-terminal half of the insert and the rest of the extracellular domain was generated by PCR using the following primers: 5′-GGGGGATCCGCGTCAGGATTGCTGGTGTTATA-3′ (forward) and 3′-CTGTAATAGGACTGCCCACTGAGAACATATATTAATAACGACATGATAGTC-5′ (reverse). Both PCR products were purified, mixed together, and a final round of PCR was performed using the following "outside" primers: 5′-CCCAAGCTTGCCGCCACCATGGGTCGGGGGCTGCTCAGG-3′ (forward) and the 3′-CTGTAATAGGACTGCCCACTGAGAACATATATTAATAACGACATGATAGTC-5′ (reverse). The resultant PCR product was purified, digested, and ligated in-frame into the restriction sites BamHI and HindIII (3′) of the vector pIg-Tail to generate the sTβRII-B.Fc mammalian expression construct. The extracellular domain (ECD) of human TβRII-B was then subcloned into full-length human TβRII to generate full-length TβRII-B. cDNAs were sequenced on both strands to confirm the fidelity of the construct.Mammalian Cell Expression—HEK 293 cells (ATCC number CRL-1573) were cultured in Dulbecco's modified of Eagle's medium (Cellgro, Mediatech, VA) supplemented with 10% fetal bovine serum. All transfections were performed with LipofectAMINE 2000 (Invitrogen). Stably transfected cells were selected and cultured in Dulbecco's modified of Eagle's medium supplemented with 10% ultra-low IgG fetal bovine serum (Invitrogen) and 1 mg/ml G418 (Invitrogen) in 175-cm2 multi-floor flasks (Sarstedt).Protein A Purification of sTβRII.Fc and sTβRII-B.Fc—The human recombinant receptors were purified by one-step Protein A affinity chromatography. Tissue culture medium was filtered through a vacuum-driven 0.22-μm, Durapore Membrane Unit (Millipore Corp., Bedford, MA). The pH of the media was adjusted to pH 8.2 by addition of Tris base and the media was applied to HiTrap rProtein A FF columns (Amersham Biosciences) previously equilibrated with phosphate-buffered saline (Invitrogen). After protein loading, the columns were washed with binding buffer (phosphate-buffered saline) to remove non-specifically bound proteins. Human soluble receptors were eluted with 3 volumes of 100 mm glycine buffer, pH 3.0. The pH of eluted fractions was immediately neutralized by addition of a 1/10 volume of 1 m Tris/HCl, pH 9.0. Eluted protein was stored at -20 °C. Purity of the protein was determined by 4-12% SDS-PAGE using pre-cast mini gels (Novex) followed by silver staining (Bio-Rad). Amounts of proteins eluted were quantified by the bovine serum albumin protein assay (Pierce) and confirmed by amino acid analysis (MGH-Protein Core Facility). Yields were ∼6 μg of soluble protein per 10-cm tissue culture plates.Soluble Receptor Deglycosylation—25 μg of purified sTβRII.Fc and sTβRII-B.Fc were denatured in 0.5% SDS, 1% β-mercaptoethanol at 100 °C for 10 min and incubated in 50 nm sodium phosphate supplemented with 1% Nonidet P-40 at 37 °C. Denatured proteins were then incubated either in the presence or absence of 5,000 units of N-glycosidase F (New England Biolabs) at 37 °C for 2 h and analyzed by SDS-PAGE and silver staining as described above.Protein Analysis with Western Blot—Recombinant human receptors eluted from the HiTrap Protein A column were separated by 4-12% gradient SDS-PAGE pre-cast minigels (Novex), then transferred to polyvinylidone difluoride transfer membrane (Schleicher & Shuell). After transfer, the membrane was washed in Tris-buffered saline supplemented with 0.1% Tween 20 (TBST), and blocked overnight in 8% powdered milk in TBST. The membrane was then incubated with goat anti-human TβRII antibodies (R&D Systems) or goat anti-human Fc-specific IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), followed by donkey anti-goat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The chemiluminescence immunoassay was performed with Renaissance Western blot chemiluminescence reagent (Amersham Biosciences). Polyclonal antibodies directed against a peptide of human TβRII-B (QKDEIICPSCNRTAHPLRHI, Peptide Core Facility, MGH, MA) were raised in goats (SIGMA-Gemosys, The Woodlands, TX) and employed to specifically detect human recombinant sTβRII-B.Fc compared with human recombinant sTβRII.Fc. Mouse sTβRI.Fc was purchased from R&D Systems.Ligand Iodination—Carrier-free human TGF-β1, -β2, and -β3 were purchased from R&D Systems. Two μg of ligand per reaction was iodinated with [125I] by the modified chloramine-T method as previously described (32Frolik C.A. Wakefield L.M. Smith D.M. Sporn M.B. J. Biol. Chem. 1984; 259: 10995-11000Abstract Full Text PDF PubMed Google Scholar).Binding Assays on Protein A Plates—Soluble recombinant human receptors were diluted in TBS/casein blocking buffer (BioFX, Owings Mills, MD) and incubated on Protein A-coated 96-well plates (Pierce) overnight. Plates were then washed with wash buffer (BioFX) and blocked 2 h at room temperature with TBS/casein buffer. For competition binding assays, fixed amounts of radioligands (50,000-100,000 counts) were added to the plates together with increasing amounts (2 pm to 500 nm) of homologous or heterologous non-radioactive ligands.Binding Assays in Solution—Soluble recombinant human receptors were diluted in TBS/casein blocking buffer (BioFX) and incubated overnight in the presence or absence of ligand. For competition binding assays, fixed amounts of radioligands (50,000-100,000 counts) were added to the samples together with increasing amounts (2 pm to 500 nm) of homologous or heterologous non-radioactive ligands. Samples were then placed on Protein A-coated 96-well plates (Pierce) for 90 min, washed 3 times with wash buffer (BioFX), and counted using a γ-counter.Luciferase Reporter Assay—Mink Lung cells (Mv1Lu) were transiently transfected with the (CAGA)12MPL-Luc reporter construct (33Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1573) Google Scholar) and with a pRL-TK vector (Promega) in a ratio of 10:1 to control for transfection efficiency. Cells were then serum starved for 6 h before treatment with varying amounts of TGF-β ligands in the presence or absence of varying amounts of soluble receptor for 16 h. Experiments were performed in triplicate wells. Cells were lysed and luciferase activity was determined with the Dual Reporter Assay (Promega). Relative light units were calculated as ratios of Firefly (reporter) and Renilla (transfection control) values. Alternatively, Mv1Lu cells were used that had been stably transfected with the (CAGA)12MPL-Luc reporter construct. In this case, relative light units were corrected for total amount of protein in the lysate as determined by a bovine serum albumin protein assay (Pierce). Rat myoblast L6 cells were transfected with the (CAGA)12MPL-Luc reporter construct and with a pRL-TK vector and in addition with empty vector or full-length TGF-β receptors constructs using LipofectAMINE 2000. The same protocol as for the Mv1Lu cells was then followed.Data Analysis—Each experiment was repeated at least three times and different preparations of sTβRII.Fc and sTβRII-B.Fc were tested and used. Data are expressed as mean ± S.E. The Ligand Program from the National Institutes of Health was used to fit binding curves for the binding data (34Schneyer A. Schoen A. Quigg A. Sidis Y. Endocrinology. 2003; 144: 1671-1674Crossref PubMed Scopus (61) Google Scholar). The Student's t test was used with a p value of <0.05 to determine statistical significance.RESULTSProduction and Characterization of Soluble Type II-B.Fc (sTβRII-B.Fc) and Soluble Type II.Fc (sTβRII.Fc) Chimeric Proteins—cDNA encoding the ECDs of either TβRII or TβRII-B were fused to the Fc portion of human IgG and transfected into HEK 293 cells to generate sTβRII.Fc and sTβRII-B.Fc as described under "Experimental Procedures" and shown schematically in Fig. 1A. The ECD of human TβRII-B contains the 26-amino acid insert that replaces Val32 of TβRII (14Suzuki A. Shioda N. Maeda T. Tada M. Ueno N. FEBS Lett. 1994; 335: 19-22Crossref Scopus (36) Google Scholar, 15Hirai R. Fijita T. Exp. Cell Res. 1996; 223: 135-141Crossref PubMed Scopus (27) Google Scholar, 16Rotzer D. Roth M. Lutz M. Lindemann D. Sebald W. Knaus P. EMBO J. 2001; 20: 480-490Crossref PubMed Scopus (78) Google Scholar).Analysis of soluble receptor proteins purified by one-step Protein A affinity chromatography with SDS-PAGE and silver staining showed that the sTβRII.Fc protein was ∼50 kDa, whereas the sTβRII-B.Fc protein was ∼55 kDa (Fig. 1B, lanes 1 and 3), consistent with the presence of the 26-amino acid insert in sTβRII-B.Fc. Under non-reducing conditions, protein bands of ∼100 kDa for sTβRII.Fc and ∼110 kDa for sTβRIIB.Fc were visualized, reflecting the disulfide bond formation of the dimeric Fc domain (data not shown).Both sTβRII.Fc and sTβRII-B.Fc proteins were sensitive to N-glycosidase F treatment (Fig. 1B), indicating that both proteins are N-glycosylated. The molecular mass of the deglycosylated receptors, ∼40 kDa (sTβRII.Fc, lane 2) and ∼42 kDa (sTβRII-B.Fc, lane 4), correspond to the predicted molecular masses of the core protein of each chimeric protein.Western blot analysis of soluble recombinant receptor chimeric proteins shown in Fig. 1C confirmed that the soluble receptor proteins contained both the human Fc domain (Fig. 1C, left), and the extracellular domain of the type II receptor (Fig. 1C, middle), using an anti-human Fc antibody (αFC) and an anti-type II receptor ECD domain antibody (αRII). As expected, a rabbit polyclonal antibody raised against the peptide encoding the 26-amino acid insertion sequence of TβRII-B recognized only the sTβRII-B.Fc protein with no detectable cross-reactivity to sTβRII.Fc protein (α26aa, Fig. 1C, right). The anti-human Fc antibody also recognized sβTRI.Fc (from R&D Systems, Fig. 1D).sTβRII.Fc and sTβRII-B.Fc Can Bind TGF-β1 and -β3, but Not TGF-β2—Radioligand competition experiments were performed to determine the selectivity and affinity of sTβRII-B.Fc and sTβRII.Fc proteins for different TGF-β isoforms. A non-saturating amount of soluble receptor was incubated overnight with 125I-labeled TGF-β1, -β2, or -β3 with or without serial dilutions of unlabeled TGF-β1, -β2, or -β3 at final concentrations from 2 pm to 500 nm, as indicated. The amount of competitor that inhibited 50% of 125I-labeled TGF-βs was defined as the effective dose (ED50). The ED50, relative potency, and calculated dissociation constants (Kd) averaged from at least three separate experiments are summarized in Table I (all slopes were parallel). For sTβRII-B.Fc, the ED50 (in ng) obtained when 125I-TGF-β1 was competed with unlabeled TGF-β1 and -β3 was 0.58 ± 0.1 and 2.2 ± 0.4, respectively, and for sTβRII.Fc, 0.64 ± 0.16 and 1.7 ± 0.4, respectively. When 125I-TGF-β3 was competed with unlabeled TGF-β1 and -β3, the ED50 (in nanograms) for sTβRII-B.Fc was 1.1 ± 0.3 and 2.3 ± 0.8, respectively, and for sTβRII.Fc, 2.0 ± 0.36 and 3.2 ± 1.1, respectively. The relative potency was calculated by comparing ED50 values and showed that TGF-β1 was more effective than TGF-β3 in all cases. Cold TGF-β2 was unable to compete with 125I-TGF-β1 or 125I-TGF-β3 binding to the soluble receptors, even at a concentration of 1250 ng/ml (500 nm).Table IED50 relative potency, and Kd for sTβRII-B.Fc and sTβRII. Fc binding to TGF-β1, -β2, and -β3125I-LigandCold ligandsTβRII-B.FcsTβRII.FcED50 ± S.E.Relative potencyKd ± S.E.ED50 ± S.E.Relative potencyKd ± S.E.ngpmngpm*β1β10.58 ± 0.1131.7 ± 22.80.64 ± 0.161112 ± 38β2No competitionNo competitionβ32.2 ± 0.40.26Not done1.7 ± 0.40.38Not done*β2β1No bindingβ2β3*β3β11.1 ± 0.32.1Not done2.0 ± 0.361.6Not doneβ2No competitionNo competitionβ32.3 ± 0.75174.6 ± 15.83.2 ± 1.11375 ± 185 Open table in a new tab Binding affinity values were calculated using Scatchard analysis of the binding data. A Scatchard analysis from one representative experiment is shown in Fig. 2, and averages from at least three separate experiments are summarized in Table I. sTβRII-B.Fc and sTβRII.Fc proteins had high affinity for TGF-β1 and -β3, with Kd values in the picomolar range. sTβRII-B.Fc had a severalfold higher affinity for TGF-β1 and -β3 than sTβRII.Fc, but the difference in calculated affinities was not statistically significant. When 125I-TGF-β2 was employed, no binding could be detected, even when the amount of soluble receptor per well was increased to 100 ng/well.Fig. 2Measurement of sTβRII.Fc and sTβRII-B.Fc binding affinities. sTβRII-B.Fc and sTβRII.Fc were incubated on Protein A-coated plates overnight followed by 100,000 counts of 125ITGF-β1 or -β3 in the presence of increasing amounts (2 pm to 500 nm) of homologous non-radioactive ligand. Samples were washed and counted using a standard γ-counter. Panels show a representative inhibition curve and Scatchard plot for sTβRII-B.Fc (upper panel) and sTβRII-B.Fc (lower panel) binding to TGF-β1 (closed circles) or -β3 (open circles) from one of three experiments.View Large Image Figure ViewerDownload (PPT)sTβRII.Fc and sTβRII-B.Fc Can Inhibit TGF-β1 and -β3, but Not TGF-β2 Biological Activity—Next we tested whether sTβRII-B.Fc and sTβRII.Fc could block TGF-β activity by performing a biological inhibition assay using a TGF-β responsive luciferase reporter assay (Fig. 3). Mv1Lu cells transfected with the (CAGA)12MPL-Luc reporter construct were treated with 100 pm TGF-β1, -β2, or -β3 in combination with increasing amounts (30-1300 pm) of purified sTβRII-B.Fc (Fig. 3A) or sTβRII.Fc (Fig. 3B). The relative luciferase activity induced by TGF-β1 (Fig. 3, diamonds) and TGF-β3 (Fig. 3, triangles) was decreased in a dose-dependent manner by either sTβRII-B.Fc or sTβRII.Fc. The ED50 was 360 pm for sTβRII-B.Fc to both TGF-β1 and TGF-β3. For sTβRII.Fc, the ED50 was 664 pm for TGF-β1 and 501 pm for TGF-β3. In contrast, there was no inhibition of TGF-β2-induced luciferase activity by either sTβRII-B.Fc or sTβRII.Fc (Fig. 3, squares).Fig. 3sTβRII.Fc or sTβRII-B.Fc can inhibit TGF-β1 and -β3 but not TGF-β2 signaling activity. Mv1Lu cells stably transfected with the TGF-β respons
Referência(s)