Transforming Growth Factor (TGF-beta )-specific Signaling by Chimeric TGF-beta Type II Receptor with Intracellular Domain of Activin Type IIB Receptor*

(Received for publication, April 9, 1997, and in revised form, June 12, 1997)

Urban Persson Dagger , Serhiy Souchelnytskyi , Petra Franzén , Kohei Miyazono §, Peter ten Dijke and Carl-Henrik Heldin

From the Ludwig Institute for Cancer Research, Box 595, S-751 24 Uppsala, Sweden and § The Cancer Institute, Tokyo, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Members of the transforming growth factor-beta (TGF-beta ) 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-beta superfamily receptors, i.e. Tbeta R-I/BMPR-IB, containing the extracellular domain of TGF-beta type I receptor (Tbeta R-I) and the intracellular domain of bone morphogenetic protein type IB receptor (BMPR-IB), and Tbeta R-II/ActR-IIB, containing the extracellular domain of TGF-beta type II receptor (Tbeta R-II) and the intracellular domain of activin type IIB receptor (ActR-IIB). In the presence of TGF-beta 1, Tbeta R-I/BMPR-IB and Tbeta R-II/ActR-IIB formed heteromeric complexes with wild-type Tbeta R-II and Tbeta R-I, respectively, upon stable transfection in mink lung epithelial cell lines. We show that Tbeta R-II/ActR-IIB restored the responsiveness upon transfection in mutant cell lines lacking functional Tbeta R-II with respect to TGF-beta -mediated activation of a transcriptional signal, extracellular matrix formation, growth inhibition, and Smad phosphorylation. Moreover, Tbeta R-I/BMPR-IB and Tbeta R-II/ActR-IIB formed a functional complex in response to TGF-beta and induced phosphorylation of Smad1. However, complex formation is not enough for signal propagation, which is shown by the inability of Tbeta R-I/BMPR-IB to restore responsiveness to TGF-beta in cell lines deficient in functional Tbeta R-I. The fact that the TGF-beta 1-induced complex between Tbeta R-II/ActR-IIB and Tbeta R-I stimulated endogenous Smad2 phosphorylation, a TGF-beta -like response, is in agreement with the current model for receptor activation in which the type I receptor determines signal specificity.


INTRODUCTION

Transforming growth factor-beta s (TGF-beta s),1 activins, and bone morphogenetic proteins (BMPs) are structurally related proteins that play important roles in intercellular communication (reviewed in Refs. 1-4). TGF-beta 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. 2, 3, and 5-7). Multiple type I and type II receptors have been identified and matched with their corresponding ligands in the TGF-beta 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 Tbeta R-I (termed R mutants) or Tbeta R-II (termed DR mutants) fail to respond to TGF-beta (8). Their responsiveness to TGF-beta is restored upon ectopic expression of Tbeta R-I in R mutant and Tbeta R-II in DR mutant cells, but not by other type I or type II receptors (9-12). Whereas the type II receptors for TGF-beta and activin can bind ligand by themselves (13-15), type I receptors need type II receptors for ligand binding (9). In contrast, BMPs show weak binding to type II as well as to type I receptors individually, and high affinity binding is found when both receptors are present (16-18). However, similar to the case for TGF-beta and activin, a BMP-induced transcriptional activation signal requires cotransfection of both type I and type II receptors, and signal transduction is not observed when one receptor type is singly transfected into Mv1Lu cells (16, 17). Genetic analysis in Drosophila also indicates that both type I and type II receptors are essential for signaling by decapentaplegic, a BMP-like factor (19).

Recent studies have led to the formulation of a common model for signal transduction via serine/threonine kinase receptors (20). TGF-beta type II receptor (Tbeta R-II) and activin type II receptors (ActR-IIs) are constitutively active kinases (20-23). The type I receptor is recruited into the complex upon ligand binding to the type II receptor; the type I receptor is thereafter phosphorylated by the type II receptor, predominantly on serine and threonine residues in the GS domain (20, 24). The importance of Tbeta R-I phosphorylation in signaling was shown by the impairment of TGF-beta signaling when multiple serine and threonine residues in the GS domain of Tbeta R-I were mutated (25, 26), by activation-defective mutants of Tbeta R-I (27), and by an activation-defective Tbeta R-II mutant that fails to recognize Tbeta R-I as a substrate (28). The notion that type I receptors act downstream of type II receptors is supported by the ability of type I receptors to determine distinct signaling responses (29, 30) and by constitutively active mutants of Tbeta R-I and ActR-IB to signal in the absence of ligand and type II receptor (22, 24, 25).

Members of the Smad family of proteins have been shown to play a key role in the intracellular signaling pathways of TGF-beta superfamily members. After activation by serine/threonine kinase receptors, Smads proteins become phosphorylated and translocate to the nucleus, where they may play a role in the transcriptional regulation (31-35). Smad1 and Smad5 are phosphorylated and translocated into the nucleus upon BMP receptor activation (34, 35),2 whereas TGF-beta and activin induce the phosphorylation and translocation of both Smad2 and Smad3 (36-38).3

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 (39-43). 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-beta were replaced with the corresponding domains of BMPR-IB and ActR-IIB, respectively. Our data indicate that the intracellular domain of Tbeta R-II can be replaced by the intracellular domain of ActR-IIB, which is a receptor for activin as well as for BMP (44), with retained ability to activate Tbeta R-I and to induce TGF-beta -like responses. In contrast, Tbeta R-I/BMPR-IB was unable to induce any signal in complex with Tbeta R-II, although it was shown to be functional in complex with Tbeta R-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 Tbeta R-II/ActR-IIB in complex with Tbeta R-I confirmed the notion that the signal specificity is controlled by the type I receptor.


EXPERIMENTAL PROCEDURES

Cell Lines

COS-1 cells and Mv1Lu cells were obtained from American Type Culture Collection. Mv1Lu cells that lack functionally active Tbeta R-I (R 4-2 mutant cells) or Tbeta R-II (DR 26 mutant cells) (8) 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.

Construction of Chimeric and Wild-type Receptors

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 Tbeta R-I (10), mouse BMPR-IB (12), human Tbeta R-II (13), and mouse ActR-IIB1 (15) were used as templates. For the Tbeta R-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 Tbeta R-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, a EcoRI-XmaI fragment of Tbeta R-I/pSV7d encoding the N-terminal part of Tbeta R-I, a XmaI-SacI fragment of chimeric type I PCR recombinant, and a SacI-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 Tbeta R-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 Tbeta R-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 Tbeta R-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 Tbeta R-II, mouse ActR-IIB, and human Tbeta R-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.


Fig. 1. Schematic representation of the chimeric receptors used in the present study. Extracellular domains, transmembrane regions, and intracellular domains are indicated. Tbeta R-II and ActR-IIB share 33.3% sequence identity, and Tbeta R-I and BMPR-IB share 59.8% sequence identity in their intracellular domains. aa, amino acids.
[View Larger Version of this Image (19K GIF file)]

Transfection

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 ZnCl2 treatment.

Binding, Affinity Cross-linking, and Immunoprecipitation

TGF-beta 1 was iodinated by the chloramine T method (45). Binding and affinity cross-linking using disuccinimidyl suberate (Pierce) were performed as described (10). Lysates were prepared from affinity cross-linked cells and subjected to immunoprecipitation, using antisera against Tbeta R-I (VPN) (10), Tbeta R-II (DRL) (46), ActR-IIB (47), BMPR-IB (DET) (12), hemagglutinin epitope (12CA5, Babco; Ref. 48), 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.

Growth Inhibition Assay

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 µM ZnCl2, and cells were then incubated with different concentrations of TGF-beta 1 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 0.1 M NaOH. 3H radioactivity was measured in a liquid scintillation beta -counter using Ecoscint (National Diagnostics).

Extracellular Matrix Formation Assay

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-beta 1 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 (49).

Transcriptional Response Assay

Stable transfectants were transiently transfected with p3TP-Lux (28), 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-beta 1 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).

[32P]Orthophosphate Labeling of Cells

TGF-beta 1-dependent phosphorylation of endogenous Smad2 and Smad5 was analyzed using R 4-2 mutant cells stably transfected with Tbeta R-I or Tbeta R-I/BMPR-IB and DR 26 mutant cells stably transfected with Tbeta R-II or Tbeta R-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-beta 1, lysed, and subjected to immunoprecipitation with antiserum against Smad2 (SED) (37) 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 Tbeta R-I/BMPR-IB, Tbeta R-II/ActR-IIB, and Smad1 or Smad2 expression constructs (37). 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.


RESULTS

Ligand Binding Properties of Chimeric Receptors

Two chimeric receptors were constructed, i.e. Tbeta R-I/BMPR-IB, containing the extracellular domain of Tbeta R-I and intracellular domain of BMPR-IB, and Tbeta R-II/ActR-IIB, containing the extracellular domain of Tbeta R-II and the intracellular domain of ActR-IIB (Fig. 1). To investigate the 125I-TGF-beta 1 binding properties of the chimeric receptors, we used Mv1Lu cell lines that lack functional Tbeta R-I (R 4-2 mutant cells) or Tbeta R-II (DR 26 mutant cells) as host cells for transfection. Tbeta R-I/BMPR-IB was stably transfected into R 4-2 mutant cells, along with Tbeta R-I and BMPR-IB as positive and negative controls, respectively. Tbeta R-II/ActR-IIB was stably transfected in DR 26 mutant cells, along with Tbeta R-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. 2A). 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. 2A). Expression of type II receptors could not be analyzed by metabolic labeling due to the weak affinity of the antiserum.


Fig. 2. Expression and ligand binding to wild-type and chimeric receptors. A, R 4-2 mutant cells were stably transfected with wild-type Tbeta R-I (hemagglutinin-tagged), Tbeta R-I/BMPR-IB, and BMPR-IB in pMEP4. Cell pools were 35S-labeled, treated with 100 µM ZnCl2, and immunoprecipitated with anti-Tbeta R-I or anti-BMPR-IB antibodies. Immunoprecipitates were resolved by SDS-PAGE followed by fluorography. The binding of 125I-TGF-beta 1 to Tbeta R-I and Tbeta R-I/BMPR-IB in stably transfected R 4-2 mutant cells (B) and to Tbeta R-II and Tbeta R-II/ActR-IIB in stably transfected DR 26 mutant cells (C) and the binding of 125I-BMP-7/OP-1 to ActR-IIB and BMPR-IB in stably transfected DR 26 mutant cells and R 4-2 mutant cells (D), respectively, were analyzed (without and with ZnCl2 treatment) by affinity cross-linking and subsequent immunoprecipitation using the indicated antisera followed by SDS-PAGE and visualization using a Fuji-X BioImager. I and II indicate the position of type I and type II receptors, respectively.
[View Larger Version of this Image (85K GIF file)]

Affinity cross-linking with 125I-TGF-beta 1 of Tbeta R-I/BMPR-IB cells revealed that Tbeta R-I/BMPR-IB bound 125I-TGF-beta 1 with similar efficiency as Tbeta R-I. Immunoprecipitation with anti-BMPR-IB antisera not only brought down the Tbeta R-I/BMPR-IB, but also Tbeta R-II, illustrating that Tbeta R-II formed a complex with Tbeta R-I/BMPR-IB (Fig. 2B). We were not able to detect neither Tbeta R-II nor Tbeta R-I/BMPR-IB with Tbeta R-II antiserum. The reason for this is unclear, since Tbeta R-I/BMPR-IB is most likely able to bind TGF-beta only in the presence of Tbeta R-II. As expected, in Tbeta R-I-transfected cells cross-linked Tbeta R complexes could be demonstrated by immunoprecipitation using antisera against Tbeta R-I or Tbeta R-II, albeit with less efficiency of complex precipitation with Tbeta R-II antiserum. In nontransfected R 4-2 mutant cells, no type I receptor cross-linked complex was immunoprecipitated with anti-Tbeta R-I or anti-BMPR-IB antisera (Ref. 10 and data not shown). Although we have reported that BMPR-IB can bind TGF-beta 1 when overexpressed in COS-1 cells (12), the expression levels in transfected Mv1Lu cells were too low for this interaction to occur (data not shown).

Affinity cross-linking with 125I-TGF-beta 1 of cells transfected with Tbeta R-II/ActR-IIB or Tbeta R-II revealed that Tbeta R-II/ActR-IIB bound 125I-TGF-beta 1 equally efficient as Tbeta R-II upon induction of their expression with ZnCl2 treatment. Tbeta R-II/ActR-IIB, like Tbeta R-II, rescued the binding to endogenously expressed Tbeta R-I (Fig. 2C). In Tbeta R-II/ActR-IIB cells, cross-linked complexes of Tbeta R-II/ActR-IIB and Tbeta R-I were immunoprecipitated with antisera against either ActR-IIB or Tbeta R-I, indicating that both receptors are part of a common TGF-beta -induced receptor complex (Fig. 2C). In the nontransfected DR mutant cells, no type II cross-linked complex could be immunoprecipitated with antisera against Tbeta R-II or ActR-IIB (data not shown). As expected, no binding of TGF-beta 1 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. 2D).

Signaling Properties of Chimeric Receptors

The signaling activity of the chimeric receptors was investigated using the p3TP-Lux transcriptional activation assay, which scores positive after stimulation with TGF-beta (9) 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 (16). No transcriptional response could be observed after TGF-beta 1 stimulation when the reporter plasmid was transfected alone into COS-1 cells (Fig. 3A). However, when we co-transfected Tbeta R-I/BMPR-IB together with Tbeta R-II/ActR-IIB and p3TP-Lux into COS-1 cells, we observed a TGF-beta -dependent signal (Fig. 3A). To elucidate the ability of the complex between Tbeta R-I/BMPR-IB and Tbeta R-II/ActR-IIB to induce Smad phosphorylation, COS-1 cells were transiently co-transfected with Tbeta R-I/BMPR-IB and Tbeta R-II/ActR-IIB together with Smad1 or Smad2. TGF-beta 1 induced the phosphorylation of Smad1, but not of Smad2 (Fig. 3B). This indicates that Tbeta R-I/BMPR-IB and Tbeta R-II/ActR-IIB are functionally intact and can form a TGF-beta -mediated complex, which signals a BMP-like response.


Fig. 3. Signaling activity of co-transfected chimeric receptors. A, Tbeta R-I/BMPR-IB and Tbeta R-II/ActR-IIB were transiently transfected with p3TP-Lux reporter plasmid into COS-1 cells. Thirty-six h after transfection, cells were treated with TGF-beta 1 for 16 h at the indicated concentrations. The transcriptional response was determined by measuring the luciferase activity. B, COS-1 cells transiently transfected with Smad1 or Smad2 together with Tbeta R-I/BMPR-IB and Tbeta R-II/ActR-IIB were labeled with [32P]orthophosphate and treated where indicated with 10 ng/ml TGF-beta 1. Cells were lysed and immunoprecipitated with indicated antiserum and subjected to SDS-PAGE, followed by autoradiography. The arrow indicates the position of Smad1 or Smad2.
[View Larger Version of this Image (34K GIF file)]

We then investigated whether Tbeta R-I/BMPR-IB and Tbeta R-II/ActR-IIB could substitute for Tbeta R-I and Tbeta R-II, respectively, using TGF-beta -induced plasminogen activator inhibitor-1 (PAI-1) production as an assay. Wild-type Mv1Lu cells responded to TGF-beta 1 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-beta 1 (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 Tbeta R-I/BMPR-IB R 4-2 mutant cells did not produce the PAI-1 protein upon induction of receptor expression by ZnCl2 and TGF-beta 1 stimulation (Fig. 4A). TGF-beta 1 stimulation of the R 4-2 mutant cells transfected with Tbeta R-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 (10, 12). However, Tbeta R-II/ActR-IIB restored the TGF-beta -mediated PAI-1 response in DR 26 mutant cells to a similar extent as Tbeta R-II, i.e. 5- and 6-fold increases, respectively (Fig. 4B). ActR-IIB, which cannot bind TGF-beta 1, was unable to complement the defect in DR 26 mutant cells (Fig. 4B). 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-beta 1 stimulation.


Fig. 4. Signaling activity of wild-type and chimeric receptors using a PAI-1 assay. R 4-2 mutant cells stably transfected with expression constructs for wild-type Tbeta R-I, wild-type BMPR-IB, or Tbeta R-I/BMPR-IB (A) and DR 26 mutant cells stably transfected with wild-type Tbeta R-II, wild-type ActR-IIB, or Tbeta R-II/ActR-IIB (B) were analyzed with respect to induction of PAI-1 protein levels upon TGF-beta 1 stimulation in the absence or presence of ZnCl2 treatment. Cells were 35S-labeled with "Promix" for the last 2 h of incubation, and production of 45 kDa PAI-1 protein in extracellular matrix was analyzed by SDS-PAGE followed by fluorography and quantification using a Fuji-X BioImager.
[View Larger Version of this Image (91K GIF file)]

In addition, we measured the ability of both chimeras to mediate growth-inhibitory responses upon stimulation with TGF-beta 1. Similar to the p3TP-Lux and PAI-1 assays, Tbeta R-II/ActR-IIB was able to replace Tbeta R-II, but Tbeta R-I/BMPR-IB was not able to replace Tbeta R-I, with respect to the antiproliferative response upon TGF-beta 1 stimulation (Fig. 5). In a few experiments, we observed that the degree of growth inhibition was less with Tbeta R-II/ActR-IIB compared with Tbeta R-II.


Fig. 5. Signaling activity of wild-type and chimeric receptors using a growth inhibition assay. Untransfected R 4-2 mutant cells (bullet ------bullet ) or R 4-2 mutant cells stably transfected with expression constructs for wild-type Tbeta R-I (open circle ------open circle ), wild-type BMPR-IB (black-diamond ------black-diamond ), or Tbeta R-I/BMPR-IB (diamond ------diamond ) (A) and untransfected DR 26 mutant cells (bullet ------bullet ) or DR 26 mutant cells stably transfected with wild-type Tbeta R-II (open circle ------open circle ), wild-type ActR-IIB (black-diamond ------black-diamond ), or Tbeta R-II/ActR-IIB (diamond ------diamond ) (B) were subjected to a [3H]thymidine incorporation assay in the absence or presence of different concentrations of TGF-beta 1. Receptor expression was induced by ZnCl2 treatment of the cells. The percentage of growth inhibition was calculated by taking as 100% the [3H]thymidine incorporation into cells in the absence of TGF-beta 1.
[View Larger Version of this Image (14K GIF file)]

Different Smads have been shown to act downstream of BMP and TGF-beta receptors (34, 37). Thus, we used differential activation of Smad2 or Smad5 to distinguish TGF-beta versus BMP receptor-mediated signaling, respectively. Using the stably transfected cell lines, the effect of ligand-induced complex formation between Tbeta R-II and Tbeta R-I/BMPR-IB, or between Tbeta R-II/ActR-IIB and Tbeta R-I, on endogenous Smad2 or Smad5 phosphorylation was analyzed (Fig. 6). Phosphorylation of Smad2, but not Smad5, was induced by the activated complex between Tbeta R-II/ActR-IIB and Tbeta R-I, whereas ligand-induced complex formation between Tbeta R-I/BMPR-IB and Tbeta R-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.


Fig. 6. Smad phosphorylation by wild-type TGF-beta and chimeric receptors. Stably transfected cells were labeled with [32P]orthophosphate and treated where indicated with 10 ng/ml TGF-beta 1. Cell lysates were subjected to immunoprecipitation with indicated antiserum, and the immunoprecipitates were analyzed by SDS-PAGE and autoradiography. The arrow indicates the position of Smad2 or Smad5.
[View Larger Version of this Image (38K GIF file)]

Thus, our results indicate that complex formation between the intracellular domains of ActR-IIB and Tbeta R-I leads to the transduction of TGF-beta -like signals, whereas complex formation between the intracellular domains of Tbeta R-II and BMPR-IB does not lead to any measurable signaling event (Fig. 7).


Fig. 7. Schematic illustration of the signaling responses by the wild-type and chimeric receptors. For an explanation, see "Discussion."
[View Larger Version of this Image (27K GIF file)]


DISCUSSION

TGF-beta 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 (20), and that phosphorylation and activation of the type I receptor is sufficient and necessary for downstream signaling activities (25, 26). 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-beta superfamily were exchanged. The purpose was to investigate whether the intracellular domains of TGF-beta 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 Tbeta R-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 Tbeta R-II induced no observable signal (Fig. 7).

Tbeta R-II/ActR-IIB can complement the lack of Tbeta R-II in DR 26 mutant cells. The intracellular domain of ActR-IIB shares 33.3% sequence identity with Tbeta R-II and is thus apparently sufficiently similar to phosphorylate and transactivate Tbeta R-I in a similar way as Tbeta R-II. Previously, we and others have shown that the intracellular domain of Tbeta R-I cannot replace the intracellular domains of Tbeta R-II in this respect (39-42). Our findings are in agreement with those of Muramatsu et al. (43), 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 Tbeta R-I/BMPR-IB to signal in combination with Tbeta R-II is unlikely to be due to a perturbation of the conformation of Tbeta R-II extracellular domain or BMPR-IB intracellular domain, since Tbeta R-I/BMPR-IB bound TGF-beta and formed a heteromeric complex with Tbeta R-II and since Tbeta R-I/BMPR-IB together with Tbeta R-II/ActR-IIB transduced a TGF-beta -dependent transcriptional activation signal and induced Smad1 phosphorylation, a BMP-like signal. It is possible that the inability of Tbeta R-I/BMPR-IB to mediate a growth-inhibitory response may be in an inherent property of the receptor, since Mv1Lu cells, which express Tbeta R-II, Tbeta R-I, ActR-II, and BMPR-IB among other receptors, are at least 100-fold more sensitive to TGF-beta 1 than BMP-7/OP-1 with respect to growth inhibition. On the other hand, TGF-beta -induced complex formation between Tbeta R-II and Tbeta R-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-beta -induced complex formation between Tbeta R-I and Tbeta R-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 Tbeta R-I need to be substituted for analogous regions/residues in the intracellular domain of BMPR-IB to allow for a Tbeta R-I/BMPR-IB chimera to transduce signals when activated by Tbeta R-II.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Ludwig Institute for Cancer Research, Box 595, S-751 24 Uppsala, Sweden. Tel.: 46-18-160400; Fax: 46-18-160420; E-mail: Urban.Persson{at}LICR.uu.se.
1   The abbreviations used are: TGF-beta , transforming growth factor-beta ; Tbeta R, TGF-beta 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.
2   S. Souchelnytskyi, unpublished data.
3   Nakao, A., Imamura, T., Souchelnytskyi, S., Kawabata, M., Ishisaki, A., Oeda, E., Tamaki, K., Hanai, J., Heldin, C.-H., Miyazono, K., and ten Dijke, P. (1997) EMBO J., in press.

ACKNOWLEDGEMENTS

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-beta 1, 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.


REFERENCES

  1. Roberts, A. B., and Sporn, M. B. (1990) in Peptide Growth Factors and Their Receptors, Part I (Sporn, M. B., and Roberts, A. B., eds), Vol. 95, pp. 419-472, Springer-Verlag, Berlin
  2. Mathews, L. S. (1994) Endocr. Rev. 15, 310-325 [Medline] [Order article via Infotrieve]
  3. Kingsley, D. M. (1994) Genes & Dev. 8, 133-146 [CrossRef][Medline] [Order article via Infotrieve]
  4. Reddi, A. H. (1994) Curr. Opin. Genet. Dev. 4, 737-744 [Medline] [Order article via Infotrieve]
  5. ten Dijke, P., Miyazono, K., and Heldin, C.-H. (1996) Curr. Opin. Cell Biol. 8, 139-145 [CrossRef][Medline] [Order article via Infotrieve]
  6. Derynck, R., and Zhang, Y. (1996) Curr. Biol. 6, 1226-1229 [Medline] [Order article via Infotrieve]
  7. Massagué, J. (1996) Cell 85, 947-950 [Medline] [Order article via Infotrieve]
  8. Laiho, M., Weis, F. M. B., and Massagué, J. (1990) J. Biol. Chem. 265, 18518-18524 [Abstract/Free Full Text]
  9. Wrana, J. L., Attisano, L., Cárcamo, J., Zentella, A., Doody, J., Laiho, M., Wang, X.-F., and Massagué, J. (1992) Cell 71, 1003-1014 [Medline] [Order article via Infotrieve]
  10. Franzén, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schulz, P., Heldin, C.-H., and Miyazono, K. (1993) Cell 75, 681-692 [Medline] [Order article via Infotrieve]
  11. Bassing, C. H., Howe, D. J., Segarini, P. R., Donahoe, P. K., and Wang, X.-F. (1994) J. Biol. Chem. 269, 14861-14864 [Abstract/Free Full Text]
  12. ten Dijke, P., Yamashita, H., Ichijo, H., Franzén, P., Laiho, M., Miyazono, K., and Heldin, C.-H. (1994) Science 264, 101-104 [Medline] [Order article via Infotrieve]
  13. Lin, H. Y., Wang, X.-F., Ng Eaton, E., Weinberg, R. A., and Lodish, H. F. (1992) Cell 68, 775-785 [Medline] [Order article via Infotrieve]
  14. Mathews, L. S., and Vale, W. W. (1991) Cell 65, 973-982 [Medline] [Order article via Infotrieve]
  15. Attisano, L., Wrana, J. L., Cheifetz, S., and Massagué, J. (1992) Cell 68, 97-108 [Medline] [Order article via Infotrieve]
  16. Rosenzweig, B. L., Imamura, T., Okadome, T., Cox, G. N., Yamashita, H., ten Dijke, P., Heldin, C.-H., and Miyazono, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7632-7636 [Abstract]
  17. Liu, F., Ventura, F., Doody, J., and Massagué, J. (1995) Mol. Cell. Biol. 15, 3479-3486 [Abstract]
  18. Nohno, T., Ishikawa, T., Saito, T., Hosokawa, K., Noji, S., Wolsing, D. H., and Rosenbaum, J. S. (1995) J. Biol. Chem. 270, 22522-22526 [Abstract/Free Full Text]
  19. Ruberte, E., Marty, T., Nellen, D., Affolter, M., and Basler, K. (1995) Cell 80, 889-897 [Medline] [Order article via Infotrieve]
  20. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massagué, J. (1994) Nature 370, 341-347 [CrossRef][Medline] [Order article via Infotrieve]
  21. Mathews, L. S., and Vale, W. W. (1993) J. Biol. Chem. 268, 19013-19018 [Abstract/Free Full Text]
  22. Attisano, L., Wrana, J. L., Montalvo, E., and Massagué, J. (1996) Mol. Cell. Biol. 16, 1066-1073 [Abstract]
  23. Chen, F., and Weinberg, R. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1565-1569 [Abstract]
  24. Willis, S. A., Zimmerman, C. M., Li, L., and Mathews, L. S. (1996) Mol. Endocrinol. 10, 367-379 [Abstract]
  25. Wieser, R., Wrana, J. L., and Massagué, J. (1995) EMBO J. 14, 2199-2208 [Abstract]
  26. Franzén, P., Heldin, C.-H., and Miyazono, K. (1995) Biochem. Biophys. Res. Commun. 207, 682-689 [CrossRef][Medline] [Order article via Infotrieve]
  27. Weis-Garcia, F., and Massagué, J. (1996) EMBO J. 15, 276-289 [Abstract]
  28. Cárcamo, J., Zentella, A., and Massagué, J. (1995) Mol. Cell. Biol. 15, 1573-1581 [Abstract]
  29. Cárcamo, J., Weis, F. M., Ventura, F., Wieser, R., Wrana, J. L., Attisano, L., and Massagué, J. (1994) Mol. Cell. Biol. 14, 3810-3821 [Abstract]
  30. Tsuchida, K., Vaughan, J. M., Wiater, E., Gaddy Kurten, D., and Vale, W. W. (1995) Endocrinology 136, 5493-5503 [Abstract]
  31. Lechleider, R. J., de Caestecker, M. P., Dehejia, A., Polymeropoulos, M. H., and Roberts, A. B. (1996) J. Biol. Chem. 271, 17617-17620 [Abstract/Free Full Text]
  32. Yingling, J. M., Das, P., Savage, C., Zhang, M., Padgett, R. W., and Wang, X.-F. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8940-8944 [Abstract/Free Full Text]
  33. Lagna, G., Hata, A., Hemmati-Brivanlou, A., and Massagué, J. (1996) Nature 383, 832-836 [CrossRef][Medline] [Order article via Infotrieve]
  34. Hoodless, P. A., Haerry, T., Abdollah, S., Stapleton, M., O'Connor, M. B., Attisano, L., and Wrana, J. L. (1996) Cell 85, 489-500 [Medline] [Order article via Infotrieve]
  35. Liu, F., Hata, A., Baker, J. C., Doody, J., Cárcamo, J., Harland, R. M., and Massagué, J. (1996) Nature 381, 620-623 [CrossRef][Medline] [Order article via Infotrieve]
  36. Macías-Silva, M., Abdollah, S., Hoodless, P. A., Pirone, R., Attisano, L., and Wrana, J. L. (1996) Cell 87, 1215-1224 [Medline] [Order article via Infotrieve]
  37. Nakao, A., Röijer, E., Imamura, T., Souchelnytskyi, S., Stenman, G., Heldin, C.-H., and ten Dijke, P. (1997) J. Biol. Chem. 272, 2896-2900 [Abstract/Free Full Text]
  38. Zhang, Y., Feng, X.-H., Wu, R. Y., and Derynck, R. (1996) Nature 383, 168-172 [CrossRef][Medline] [Order article via Infotrieve]
  39. Okadome, T., Yamashita, H., Franzén, P., Morén, A., Heldin, C.-H., and Miyazono, K. (1994) J. Biol. Chem. 269, 30753-30756 [Abstract/Free Full Text]
  40. Vivien, D., Attisano, L., Wrana, J. L., and Massagué, J. (1995) J. Biol. Chem. 270, 7134-7141 [Abstract/Free Full Text]
  41. Feng, X.-H., Filvaroff, E. H., and Derynck, R. (1995) J. Biol. Chem. 270, 24237-24245 [Abstract/Free Full Text]
  42. Anders, R. A., and Leof, E. B. (1996) J. Biol. Chem. 271, 21758-21766 [Abstract/Free Full Text]
  43. Muramatsu, M., Yan, J., Eto, K., Tomoda, T., Yamada, R., and Arai, K. (1997) Mol. Biol. Cell 8, 469-480 [Abstract]
  44. Yamashita, H., ten Dijke, P., Huylebroeck, D., Sampath, T. K., Andries, M., Smith, J. C., Heldin, C.-H., and Miyazono, K. (1995) J. Cell Biol. 130, 217-226 [Abstract]
  45. Frolik, C. A., Wakefield, L. M., Smith, D. M., and Sporn, M. B. (1984) J. Biol. Chem. 259, 10995-11000 [Abstract/Free Full Text]
  46. Yamashita, H., ten Dijke, P., Franzén, P., Miyazono, K., and Heldin, C.-H. (1994) J. Biol. Chem. 269, 20172-20178 [Abstract/Free Full Text]
  47. Verschueren, 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., and Huylebroeck, D. (1995) Mech. Dev. 52, 109-123 [CrossRef][Medline] [Order article via Infotrieve]
  48. Souchelnytskyi, S., ten Dijke, P., Miyazono, K., and Heldin, C.-H. (1996) EMBO J. 15, 6231-6240 [Abstract]
  49. Laiho, M., Rönnstrand, L., Heino, J., Decaprio, J. A., Ludlow, J. W., Livingston, D. M., and Massagué, J. (1991) Mol. Cell. Biol. 11, 972-978 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.