©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Signaling Activity of Homologous and Heterologous Transforming Growth Factor- Receptor Kinase Complexes (*)

(Received for publication, November 8, 1994)

Denis Vivien (§) Liliana Attisano Jeffrey L. Wrana (¶) Joan Massagué(**)

From the Cell Biology and Genetics Program and the Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor-beta (TGF-beta) signaling in Mv1Lu lung epithelial cells requires coexpression of TGF-beta receptors I (TbetaR-I) and II (TbetaR-II), two distantly related transmembrane serine/threonine kinases that form a heteromeric complex upon ligand binding. Here, we examine the formation of TGF-beta receptor homooligomers and their possible contribution to signaling. TbetaR-I can contact ligand bound to TbetaR-II, but not ligand free in the medium, and thus cannot form ligandinduced homo-oligomers. TbetaR-II, which binds ligand on its own, formed oligomeric complexes when overexpressed in transfected COS cells. However, these complexes were largely ligand-independent and involved immature receptor protein. Since ligand-induced homo-oligomers could not be obtained with the wild-type TGF-beta receptors, we studied receptor cytoplasmic domain homo-oligomerization by using receptor chimeras. The extracellular domain of TbetaR-II was fused to the transmembrane and cytoplasmic domains of TbetaR-I, yielding TbetaR-II/I, and the extracellular domain of TbetaR-I was fused to the transmembrane and cytoplasmic domains of TbetaR-II, yielding TbetaR-I/II. When cotransfected with wild-type receptors and exposed to ligand, TbetaR-II/I formed a complex with TbetaR-I, and TbetaR-I/II formed a complex with TbetaR-II, thus yielding complexes with homologous cytoplasmic domains. TbetaR-II/I transfected alone or with TbetaR-I did not restore TGF-beta responsiveness in TbetaR-II-defective cell mutants. Furthermore, TbetaR-II/I acted in a dominant negative fashion, inhibiting restoration of TGF-beta responsiveness by a cotransfected TbetaR-II in TbetaR-II-defective cells and by a cotransfected TbetaR-I in TbetaR-I-defective cells. Similarly, TbetaR-I/II transfected alone or with TbetaR-II did not restore TGF-beta responsiveness and acted in a dominant negative fashion against TbetaR-I. Together with previous genetic and biochemical evidence, these results suggest that TGF-beta mediates transcriptional and antiproliferative responses through the heteromeric TbetaR-IbulletTbetaR-II complex and not through homo-oligomeric TbetaR-I or TbetaR-II complexes.


INTRODUCTION

Transforming growth factor-beta (TGF-beta) (^1)is a multifunctional cytokine that binds to various membrane proteins(1, 2) . Genetic and biochemical evidence indicates that two of these proteins, known as receptor types I and II, are involved in signaling(3, 4, 5, 6, 7, 8) . Both receptors are transmembrane serine/threonine kinases and consist of a short extracellular domain, a single transmembrane region, a kinase domain, and, in the type II receptors, a serine/threonine-rich tail. Despite their similar domain structure, TGF-beta receptors I and II show only 40% amino acid sequence identity in the kinase domain and even lower similarity outside this domain. The same holds true for receptors that bind other TGF-beta-related factors such as activin and bone morphogenetic proteins (BMPs). The type I receptors for these various factors are more similar to each other than they are to their corresponding type II receptors, generating two distinct subfamilies of transmembrane serine/threonine kinases(1) .

Evidence that receptors I and II are essential for signaling derived originally from chemically induced cell mutants selected for resistance to TGF-beta action(3, 9, 10) . Some of the mutants lack binding to receptor I, and others lack binding to receptors I and II. Hybrids created by fusion of cells from one group with cells from the other recovered receptors and full responsiveness to TGF-beta(3) . These observations suggested a model in which receptor I requires receptor II for ligand binding and both receptors act in concert to elicit transcriptional and antiproliferative responses(3) . This model was recently confirmed in studies with the cloned receptors. The type II receptors identified to date can bind ligand on their own(5, 11, 12, 13, 14) , but cannot signal independently of type I receptors(4, 6, 7, 8) . The type I receptors for TGF-beta and activin can contact ligand bound to receptor II, but not ligand that is free in the medium(6, 7, 15, 16, 17) . This interaction leads to formation of a tight trimolecular complex between ligand, receptor I, and receptor II. Certain BMP type I receptors can bind ligand when transfected alone into test cells; however, they associate with cotransfected certain type II receptors and bind ligand more effectively in their presence (18, 19, 20, 21) . (^2)

The type II receptor for a given ligand may interact with different type I receptor isoforms, forming combinations of different signaling capacity(8, 22) . Antiproliferative and extracellular matrix transcriptional responses to TGF-beta in Mv1Lu mink lung epithelial cells are mediated by the TGF-beta type II receptor (TbetaR-II) in concert with the TGF-beta type I receptor (TbetaR-I)(6, 7, 8) . No TGF-beta responses are observed when TGF-beta binds to TbetaR-II expressed in the absence of TbetaR-I (4, 6, 7, 8) or to TbetaR-I via a TbetaR-II construct lacking the cytoplasmic domain (23) or when TbetaR-I and TbetaR-II are expressed in the same cell, but one of the two harbors a mutation that disrupts kinase activity(4, 7, 8) .

The interaction between TbetaR-I and TbetaR-II suggests two alternative models for TGF-beta signal transduction. In one model, TbetaR-I and TbetaR-II might signal separately, initiating distinct pathways whose cooperation produces TGF-beta responses(15) . After contacting the ligand, TbetaR-I and TbetaR-II could each signal as monomers or, by analogy with the tyrosine kinase receptors(24) , as ligand-induced homodimers. Alternatively, the signaling entity could be the heteromeric TbetaR-IbulletTbetaR-II complex. Formation of this complex might be necessary to allow each receptor access to its substrates or to allow one receptor kinase to stimulate the other, which then propagates the signal to downstream substrates. Support for the latter model comes from the recent demonstration that TbetaR-II is a constitutively active kinase that, upon ligand binding, recruits and phosphorylates TbetaR-I as a necessary step for signal propagation via the TbetaR-I kinase(25) . However, the evidence to date does not exclude the possibility that ligand-induced homologous receptor interactions might participate in TGF-beta signaling.

To investigate this possibility, we have sought evidence for ligand-induced homo-oligomeric TbetaR-I or TbetaR-II complexes. In addition, we forced homo-oligomerization of receptor cytoplasmic domains by using chimeras that contain the extracellular domain of one receptor and the transmembrane and cytoplasmic domains of the other. The results of studies using these constructs argue that homologous receptor kinase interactions do not mediate TGF-beta signals.


MATERIALS AND METHODS

Receptor Constructs

Chimeric forms of human TbetaR-II and TbetaR-I were generated by polymerase chain reaction using as templates the human TbetaR-I cDNA (6) and a cDNA encoding human TbetaR-II (5) tagged with a hexahistidine sequence at the C terminus(4) . cDNA fragments encoding the extracellular domain of TbetaR-II (amino acid residues 1-158), the transmembrane and cytoplasmic domains of TbetaR-II/His (amino acids 159-567), the extracellular domain of TbetaR-I (amino acid residues 1-124), and the transmembrane and cytoplasmic domains of TbetaR-I (amino acids 125-485) were generated by polymerase chain reaction using appropriate primers. Polymerase chain reaction conditions were 15 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C for 25 cycles. Gel-purified reaction products were then combined pairwise (TbetaR-I ectodomains with TbetaR-II transmembrane-cytoplasmic domains and vice versa) and reamplified with the corresponding primers to create cDNAs encoding the chimeras TbetaR-I/II and TbetaR-II/I. Products of these reactions were cloned into pBluescript using convenient internal restriction sites, verified by DNA sequencing, and subcloned into pCMV5 for transient transfection.

Cell Lines and Transfections

R-1B and DR-26 cells were derived in earlier studies by chemical mutagenesis of the mink lung epithelial cell line Mv1Lu (CCL-64; American Type Culture Collection) and selection for resistance to TGF-beta(9, 10) . These cells were maintained in minimal essential medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS), nonessential amino acids, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 25 ng/ml Fungizone (Life Technologies, Inc.). L(6)E(9) rat skeletal myoblasts (obtained from B. Nadal-Ginard) were maintained in minimal essential medium supplemented with 10% fetal bovine serum. COS-1 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% FBS. Cells were transfected using 100 µM chloroquine and 400 µM DEAE-dextran(26) .

Cell Labeling

TGF-beta1 from human platelets (R& Systems, Minneapolis, MN) was iodinated by the chloramine-T method(27) . Cell monolayers were affinity-labeled by cross-linking to receptor-bound I-TGF-beta1 as described previously (28) using 250 pMI-TGF-beta1 unless otherwise indicated. Labeled cell monolayers were solubilized with buffer containing Triton X-100 and protease inhibitors(28) , and soluble extracts were electrophoresed on SDS-polyacrylamide gels in the presence of 1 mM dithiothreitol.

For metabolic labeling, transiently transfected cells were incubated in methionine-free minimal essential medium containing 50 µCi/ml [S]methionine (TranS-label, ICN) for 2.5 h at 37 °C followed by 1 h at 4 °C in the presence or absence of TGF-beta (1 nM). Cell monolayers were then washed once in ice-cold phosphate-buffered saline and lysed in buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% (v/v) Triton X-100, and protease inhibitor mixture).

Immunoprecipitation

Labeled cells were solubilized in 0.05 ml/cm^2 lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.5% Triton X-100) in the presence of protease inhibitors at 4 °C for 30 min. Insoluble debris was removed by microcentrifugation at 10,000 rpm for 5 min. HA-tagged receptors were immunoprecipitated for 1 h at 4 °C with 5 µg/ml monoclonal antibody 12CA5 (BABCO), followed by adsorption to protein G-Sepharose (Pharmacia Biotech Inc.). Histidine-tagged receptors were collected by binding to Ni-NTA-agarose (QIAGEN Inc.) for 1 h at 4 °C. Two-step precipitation was done by eluting material bound to Ni-NTA-agarose beads with 0.25 M imidazole at room temperature for 10 min, followed by immunoprecipitating the eluate with anti-HA antibody. Agarose-bound receptors were eluted by heating in SDS-PAGE sample buffer.

Luciferase Assay

Exponentially growing cells plated and grown for 48 h to 75% confluence were transfected with receptor constructs of interest and/or vector alone (up to 3 µg of total plasmid DNA) plus 3 µg of the reporter plasmid p3TP-Lux(8) . 24 h later, cells were incubated for 3 h in medium containing 0.2% FBS, followed by the addition of TGF-beta1. Cells were harvested 20 h later and assayed for luciferase activity using a commercial system as described by the manufacturer (Promega). Total light emission during the initial 20 s of the reaction was measured with a luminometer (Berthold Lumat LB 9501).

DNA Synthesis Assay

Cells were plated 24 h after transfection into 24-well plates containing medium with 10% FBS. After 3 h, the medium was replaced with medium containing 0.2% FBS. After 4 h, TGF-beta1 was added for 17 h. Cells were labeled with 0.5 µCi/ml [I]iododeoxyuridine (DuPont NEN) for the last 4 h of incubation. Cells were washed three times with cold phosphate-buffered saline, fixed with 95% methanol for 1 h at 4 °C, washed twice with phosphate-buffered saline, and extracted with 1 N NaOH for 30 min at 4 °C. The extracts were collected and counted in a -counter.


RESULTS

TGF-beta Type II Receptor Complexes in COS Cells

To determine whether TbetaR-II can form homo-oligomers, COS cells were cotransfected with expression vectors encoding human TbetaR-II forms tagged with two different retrieval sequences. TbetaR-II was tagged at the C terminus with either the influenza virus HA epitope (TbetaR-II/HA) or a hexahistidine sequence (TbetaR-II/His) that can bind to Ni chelated onto agarose (Ni-NTA-agarose)(4, 8) . Like wild-type TbetaR-II, these tagged TbetaR-II forms bind ligand on the cell surface as determined by cross-linking to I-TGF-beta1 (the isoform used throughout this study)(4) , are able to form heteromeric complexes with TbetaR-I as determined by coprecipitation from cell lysates(4, 6) , and have normal signaling capacity as determined by their ability to restore TGF-beta responsiveness in TbetaR-II-deficient cell lines(4) .

To determine the relative levels of functional TbetaR-II/HA and TbetaR-II/His expressed on the surface of cotransfected COS cells, receptors were affinity-labeled by cross-linking to bound I-TGF-beta1 and precipitated from cell lysates with anti-HA antibody or Ni-NTA-agarose beads, respectively (Fig. 1A). The yield of labeled TbetaR-II/HA was 3-fold less than the yield of labeled TbetaR-II/His; however, this difference is attributable to different recoveries in the two isolation procedures. Previous work indicated that precipitation with anti-HA antibody recovers only 30% of the total affinity-labeled TbetaR-II/HA, whereas precipitation with Ni-NTA-agarose recovers TbetaR-II/His almost quantitatively(16, 25) . Taking this difference into account, the results indicated that functional TbetaR-II/HA and TbetaR-II/His are expressed at equivalent levels on the surface of COS cells.


Figure 1: TGF-beta type II receptors do not form ligand-dependent homo-oligomers. A, COS cells were transiently transfected with HA-tagged TbetaR-II alone, His-tagged TbetaR-II alone, both receptor vectors, or empty pCMV5 vector. Lysates from transfected cells affinity-labeled with I-TGF-beta1 were precipitated with anti-HA antibody or Ni-NTA-agarose beads as indicated. Another aliquot was incubated with Ni-NTA-agarose, and the eluate from these beads was precipitated with anti-HA antibody. Precipitated material was visualized by SDS-PAGE and autoradiography. B, COS cells, Mv1Lu cells, and L(6)E(9) cells were transiently transfected with HA-tagged TbetaR-II and metabolically labeled with [S]methionine in methionine-free medium for 30 (COS) or 10 (L(6)E(9) and Mv1Lu) min, and the label was chased with regular medium for the indicated periods. Cell lysates were precipitated with HA antibody, and the precipitate was subjected to SDS-PAGE and fluorography. The immature TbetaR-II core and the mature TbetaR-II product are indicated. C, COS cells transfected with the indicated TbetaR-II constructs or empty vector were metabolically labeled with [S]methionine for 2.5 h and then incubated for 1 h at 4 °C in the presence or absence of 1 nM TGF-beta1. Cell lysates were precipitated with anti-HA antibody either directly or after one cycle of binding and elution from Ni-NTA-agarose.



More important, when these cell lysates were bound to Ni-NTA-agarose and eluted with imidazole, and the eluates were precipitated with anti-HA antibody, little or no ligand-labeled TbetaR-II was recovered (Fig. 1A). This two-step precipitation method can readily recover ligand-induced complexes of TbetaR-II/His and TbetaR-I/HA from COS cells (see Fig. 3)(25) . The present results suggested that TbetaR-II did not form stable ligand-induced homo-oligomers in COS cells.


Figure 3: Cell-surface expression of TbetaR-II/I and TbetaR-I/II and association with wild-type receptors. Confluent monolayers of COS-1 cells (A and B) or R-1B cells (C and D) transfected with the indicated receptor combinations or empty pCMV5 vector were affinity-labeled by sequential incubation with I-TGF-beta1. Cell lysates were subjected to SDS-PAGE and autoradiography either directly (A and C) or after precipitation with a monoclonal antibody against the HA epitope tagged onto TbetaR-I or with Ni-NTA-agarose beads that bind hexahistidine sequences tagged onto TbetaR-II or TbetaR-I/II (B and D). Labeled material was visualized by PAGE and autoradiography.



Since COS cells have been used in many TGF-beta receptor studies, we examined the interaction of transfected TbetaR-II in more detail by immunoprecipitation of metabolically labeled receptor. Pulse-chase labeling with [S]methionine revealed that TbetaR-II was synthesized as a 70-kDa product that was converted with time into a heterogeneous product of 80 kDa (Fig. 1B). Based on previous studies(30) , these products are identified as the newly synthesized TbetaR-II core before N-linked glycan chain maturation and the mature TbetaR-II glycoprotein, respectively. Metabolically labeled receptor began to disappear 2 h after the start of the chase (Fig. 1B), at which time only about half of the receptor population had reached maturity. Surface labeling of TbetaR-II-transfected COS cells indicated that immature TbetaR-II did not reach the cell surface (data not shown; see (25) ). Continued labeling with [S]methionine confirmed that only about half of the steady-state receptor population was the mature form (Fig. 1C). COS cells support episomal replication of vectors that contain an SV40 origin of replication(29) , resulting in vast overexpression of products encoded by these vectors. In contrast to COS cells, Mv1Lu mink lung epithelial cells and L(6)E(9) rat skeletal myoblasts, which expressed stably transfected TbetaR-II at modest levels, completely processed this receptor within 60 min after synthesis (Fig. 1B). Collectively, these results argue that transiently transfected COS cells express more TbetaR-II protein than they can fully process.

To search for homo-oligomeric TbetaR-II interactions, COS cells transfected with TbetaR-II/HA, TbetaR-II/His, or the two receptors were metabolically labeled and subjected to the two-step precipitation protocol described above to recover complexes containing both tagged receptor forms. As expected, lysates from singly transfected cells yielded no labeled products with this protocol (Fig. 1C). Two-step precipitation of lysates from doubly transfected cells yielded a large amount of immature TbetaR-II core and very little mature receptor form (Fig. 1C). The amount of immature TbetaR-II form recovered in this process was approximately half of the total amount, as determined from the autoradiographic signals (Fig. 1C, compare the second and fifthlanes). The receptor yield was only marginally increased by incubation of cells with a high concentration (1 nM) of TGF-beta1 (Fig. 1C). These results indicate that in COS cells overexpressing TbetaR-II, a large proportion of excess immature TbetaR-II and a small proportion of mature TbetaR-II form ligand-independent complexes and that the addition of ligand has little effect on the yield of these complexes. Similar experiments performed with Mv1Lu cells did not yield ligand-independent or -dependent TbetaR-II complexes (data not shown).

Binding Activity and Interactions of Chimeric TGF-beta Receptors

Since neither wild-type TbetaR-I nor TbetaR-II appeared to form ligand-induced homo-oligomers, to investigate the activity of homologous TGF-beta receptor kinase interactions we forced their formation using chimeric receptor constructs. The chimera TbetaR-I/II was created by fusing the entire extracellular domain of human TbetaR-I (up to Val(6) ) with the transmembrane and intracellular domains of human TbetaR-II (from Glu(5) ) (Fig. 2). The reciprocal chimera, TbetaR-II/I, was generated by fusing the extracellular domain of TbetaR-II with the transmembrane and cytoplasmic domains of TbetaR-I (Fig. 2). We reasoned that TbetaR-I/II might be able to form ligand-induced complexes with TbetaR-II, thus leading to the association of two or more TbetaR-II cytoplasmic domains. Likewise, TbetaR-II/I might form ligand-induced complexes with TbetaR-I that should contain multiple TbetaR-I cytoplasmic domains.


Figure 2: Schematic representation of TGF-beta receptor chimeras. Boxes represent the receptor kinase domains. The amino acid positions of each receptor portion at the chimera break points are indicated by single letter code.



When transfected into COS cells, TbetaR-II/I had the ligand binding and receptor interaction properties of wild-type TbetaR-II, as determined by affinity labeling with I-TGF-beta. TbetaR-II/I bound ligand when expressed alone (Fig. 3A), supported ligand binding to a cotransfected TbetaR-I (Fig. 3A), and formed a coprecipitating complex with TbetaR-I (Fig. 3B). Thus, TbetaR-II/I was able to bind ligand in concert with TbetaR-I, generating oligomeric receptor complexes with multiple TbetaR-I cytoplasmic domains. Similar results were obtained by transfection of TbetaR-II/I and TbetaR-I into R-1B cells (Fig. 3, C and D). R-1B cells are a chemically induced mutant derivative of the Mv1Lu cell line and lack TGF-beta responses because of a defect in endogenous TbetaR-I(9) . Untransfected R-1B cells express endogenous TbetaR-II at levels much lower than those of transfected TbetaR-II, hence the lack of TbetaR-II signal in the autoradiographs shown here.

Like TbetaR-I, the chimera TbetaR-I/II did not bind TGF-beta when transfected alone into COS or R-1B cells (Fig. 3, A and C). Cotransfection of a hexahistidine-tagged TbetaR-I/II with TbetaR-II, followed by affinity labeling and precipitation with Ni-NTA-agarose, showed coprecipitation of TbetaR-II with TbetaR-I/II (Fig. 3, B and D). This complex was ligand-dependent as demonstrated by two-step precipitation of metabolically labeled receptors from cells expressing TbetaR-II/HA and hexahistidine-tagged TbetaR-I/II (Fig. 4A). Under these conditions, TbetaR-I/II did not become efficiently labeled by cross-linking to bound I-TGF-beta (Fig. 3). However, TbetaR-I/II labeling was detected when a higher concentration of I-TGF-beta (1 nM instead of 250 pM) was used in the assays (Fig. 4B). The low labeling efficiency could be due to formation of anomalous contacts between TGF-beta and TbetaR-I/II, diminishing the efficiency of the cross-linking reaction. Nevertheless, TbetaR-I/II was clearly capable of forming ligand-induced complexes with TbetaR-II, generating oligomers containing multiple TbetaR-II cytoplasmic domains.


Figure 4: Ligand-dependent interaction of TbetaR-I/II with TbetaR-II. A, R-1B cells cotransfected with hexahistidine-tagged TbetaR-I/II and HA epitope-tagged TbetaR-II were metabolically labeled with [S]methionine and then incubated for 1 h at 4 °C in the presence or absence of 1 nM TGF-beta1. Cell lysates were precipitated with Ni-NTA-agarose beads, the beads were eluted with imidazole, and the eluate was precipitated with HA antibody. Immunoprecipitates were subjected to SDS-PAGE and autoradiography, which demonstrated a ligand-dependent interaction between the two receptors. B, R-1B cells cotransfected with TbetaR-I/II and TbetaR-II were affinity-labeled with the indicated concentrations of I-TGF-beta1, revealing labeling of TbetaR-I/II only at the high I-TGF-beta1 concentration.



Signaling Activity of Chimeric TGF-beta Receptors

We determined the signaling properties of TGF-beta receptor complexes by using the R-1B and DR-26 cell lines. DR-26 cells are resistant to TGF-beta due to a nonsense mutation in the transmembrane region of endogenous TbetaR-II(4) . As a result, DR-26 cells lack TbetaR-II on the surface and are unable to bind TGF-beta through endogenous TbetaR-I. TGF-beta binding and responsiveness are fully restored by transfection of wild-type TbetaR-II into DR-26 cells (4) or by transfection of wild-type TbetaR-I into R-1B cells(6, 7, 8) .

TGF-beta receptors, singly or in relevant combinations, were cotransfected with the TGF-beta-inducible construct p3TP-Lux, which contains a luciferase reporter gene(4) . Transfection of TbetaR-I alone or in combination with TbetaR-II restored TGF-beta responsiveness in R-1B cells, whereas transfection of TbetaR-II/I did not (Fig. 5A). Cotransfection of TbetaR-II/I prevented TbetaR-I from restoring this TGF-beta response (Fig. 5A). Similar results were obtained with the antimitogenic effect of TGF-beta under conditions (16) in which 60% of the R-1B cell population takes up transfected TbetaR-I cDNA and becomes inhibited in response to TGF-beta (Fig. 6). As determined by [I]iododeoxyuridine incorporation into DNA, the ability of R-1B cells to respond to TGF-beta was not diminished by cotransfection of TbetaR-II, but was blocked by cotransfection of TbetaR-II/I (Fig. 6).


Figure 5: Dominant negative effect of TbetaR-II/I on TGF-beta transcriptional responses. R-1B cells (A) or DR-26 cells (B) were transiently cotransfected with the indicated receptor vectors and/or empty pCMV5 to equalize the amount of DNA in the transfections. A fixed amount of the reporter vector p3TP-Lux was included in all transfections. TGF-beta1 (250 pM) was added 36 h later. Cell extracts were prepared 20 h later, and luciferase activity was determined in a luminometer.




Figure 6: Dominant negative effect of TbetaR-II/I on TGF-beta antiproliferative response. R-1B cells were transfected with the indicated receptor vectors under conditions that yield 60% transient transfection efficiency. TGF-beta1 (250 pM) was added 36 h later for 20 h. [I]Iododeoxyuridine (I-dU) was added during the last 4 h of incubation, and its incorporation into DNA was determined. Data are presented as the percent inhibition of [I]iododeoxyuridine incorporation relative to controls that did not receive TGF-beta. Results are the means ± S.D. of triplicate determinations.



These results indicate that ligand-induced association of TbetaR-I intracellular domains via the TbetaR-I and TbetaR-II/I combination does not generate transcriptional or antimitogenic responses to TGF-beta in R-1B cells. Instead, TbetaR-II/I acted in a dominant negative fashion presumably by competing with endogenous TbetaR-II for transfected TbetaR-I or by titrating out a substrate. TbetaR-II/I was also unable to signal when transfected alone or with TbetaR-I into DR-26 cells (Fig. 5B) and inhibited the rescue of TGF-beta responsiveness by TbetaR-II in these cells (Fig. 5B). This effect was probably due to competition for the limited amount of endogenous TbetaR-I. These results provide further evidence that complexes of TbetaR-II/I with either endogenous or cotransfected TbetaR-I lack the signaling capacity of heteromeric TbetaR-IbulletTbetaR-II complexes.

We determined the signaling capacity of TbetaR-I/II by transfection of this construct into R-1B cells. In these transfectants, TGF-beta concentrations up to 2 nM did not elicit a transcriptional response (Fig. 7) or an antiproliferative response (data not shown). Furthermore, the ability of transfected TbetaR-I to restore responsiveness in R-1B cells, which was increased by cotransfection with TbetaR-II in some experiments, was decreased by cotransfection with TbetaR-I/II (Fig. 7). This dominant negative effect was most evident at the higher TGF-beta concentration range (Fig. 7), where the ability of transfected TbetaR-I/II to compete for the limited pool of endogenous TbetaR-II would be greater. Thus, ligand-induced association of TbetaR-II intracellular domains via the TbetaR-II and TbetaR-I/II combination was ineffective as a mediator of TGF-beta responses in Mv1Lu cells.


Figure 7: Effect of TbetaR-I/II on the TGF-beta transcriptional response. R-1B cells were transiently transfected with empty pCMV5 (open circles), TbetaR-I vector (closed circles), TbetaR-I and TbetaR-II vectors (open squares), TbetaR-I/II vector (closed squares), or TbetaR-I and TbetaR-I/II vectors (closed triangles). The amount of DNA in all transfections was equalized by the addition of an appropriate amount of empty pCMV5. The p3TP-Lux vector was included in all transfections. 36 h after transfection, TGF-beta1 was added at the indicated concentrations. Cell extracts were prepared after 20 h, and luciferase activity was determined in a luminometer. Data are the average of triplicate determinations, with standard deviations smaller than the size of the symbols.



Cotransfection of TbetaR-I/II and TbetaR-II/I into R-1B cells and incubation with up to 1 nMI-TGF-beta yielded a very low level of receptor complex, as determined by the two-step precipitation assay (data not shown). The untagged versions of these two chimeras did not restore TGF-beta responsiveness when cotransfected into R-1B cells (data not shown). However, the weak interaction of these two constructs precluded interpretation of these results.


DISCUSSION

Tyrosine kinase receptors are usually activated by ligand-induced homodimerization(24) . In contrast, the serine/threonine kinase receptors for TGF-beta and related factors form ligand-induced heterodimers. The type II receptors for TGF-beta and activin bind ligand when expressed alone, whereas the type I receptors contact ligand only when coexpressed with the corresponding type II receptors, with both receptors forming a tight heteromeric complex(1, 4, 6, 7, 25) . Certain type I receptors for BMP deviate from this rule since they bind ligand when transfected alone into COS cells(19, 20, 21) . However, they still form ligand-induced complexes with a coexpressed BMP-type II receptor (19, 20, 21) .^2 The existence of these heterologous interactions, together with genetic evidence that TGF-beta signaling requires the presence of receptor types I and II in the same cell(3) , led to the recent demonstration that TbetaR-II is a constitutively active kinase that can phosphorylate an associated TbetaR-I, which then propagates the signal to downstream substrates(25) .

Although the evidence for TGF-beta-induced heteromeric receptor complexes is extensive, the homodimeric nature of TGF-beta raises the possibility that its receptors might also form ligand-induced homodimers. In the case of TbetaR-II, this possibility is in principle compatible with the ability of this receptor to bind ligand on its own. However, the present results argue against the existence of ligand-induced TbetaR-II homo-oligomers. We searched for such receptor complexes using a two-step precipitation method that readily reveals ligand-induced association of TbetaR-I with TbetaR-II(25) . This method relies on the retrieval of receptors via sequences tagged onto their C termini. This modification is not likely to interfere with TbetaR-II complex formation since the C-terminal tail of TbetaR-II is dispensable for signaling(23) , and its extension with short sequence tags does not disrupt association with TbetaR-I or signaling activity(25) .

Using this approach, a large proportion of TbetaR-II in transfected COS cells was found as oligomers. However, these oligomers are ligand-independent and mostly include immature receptor protein. COS cells constitutively express SV40 large T antigen, which allows episomal replication of expression vectors containing an SV40 origin of replication, resulting in vast overexpression of the encoded protein (29) . Our results show that the amount of TbetaR-II expressed by this method in COS cells saturates the cell's capacity to properly process the protein and forward it to the plasma membrane. As a result, a large proportion of TbetaR-II in transfected COS cells accumulates as immature core. In contrast, Mv1Lu mink lung epithelial cells and L(6)E(9) rat skeletal myoblasts, expressing transfected TbetaR-II at lower levels than COS cells, showed less accumulation of the immature TbetaR-II form or formation of ligand-independent complexes.

Collectively, the evidence suggests that formation of TbetaR-II homo-oligomers in COS cells is ligand-independent and results largely from receptor overexpression. In agreement with recent observations (32, 33) , we found that a proportion of mature TbetaR-II protein forms ligand-independent complexes. The proportion of mature TbetaR-II that we detected in complexes was very small, a result that is in contrast with the more substantial levels reported by others(32, 33) . This discrepancy could be due to differences in receptor expression levels or in stability of the receptor complex under the different precipitation conditions.

Although TbetaR-I contains a ligand-binding domain, the possibility of ligand-induced TbetaR-I homo-oligomerization is precluded by the fact that this receptor requires the presence of TbetaR-II in order to bind ligand(4, 6, 7, 15, 25) . At variance with this notion, it has been reported that human kidney 293 cells appear to bind TGF-beta solely through the type I receptor in receptor affinity labeling assays(31) . However, immunoprecipitation of affinity-labeled cell lysates with anti-TbetaR-II antibodies has demonstrated the presence of TbetaR-II in 293 cells and its association with TbetaR-I. (^3)

The stoichiometry of the TbetaR-IbulletTbetaR-II complex has not been established yet. However, since the ligand is itself a disulfide-linked homodimer, the stoichiometry of the receptor complex could be higher than 1:1. Evidence supporting this notion has been provided by recent receptor cross-linking experiments(34) . This raises the possibility of interactions between homologous kinase domains within the ligand-induced heteromeric receptor complex. Therefore, we probed the signaling capacity of ligand-induced homologous kinase complexes by using chimeras containing the extracellular domain of one receptor and the transmembrane and cytoplasmic domains of the other. The binding properties of these chimeras are those expected from the extracellular domains they contain. The chimera TbetaR-II/I containing the extracellular domain of TbetaR-II and the transmembrane and cytoplasmic domains of TbetaR-I binds TGF-beta when transfected alone and recruits a cotransfected TbetaR-I into a complex with ligand. This complex contains TbetaR-I cytoplasmic domains contributed by both TbetaR-I and TbetaR-II/I. However, this complex does not generate TGF-beta responses when expressed in receptor-defective cells. In fact, TbetaR-II/I acts in a dominant negative fashion, preventing wild-type receptors from signaling. This dominant negative effect presumably results from competition of TbetaR-II/I with endogenous TbetaR-II for transfected TbetaR-I or from competition with TbetaR-I for a rate-limiting substrate. These results indicate that homo-oligomerization of the TbetaR-I cytoplasmic domain is not sufficient to generate TGF-beta responses. This phenomenon is of particular interest since the TbetaR-I kinase domain is the downstream signaling element in the heteromeric receptor complex(25, 35) .

An analogous conclusion can be drawn from our results with the TbetaR-I/II chimera. TGF-beta readily induces association of TbetaR-I/II with TbetaR-II, yielding a complex that contains TbetaR-II cytoplasmic domains contributed by both TbetaR-II and TbetaR-I/II. Formation of this complex does not lead to signaling activity, but rather interferes with TbetaR-I in a dominant negative fashion. The limited ability of TbetaR-I/II and TbetaR-II/I to form a complex in the presence of ligand did not allow us to determine if their intracellular domains can generate a response when brought together by ligand via heterologous ectodomains (but see note added in proof).

In conclusion, wild-type TbetaR-I and TbetaR-II do not appear to form stable ligand-induced homo-oligomers even in cells overexpressing this receptor. Furthermore, when ligand-induced homo-oligomerization of TGF-beta receptor cytoplasmic domains is enforced with the use of receptor chimeras, it is not sufficient to generate a signal. These observations are consistent with the general conclusion that TGF-beta signaling is the result of an interaction between TbetaR-I and TbetaR-II. Together with the observation that TbetaR-II is a constitutively active kinase(25) , these results argue that the ligand-induced homodimerization/activation phenomenon characteristic of tyrosine kinase receptors (24) is not shared by the TGF-beta receptors. The main role of the ligand appears to be the induction or stabilization of a complex between TbetaR-II and its substrate, TbetaR-I, allowing phosphorylation and activation of TbetaR-I as a requirement for signal propagation.


FOOTNOTES

*
This work was supported in part by Grant CA34610 from the National Institutes of Health (to J. M.) and Cancer Center Support Grant P30-CA08748. 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.

§
Recipient of a postdoctoral fellowship from the Association for Cancer Research (France).

Medical Research Council of Canada Postdoctoral Fellow.

**
Howard Hughes Medical Institute Investigator. To whom correspondence should be addressed: Box 116, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212-639-8975; Fax: 212-717-3298.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; BMP, bone morphogenetic protein; TbetaR, TGF-beta receptor; FBS, fetal bovine serum; Ni-NTA, nickel-nitrilotriacetic acid; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis.

(^2)
Letsou, A., Arora, K., Wrana, J. L., Simin, K., Twombly, V., Jamal, J., Staehling-Hampton, K., Hoffmann, F. M., Gelbart, W. M., Massagué, J., and O'Connor, M. B.(1995) Cell, in press.

(^3)
F. Ventura and J. Massagué, unpublished observations.


ACKNOWLEDGEMENTS

We thank K. Miyazono and C.-H. Heldin for the TbetaR-I cDNA and H. Hernandez and E. Montalvo for expert technical assistance.

Note Added in Proof-After submission of this manuscript, Okadome et al.(36) reported an experimental strategy, results, and conclusions similar to those reported here, and additionally reported signaling by TbetaR-I/II cotransfected with TbetaR-II/I.


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