©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inactive Type II and Type I Receptors for TGF Are Dominant Inhibitors of TGF-dependent Transcription (*)

(Received for publication, November 28, 1994)

Thomas Brand (§) Michael D. Schneider (¶)

From the Molecular Cardiology Unit, Departments of Medicine, Cell Biology, and Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Although transforming growth factor-beta (TGFbeta) is implicated in differentiation and disease, proof of in vivo function requires specific inhibitors of the TGFbeta cascade. TGFbeta binds a family of type I and type II receptors (TbetaRI, TbetaRII), containing a cytoplasmic serine/threonine kinase domain. We previously reported that kinase-deficient TbetaRII (DeltakTbetaRII) blocks TGFbeta-dependent transcription in cardiac myocytes. It is controversial whether both receptors are needed in all cells for gene regulation by TGFbeta or whether they mediate distinct subsets of TGFbeta-dependent events. To resolve this uncertainty, TGFbeta-dependent transcription was investigated in cardiac myocytes versus mink lung epithelial cells. 1) DeltakTbetaRII inhibits induction of a TGFbeta-responsive reporter gene, in both cell backgrounds. 2) Charged-to-alanine mutations of key residues of the TbetaRII kinase, including consensus ATP binding and amino acid recognition motifs, are competent for binding but not transcriptional activation. Each inactive receptor inhibits TGFbeta-dependent transcription in both cell types. 3) Kinase-deficient TbetaRI (DeltakTbetaRI) likewise impairs TGFbeta-dependent transcription, less completely than DeltakTbetaRII; kinase-deficient activin type I receptor has no effect. 4) TGFbeta-binding proteins in cardiac cells and Mv1Lu cells are comparable by affinity labeling and immunoprecipitation; however, Mv1Lu cells express up to 3-fold higher levels of TbetaRII and TbetaRI. Thus, the model inferred from TGFbeta-resistant cell lines (that TbetaRII and TbetaRI are necessary in tandem for the TGFbeta-signaling complex to regulate transcription) is valid for cardiac myocytes, the cell type most prominently affected in TGFbeta-deficient animals.


INTRODUCTION

Cytokines of the type beta transforming growth factor (TGFbeta) (^1)superfamily participate in remarkably diverse aspects of development, including pattern formation, organogenesis, and tissue-specific transcription, as well as in human disease(1) . For the cardiovascular system, TGFbeta has been implicated in cardiac myogenesis itself, formation of the heart valves, protection of contractility from depressant effects of interleukin 1beta, atherosclerosis in apolipoprotein(a) transgenic mice, and activation of a ``fetal'' program of transcription during work-induced hypertrophy (2, 3, 4, 5, 6, 7) . Despite multiple roles for TGFbeta anticipated from embryonal stem cells and other model systems, both embryonic development and perinatal survival are normal in mice homozygous for a null mutation of TGFbeta1(8, 9) . While, a priori, such a discrepancy might be explained by functional redundancy among the three mammalian isoforms of TGFbeta, a more direct explanation possibly is furnished by the unexpected finding that maternal TGFbeta crosses the placenta and also can be conferred by breast milk(10) . The TGFbeta1-null offspring of heterozygous mothers, which initially are normal, soon develop a multifocal inflammatory response primarily affecting the heart and lungs(8, 9) , with enhanced expression of major histocompatibility antigens, resembling autoimmune disease(11) . By contrast, the homozygous offspring of a mother homozygous for the null mutation die uniformly in the first day of life, with gross abnormalities of only ventricular myocardium and the atrioventricular valves(10) . Thus, the heart is reported to be the predominant, if not the exclusive, target organ affected by TGFbeta1 deficiency in the embryo and newborn.

Affinity labeling with radioiodinated TGFbeta has identified three major TGFbeta receptors in most mammalian lineages, designated type III (betaglycan; mass 200-400 kDa), type II (TbetaRII; mass 70-80 kDa), and type I (TbetaRI; mass 53 kDa); however, additional membrane proteins, of less certain function, also bind TGFbeta in various cell types(12, 13) . Betaglycan, a membrane-spanning proteoglycan with an extremely short cytoplasmic domain, is absent from certain TGFbeta-responsive cells and is thus dispensable for TGFbeta signal generation. Soluble fragments of betaglycan have been identified that interfere with access of TGFbeta to the signaling receptors, whereas membrane-anchored betaglycan enhances the action of TGFbeta via increased binding of TGFbeta, particularly TGFbeta2, to the type II and type I receptors(14, 15) . In the presence of ligand, betaglycan associates with the type I and type II receptors, forming a ternary complex(14) . However, homodimers of betaglycan are the predominant type III receptor complex present on the cell surface in both the absence and presence of TGFbeta(16) .

TbetaRII, first identified by expression cloning and subsequently isolated in several species, is a transmembrane protein characterized by a single transmembrane segment and an intracellular domain comprising a highly conserved serine/threonine kinase, the hallmark of this emerging superfamily of receptors for TGFbeta, activins, bone morphogenetic proteins, and related cytokines(17, 18, 19, 20) . The protein kinase domain itself is characterized by two inserts, positioned between subdomains VIa and VIb (insert I) and between subdomains X and XI (insert II), while carboxyl-terminal to the kinase domain is a serine/threonine-rich cytoplasmic tail. The ligand-binding domain is abundant in cysteine residues, and an especially conserved motif, the Cys box, is found in all members of the receptor serine/threonine kinase superfamily. A distinguishable subfamily of type I receptors has been isolated, based upon sequence homologies within the kinase domains, which show high homology to each other (60-90% amino acid sequence identity) but lesser homology to the kinase domains of the known type II receptors (30-40%)(21, 22, 23, 24) . Features common to the type I receptors include the spacing of extracellular cysteine residues, and a glycine- and serine-rich motif amino-terminal to the kinase domain.

One distinction between the cloned type I and type II receptors is the inability of type I receptors to bind TGFbeta in the absence of type II receptors, a feature predicted from mutant cells unresponsive to TGFbeta (25) . Conversely, the type II receptor is fully sufficient for ligand binding, yet not for signal transduction. Antibodies directed against the type II receptor coprecipitate type I receptor from cells treated with TGFbeta, suggesting that type I and type II receptors form a heteromeric complex in the presence of ligand(18) . Hence, the genetic evidence from TGFbeta-resistant cell lines implies that only a heteromeric complex comprising type I and type II receptor in tandem is competent to support TGFbeta signal transduction(18) . Although TGFbeta-related growth factors bind with promiscuity to a variety of type I receptors under the conditions of forced expression in COS cells, physical association of the respective endogenous type I and type II receptors exhibits greater specificity, as does the ability of type I receptors to support biological responses(26, 27, 28, 29) . Of six type I receptors, only one (ALK-5/R4) has been proven to rescue gene induction by TGFbeta when introduced into type I receptor-deficient cells(26, 30) . Thus, ALK-5/R4 is a functional type I receptor for TGFbeta. Activity of the TbetaRII kinase is constitutive even in the absence of TGFbeta, and ligand binding is understood to initiate the TGFbeta signal transduction cascade through the physical recruitment of TbetaRI into a receptor heterodimer and, consequently, the asymmetric phosphorylation of TbetaRI by TbetaRII(31) .

We demonstrated previously that a truncation of TbetaRII, lacking the serine/threonine kinase domain (DeltakTbetaRII), introduced into cardiac muscle cells by transient transfection, is sufficient to suppress TGFbeta-dependent activation of the skeletal alpha-actin promoter, by all three isoforms of the peptide, and to block down-regulation of the alpha-myosin heavy chain promoter(32) . Thus, by itself, the truncated type II receptor is sufficient to block both positive and negative control of transcription by TGFbeta, as predicted from models requiring the action of TbetaRII and TbetaRI in concert(18, 27, 31, 33) . This model for signal generation via a heteromeric complex of TbetaRI and TbetaRII is disputed, however. Differing conclusions for a dominant-negative type II receptor have been drawn in Mv1Lu mink lung epithelial cells, used extensively for the analysis of TGFbeta signaling mechanisms: a related kinase-defective truncation of the human type II receptor failed to suppress three representative TGFbeta-dependent genes, including plasminogen activator inhibitor-1 (PAI-1), yet was capable of disrupting growth inhibition by TGFbeta(34) . These results lend support to an alternative model-that inactivation of the type II receptor exclusively impairs a pathway important for growth control, whereas transcriptional control might be mediated by the type I receptor alone.

To address these uncertainties regarding the comparative role of TbetaRI and TbetaRII in gene regulation, in the present study we have developed three independent lines of evidence. First, we transfected Mv1Lu cells with our kinase-defective truncation of the type II TGFbeta receptor, together with a TGFbeta-inducible reporter construct derived from the PAI-1 promoter. In concordance with our prior observations using ventricular myocytes, expression of DeltakTbetaRII inhibited induction of the reporter gene by TGFbeta in the Mv1Lu cell background. Second, charged-to-alanine point mutations were engineered at conserved residues within subdomains II, VI-B, and kinase insert II of the TbetaRII kinase, which are associated with ATP binding, amino acid recognition, and protein kinase activity. Each substitution failed to rescue receptor-deficient cells and was fully as active as DeltakTbetaRII for inhibiting TGFbeta-dependent gene induction both in normal Mv1Lu cells and in cardiac myocytes. Third, we compared the ability of a kinase-deficient truncation of the type I receptor (DeltakTbetaRI) to likewise block TGFbeta signaling events that mediate gene activation. Overexpression of DeltakTbetaRI in both cardiac myocytes and mink lung cells blocked gene induction by TGFbeta, although less effectively than did DeltakTbetaRII. Although ALK-2, a type I receptor for activin, can bind TGFbeta both in its full-length and truncated form(35) , neither full-length ALK-2 nor a corresponding truncation of ALK-2 interfered with TGFbeta-dependent transcription. Thus, our data concur with the model inferred from TGFbeta receptor-deficient cells, that the function of type I and type II receptors is necessary in tandem for the TGFbeta-signaling complex to regulate gene transcription.


EXPERIMENTAL PROCEDURES

Plasmids

Three sets of TGFbeta receptor mutations were engineered for these investigations: the cytoplasmic domain deletion mutant of TbetaRII (DeltakTbetaRII), site-directed mutations within the kinase domain of full-length TbetaRII, and truncations of the cytoplasmic kinase domain of two type I receptors (Fig. 1). Construction of DeltakTbetaRII has been reported previously(32) . A wild-type human TbetaRII construct, lacking most of the 5` and 3` untranslated sequences, was produced by the polymerase chain reaction (PCR) using primers 1 and 6, as sense and antisense primers within subdomain I and downstream of the coding region, respectively (Table 1). The resulting product, comprising nucleotides 894-2056 of the human TbetaRII coding sequence, was subcloned as a BglII-HindIII fragment into BglII-HindIII sites of DeltakTbetaRII, restoring the kinase domain.


Figure 1: Structure of the TbetaRII mutations. Subdomains denote the consensus subdomains conserved within the serine/threonine and tyrosine kinase superfamilies(39) . The schematic representation of TbetaRII indicates the extracellular cysteines as verticallines at the left of the figure and the transmembrane domain as a solidbar. The whitebar denotes subdomains II-XI of the cytoplasmic serine/threonine kinase domain; the hatchedbar indicates the amino-terminal remnant of the kinase domain retained in DeltakTbetaRII; graybars indicate kinase inserts 1 and 2. Sense and antisense primers used in conjunction with the mutagenic oligonucleotides are illustrated below. See ``Experimental Procedures'' and Table 1for details.





For the purpose of subcloning mutagenized sequences of TbetaRII into the wild-type receptor background, full-length TbetaRII was first subcloned into pGem4Z. Site-directed charged-to-alanine mutations of the human TbetaRII cDNA were generated by PCR, using H2-3FF (17) as the template. To generate each point mutation of the TbetaRII kinase domain, two PCR reactions were performed using overlapping primer pairs: e.g. primer 1 or 2, respectively, with the antisense or sense primers for E272A and K277A (Fig. 1; Table 1). The resulting PCR products for each primer pair were gel-purified, pooled, and subjected to another round of PCR amplification using primers 1 and 2. The final product was subcloned using the HpaI-SmaI (K277A) or HpaI-BglII (E272A) sites of human TbetaRII.

For all other point mutations, a similar three-step PCR mutagenesis protocol was employed. Primers 3 and 4, respectively, were used in conjunction with antisense or sense primers for H362A, K372A/H377A, R378A/D379A, K381A, and D397A; secondary amplification was performed using primers 3 and 4, and the final product was subcloned into the SmaI-AccI sites of TbetaRII-pGem4Z. Primers 3 and 6 were utilized for amplification of D446A. Primers 5 and 6 were used, respectively, with the antisense or sense primer for R497/H507 and R528A; secondary amplification was performed using primers 5 and 6, and the final product was subcloned into AccI-HindIII sites of TbetaRII-pGem4Z. The truncation of the carboxyl-terminal serine-rich tail was generated by amplifying H2-3FF using primers 1 and Deltatail (Table 1) and was subcloned as a BglII-HindIII fragment into the BglII-HindIII sites of wild-type TbetaRII. Reaction conditions were 1 min each at 94, 55, and 72 °C for 12 cycles. All constructs were authenticated by dideoxy sequencing (U. S. Biochemical Corp., Sequenase version 1) and were subcloned for expression in eukaryotic cells into the vector pcDNA-1 (Invitrogen; Fig. 3) or pSV-Sport-1 (Life Technologies, Inc.; Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8). Transfections in each experiment were balanced for promoter and total DNA content using the corresponding empty vector control, and all comparisons between receptor constructs used the identical promoter for each.


Figure 3: The truncated type II receptor, DeltakTbetaRII, is a dominant inhibitor of TGFbeta-dependent transcription in mink lung epithelial cells. A, Mv1Lu cells transfected with DeltakTbetaRII and full-length TbetaRII cDNA in the amounts indicated were analyzed for the activity of p3TP-lux and CMV-beta-galactosidase reporter genes. Results (mean ± S.E.) are shown for vehicle-treated (circle) and TGFbeta1-treated cells (). Levels of luciferase expression, corrected for transfection efficiency, are expressed relative to the vehicle-treated, vector-transfected cells. B, Mv1Lu cells were transfected with a fixed amount (10 µg) of DeltakTbetaRII () or the vector (&cjs2113;), together with decreasing amounts (7.5, 5, 2.5, or 1 µg) of the p3TP-lux reporter gene; the ratio of expression vector to p3TP-lux was thus 1.3, 2, 4, or 10, respectively. Cells were incubated for 16 h in the absence(-) or presence (+) of TGFbeta1 and were assayed for luciferase and beta-galactosidase activity. Results (mean values ± S.E.) are expressed relative to p3TP-lux expression in vehicle-treated, vector transfected cells.




Figure 4: TGFbeta receptors in cardiac myocyte and Mv1Lu cell cultures. A, cardiac myocytes, Mv1Lu cells, and the receptor-deficient derivatives shown were affinity-labeled with 100 pMI-TGFbeta1 ± 60 nM unlabeled TGFbeta1. B, Mv1Lu cells and cardiac myocytes were affinity-labeled with I-TGFbeta1, and the cell lysates were subjected to immunoprecipitation with polyclonal antibody directed against TbetaRII (left) or TbetaRI (right). For reference, lysate not subjected to immunoprecipitation is shown at the left of panelB. Immunoprecipitation with antibody directed against TbetaRI was performed in the absence or presence, as indicated, of synthetic TbetaRI peptide. Specificity of immunoprecipitation with antibody directed against TbetaRII was corroborated, analogously, using synthetic TbetaRII peptide, in additional experiments not illustrated here.




Figure 5: Dominant-negative activity of point mutations of the kinase domain of TbetaRII. Cardiac myocytes (A), Mv1Lu cells (B), and TbetaRII-deficient DR-26 cells (C) were transfected with vector, Deltatail, or the missense mutations indicated. Cell lysates were analyzed for the activity of skeletal alpha-actin- (A) or p3TP-luciferase (B and C) and CMV-beta-galatosidase reporter genes. Results are shown for cells cultured in the absence (box) and presence () of 1 ng/ml TGFbeta1. Luciferase activity (mean ± S.E.) is expressed relative to that of the p3TP-lux activity in vehicle, vector-transfected cells.




Figure 6: Cell surface expression of point mutations of TbetaRII. COS-1 cells were transfected with the indicated charged-to-alanine substitutions of TbetaRII, wild-type TbetaRII, or the vector alone, and were then affinity-labeled with 100 pMI-TGFbeta1 in the absence(-) or presence (+) of 60 nM unlabeled TGFbeta1 as competitor. For comparison as a positive control, Mv1Lu cells were included. Cell lysates were subjected to gel electrophoresis and autoradiography.




Figure 7: A deletion of the TGFbeta type I receptor kinase specifically inhibits TGFbeta-dependent transcription. Mv1Lu cells were transfected with vector, DeltakTbetaRII, DeltakTbetaRI, wild-type ActRI, or DeltakActRI. Cell lysates were analyzed for the activity of p3TP-lux and CMV-beta-galatosidase reporter genes. Results are shown for cells cultured in the absence (box) and presence () of 1 ng/ml TGFbeta1. Luciferase activity (mean ± S.E.) is expressed relative to that in vehicle, vector-transfected cells.




Figure 8: Truncated type II receptor inhibits TGFbeta-dependent transcription at least as effectively as the truncated type I receptor. Mv1Lu cells (A) and cardiac myocytes (B) were transfected with DeltakTbetaRII, (circle, bullet), DeltakTbetaRI (box, ), or DeltakTbetaRII + DeltakTbetaRI (up triangle, ) expression vectors at the concentrations shown, in the absence (circle, box, up triangle) or presence (bullet, , ) of 1 ng/ml TGFbeta1. For cotransfection of DeltakTbetaRII + DeltakTbetaRI, the ordinate indicates the sum of the two receptor plasmids (e.g.4 µg denotes 2 µg of each). Cell lysates were analyzed for the activity of p3TP- (A) or skeletal alpha-actin-luciferase (B) and CMV-beta-galatosidase reporter genes. Mean luciferase activity is expressed relative to that in vehicle-treated, vector-transfected cells.



A kinase-deficient truncation of the TGFbeta type I receptor, DeltakTbetaRI, was constructed using PCR primers DeltakTbetaRI(S) and DeltakTbetaRI(A) (Table 1) and human ALK-5-pSV7D (26) as the template. Primers upstream of the start codon were problematic, given the unusually high GC content in this region. Therefore, a sense-strand primer was used, positioned 135 nucleotides 3` to the start site. The resulting fragment was subcloned into the EcoRI-HindIII sites of pSV-Sport-1. The missing 5` end was rescued by subcloning an EcoRI/XbaI fragment of ALK-5-pSV7D into the EcoRI-XbaI sites of DeltakTbetaRI-pSV-Sport-1. The truncated, kinase-deficient activin type I receptor, DeltakActRI, was produced by subcloning a 1.0-kilobase pair EcoRI/BglII fragment of SKR1/ALK-2 (22) into the EcoRI and BglII sites of DeltakTbetaRII-pSV-Sport-1, thereby substituting the DeltakActRI coding sequence for DeltakTbetaRII. For construction of wild-type ActRI, a SmaI/HindIII fragment of SKR1 in pBluescript was subcloned into pSV-Sport-1.

The TGFbeta-responsive reporter gene analyzed in cardiac myocytes, -394/+24SkALuc(32) , contains nucleotides -394 to +24 of the chicken SkA gene as an RsaI-HindIII fragment between the SmaI and HindIII sites of the firefly luciferase reporter expression vector pXP1(36) ; activation of the full-length promoter by TGFbeta is contingent on serum response factor and the SV40 enhancer-binding protein, TEF-1, in concert(37) . p3TP-lux, used for the mink lung epithelial cells, is a derivative of the human PAI-1 promoter containing (5` to 3`) three copies of the tetradecanoyl phorbol acetate-responsive element from the human collagenase promoter (nucleotides -73 to -42), a TGFbeta-responsive element of the PAI-1 promoter (nucleotides -740 to -636), and the the adenovirus E4 promoter sequence (-38 to +38), cloned into pGL2 (Promega)(18) . As a constitutive reporter plasmid, CMVbeta-gal was used as detailed previously(32) .

Cell Culture and Transfection

Wild-type Mv1Lu mink lung epithelial cells (CCL-64; American Type Culture Collection) and the TGFbeta-resistant, TbetaRII-deficient derivative DR-26 (provided by J. Massagué) (18, 25) were grown in alpha-minimum essential medium containg 10% fetal bovine serum. COS-1 cells were cultured in Dulbecco's modified essential medium (DMEM) containing 10% fetal bovine serum. Isolation and culture of neonatal rat cardiac myocytes were performed as described previously(32) . For transfection, cells were seeded at 2 times 10^5 cells/35-mm well, in six-well plates. Cells were transfected 16-24 h after plating by the diaminoethyl-dextran sulfate (DEAE-dextran) method(32) .

Briefly, 10 µg of receptor construct (except as noted in the text) versus the empty expression vector, 5 µg of the appropriate luciferase reporter plasmid, and 2.5 µg of CMVbeta-gal were mixed with 1 ml of DMEM, 10% NuSerum (HyClone), and 200 µg/ml DEAE-dextran. Cells were incubated for 4 h with DNA/DEAE-dextran complexes, and then shocked for 90 s with 10% dimethyl sulfoxide in DMEM. The cells were cultured overnight in alpha-minimum essential medium containing 0.2% fetal bovine serum. The following day, the medium was replaced, and 1 ng/ml purified porcine TGFbeta1 (R& Systems) or the vehicle was added, as described(32) . Sixteen h after addition of the growth factor, cells were harvested and assayed for luciferase and beta-galactosidase activity(32) . Three to six transfections were performed for each condition tested, using at least two independent DNA preparations. Results were compared by analysis of variance and the Student-Newman-Keuls multiple comparison test, using a significance level of p < 0.05.

Receptor Affinity Labeling

COS-1 cells were plated at a density 1 times 10^6 cells/60-mm dish in growth medium (DMEM, 25 mM HEPES (pH 7.4), 10% fetal calf serum), 18 h prior to transfection. Cells were transfected with 10 µg of receptor cDNA, using DEAE-dextran, for 6 h. Cells were cultured after transfection for 48 h in growth medium. Cell monolayers were washed twice with binding buffer (DMEM, 25 mM HEPES (pH 7.4), 2 mg/ml bovine serum albumin), then were incubated for 4 h at 4 °C with 100 pMI-TGFbeta1 (Bolton-Hunter-labeled human recombinant TGFbeta1, specific activity 6,000 kBq/µg; DuPont NEN) in the absence or presence of 60 nM unlabeled TGFbeta1, as indicated. After washing the cells extensively with albumin-free binding buffer, ligand was cross-linked for 15 min at 4 °C using 300 µM disuccinimidyl suberate (Pierce) in dimethyl sulfoxide. Cells were washed twice in 0.25 M sucrose, 10 mM Tris, 1 mM EDTA (pH 7.4), 0.3 mM phenylmethylsulfonyl fluoride, and harvested using a rubber policeman. After brief centrifugation at 12,000 times g, the pellet was solubilized in 125 mM NaCl, 10 mM Tris-HCl (pH 7.0), 1 mM EDTA, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml antipain, 50 µg/ml aprotinin, 100 µg/ml soybean trypsin inhibitor, 100 µg/ml benzamidine hydrochloride, 10 µg/ml pepstatin (Sigma), 1 mg/ml bestatin (Peninsula), 0.3 mM phenylmethylsulfonyl fluoride, by rotary inversion for 40 min at 4 °C. Insoluble debris was pelleted by centrifugation at 12,000 times g for 15 min at 4 °C. Solubilized proteins were mixed with 2times electrophoresis sample buffer (100 mM Tris-HCl (pH 6.8), 20% glycerol, 2% SDS, 0.05% bromphenol blue, 100 mM dithiothreitol) and boiled for 5 min. Proteins were electrophoresed on a 10% or 4-15% gradient sodium dodecyl sulfate-polyacrylamide gel.

Immunoprecipitation

For immunoprecipitation, affinity-labeled cells in 100-mm plates were solubilized at 4 °C for 40 min in 1 ml of lysis buffer (150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 0.5% Triton X-100, 1 mM EDTA), in the presence of protease inhibitors as indicated for affinity labeling. Insoluble debris was removed by centrifugation. Lysates were preincubated for 1 h with 20 µl of protein A/G-agarose (Santa Cruz). After centrifugation, 1 ml of the supernatant was incubated at 4 °C for 1 h with 5 µg of rabbit antibody to TbetaRI (V-22 directed against amino acids 158-179 of human ALK-5; Santa Cruz Biotechnology), or 1 µg of rabbit antibody to TbetaRII (L-21 directed against amino acids 246-266 of human TbetaRII; Santa Cruz Biotechnology). For immunoadsorption, 20 µl of protein A/G-agarose was added, the incubation was continued for an additional hour, and the beads were washed five times with lysis buffer containing 0.1% Triton X-100. Bound proteins were eluted by boiling for 5 min in 1 times electrophoresis sample buffer. To establish competition by the authentic immunogen, cell lysate was incubated with antibody in the presence of 25 µg of the appropriate TbetaRI or TbetaRII peptide.


RESULTS

The Kinase-deficient Truncation, DeltakTbetaRII, Blocks TGFbeta-dependent Gene Expression in Mv1Lu Cells

As biological differences between lineages in the TGFbeta receptors themselves, the signaling intermediaries, or the end points examined might explain the apparent lack of dependence on TbetaRII shown for TGFbeta-regulated gene expression in Mv1Lu cells(34) , we first investigated the generality of our previous observation, that a truncation of the kinase domain of TbetaRII could block TGFbeta-dependent transcription in cardiac myocytes (32) . In neonatal rat ventricular myocytes, both positive and negative regulation of gene expression is efficiently blocked by the cytoplasmic deletion mutation of the type II TGFbeta receptor, DeltakTbetaRII, truncated NH(2)-terminal to the kinase domain (shown schematically, together with point mutations to be discussed below, in Fig. 1). Ligand binding was determined in COS-1 cells transfected with DeltakTbetaRII and wild-type TbetaRII constructs (Fig. 2). Vector-transfected COS-1 cells and untransfected Mv1Lu cells were analyzed, as negative and positive controls, respectively. Cells were affinity-labeled with I-TGFbeta1 as described under ``Experimental Procedures.'' Vector-transfected COS-1 cells showed only minor levels of betaglycan and type I receptor (Fig. 2), whereas intense additional bands were readily visible in COS-1 cells transfected with wild-type TbetaRII and DeltakTbetaRII. The human wild-type receptor migrates more slowly than the mink type II receptor in Mv1Lu cells(18) , and has an apparent mass of 95 kDa. Transfection of DeltakTbetaRII produced a truncated protein of 55-60 kDa, the expected size of affinity-labeled DeltakTbetaRII, given that 279 of the 567 amino acids of the wild-type human receptor are retained in the kinase-deficient construct. Thus, the ligand-binding domain of the truncated TbetaRII protein is appropriately expressed and is accessible to the extracellular ligand.


Figure 2: Cell surface expression of TbetaRII and DeltakTbetaRII. COS-1 cells transfected with TbetaRII, DeltakTbetaRII, or vector were affinity-labeled with I-TGFbeta1 and disuccinimidyl suberate. Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Mv1Lu cells are included for comparison at the right.



We transfected DeltakTbetaRII into Mv1Lu mink lung epithelial cells using p3TP-lux as the reporter gene. Mv1Lu cells are known to be unusually sensitive to TGFbeta stimulation, and the reporter gene p3TP-lux, a derivative of the PAI-1 promoter, is in turn a construct that is highly responsive to TGFbeta, induced up to 20-fold ((18) and Fig. 3). Thus, this cell/reporter pair provides an excellent responder system with which to evaluate the potential trans-dominant effects of TGFbeta receptor mutations. Forced expression of DeltakTbetaRII resulted in up to 80% inhibition of p3TP-lux activity, compared to vector transfected cells (p = 0.0009). The cotransfected constitutive control, CMV-lacZ, by contrast, was unaffected by the receptor mutation. Thus, the cytoplasmic deletion mutant of TbetaRII functions as a dominant inhibitor of TGFbeta-induced gene expression in Mv1Lu cells, without need for concurrent mutation of TbetaRI.

Overexpression of Wild-type TbetaRII Can Rescue the Dominant-negative Phenotype Produced by DeltakTbetaRII in Mv1Lu Cells

One stringent test for specificity of the inhibitory action of DeltakTbetaRII is whether its dominant-negative phenotype can be rescued by overexpressing wild-type receptor (Fig. 3A). DeltakTbetaRII plasmid concentration was kept constant at 10 µg/plate, and increasing concentrations of wild-type TbetaRII were cotransfected. Progressive introduction of TbetaRII led to a dose-dependent increase in the TGFbeta-dependent transcription of p3TP-lux (p = 0.0052), equalling at 7.5 µg/plate the level seen in control, vector-transfected cells. Further increase in p3TP-lux activity was obtained when wild-type TbetaRII cDNA was held constant at 10 µg/plate and the concentration of DeltakTbetaRII was diminished. Thus, wild-type TbetaRII can rescue the dominant-negative phenotype of DeltakTbetaRII. The dominant-negative impact of DeltakTbetaRII on gene expression, which we first described in primary cultures of neonatal ventricular myocytes (32) is also detected in an altogether unrelated cell type using an unrelated promoter, and is thus a generalized, if not global, property of the kinase-deleted TbetaRII.

Potentially, one reason for the residual induction seen in the Mv1Lu cell type, but not in ventricular myocytes, might be that an incomplete fraction of cells that have incorporated the reporter gene have also taken up the DeltakTbetaRII construct. To circumvent the risk of excessive DNA content per transfected culture, we therefore transfected Mv1Lu cells with decreasing ratios of the p3TP-lux reporter relative to DeltakTbetaRII. However, even using a 1:10 ratio of reporter to receptor, residual TGFbeta-dependent activity remained, compared to control, vehicle-treated cells receiving DeltakTbetaRII (p = 0.0003). Thus, residual induction in Mv1Lu cells cannot be accounted for by failure to deliver DeltakTbetaRII to cells receiving the luciferase reporter.

As inhibition by the dominant-negative receptor could be reversed by titratable addition of wild-type receptor, in both Mv1Lu cells (Fig. 3) and cardiac myocytes(32) , we tested the alternative possibility that differences in residual receptor function might be explained by the respective levels of endogenous TGFbeta receptor. The molecular profile of TGFbeta receptors in mink lung epithelial cells and neonatal rat cardiac myocytes was compared by receptor affinity-labeling and immunoprecipitation (Fig. 4). Both cell types expressed all three receptor components, betaglycan, type II, and type I receptor, in concordance with prior studies of the cardiac myocyte(6) . Whereas the levels of betaglycan were comparable in both cell backgrounds, Mv1Lu cells contained up to 3-fold higher content of TbetaRII and TbetaRI (Fig. 4A). The identity of TbetaRII and ALK-5, respectively, as the type II and type I receptors in cardiac myocyte cultures was corroborated by coimmunoprecipitation (Fig. 4B). Thus, by the criteria detailed here, both cell types have an equivalent set of TGFbeta-binding proteins, but the higher content of signaling receptor in Mv1Lu cells may account, at least in part, for the observed differences in residual TGFbeta-induced transcription.

Point Mutations at Essential Residues of the TbetaRII Kinase Domain Disrupt TGFbeta-dependent Transcription

To gain further insight into the mechanisms underlying the dominant-negative action of DeltakTbetaRII, we mutated invariant or highly conserved amino acids in the kinase domain of full length TbetaRII ( Fig. 1and 5). Charged amino acids in subdomain II, subdomain VI-B and kinase insert II were converted to the nonpolar residue, alanine. This mutagenesis strategy has been used previously to characterize the functional domains of cyclic AMP-dependent protein kinase, which is paradigmatic for the serine/threonine kinases(38) . To summarize the rationale for this approach, basic and acidic side chains are likeliest to be exposed in the protein's tertiary structure. Consequently, these residues are more likely than internal hydrophobic ones to interact with protein substrates or cofactors. Conversely, their substitution is less likely than internal ones' to disrupt the global conformation of the protein. Finally, unlike glycine, alanine retains a beta-CH(3) group and is less likely to perturb higher-order structure. Lys-277 in subdomain II of the kinase domain of TbetaRII represents an invariant residue in all characterized protein kinase molecules (39) and is involved in the phosphotransfer reaction(40) . Three neighboring mutations were made in domain VI-B, which forms the catalytic loop of the cyclic AMP-dependent protein kinase domain and is involved in substrate binding and phosphate transfer: Arg-378 and Asp-379 are invariant across the protein kinase superfamily(41) ; Lys-381 is specific to and conserved in all serine/threonine kinases; Lys-372 and His-377 are invariant among the receptor serine/threonine kinases. Other residues selected for mutation, which are invariant among receptor serine/threonine kinases, cyclic AMP-dependent protein kinase, and the tyrosine kinase superfamily, were Asp-397, Asp-446, and Arg-528. His-362 is invariant among receptor serine-threonine kinases, and Glu-272 is highly conserved (excepting Daf-4 and the type IB and IIB activin receptors). Finally, Arg-497 and His-507 are located within kinase insert II, which has been shown recently to be necessary for the intrinsic kinase activity of TbetaRII: an internal deletion of insert II, but not insert I, abrogates kinase activity and renders the receptor non-functional(42) . In agreement with the predicted function of these residues each of these point mutations was inactive at restoring TGFbeta-dependent transcription to DR-26 cells lacking functional TbetaRII; a deletion of the serine-rich tail of the receptor, which is superfluous for receptor function(42) , is included for comparison (Fig. 5C).

Transfection of each point mutation of TbetaRII into cardiac myocytes (Fig. 5A) and Mv1Lu cells (Fig. 5B) resulted in strong inhibition of TGFbeta-dependent transcription, to a similar or greater extent than the cytoplasmic deletion mutant, DeltakTbetaRII (in cardiac myocytes, p < 0.01 for each construct, versus vector-transfected cells; in Mv1Lu cells, p < 0.05). Basal transcription of the skeletal alpha-actin promoter was affected neither by full-length TbetaRII nor by any of the point mutations, consistent with the prior finding that endogenous TGFbeta is expressed by neonatal ventricular muscle cells preponderantly in the latent, inactive form(6) . By contrast, in Mv1Lu cells, basal transcription of the 3TP-luciferase gene was augmented by wild-type receptor and inhibited by signal-defective receptors, suggesting, in this cell background, that basal activity of the TGFbeta-responsive promoter is mediated at least in part by autocrine TGFbeta. In substantiation of this inference, 3TP-luciferase activity in Mv1Lu cells was inhibited 60% (p = 0.0149) by cotransfection with 10 µg of plasmid encoding the NH(2)-terminal remnant of TGFbeta1 precursor protein, beta1 latency-associated peptide. (^2)Thus, basal activity of this TGFbeta-inducible reporter gene was repressed by a high-affinity antagonist of mature TGFbeta(43) .

To ensure the proper translation and comparable expression of the mutant receptors, COS-1 cells were transfected with representative point mutations in parallel with wild-type TbetaRII and were subjected to cross-linking with I-TGFbeta1 (Fig. 6). In each case, a 95-kDa protein band was affinity-labeled, which was specifically displaced by excess unlabeled ligand. Thus, the missense mutations were expressed, transported across the surface membrane, and competent to bind TGFbeta1, to a similar extent as the wild-type receptor (Fig. 6). Importantly, each missense mutation of TbetaRII also was competent for presentation of ligand to TbetaRI. As the extent of I-TGFbeta1 binding and the efficacy for competition by unlabeled ligand each were comparable to cells transfected with the full-length receptor, no evidence was found to suggest that the dominant-negative deletion and substitution mutants might merely bind TGFbeta more avidly than wild-type receptor. Thus, all mutations that specifically disrupt the signaling activity of TbetaRII resulted in the formation of a type II receptor that is dominant-negative for gene induction.

A Kinase-deficient Cytoplasmic Deletion Mutant of Type I TGFbeta Receptor Blocks TGFbeta-dependent Transcription

The recent cloning of six related type I receptors by several laboratories and identification of ALK-5/R4 as the signal-transducing type I TGFbeta receptor (26, 30, 33) has enabled more direct tests, to compare the putative roles assigned to TbetaRII versus TbetaRI by analysis of a corresponding deletion in the serine/threonine kinase of TbetaRI (DeltakTbetaRI). Forced expression of DeltakTbetaRI inhibited TGFbeta-induced transcription of p3TP-lux in Mv1Lu cells, whereas a kinase-deleted truncation (DeltakActRI) of the related activin type I receptor (ALK-2; ActRI) and full-length ActRI (21, 22) produced no discernible effect on p3TP-lux activity (Fig. 7). The specificity is noteworthy, given the potential for nonselective binding of TGFbeta to overexpressed, exogenous type I receptors(27) , as well as evidence that the murine homologue of ActRI (Tsk7L) can interfere with TGFbeta binding to TbetaRII under some circumstances(24) .

To compare the dominant-negative activities of DeltakTbetaRII and DeltakTbetaRI in more detail, we transfected each truncated receptor into Mv1Lu cells at multiple concentrations from 1 to 16 µg/culture, using the identical SV40-driven expression vector, and using p3TP-lux as the reporter. Both kinase truncations resulted in inhibition of TGFbeta-dependent transcription (Fig. 8); however, DeltakTbetaRI was significantly less effective than DeltakTbetaRII (p = 0.0001); a similar distinction was observed in cardiac muscle cells (p = 0.0258). In Mv1Lu cells, maximal inhibition at 16 µg of each construct was 88% for DeltakTbetaRII, versus 66% for DeltakTbetaRI; half-maximal inhibition was achieved with 3 µg of DeltakTbetaRII, versus 8 µg of DeltakTbetaRI. We further tested whether cotransfection of both kinase-truncated receptors might result in synergistic or less-than-additive inhibition. At each plasmid concentration examined, cotransfection of DeltakTbetaRI and DeltakTbetaRII plasmids together inhibited p3TP-lux as efficiently as the same total plasmid concentration of DeltakTbetaRII, but was significantly greater than inhibition produced by DeltakTbetaRI alone (p = 0.0001).

In summary, kinase-defective deletions of both the type I and type II TGFbeta receptors, as well as point mutations in essential residues of the TbetaRII kinase domain, are dominant-acting inhibitors of signal transduction by the TGFbeta receptor complex, for events that culminate in TGFbeta-dependent gene expression. Taken together, the results reported here indicate that loss of function for either type I or type II TGFbeta receptor is sufficient for dominant interference with the cascade for transcriptional activation (Fig. 9). Indeed, under the conditions we have examined, all functionally inactive receptors serve as dominant-negative inhibitors of this pathway.


Figure 9: Three classes of inactive TGFbeta receptor are dominant inhibitors of TGFbeta-dependent transcription. The prevailing genetic and biochemical evidence supports the model, shown at the left, for TGFbeta signal transduction via a heteromeric complex enabling directional phosphorylation of TbetaRI by TbetaRII(31) . Dominant inhibitors for gene regulation by TGFbeta include, from left to right, the kinase-deficient truncation of TbetaRII, all inactivating point mutations of TbetaRII analyzed thus far, and the kinase-deficient truncation of TbetaRI.




DISCUSSION

Three Classes of Inactive TGFbeta Receptor Are Dominant Inhibitors of TGFbeta-dependent Gene Expression

In the present study we have established that kinase-deficient cytoplasmic domain truncations of type II and type I TGFbeta receptors, as well as charged-to-alanine mutations at critical residues of the TbetaRII kinase domain, each can block signal transduction mediating gene regulation by TGFbeta in mink lung epithelial cells and cardiac myocytes. For the superfamily of receptor tyrosine kinases, kinase-defective mutations are known to inhibit the function of the corresponding wild-type receptors, presumably by a block to intermolecular autophosphorylation after ligand-induced dimerization, by competition for downstream effector molecules, or by a combination of these mechanisms(44, 45, 46) . We previously engineered a truncation of the type II TGFbeta receptor, devoid of the serine/threonine kinase domain, as a predicted dominant-inhibitor which might be utilized to explore the biological functions of TGFbeta. In rat ventricular muscle cells, the truncated type II TGF-beta receptor (DeltakTbetaRII) suppressed the induction of the SkA promoter by TGFbeta1, -beta2, and -beta3(32) . Moreover, DeltakTbetaRII specifically blocked TGFbeta-dependent inhibition of the alpha-myosin heavy chain gene, while sparing control of alpha-myosin heavy chain by thyroid hormone. Thus, the truncated type II receptor was sufficient, by itself, to interfere with gene regulation by all three mammalian isoforms of TGF-beta, and to block both positive and negative control of transcription(32).

Divergent conclusions have been inferred, previously, using stably transfected Mv1Lu mink lung epithelial cells. A kinase-defective truncation of the human type II receptor was competent to disrupt growth inhibition by TGFbeta but failed to suppress three representative TGFbeta-inducible genes including PAI-1(34) , suggesting that inactivation of TbetaRII might impair only pathways important for growth control, whereas gene regulation might be mediated exclusively by TbetaRI, a model that is incompatible with our results and those of Wieser et al.(42) , for a kinase-deficient truncation of TbetaRII. As our equivalent results in Mv1Lu cells and cardiac myocytes make clear, neither the use of different genes as end points nor differences in the recipient cell background account for this disparity. Although the potential for compensatory responses or fortuitous effects in stably transfected cell lines cannot be excluded, a more plausible explanation may be titratable differences in the levels of dominant-negative receptor protein achieved in transiently versus stably transfected cells.

We have identified three classes of inactive TGFbeta receptor that block the signals for control of gene expression by TGFbeta. In Mv1Lu cells, as in ventricular myocytes, a truncation of the type II TGFbeta receptor interferes with ligand-dependent activation of a TGFbeta-responsive gene. Beyond this finding, seen recently by others(42) , we have engineered 10 point mutations of the TbetaRII kinase that are defective for signaling activity in receptor-deficient cells; in primary cultures of cardiac myocytes, as well as in mink lung epithelial cells, all 10 receptors are dominant inhibitors of TGFbeta-dependent transcription. Finally, we have shown that a kinase-defective truncation of TbetaRI likewise can impair TGFbeta control of gene expression in two unrelated lineages, with no greater inhibition of these end points by the mutation of type I receptor than by the homologous mutation of TbetaRII. Thus, we find no evidence to support a ``two-pathway'' model. Although kinase-inactive variants of TbetaRII are unable to rescue the mutant phenotype in TbetaRII-deficient cells, the kinase activity of TbetaRII is not required for ligand recognition by the type I receptor (this study and (18) ). Thus, neither a block to ligand-binding by TbetaRI nor a block to appropriate expression of TbetaRI is tenable as a mechanism to account for the dominant-negative phenotype produced by the TbetaRII mutations.

The finding that a kinase-defective truncation of TbetaRII was at least as effective as truncated TbetaRI as a dominant inhibitor of TGFbeta-dependent transcription is compatible with the recent model for activation of the TGFbeta receptor complex by directional phosphorylation of TbetaRI by TbetaRII(31) . First, ligand binding by TbetaRI is contingent on presentation of TGFbeta by TbetaRII; thus, the truncated type I receptor is expected to function as a dominant-negative receptor only upon incorporation into a heteromeric complex. By contrast, the truncated type II receptor, and the inactive point mutations of TbetaRII, might act in either of two ways: by sequestering TbetaRI, in the presence of ligand, into a functionally inactive complex, or, alternatively, by competing with wild-type receptor for TGFbeta itself. As the majority of type II receptor may exist as homo-oligomers in both the presence or absence of ligand(16) , a third possibility is sequestration of wild-type TbetaRII. Finally, the relative activity of TbetaRI and TbetaRII mutations is expected to depend, in part, on relative levels of the respective endogenous receptors; the abundance of TbetaRI inferred from affinity labeling data is an estimate that reflects only the fraction of TbetaRI to which ligand has been presented by TbetaRII. Many residues of the TbetaRII kinase domain that were selected for mutagenesis (Lys-277, Arg-378/Asp-379, Lys-381, Asp-397, Arg-528) are known to be essential for activity of cyclic AMP-dependent protein kinase, the best characterized serine/threonine kinase(38) ; one exception is Asp-446, which was reportedly dispensable, notwithstanding its invariant presence throughout the protein kinase superfamily.

Thus, all mutations which failed to restore gene induction to receptor-deficient cells were inhibitors of TGFbeta-dependent transcription in wild-type mink lung epithelial cells and in cardiac myocytes. Measurements of receptor protein kinase activity will be needed to distinguish between inactivation of the kinase itself and interference with a potential effector domain. However, the meager efficiency for transfection into ventricular myocytes (typically, 1-5%) precludes a direct comparison of exogenous versus endogenous receptor in the two cell backgrounds we have used, and prevents our determining the impact of these receptor mutations on endogenous cardiac gene products or on cardiac growth control. Providing a potential means to overcome this impediment, we have demonstrated that virtually uniform gene transfer to cardiac myocytes can be achieved with recombinant adenoviruses(46) .

Dominant-negative Receptors Substantiate the Interdependence of TbetaRI and TbetaRII

Our investigations of TGFbeta-dependent gene expression in cardiac muscle cells corroborate the functional interdependence of type II and type I receptor, pointing to more general validity for a model that is heavily dependent on chemical mutagenesis of Mv1Lu cells, selection of derivatives for TGFbeta resistance(25) , and subsequent genetic manipulations of this system (18, 26, 33, 42) . Differing models of TGFbeta receptor function have been proposed. For example, EJ bladder carcinoma cells and SW480 colon carcinoma cells express TbetaRI and betaglycan but only minute amounts of TbetaRII; these cells are refractory to growth inhibition by TGFbeta, yet can respond to TGFbeta as assessed by induction of fibronectin, type IV collagenase, and PAI-1(47) . Fusion of EJ and SW480 cells results in the appearance of normal levels of TbetaRII and the restoration of growth control by TGFbeta. It has been postulated that, under these circumstances, receptor I may be responsible for transducing signals that mediate gene regulation, whereas the growth inhibitory effect of TGFbeta might preferentially be dependent on the type II receptor(47) . Hence, when the level of type II receptor falls below some operationally defined threshold level, growth regulation by TGFbeta is lost whereas gene induction still occurs: conceivably, such differences in levels of TbetaRII required for antiproliferative versus transcriptional effects could explain the reported preference for inhibition of growth control by the dominant-negative type II receptor (34) . Despite the plausibility of such deductions, caution must be observed, given the risk of circular reasoning. Cancer cells often acquire resistance to growth inhibition by TGFbeta, which may be in part related to observed changes in expression of the TGFbeta receptors, including loss of all three receptor types(48) , receptors I and II (49) , or the type II receptor alone(26, 47, 48) ; however, there is potential for unrelated or secondary events, which might account for loss of growth inhibition. Analogously, M40 endothelial cells express a type II receptor for TGFbeta that is diminished in size, are resistant to growth inhibition by TGFbeta, and might be construed as supporting the two-pathway model; however, the reduced molecular weight of TbetaRII results from a generalized defect in N-glycosylation of proteins, and is accompanied by resistance to growth inhibition by other cytokines as well(50) . By contrast, dominant-negative receptors can be employed transiently, to produce an acute loss of gene function, in a genetically normal cell background.

A more complex challenge to this model of the heteromeric receptor complex as obligatory for all actions of TGFbeta is suggested by the several non-transformed cell types, such as murine hematopoietic progenitor cells and human neutrophils, which express negligible levels of type II receptors, yet display type I receptor by cross-linking and respond to TGFbeta, measured by growth arrest and chemotaxis, respectively(51, 52, 53) . One potential explanation for this paradox is the existence of low levels of type II receptors, below the threshold for detection by conventional means(49) . Alternatively, although isoforms of the type I and type II receptor cDNAs for TGFbeta have not been isolated, comparable to the diversity of activin receptors(28, 54) , immunoprecipitation of the TGFbeta receptor complexes in various cell types, using antiserum against TbetaRI, indicates that multiple type I receptors may exist, which interact differentially with the one known form of TbetaRII(26) . Thus, it is conceivable that alternative type I and type II receptors, which differ in their biochemical characteristics and dimerization potential from those presently identified, may account for some of the discrepancies in the reported data and their interpretation. Despite the potential for heterologous type I receptors to bind TGFbeta, at least under some conditions of forced expression, no interference was produced by the kinase-defective truncation of SKR1 (ALK-2).

Dominant-negative Receptors for the TGFbeta Superfamily in Vivo

Interestingly, the genomic structure for another type I receptor for activin, SKR2 (ALK-4), resembles the activin type II receptor gene. By alternative mRNA splicing and poly(A) addition, SKR2 generates variants of the carboxyl terminus that would lack subdomain X or X plus XI; the presence of a consensus poladenylation signal within intron 8 suggests the further likelihood of a variant lacking subdomains VIII-XI, as found for the type II activin receptor(55) . Moreover, in Drosophila, dominant-negative mutations that block the effect of the TGFbeta-related factor, decapentaplegic, include substitutions in the kinase domain of type I receptors encoded by the saxophone and thick veins genes(56, 57) . Thus, truncated isoforms and missense mutations of receptors for the TGFbeta superfamily have been identified although not, to date, for TGFbeta itself. Given that a truncation of the entire ActRII cytoplasmic domain acts as a dominant-inhibitor of activin-induced mesoderm formation in Xenopus embryos(58, 59) , it is unexpected that the naturally occurring truncation is even more active than full-length ActRII(60) . However, recent precedents for the signaling activity of tyrosine kinase-defective receptors include engineered mutations of the epidermal growth factor (61) and insulin (62) receptors, and the requirement for erbB3, which lacks intrinsic kinase activity, in tandem with erbB2/neu as a heterodimeric signaling complex for heregulin (63

As one strategy to gain insight into the biological functions of endogenous growth factors, dominant-negative receptors for activin have been introduced into Xenopus embryos and explants, and substantiate a requirement for this growth factor in establishing the body plan and inducing mesoderm(58, 59) . Targeted expression of TGFbeta in its active form, in transgenic mice, supports the role of TGFbeta in development of the skin (64) and mammary gland(65, 66) , producing marked hypoplasia in both cell backgrounds. Given the multiple functions for TGFbeta1 proposed in mammalian development, including effects on proliferation, cell fate and gene expression, adhesion, migration, extracellular matrix production, and angiogenesis, it is particularly noteworthy that animals homozygous for a null mutation of TGFbeta1 that survive until birth possess no apparent morphological defects(8, 9) . (The premature, but incomplete, lethality of the mutation should not be overlooked.) Functional redundancy (in this case, the capacity of TGFbeta2, TGFbeta3, or more distant growth factors to substitute for TGFbeta1) is a potential and inescapable impediment to ready interpretation of ``knock-out'' mutations, illustrated by the redundancy among MyoD-like transcription factors(67, 68, 69) . Alternatively, a more direct explanation for the lack of a more obvious phenotype is offered by the fact that mice homozygous for the null mutation have been found to receive significant amounts of maternal TGFbeta1 across the placenta, which might rescue the developmental consequences of the presumptive knockout(10) . Targeted expression of dominant-negative TGFbeta receptors provides a potential means to overcome the redundancy of TGFbeta isoforms, to obviate the confounding effects of maternal protein, and to confine a loss-of-function mutation to an individual organ or single cell type-such as the cardiac myocyte. Dominant-negative receptors, in vivo, could complement information to be gained from other genetic models(70) .


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants R01 HL47567, P01 HL49953, and T32 HL07706 (to M. D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Fellow of the Deutsche Forschungsgemeinschaft. Present address: Institut für Biochemie und Biotechnologie, Abteilung für Zell- und Molekularbiologie, Technische Universität Braunschweig, D-38108 Braunschweig, Federal Republic of Germany.

To whom correspondence should be addressed: Molecular Cardiology Unit, One Baylor Plaza, Rm. 506C, Baylor College of Medicine, Houston, TX 77030. Tel.: 713-798-6683; Fax: 713-798-7437.

(^1)
The abbreviations used are: TGFbeta, type beta transforming growth factor; TbetaRI, type I TGFbeta receptor; TbetaRII, type II TGFbeta receptor; DeltakTbetaRI, kinase-deficient truncation of TbetaRI; DeltakTbetaRII, kinase-deficient truncation of TbetaRII; PAI-1, plasminogen activator inhibitor-1; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium.

(^2)
T. Brand and M. D. Schneider, unpublished results.


ACKNOWLEDGEMENTS

We gratefully acknowledge Drs. H. Lin and H. Lodish for H2-3FF; J. Wrana, J. Cárcamo, and J. Massagué for p3TP-lux and DR-26 cells; K. Matzuaki and W. McKeehan for SKR1; K. Miyazono and C.-H. Heldin for ALK-5; F. Ervin for invaluable assistance in cell culture; M.-J. Charng for contributions to the initial construction of TbetaRII point mutations; J. Hawker and M. Abdellatif for helpful discussions of the immunological methods; M. Majesky, B. French, and R. Schwartz for detailed comments on the manuscript; and R. Roberts for encouragement and support.


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