(Received for publication, April 9, 1997, and in revised form, June 12, 1997)
From the Ludwig Institute for Cancer Research, Box 595, S-751 24 Uppsala, Sweden and § The Cancer Institute, Tokyo, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan
Members of the transforming growth factor-
(TGF-
) superfamily signal via different heteromeric complexes of two
sequentially acting serine/threonine kinase receptors, i.e.
type I and type II receptors. We generated two different chimeric
TGF-
superfamily receptors, i.e. T
R-I/BMPR-IB,
containing the extracellular domain of TGF-
type I receptor
(T
R-I) and the intracellular domain of bone morphogenetic protein
type IB receptor (BMPR-IB), and T
R-II/ActR-IIB, containing the
extracellular domain of TGF-
type II receptor (T
R-II) and the
intracellular domain of activin type IIB receptor (ActR-IIB). In the
presence of TGF-
1, T
R-I/BMPR-IB and T
R-II/ActR-IIB formed
heteromeric complexes with wild-type T
R-II and T
R-I,
respectively, upon stable transfection in mink lung epithelial cell
lines. We show that T
R-II/ActR-IIB restored the responsiveness upon
transfection in mutant cell lines lacking functional T
R-II with
respect to TGF-
-mediated activation of a transcriptional signal,
extracellular matrix formation, growth inhibition, and Smad
phosphorylation. Moreover, T
R-I/BMPR-IB and T
R-II/ActR-IIB formed
a functional complex in response to TGF-
and induced phosphorylation
of Smad1. However, complex formation is not enough for signal
propagation, which is shown by the inability of T
R-I/BMPR-IB to
restore responsiveness to TGF-
in cell lines deficient in functional
T
R-I. The fact that the TGF-
1-induced complex between
T
R-II/ActR-IIB and T
R-I stimulated endogenous Smad2
phosphorylation, a TGF-
-like response, is in agreement with the
current model for receptor activation in which the type I receptor
determines signal specificity.
Transforming growth factor-s
(TGF-
s),1 activins, and
bone morphogenetic proteins (BMPs) are structurally related proteins that play important roles in intercellular communication (reviewed in
Refs. 1-4). TGF-
superfamily members regulate proliferation, differentiation, migration, and apoptosis of many cell types. Signaling
occurs via ligand-induced complex formation of two related serine/threonine kinase receptors, i.e. type I and type II
receptors (reviewed in Refs. 2, 3, and 5-7). Multiple type I and type
II receptors have been identified and matched with their corresponding
ligands in the TGF-
superfamily. The overall structures of type I
and type II receptors are similar and consist of relatively short
cysteine-rich extracellular domains, single transmembrane domains, and
intracellular parts that consist almost entirely of serine/threonine
kinase domains. Phylogenetic analysis of the serine/threonine kinase
receptor family reveals that the type I and type II receptors form two
distinct subfamilies. Type II receptors, but not type I receptors, have
carboxyl-terminal extensions of variable lengths that are rich in
serine and threonine residues; whereas type I receptors, but not type
II receptors, have a GS domain in the intracellular juxtamembrane
region, which is rich in glycine and serine residues.
Biochemical as well as genetic approaches have indicated that both type
I and type II receptors are essential for signaling. Mink lung
epithelial (Mv1Lu) cells lacking functional TR-I (termed R mutants)
or T
R-II (termed DR mutants) fail to respond to TGF-
(8). Their
responsiveness to TGF-
is restored upon ectopic expression of
T
R-I in R mutant and T
R-II in DR mutant cells, but not by other
type I or type II receptors (9-12). Whereas the type II receptors for
TGF-
and activin can bind ligand by themselves (13-15), type I
receptors need type II receptors for ligand binding (9). In contrast,
BMPs show weak binding to type II as well as to type I receptors
individually, and high affinity binding is found when both receptors
are present (16-18). However, similar to the case for TGF-
and
activin, a BMP-induced transcriptional activation signal requires
cotransfection of both type I and type II receptors, and signal
transduction is not observed when one receptor type is singly
transfected into Mv1Lu cells (16, 17). Genetic analysis in
Drosophila also indicates that both type I and type II
receptors are essential for signaling by decapentaplegic, a BMP-like
factor (19).
Recent studies have led to the formulation of a common model for signal
transduction via serine/threonine kinase receptors (20). TGF- type
II receptor (T
R-II) and activin type II receptors (ActR-IIs) are
constitutively active kinases (20-23). The type I receptor is
recruited into the complex upon ligand binding to the type II receptor;
the type I receptor is thereafter phosphorylated by the type II
receptor, predominantly on serine and threonine residues in the GS
domain (20, 24). The importance of T
R-I phosphorylation in signaling
was shown by the impairment of TGF-
signaling when multiple serine
and threonine residues in the GS domain of T
R-I were mutated (25,
26), by activation-defective mutants of T
R-I (27), and by an
activation-defective T
R-II mutant that fails to recognize T
R-I as
a substrate (28). The notion that type I receptors act downstream of
type II receptors is supported by the ability of type I receptors to
determine distinct signaling responses (29, 30) and by constitutively
active mutants of T
R-I and ActR-IB to signal in the absence of
ligand and type II receptor (22, 24, 25).
Members of the Smad family of proteins have been shown to play a key
role in the intracellular signaling pathways of TGF- superfamily
members. After activation by serine/threonine kinase receptors, Smads
proteins become phosphorylated and translocate to the nucleus, where
they may play a role in the transcriptional regulation (31-35). Smad1
and Smad5 are phosphorylated and translocated into the nucleus upon BMP
receptor activation (34,
35),2 whereas TGF-
and
activin induce the phosphorylation and translocation of both Smad2 and
Smad3 (36-38).3
Previously, we and others have used chimeras of type I and type II
receptors to demonstrate that the intracellular domains of type I and
type II receptors each serve distinct roles in signaling (39-43). In
the present study, we investigated the functional properties of
chimeric receptors in which the intracellular domains of type I or type
II receptors for TGF- were replaced with the corresponding domains
of BMPR-IB and ActR-IIB, respectively. Our data indicate that the
intracellular domain of T
R-II can be replaced by the intracellular
domain of ActR-IIB, which is a receptor for activin as well as for BMP
(44), with retained ability to activate T
R-I and to induce
TGF-
-like responses. In contrast, T
R-I/BMPR-IB was unable to
induce any signal in complex with T
R-II, although it was shown to be
functional in complex with T
R-II/ActR-IIB in both a transcriptional
activation assay and in a Smad phosphorylation assay. This indicates
that complex formation is necessary but not sufficient for signal
transduction. Moreover, the ligand-induced phosphorylation of
endogenous Smad2, but not of endogenous Smad5, by T
R-II/ActR-IIB in
complex with T
R-I confirmed the notion that the signal specificity
is controlled by the type I receptor.
COS-1 cells and Mv1Lu cells were obtained from
American Type Culture Collection. Mv1Lu cells that lack functionally
active TR-I (R 4-2 mutant cells) or T
R-II (DR 26 mutant cells)
(8) were provided by Dr. J. Massagué. Cells were cultured in 5%
CO2 at 37 °C in Dulbecco's modified Eagle's medium
(DMEM) (Life Technologies, Inc.) with 10% fetal bovine serum (FBS;
Life Technologies, Inc.), 100 units of penicillin, and 50 µg/ml
streptomycin.
We
employed a two-step polymerase chain reaction (PCR) using a
Perkin-Elmer thermal cycler with Pyrococcus furiosus DNA
polymerase (Stratagene) to generate the chimeric receptors (Fig. 1).
cDNAs for human TR-I (10), mouse BMPR-IB (12), human T
R-II
(13), and mouse ActR-IIB1 (15) were used as templates. For
the T
R-I/BMPR-IB chimera, in the first PCR step primer pairs 5a
(sense; 5
-GCTCCCGGGGGCGACGGCGTT-3
) and 5b (antisense;
5
-CTTGTGGTGTATAGGACCAAGGCCAGGTGATGA-3
) were used with
T
R-I as template, and primer pairs 6a (sense;
5
-GGCCTTGGTCCTATACACCACAAGGCCTTGCT-3
) and 6b (antisense;
5
-CCGAGCTCTGAGACTGCTCGATC-3
) with BMPR-IB as template. Primers 5b and
6a are complementary to each other (underlined sequences). In the
second PCR step, the two PCR products were used as template along with
the terminal primers 5a and 6b; the chimeric type I receptor PCR
product was subcloned, and the DNA was sequenced. Subsequently, a
EcoRI-XmaI fragment of T
R-I/pSV7d encoding the
N-terminal part of T
R-I, a XmaI-SacI fragment
of chimeric type I PCR recombinant, and a
SacI-BamHI fragment of BMPR-IB encoding the
C-terminal part of BMPR-IB were ligated together by subsequent
subcloning steps in pMEP4 (Invitrogen). For the T
R-II/ActR-IIB
chimera, in the first PCR step primer pairs 21 (sense;
5
-GCGGAATTCGGGTCTGCCATGGGTCGGGGG-3
) and 22 (antisense; 5
-AGGTTTCCGATGGCGGTAGCAGTAGAAGATGATG-3
) were used with
T
R-II as template, and primer pairs 23 (sense;
5
-CTACTGCTACCGCCATCGGAAACCTCCCTACGGC-3
) and 24 (antisense; 5
-CCGAGAGACGAGCTCCCACAG-3
) were used with ActR-IIB as
template. Primers 22 and 23 are complementary to each other (underlined
sequences), and primer 21 contains an extra 5
-EcoRI
restriction site. In the second PCR step, the two PCR products were
used as template along with the terminal primers 21 and 24; the
chimeric type II receptor PCR product was subcloned, and the DNA was
sequenced. Subsequently, the EcoRI-SacI PCR
fragment of T
R-II/ActR-IIB encoding the N-terminal part was ligated
together with the C-terminal part of ActR-IIB by subsequent subcloning into pMEP4. Wild-type mouse BMPR-IB, human T
R-II, mouse ActR-IIB, and human T
R-I were subcloned in pMEP4 using convenient restriction enzyme cutting sites. Receptor expression thereby came under the transcriptional control of the ZnCl2-inducible human
metallothionein promoter.
Transfection
R 4-2 and DR 26 mutant cells were stably transfected by the calcium phosphate precipitation method using the MBS mammalian transfection kit (Stratagene), following the manufacturer's protocol. Selection of transfectants was performed in the presence of 100 units/ml hygromycin (Sigma). We obtained cell pool cultures with similar levels of receptor expression upon ZnCl2 treatment.
Binding, Affinity Cross-linking, and ImmunoprecipitationTGF-1 was iodinated by the chloramine T
method (45). Binding and affinity cross-linking using disuccinimidyl
suberate (Pierce) were performed as described (10). Lysates were
prepared from affinity cross-linked cells and subjected to
immunoprecipitation, using antisera against T
R-I (VPN) (10),
T
R-II (DRL) (46), ActR-IIB (47), BMPR-IB (DET) (12), hemagglutinin
epitope (12CA5, Babco; Ref. 48), or His epitope (HSV) (gift from Dr.
T. K. Sampath); samples were analyzed by SDS-PAGE using 4-15%
gradient gels and visualized using a Fuji-X BioImager.
Cells were seeded at a density of
1.5 × 104 cells/well in 24-well plates in DMEM with
10% FBS. After 24 h, cells were washed once, and medium was
changed to DMEM with 3% FBS and 100 µM
ZnCl2, and cells were then incubated with different
concentrations of TGF-1 for 22-24 h; during the last 2 h,
cells were labeled with 1 µCi/ml [3H]thymidine
(Amersham Corp.). Thereafter, the cells were fixed in 5% ice-cold
trichloroacetic acid for 20 min, washed with 5% trichloroacetic acid
followed by water and 70% ethanol, and finally solubilized in 0.1 M NaOH. 3H radioactivity was measured in a
liquid scintillation
-counter using Ecoscint (National
Diagnostics).
Cells were seeded in
six-well plates at a density of 1 × 105 cells/well.
After 18-24 h, the medium was changed to DMEM supplemented with 0.1%
FBS, with or without 100 µM ZnCl2. After
15 h, the medium was changed to methionine-free MCDB medium (SVA,
Sweden) with different concentrations of TGF-1 and incubation
prolonged for 6 h; during the last 2 h, cells were incubated
with 25 µCi/ml 35S-labeling mixture "ProMix"
(Amersham Corp.). For extracellular matrix isolation, the cells were
removed by washing on ice; once in phosphate-buffered saline, three
times in 10 mM Tris-HCl, pH 8.0, 0.5% sodium deoxycholate,
1 mM phenylmethylsulfonyl fluoride; two times in 20 mM Tris-HCl, pH 8.0; and once in phosphate-buffered saline.
Extracellular matrix proteins were scraped off and extracted into SDS
sample buffer containing 10 mM dithiothreitol. Secreted proteins and extracellular matrix proteins were analyzed by SDS-PAGE, followed by fluorography using Amplify (Amersham Corp.) and
quantification using a Fuji-X BioImager. PAI-1 was identified as a
45-kDa protein in the extracellular matrix fraction (49).
Stable transfectants
were transiently transfected with p3TP-Lux (28), as described above.
The following day, cells were washed extensively with
phosphate-buffered saline to remove calcium phosphate precipitates.
Subsequently, the cells were incubated in DMEM with 10% FBS for 16-20
h. Thereafter, the receptor expression was induced by treatment of the
cells with 100 µM ZnCl2 in DMEM supplemented
with 0.1% FBS for 5 h, after which TGF-1 was added. Luciferase
activity in cell lysate was measured after 22-24 h using the
luciferase assay system (Promega Biotec), according to the
manufacturer's protocol using an LKB Luminometer (LKB-Bromma).
TGF-1-dependent phosphorylation of
endogenous Smad2 and Smad5 was analyzed using R 4-2 mutant cells
stably transfected with T
R-I or T
R-I/BMPR-IB and DR 26 mutant
cells stably transfected with T
R-II or T
R-II/ActR-IIB. Cells were
labeled for 3 h in phosphate-free medium supplemented with 0.7 mCi/ml [32P]orthophosphate. Cells were incubated in the
absence or presence of TGF-
1, lysed, and subjected to
immunoprecipitation with antiserum against Smad2 (SED) (37) or Smad5
(SSN) (gift from Dr. K. Tamaki). The precipitates were analyzed by
SDS-PAGE followed by autoradiography. In addition, COS-1 cells were
transiently transfected, as described above, with T
R-I/BMPR-IB,
T
R-II/ActR-IIB, and Smad1 or Smad2 expression constructs (37). Cells
were then treated in the same way as the stable transfectants and
immunoprecipitated with antiserum against Smad1 (QWL) (gift from Dr. A. Nakao) or Smad2 (SED). The precipitates were subjected to SDS-PAGE and
autoradiography.
Two chimeric
receptors were constructed, i.e. TR-I/BMPR-IB, containing
the extracellular domain of T
R-I and intracellular domain of
BMPR-IB, and T
R-II/ActR-IIB, containing the extracellular domain of
T
R-II and the intracellular domain of ActR-IIB (Fig. 1). To investigate the
125I-TGF-
1 binding properties of the chimeric receptors,
we used Mv1Lu cell lines that lack functional T
R-I (R 4-2 mutant
cells) or T
R-II (DR 26 mutant cells) as host cells for transfection. T
R-I/BMPR-IB was stably transfected into R 4-2 mutant cells, along
with T
R-I and BMPR-IB as positive and negative controls, respectively. T
R-II/ActR-IIB was stably transfected in DR 26 mutant
cells, along with T
R-II and ActR-IIB as positive and negative controls, respectively. All receptor expression constructs were placed
under the transcriptional control of the metallothionein promoter,
which can be induced by ZnCl2. Expression of type I receptors in stable transfectants was analyzed by metabolic labeling using specific antibodies, directed against the intracellular domains
of the receptors. For all transfectants, proteins with expected
molecular weights were observed upon the addition of 100 µM ZnCl2 (Fig.
2A). As observed before, due
to the leaky character of the metallothionein promoter, the receptors
were also expressed, but at lower levels, in the absence of
ZnCl2 treatment. We observed no co-immunoprecipitation of
endogenous type II receptors with anti-type I antibodies, suggesting
that there was no ligand-independent type I-type II complex formed as a
result of overexpression (Fig. 2A). Expression of type II
receptors could not be analyzed by metabolic labeling due to the weak
affinity of the antiserum.
Affinity cross-linking with 125I-TGF-1 of
T
R-I/BMPR-IB cells revealed that T
R-I/BMPR-IB bound
125I-TGF-
1 with similar efficiency as T
R-I.
Immunoprecipitation with anti-BMPR-IB antisera not only brought down
the T
R-I/BMPR-IB, but also T
R-II, illustrating that T
R-II
formed a complex with T
R-I/BMPR-IB (Fig. 2B). We were not
able to detect neither T
R-II nor T
R-I/BMPR-IB with T
R-II
antiserum. The reason for this is unclear, since T
R-I/BMPR-IB is
most likely able to bind TGF-
only in the presence of T
R-II. As
expected, in T
R-I-transfected cells cross-linked T
R complexes
could be demonstrated by immunoprecipitation using antisera against
T
R-I or T
R-II, albeit with less efficiency of complex
precipitation with T
R-II antiserum. In nontransfected R 4-2 mutant
cells, no type I receptor cross-linked complex was immunoprecipitated
with anti-T
R-I or anti-BMPR-IB antisera (Ref. 10 and data not
shown). Although we have reported that BMPR-IB can bind TGF-
1 when
overexpressed in COS-1 cells (12), the expression levels in transfected
Mv1Lu cells were too low for this interaction to occur (data not
shown).
Affinity cross-linking with 125I-TGF-1 of cells
transfected with T
R-II/ActR-IIB or T
R-II revealed that
T
R-II/ActR-IIB bound 125I-TGF-
1 equally efficient as
T
R-II upon induction of their expression with ZnCl2
treatment. T
R-II/ActR-IIB, like T
R-II, rescued the binding to
endogenously expressed T
R-I (Fig. 2C). In
T
R-II/ActR-IIB cells, cross-linked complexes of T
R-II/ActR-IIB
and T
R-I were immunoprecipitated with antisera against either
ActR-IIB or T
R-I, indicating that both receptors are part of a
common TGF-
-induced receptor complex (Fig. 2C). In the
nontransfected DR mutant cells, no type II cross-linked complex could
be immunoprecipitated with antisera against T
R-II or ActR-IIB (data
not shown). As expected, no binding of TGF-
1 to cells stably
transfected with ActR-IIB was observed (data not shown).
Affinity cross-linking with 125I-BMP-7/OP-1 of cells transfected with BMPR-IB or ActR-IIB revealed that both receptors were expressed and able to bind ligand upon ZnCl2 treatment (Fig. 2D).
Signaling Properties of Chimeric ReceptorsThe signaling
activity of the chimeric receptors was investigated using the p3TP-Lux
transcriptional activation assay, which scores positive after
stimulation with TGF- (9) and, albeit less efficiently, after BMP or
activin stimulation. Previously, we have shown that ActR-IIs and
BMPR-IB can form a BMP-7/OP-1-induced heteromeric complex, which can
mediate a p3TP-Lux signal (16). No transcriptional response could be
observed after TGF-
1 stimulation when the reporter plasmid was
transfected alone into COS-1 cells (Fig.
3A). However, when we
co-transfected T
R-I/BMPR-IB together with T
R-II/ActR-IIB and
p3TP-Lux into COS-1 cells, we observed a TGF-
-dependent
signal (Fig. 3A). To elucidate the ability of the complex
between T
R-I/BMPR-IB and T
R-II/ActR-IIB to induce Smad
phosphorylation, COS-1 cells were transiently co-transfected with
T
R-I/BMPR-IB and T
R-II/ActR-IIB together with Smad1 or Smad2.
TGF-
1 induced the phosphorylation of Smad1, but not of Smad2 (Fig.
3B). This indicates that T
R-I/BMPR-IB and
T
R-II/ActR-IIB are functionally intact and can form a
TGF-
-mediated complex, which signals a BMP-like response.
We then investigated whether TR-I/BMPR-IB and T
R-II/ActR-IIB
could substitute for T
R-I and T
R-II, respectively, using TGF-
-induced plasminogen activator inhibitor-1 (PAI-1) production as
an assay. Wild-type Mv1Lu cells responded to TGF-
1 by producing a
45-kDa PAI-1 protein, whereas the R 4-2 mutant and DR 26 mutant cell
lines did not produce PAI-1 after stimulation by TGF-
1 (data not
shown). Sometimes the PAI-1 protein appeared as two discrete bands of
45 and 43 kDa, of which the latter is likely to be a proteolytic
breakdown product of the 45-kDa protein. Stably transfected T
R-I/BMPR-IB R 4-2 mutant cells did not produce the PAI-1 protein upon induction of receptor expression by ZnCl2 and TGF-
1
stimulation (Fig. 4A).
TGF-
1 stimulation of the R 4-2 mutant cells transfected with
T
R-I, but not BMPR-IB, led to a 2.5-fold increase, determined by
densitometric scanning, in the production of PAI-1 in response to
ZnCl2 treatment, as reported before (10, 12). However, T
R-II/ActR-IIB restored the TGF-
-mediated PAI-1 response in DR 26 mutant cells to a similar extent as T
R-II, i.e. 5- and 6-fold increases, respectively (Fig. 4B). ActR-IIB, which
cannot bind TGF-
1, was unable to complement the defect in DR 26 mutant cells (Fig. 4B). In ActR-IIB-transfected cells
treated with ZnCl2, a weak 45-kDa protein was observed.
However, the production of this protein was not induced upon TGF-
1
stimulation.
In addition, we measured the ability of both chimeras to mediate
growth-inhibitory responses upon stimulation with TGF-1. Similar to
the p3TP-Lux and PAI-1 assays, T
R-II/ActR-IIB was able to replace
T
R-II, but T
R-I/BMPR-IB was not able to replace T
R-I, with
respect to the antiproliferative response upon TGF-
1 stimulation
(Fig. 5). In a few experiments, we
observed that the degree of growth inhibition was less with
T
R-II/ActR-IIB compared with T
R-II.
Different Smads have been shown to act downstream of BMP and TGF-
receptors (34, 37). Thus, we used differential activation of Smad2 or
Smad5 to distinguish TGF-
versus BMP receptor-mediated signaling, respectively. Using the stably transfected cell lines, the
effect of ligand-induced complex formation between T
R-II and
T
R-I/BMPR-IB, or between T
R-II/ActR-IIB and T
R-I, on
endogenous Smad2 or Smad5 phosphorylation was analyzed (Fig.
6). Phosphorylation of Smad2, but not
Smad5, was induced by the activated complex between T
R-II/ActR-IIB
and T
R-I, whereas ligand-induced complex formation between
T
R-I/BMPR-IB and T
R-II induced no Smad2 or Smad5 phosphorylation.
BMP-7/OP-1 was found to induce the phosphorylation of Smad5 in Mv1Lu
cells; phosphorylated Smad2 and Smad5 were found to co-migrate on
SDS-PAGE.2 Smad5 antibody also brought down a
phosphoprotein larger than Smad5, the identity of which is unknown.
Thus, our results indicate that complex formation between the
intracellular domains of ActR-IIB and TR-I leads to the transduction of TGF-
-like signals, whereas complex formation between the
intracellular domains of T
R-II and BMPR-IB does not lead to any
measurable signaling event (Fig. 7).
TGF- superfamily members exert their cellular effects through
formation of hetero-oligomeric complexes of type I and type II
serine/threonine kinase receptors. Previous reports have shown that the
type II receptor has a constitutively active kinase domain (20), and
that phosphorylation and activation of the type I receptor is
sufficient and necessary for downstream signaling activities (25, 26).
Here we have characterized the properties of two chimeric receptors in
which the intracellular domains of different type I or type II
receptors within the TGF-
superfamily were exchanged. The purpose
was to investigate whether the intracellular domains of TGF-
type I
and type II receptors could be replaced by the corresponding domains of
BMPR-IB and ActR-IIB, respectively. The presented data show that the
intracellular domain of ActR-IIB can functionally substitute for the
intracellular domain of T
R-II with respect to the induction of a
transcriptional activation signal, stimulation of PAI-1 production,
growth inhibition, and Smad2 activation. In contrast, a complex between
the intracellular domains of BMPR-IB and T
R-II induced no observable
signal (Fig. 7).
TR-II/ActR-IIB can complement the lack of T
R-II in DR 26 mutant
cells. The intracellular domain of ActR-IIB shares 33.3% sequence
identity with T
R-II and is thus apparently sufficiently similar to
phosphorylate and transactivate T
R-I in a similar way as T
R-II.
Previously, we and others have shown that the intracellular domain of
T
R-I cannot replace the intracellular domains of T
R-II in this
respect (39-42). Our findings are in agreement with those of Muramatsu
et al. (43), who reported the signaling activities of
chimeric human granulocyte-macrophage colony-stimulating factor receptor/ActR-II and human granulocyte-macrophage colony-stimulating factor receptor/BMPR-II.
The failure of TR-I/BMPR-IB to signal in combination with T
R-II
is unlikely to be due to a perturbation of the conformation of T
R-II
extracellular domain or BMPR-IB intracellular domain, since
T
R-I/BMPR-IB bound TGF-
and formed a heteromeric complex with
T
R-II and since T
R-I/BMPR-IB together with T
R-II/ActR-IIB transduced a TGF-
-dependent transcriptional activation
signal and induced Smad1 phosphorylation, a BMP-like signal. It is
possible that the inability of T
R-I/BMPR-IB to mediate a
growth-inhibitory response may be in an inherent property of the
receptor, since Mv1Lu cells, which express T
R-II, T
R-I, ActR-II,
and BMPR-IB among other receptors, are at least 100-fold more sensitive
to TGF-
1 than BMP-7/OP-1 with respect to growth inhibition. On the other hand, TGF-
-induced complex formation between T
R-II and T
R-I/BMPR-IB did not lead to Smad2 or Smad5 phosphorylation, suggesting that this is an inactive complex.
Phosphorylation of endogenous Smad2 was stimulated by TGF--induced
complex formation between T
R-I and T
R-II/ActR-IIB, indicating that type I receptor is the determinant for signal specificity. Our
future studies will be aimed at elucidating which minimal specific
regions/residues in the intracellular domain of T
R-I need to be
substituted for analogous regions/residues in the intracellular domain
of BMPR-IB to allow for a T
R-I/BMPR-IB chimera to transduce signals
when activated by T
R-II.
We thank Dr. Atsuhito Nakao for Smad1
and Smad2 expression constructs and antisera against Smad1 and Smad2,
Dr. Kiyoshi Tamaki for antiserum against Smad5, Christer Wernstedt for
preparing oligonucleotides, Hideya Ohashi for recombinant TGF-1, Dr.
T. K. Sampath for BMP-7/OP-1 and antiserum against His epitope,
Kristin Verschueren for an antiserum against ActR-II intracellular
domain, and Joan Massagué for Mv1Lu mutant cell lines,
ActR-IIB1 cDNA, and p3TP-Lux plasmid.