Title: Transforming Growth Factor (TGF-β)-specific Signaling by Chimeric TGF-β Type II Receptor with Intracellular Domain of Activin Type IIB Receptor
Abstract: Members of the transforming growth factor-β (TGF-β) superfamily signal via different heteromeric complexes of two sequentially acting serine/threonine kinase receptors, i.e.type I and type II receptors. We generated two different chimeric TGF-β superfamily receptors, i.e. TβR-I/BMPR-IB, containing the extracellular domain of TGF-β type I receptor (TβR-I) and the intracellular domain of bone morphogenetic protein type IB receptor (BMPR-IB), and TβR-II/ActR-IIB, containing the extracellular domain of TGF-β type II receptor (TβR-II) and the intracellular domain of activin type IIB receptor (ActR-IIB). In the presence of TGF-β1, TβR-I/BMPR-IB and TβR-II/ActR-IIB formed heteromeric complexes with wild-type TβR-II and TβR-I, respectively, upon stable transfection in mink lung epithelial cell lines. We show that TβR-II/ActR-IIB restored the responsiveness upon transfection in mutant cell lines lacking functional TβR-II with respect to TGF-β-mediated activation of a transcriptional signal, extracellular matrix formation, growth inhibition, and Smad phosphorylation. Moreover, TβR-I/BMPR-IB and TβR-II/ActR-IIB formed a functional complex in response to TGF-β and induced phosphorylation of Smad1. However, complex formation is not enough for signal propagation, which is shown by the inability of TβR-I/BMPR-IB to restore responsiveness to TGF-β in cell lines deficient in functional TβR-I. The fact that the TGF-β1-induced complex between TβR-II/ActR-IIB and TβR-I stimulated endogenous Smad2 phosphorylation, a TGF-β-like response, is in agreement with the current model for receptor activation in which the type I receptor determines signal specificity. Members of the transforming growth factor-β (TGF-β) superfamily signal via different heteromeric complexes of two sequentially acting serine/threonine kinase receptors, i.e.type I and type II receptors. We generated two different chimeric TGF-β superfamily receptors, i.e. TβR-I/BMPR-IB, containing the extracellular domain of TGF-β type I receptor (TβR-I) and the intracellular domain of bone morphogenetic protein type IB receptor (BMPR-IB), and TβR-II/ActR-IIB, containing the extracellular domain of TGF-β type II receptor (TβR-II) and the intracellular domain of activin type IIB receptor (ActR-IIB). In the presence of TGF-β1, TβR-I/BMPR-IB and TβR-II/ActR-IIB formed heteromeric complexes with wild-type TβR-II and TβR-I, respectively, upon stable transfection in mink lung epithelial cell lines. We show that TβR-II/ActR-IIB restored the responsiveness upon transfection in mutant cell lines lacking functional TβR-II with respect to TGF-β-mediated activation of a transcriptional signal, extracellular matrix formation, growth inhibition, and Smad phosphorylation. Moreover, TβR-I/BMPR-IB and TβR-II/ActR-IIB formed a functional complex in response to TGF-β and induced phosphorylation of Smad1. However, complex formation is not enough for signal propagation, which is shown by the inability of TβR-I/BMPR-IB to restore responsiveness to TGF-β in cell lines deficient in functional TβR-I. The fact that the TGF-β1-induced complex between TβR-II/ActR-IIB and TβR-I stimulated endogenous Smad2 phosphorylation, a TGF-β-like response, is in agreement with the current model for receptor activation in which the type I receptor determines signal specificity. Transforming growth factor-βs (TGF-βs), 1The abbreviations used are: TGF-β, transforming growth factor-β; TβR, TGF-β receptor; ActR, activin receptor; BMP, bone morphogenetic protein; BMPR, BMP receptor; GS domain, glycine-serine-rich domain; FBS, fetal bovine serum; OP, osteogenic protein; PAGE, polyacrylamide gel electrophoresis; PAI, plasminogen activator inhibitor; PCR, polymerase chain reaction; Smad, Sma and MAD-related protein; DMEM, Dulbecco's modified Eagle's medium. activins, and bone morphogenetic proteins (BMPs) are structurally related proteins that play important roles in intercellular communication (reviewed in Refs. 1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Peptide Growth Factors and Their Receptors, Part I. 95. Springer-Verlag, Berlin1990: 419-472Google Scholar, 2Mathews L.S. Endocr. Rev. 1994; 15: 310-325Crossref PubMed Scopus (274) Google Scholar, 3Kingsley D.M. Genes & Dev. 1994; 8: 133-146Crossref PubMed Scopus (1731) Google Scholar, 4Reddi A.H. Curr. Opin. Genet. Dev. 1994; 4: 737-744Crossref PubMed Scopus (333) Google Scholar). TGF-β superfamily members regulate proliferation, differentiation, migration, and apoptosis of many cell types. Signaling occurs via ligand-induced complex formation of two related serine/threonine kinase receptors, i.e. type I and type II receptors (reviewed in Refs. 2Mathews L.S. Endocr. Rev. 1994; 15: 310-325Crossref PubMed Scopus (274) Google Scholar, 3Kingsley D.M. Genes & Dev. 1994; 8: 133-146Crossref PubMed Scopus (1731) Google Scholar, and 5ten Dijke P. Miyazono K. Heldin C.-H. Curr. Opin. Cell Biol. 1996; 8: 139-145Crossref PubMed Scopus (237) Google Scholar, 6Derynck R. Zhang Y. Curr. Biol. 1996; 6: 1226-1229Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 7Massagué J. Cell. 1996; 85: 947-950Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar). Multiple type I and type II receptors have been identified and matched with their corresponding ligands in the TGF-β superfamily. The overall structures of type I and type II receptors are similar and consist of relatively short cysteine-rich extracellular domains, single transmembrane domains, and intracellular parts that consist almost entirely of serine/threonine kinase domains. Phylogenetic analysis of the serine/threonine kinase receptor family reveals that the type I and type II receptors form two distinct subfamilies. Type II receptors, but not type I receptors, have carboxyl-terminal extensions of variable lengths that are rich in serine and threonine residues; whereas type I receptors, but not type II receptors, have a GS domain in the intracellular juxtamembrane region, which is rich in glycine and serine residues. Biochemical as well as genetic approaches have indicated that both type I and type II receptors are essential for signaling. Mink lung epithelial (Mv1Lu) cells lacking functional TβR-I (termed R mutants) or TβR-II (termed DR mutants) fail to respond to TGF-β (8Laiho M. Weis F.M.B. Massagué J. J. Biol. Chem. 1990; 265: 18518-18524Abstract Full Text PDF PubMed Google Scholar). Their responsiveness to TGF-β is restored upon ectopic expression of TβR-I in R mutant and TβR-II in DR mutant cells, but not by other type I or type II receptors (9Wrana J.L. Attisano L. Cárcamo J. Zentella A. Doody J. Laiho M. Wang X.-F. Massagué J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1372) Google Scholar, 10Franzén P. ten Dijke P. Ichijo H. Yamashita H. Schulz P. Heldin C.-H. Miyazono K. Cell. 1993; 75: 681-692Abstract Full Text PDF PubMed Scopus (716) Google Scholar, 11Bassing C.H. Howe D.J. Segarini P.R. Donahoe P.K. Wang X.-F. J. Biol. Chem. 1994; 269: 14861-14864Abstract Full Text PDF PubMed Google Scholar, 12ten Dijke P. Yamashita H. Ichijo H. Franzén P. Laiho M. Miyazono K. Heldin C.-H. 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Previously, we and others have used chimeras of type I and type II receptors to demonstrate that the intracellular domains of type I and type II receptors each serve distinct roles in signaling (39Okadome T. Yamashita H. Franzén P. Morén A. Heldin C.-H. Miyazono K. J. Biol. Chem. 1994; 269: 30753-30756Abstract Full Text PDF PubMed Google Scholar, 40Vivien D. Attisano L. Wrana J.L. Massagué J. J. Biol. Chem. 1995; 270: 7134-7141Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 41Feng X.-H. Filvaroff E.H. Derynck R. J. Biol. Chem. 1995; 270: 24237-24245Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 42Anders R.A. Leof E.B. J. Biol. Chem. 1996; 271: 21758-21766Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 43Muramatsu M. Yan J. Eto K. Tomoda T. Yamada R. Arai K. Mol. Biol. Cell. 1997; 8: 469-480Crossref PubMed Scopus (18) Google Scholar). In the present study, we investigated the functional properties of chimeric receptors in which the intracellular domains of type I or type II receptors for TGF-β were replaced with the corresponding domains of BMPR-IB and ActR-IIB, respectively. Our data indicate that the intracellular domain of TβR-II can be replaced by the intracellular domain of ActR-IIB, which is a receptor for activin as well as for BMP (44Yamashita H. ten Dijke P. Huylebroeck D. Sampath T.K. Andries M. Smith J.C. Heldin C.-H. Miyazono K. J. Cell Biol. 1995; 130: 217-226Crossref PubMed Scopus (462) Google Scholar), with retained ability to activate TβR-I and to induce TGF-β-like responses. In contrast, TβR-I/BMPR-IB was unable to induce any signal in complex with TβR-II, although it was shown to be functional in complex with TβR-II/ActR-IIB in both a transcriptional activation assay and in a Smad phosphorylation assay. This indicates that complex formation is necessary but not sufficient for signal transduction. Moreover, the ligand-induced phosphorylation of endogenous Smad2, but not of endogenous Smad5, by TβR-II/ActR-IIB in complex with TβR-I confirmed the notion that the signal specificity is controlled by the type I receptor. COS-1 cells and Mv1Lu cells were obtained from American Type Culture Collection. Mv1Lu cells that lack functionally active TβR-I (R 4–2 mutant cells) or TβR-II (DR 26 mutant cells) (8Laiho M. Weis F.M.B. Massagué J. J. Biol. Chem. 1990; 265: 18518-18524Abstract Full Text PDF PubMed Google Scholar) were provided by Dr. J. Massagué. Cells were cultured in 5% CO2 at 37 °C in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.) with 10% fetal bovine serum (FBS; Life Technologies, Inc.), 100 units of penicillin, and 50 μg/ml streptomycin. We employed a two-step polymerase chain reaction (PCR) using a Perkin-Elmer thermal cycler with Pyrococcus furiosus DNA polymerase (Stratagene) to generate the chimeric receptors (Fig. 1). cDNAs for human TβR-I (10Franzén P. ten Dijke P. Ichijo H. Yamashita H. Schulz P. Heldin C.-H. Miyazono K. Cell. 1993; 75: 681-692Abstract Full Text PDF PubMed Scopus (716) Google Scholar), mouse BMPR-IB (12ten Dijke P. Yamashita H. Ichijo H. Franzén P. Laiho M. Miyazono K. Heldin C.-H. Science. 1994; 264: 101-104Crossref PubMed Scopus (511) Google Scholar), human TβR-II (13Lin H.Y. Wang X.-F. Ng Eaton E. Weinberg R.A. Lodish H.F. Cell. 1992; 68: 775-785Abstract Full Text PDF PubMed Scopus (969) Google Scholar), and mouse ActR-IIB1 (15Attisano L. Wrana J.L. Cheifetz S. Massagué J. Cell. 1992; 68: 97-108Abstract Full Text PDF PubMed Scopus (459) Google Scholar) were used as templates. For the TβR-I/BMPR-IB chimera, in the first PCR step primer pairs 5a (sense; 5′-GCTCCCGGGGGCGACGGCGTT-3′) and 5b (antisense; 5′-CTTGTGGTGTATAGGACCAAGGCCAGGTGATGA-3′) were used with TβR-I as template, and primer pairs 6a (sense; 5′-GGCCTTGGTCCTATACACCACAAGGCCTTGCT-3′) and 6b (antisense; 5′-CCGAGCTCTGAGACTGCTCGATC-3′) with BMPR-IB as template. Primers 5b and 6a are complementary to each other (underlined sequences). In the second PCR step, the two PCR products were used as template along with the terminal primers 5a and 6b; the chimeric type I receptor PCR product was subcloned, and the DNA was sequenced. Subsequently, aEcoRI-XmaI fragment of TβR-I/pSV7d encoding the N-terminal part of TβR-I, a XmaI-SacI fragment of chimeric type I PCR recombinant, and aSacI-BamHI fragment of BMPR-IB encoding the C-terminal part of BMPR-IB were ligated together by subsequent subcloning steps in pMEP4 (Invitrogen). For the TβR-II/ActR-IIB chimera, in the first PCR step primer pairs 21 (sense; 5′-GCGGAATTCGGGTCTGCCATGGGTCGGGGG-3′) and 22 (antisense; 5′-AGGTTTCCGATGGCGGTAGCAGTAGAAGATGATG-3′) were used with TβR-II as template, and primer pairs 23 (sense; 5′-CTACTGCTACCGCCATCGGAAACCTCCCTACGGC-3′) and 24 (antisense; 5′-CCGAGAGACGAGCTCCCACAG-3′) were used with ActR-IIB as template. Primers 22 and 23 are complementary to each other (underlined sequences), and primer 21 contains an extra 5′-EcoRI restriction site. In the second PCR step, the two PCR products were used as template along with the terminal primers 21 and 24; the chimeric type II receptor PCR product was subcloned, and the DNA was sequenced. Subsequently, the EcoRI-SacI PCR fragment of TβR-II/ActR-IIB encoding the N-terminal part was ligated together with the C-terminal part of ActR-IIB by subsequent subcloning into pMEP4. Wild-type mouse BMPR-IB, human TβR-II, mouse ActR-IIB, and human TβR-I were subcloned in pMEP4 using convenient restriction enzyme cutting sites. Receptor expression thereby came under the transcriptional control of the ZnCl2-inducible human metallothionein promoter. R 4–2 and DR 26 mutant cells were stably transfected by the calcium phosphate precipitation method using the MBS mammalian transfection kit (Stratagene), following the manufacturer's protocol. Selection of transfectants was performed in the presence of 100 units/ml hygromycin (Sigma). We obtained cell pool cultures with similar levels of receptor expression upon ZnCl2treatment. TGF-β1 was iodinated by the chloramine T method (45Frolik 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 and affinity cross-linking using disuccinimidyl suberate (Pierce) were performed as described (10Franzén P. ten Dijke P. Ichijo H. Yamashita H. Schulz P. Heldin C.-H. Miyazono K. Cell. 1993; 75: 681-692Abstract Full Text PDF PubMed Scopus (716) Google Scholar). Lysates were prepared from affinity cross-linked cells and subjected to immunoprecipitation, using antisera against TβR-I (VPN) (10Franzén P. ten Dijke P. Ichijo H. Yamashita H. Schulz P. Heldin C.-H. Miyazono K. Cell. 1993; 75: 681-692Abstract Full Text PDF PubMed Scopus (716) Google Scholar), TβR-II (DRL) (46Yamashita H. ten Dijke P. Franzén P. Miyazono K. Heldin C.-H. J. Biol. Chem. 1994; 269: 20172-20178Abstract Full Text PDF PubMed Google Scholar), ActR-IIB (47Verschueren K. Dewulf N. Goumans M.J. Lonnoy O. Feijen A. Grimsby S. Vande Spiegle K. ten Dijke P. Morén A. Vanscheeuwijck P. Heldin C.- H. Miyazono K. Mummery C. Van Den Eijnden Van Raaij J. Huylebroeck D. Mech. Dev. 1995; 52: 109-123Crossref PubMed Scopus (104) Google Scholar), BMPR-IB (DET) (12ten Dijke P. Yamashita H. Ichijo H. Franzén P. Laiho M. Miyazono K. Heldin C.-H. Science. 1994; 264: 101-104Crossref PubMed Scopus (511) Google Scholar), hemagglutinin epitope (12CA5, Babco; Ref. 48Souchelnytskyi S. ten Dijke P. Miyazono K. Heldin C.-H. EMBO J. 1996; 15: 6231-6240Crossref PubMed Scopus (109) Google Scholar), or His epitope (HSV) (gift from Dr. T. K. Sampath); samples were analyzed by SDS-PAGE using 4–15% gradient gels and visualized using a Fuji-X BioImager. Cells were seeded at a density of 1.5 × 104 cells/well in 24-well plates in DMEM with 10% FBS. After 24 h, cells were washed once, and medium was changed to DMEM with 3% FBS and 100 μmZnCl2, and cells were then incubated with different concentrations of TGF-β1 for 22–24 h; during the last 2 h, cells were labeled with 1 μCi/ml [3H]thymidine (Amersham Corp.). Thereafter, the cells were fixed in 5% ice-cold trichloroacetic acid for 20 min, washed with 5% trichloroacetic acid followed by water and 70% ethanol, and finally solubilized in 0.1m NaOH. 3H radioactivity was measured in a liquid scintillation β-counter using Ecoscint (National Diagnostics). Cells were seeded in six-well plates at a density of 1 × 105 cells/well. After 18–24 h, the medium was changed to DMEM supplemented with 0.1% FBS, with or without 100 μm ZnCl2. After 15 h, the medium was changed to methionine-free MCDB medium (SVA, Sweden) with different concentrations of TGF-β1 and incubation prolonged for 6 h; during the last 2 h, cells were incubated with 25 μCi/ml 35S-labeling mixture "ProMix" (Amersham Corp.). For extracellular matrix isolation, the cells were removed by washing on ice; once in phosphate-buffered saline, three times in 10 mm Tris-HCl, pH 8.0, 0.5% sodium deoxycholate, 1 mm phenylmethylsulfonyl fluoride; two times in 20 mm Tris-HCl, pH 8.0; and once in phosphate-buffered saline. Extracellular matrix proteins were scraped off and extracted into SDS sample buffer containing 10 mm dithiothreitol. Secreted proteins and extracellular matrix proteins were analyzed by SDS-PAGE, followed by fluorography using Amplify (Amersham Corp.) and quantification using a Fuji-X BioImager. PAI-1 was identified as a 45-kDa protein in the extracellular matrix fraction (49Laiho M. Rönnstrand L. Heino J. Decaprio J.A. Ludlow J.W. Livingston D.M. Massagué J. Mol. Cell. Biol. 1991; 11: 972-978Crossref PubMed Google Scholar). Stable transfectants were transiently transfected with p3TP-Lux (28Cárcamo J. Zentella A. Massagué J. Mol. Cell. Biol. 1995; 15: 1573-1581Crossref PubMed Google Scholar), as described above. The following day, cells were washed extensively with phosphate-buffered saline to remove calcium phosphate precipitates. Subsequently, the cells were incubated in DMEM with 10% FBS for 16–20 h. Thereafter, the receptor expression was induced by treatment of the cells with 100 μm ZnCl2 in DMEM supplemented with 0.1% FBS for 5 h, after which TGF-β1 was added. Luciferase activity in cell lysate was measured after 22–24 h using the luciferase assay system (Promega Biotec), according to the manufacturer's protocol using an LKB Luminometer (LKB-Bromma). TGF-β1-dependent phosphorylation of endogenous Smad2 and Smad5 was analyzed using R 4–2 mutant cells stably transfected with TβR-I or TβR-I/BMPR-IB and DR 26 mutant cells stably transfected with TβR-II or TβR-II/ActR-IIB. Cells were labeled for 3 h in phosphate-free medium supplemented with 0.7 mCi/ml [32P]orthophosphate. Cells were incubated in the absence or presence of TGF-β1, lysed, and subjected to immunoprecipitation with antiserum against Smad2 (SED) (37Nakao A. Röijer E. Imamura T. Souchelnytskyi S. Stenman G. Heldin C.-H. ten Dijke P. J. Biol. Chem. 1997; 272: 2896-2900Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) or Smad5 (SSN) (gift from Dr. K. Tamaki). The precipitates were analyzed by SDS-PAGE followed by autoradiography. In addition, COS-1 cells were transiently transfected, as described above, with TβR-I/BMPR-IB, TβR-II/ActR-IIB, and Smad1 or Smad2 expression constructs (37Nakao A. Röijer E. Imamura T. Souchelnytskyi S. Stenman G. Heldin C.-H. ten Dijke P. J. Biol. Chem. 1997; 272: 2896-2900Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Cells were then treated in the same way as the stable transfectants and immunoprecipitated with antiserum against Smad1 (QWL) (gift from Dr. A. Nakao) or Smad2 (SED). The precipitates were subjected to SDS-PAGE and autoradiography. Two chimeric receptors were constructed, i.e. TβR-I/BMPR-IB, containing the extracellular domain of TβR-I and intracellular domain of BMPR-IB, and TβR-II/ActR-IIB, containing the extracellular domain of TβR-II and the intracellular domain of ActR-IIB (Fig. 1). To investigate the125I-TGF-β1 binding properties of the chimeric receptors, we used Mv1Lu cell lines that lack functional TβR-I (R 4–2 mutant cells) or TβR-II (DR 26 mutant cells) as host cells for transfection. TβR-I/BMPR-IB was stably transfected into R 4–2 mutant cells, along with TβR-I and BMPR-IB as positive and negative controls, respectively. TβR-II/ActR-IIB was stably transfected in DR 26 mutant cells, along with TβR-II and ActR-IIB as positive and negative controls, respectively. All receptor expression constructs were placed under the transcriptional control of the metallothionein promoter, which can be induced by ZnCl2. Expression of type I receptors in stable transfectants was analyzed by metabolic labeling using specific antibodies, directed against the intracellular domains of the receptors. For all transfectants, proteins with expected molecular weights were observed upon the addition of 100 μm ZnCl2 (Fig. 2 A). As observed before, due to the leaky character of the metallothionein promoter, the receptors were also expressed, but at lower levels, in the absence of ZnCl2 treatment. We observed no co-immunoprecipitation of endogenous type II receptors with anti-type I antibodies, suggesting that there was no ligand-independent type I-type II complex formed as a result of overexpression (Fig. 2 A). Expression of type II receptors could not be analyzed by metabolic labeling due to the weak affinity of the antiserum. Affinity cross-linking with 125I-TGF-β1 of TβR-I/BMPR-IB cells revealed that TβR-I/BMPR-IB bound125I-TGF-β1 with similar efficiency as TβR-I. Immunoprecipitation with anti-BMPR-IB antisera not only brought down the TβR-I/BMPR-IB, but also TβR-II, illustrating that TβR-II formed a complex with TβR-I/BMPR-IB (Fig. 2 B). We were not able to detect neither TβR-II nor TβR-I/BMPR-IB with TβR-II antiserum. The reason for this is unclear, since TβR-I/BMPR-IB is most likely able to bind TGF-β only in the presence of TβR-II. As expected, in TβR-I-transfected cells cross-linked TβR complexes could be demonstrated by immunoprecipitation using antisera against TβR-I or TβR-II, albeit with less efficiency of complex precipitation with TβR-II antiserum. In nontransfected R 4–2 mutant cells, no type I receptor cross-linked complex was immunoprecipitated with anti-TβR-I or anti-BMPR-IB antisera (Ref. 10Franzén P. ten Dijke P. Ichijo H. Yamashita H. Schulz P. Heldin C.-H. Miyazono K. Cell. 1993; 75: 681-692Abstract Full Text PDF PubMed Scopus (716) Google Scholar and data not shown). Although we have reported that BMPR-IB can bind TGF-β1 when overexpressed in COS-1 cells (12ten Dijke P. Yamashita H. Ichijo H. Franzén P. Laiho M. Miyazono K. Heldin C.-H. Science. 1994; 264: 101-104Crossref PubMed Scopus (511) Google Scholar), the expression levels in transfected Mv1Lu cells were too low for this interaction to occur (data not shown). Affinity cross-linking with 125I-TGF-β1 of cells transfected with TβR-II/ActR-IIB or TβR-II revealed that TβR-II/ActR-IIB bound 125I-TGF-β1 equally efficient as TβR-II upon induction of their expression with ZnCl2treatment. TβR-II/ActR-IIB, like TβR-II, rescued the binding to endogenously expressed TβR-I (Fig. 2 C). In TβR-II/ActR-IIB cells, cross-linked complexes of TβR-II/ActR-IIB and TβR-I were immunoprecipitated with antisera against either ActR-IIB or TβR-I, indicating that both receptors are part of a common TGF-β-induced receptor complex (Fig. 2 C). In the nontransfected DR mutant cells, no type II cross-linked complex could be immunoprecipitated with antisera against TβR-II or ActR-IIB (data not shown). As expected, no binding of TGF-β1 to cells stably transfected with ActR-IIB was observed (data not shown). Affinity cross-linking with 125I-BMP-7/OP-1 of cells transfected with BMPR-IB or ActR-IIB revealed that both receptors were expressed and able to bind ligand upon ZnCl2 treatment (Fig. 2 D). The signaling activity of the chimeric receptors was investigated using the p3TP-Lux transcriptional activation assay, which scores positive after stimulation with TGF-β (9Wrana J.L. Attisano L. Cárcamo J. Zentella A. Doody J. Laiho M. Wang X.-F. Massagué J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1372) Google Scholar) and, albeit less efficiently, after BMP or activin stimulation. Previously, we have shown that ActR-IIs and BMPR-IB can form a BMP-7/OP-1-induced heteromeric complex, which can mediate a p3TP-Lux signal (16Rosenzweig B.L. Imamura T. Okadome T. Cox G.N. Yamashita H. ten Dijke P. Heldin C.-H. Miyazono K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7632-7636Crossref PubMed Scopus (478) Google Scholar). No transcriptional response could be observed after TGF-β1 stimulation when the reporter plasmid was transfected alone into COS-1 cells (Fig. 3 A). However, when we co-transfected TβR-I/BMPR-IB together with TβR-II/ActR-IIB and p3TP-Lux into COS-1 cells, we observed a TGF-β-dependent signal (Fig. 3 A). To elucidate the ability of the complex between TβR-I/BMPR-IB and TβR-II/ActR-IIB to induce Smad phosphorylation, COS-1 cells were transiently co-transfected with TβR-I/BMPR-IB and TβR-II/ActR-IIB together with Smad1 or Smad2. TGF-β1 induced the phosphorylation of Smad1, but not of Smad2 (Fig. 3 B). This indicates that TβR-I/BMPR-IB and TβR-II/ActR-IIB are functionally intact and can form a TGF-β-mediated complex, which signals a BMP-like response. We then investigated whether TβR-I/BMPR-IB and TβR-II/ActR-IIB could substitute for TβR-I and TβR-II, respectively, using TGF-β-induced plasminogen activator inhibitor-1 (PAI-1) production as an assay. Wild-type Mv1Lu cells responded to TGF-β1 by producing a 45-kDa PAI-1 protein, whereas the R 4–2 mutant and DR 26 mutant cell lines did not produce PAI-1 after stimulation by TGF-β1 (data not shown). Sometimes the PAI-1 protein appeared as two discrete bands of 45 and 43 kDa, of which the latter is likely to be a proteolytic breakdown product of the 45-kDa protein. Stably transfected TβR-I/BMPR-IB R 4–2 mutant cells did not produce the PAI-1 protein upon induction of receptor expression by ZnCl2 and TGF-β1 stimulation (Fig. 4 A). TGF-β1 stimulation of the R 4–2 mutant cells transfected with TβR-I, but not BMPR-IB, led to a 2.5-fold increase, determined by densitometric scanning, in the production of PAI-1 in response to ZnCl2 treatment, as reported before (10Franzén P. ten Dijke P. Ichijo H. Yamashita H. Schulz P. Heldin C.-H. Miyazono K. Cell. 1993; 75: 681-692Abstract Full Text PDF PubMed Scopus (716) Google Scholar, 12ten Dijke P. Yamashita H. Ichijo H. Franzén P. Laiho M. Miyazono K. Heldin C.-H. Science. 1994; 264: 101-104Crossref PubMed Scopus (511) Google Scholar). However, TβR-II/ActR-IIB restored the TGF-β-mediated PAI-1 response in DR 26 mutant cells to a similar extent as TβR-II, i.e. 5- and 6-fold increases, respectively (Fig. 4 B). ActR-IIB, which cannot bind TGF-β1, was unable to complement the defect in DR 26 mutant cells (Fig. 4 B). In ActR-IIB-transfected cells treated with ZnCl2, a weak 45-kDa protein was observed. However, the production of this protein was not induced upon TGF-β1 stimulation. In addition, we measured the ability of both chimeras to mediate growth-inhibitory responses upon stimulation with TGF-β1. Similar to the p3TP-Lux and PAI-1 assays, TβR-II/ActR-IIB was able to replace TβR-II, but TβR-I/BMPR-IB was not able to replace TβR-I, with respect to the antiproliferative response upon TGF-β1 stimulation (Fig. 5). In a few experiments, we observed that the degree of growth inhibition was less with TβR-II/ActR-IIB compared with TβR-II. Different Smads have been shown to act downstream of BMP and TGF-β receptors (34Hoodless P.A. Haerry T. Abdollah S. Stapleton M. O'Connor M.B. Attisano L. Wrana J.L. Cell. 1996; 85: 489-500Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 37Nakao A. Röijer E. Imamura T. Souchelnytskyi S. Stenman G. Heldin C.-H. ten Dijke P. J. Biol. Chem. 1997; 272: 2896-2900Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Thus, we used differential activation of Smad2 or Smad5 to distinguish TGF-β versus BMP receptor-mediated signaling, respectively. Using the stably transfected cell lines, the effect of ligand-induced complex formation between TβR-II and TβR-I/BMPR-IB, or between TβR-II/ActR-IIB and TβR-I, on endogenous Smad2 or Smad5 phosphorylation was analyzed (Fig. 6). Phosphorylation of Smad2, but not Smad5, was induced by the activated complex between TβR-II/ActR-IIB and TβR-I, whereas ligand-induced complex formation between TβR-I/BMPR-IB and TβR-II induced no Smad2 or Smad5 phosphorylation. BMP-7/OP-1 was found to induce the phosphorylation of Smad5 in Mv1Lu cells; phosphorylated Smad2 and Smad5 were found to co-migrate on SDS-PAGE.2 Smad5 antibody also brought down a phosphoprotein larger than Smad5, the identity of which is unknown. Thus, our results indicate that complex formation between the intracellular domains of ActR-IIB and TβR-I leads to the transduction of TGF-β-like signals, whereas complex formation between the intracellular domains of TβR-II and BMPR-IB does not lead to any measurable signaling event (Fig. 7). TGF-β superfamily members exert their cellular effects through formation of hetero-oligomeric complexes of type I and type II serine/threonine kinase receptors. Previous reports have shown that the type II receptor has a constitutively active kinase domain (20Wrana J.L. Attisano L. Wieser R. Ventura F. Massagué J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2120) Google Scholar), and that phosphorylation and activation of the type I receptor is sufficient and necessary for downstream signaling activities (25Wieser R. Wrana J.L. Massagué J. EMBO J. 1995; 14: 2199-2208Crossref PubMed Scopus (596) Google Scholar, 26Franzén P. Heldin C.-H. Miyazono K. Biochem. Biophys. Res. Commun. 1995; 207: 682-689Crossref PubMed Scopus (40) Google Scholar). Here we have characterized the properties of two chimeric receptors in which the intracellular domains of different type I or type II receptors within the TGF-β superfamily were exchanged. The purpose was to investigate whether the intracellular domains of TGF-β type I and type II receptors could be replaced by the corresponding domains of BMPR-IB and ActR-IIB, respectively. The presented data show that the intracellular domain of ActR-IIB can functionally substitute for the intracellular domain of TβR-II with respect to the induction of a transcriptional activation signal, stimulation of PAI-1 production, growth inhibition, and Smad2 activation. In contrast, a complex between the intracellular domains of BMPR-IB and TβR-II induced no observable signal (Fig. 7). TβR-II/ActR-IIB can complement the lack of TβR-II in DR 26 mutant cells. The intracellular domain of ActR-IIB shares 33.3% sequence identity with TβR-II and is thus apparently sufficiently similar to phosphorylate and transactivate TβR-I in a similar way as TβR-II. Previously, we and others have shown that the intracellular domain of TβR-I cannot replace the intracellular domains of TβR-II in this respect (39Okadome T. Yamashita H. Franzén P. Morén A. Heldin C.-H. Miyazono K. J. Biol. Chem. 1994; 269: 30753-30756Abstract Full Text PDF PubMed Google Scholar, 40Vivien D. Attisano L. Wrana J.L. Massagué J. J. Biol. Chem. 1995; 270: 7134-7141Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 41Feng X.-H. Filvaroff E.H. Derynck R. J. Biol. Chem. 1995; 270: 24237-24245Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 42Anders R.A. Leof E.B. J. Biol. Chem. 1996; 271: 21758-21766Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Our findings are in agreement with those of Muramatsuet al. (43Muramatsu M. Yan J. Eto K. Tomoda T. Yamada R. Arai K. Mol. Biol. Cell. 1997; 8: 469-480Crossref PubMed Scopus (18) Google Scholar), who reported the signaling activities of chimeric human granulocyte-macrophage colony-stimulating factor receptor/ActR-II and human granulocyte-macrophage colony-stimulating factor receptor/BMPR-II. The failure of TβR-I/BMPR-IB to signal in combination with TβR-II is unlikely to be due to a perturbation of the conformation of TβR-II extracellular domain or BMPR-IB intracellular domain, since TβR-I/BMPR-IB bound TGF-β and formed a heteromeric complex with TβR-II and since TβR-I/BMPR-IB together with TβR-II/ActR-IIB transduced a TGF-β-dependent transcriptional activation signal and induced Smad1 phosphorylation, a BMP-like signal. It is possible that the inability of TβR-I/BMPR-IB to mediate a growth-inhibitory response may be in an inherent property of the receptor, since Mv1Lu cells, which express TβR-II, TβR-I, ActR-II, and BMPR-IB among other receptors, are at least 100-fold more sensitive to TGF-β1 than BMP-7/OP-1 with respect to growth inhibition. On the other hand, TGF-β-induced complex formation between TβR-II and TβR-I/BMPR-IB did not lead to Smad2 or Smad5 phosphorylation, suggesting that this is an inactive complex. Phosphorylation of endogenous Smad2 was stimulated by TGF-β-induced complex formation between TβR-I and TβR-II/ActR-IIB, indicating that type I receptor is the determinant for signal specificity. Our future studies will be aimed at elucidating which minimal specific regions/residues in the intracellular domain of TβR-I need to be substituted for analogous regions/residues in the intracellular domain of BMPR-IB to allow for a TβR-I/BMPR-IB chimera to transduce signals when activated by TβR-II. We thank Dr. Atsuhito Nakao for Smad1 and Smad2 expression constructs and antisera against Smad1 and Smad2, Dr. Kiyoshi Tamaki for antiserum against Smad5, Christer Wernstedt for preparing oligonucleotides, Hideya Ohashi for recombinant TGF-β1, Dr. T. K. Sampath for BMP-7/OP-1 and antiserum against His epitope, Kristin Verschueren for an antiserum against ActR-II intracellular domain, and Joan Massagué for Mv1Lu mutant cell lines, ActR-IIB1 cDNA, and p3TP-Lux plasmid.