From the Whitehead Institute for Biomedical Research,
Cambridge, Massachusetts 02142, the § Dana-Farber Cancer
Institute, Department of Adult Oncology, Massachusetts General Hospital
and Department of Medicine, Harvard Medical School, Boston,
Massachusetts 02115, the ** Cell Surface Recognition Group,
Biotechnology Research Institute, Montreal, Quebec H4P 2R2, Canada,
and the
Ludwig Institute for Cancer
Research, Box 595, Uppsala SE-751 24, Sweden
Received for publication, January 9, 2001, and in revised form, April 25, 2001
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ABSTRACT |
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Transforming growth factor Transforming growth factor TGF- A general mechanism for TGF- How can such a simplistic pathway regulate such a diverse array of
biology? Many groups have attempted to address this issue through
identification of type I and type II receptor-interacting proteins that
may modulate TGF- To explore the involvement of TGF- Cell Culture--
The COS-7 and L6 cell lines were obtained from
ATCC. These cell lines were maintained in Dulbecco's modified Eagle's
medium with 10% fetal calf serum. Cells were transiently transfected using expression vectors for the type III receptor and type II or type
I receptors using FuGene6 reagent under conditions described by the
manufacturer (Roche). The L6-III and L6-III-cyto stable cell lines were
selected in 0.6 mg/ml G418 and maintained in Dulbecco's modified
Eagle's medium with 10% fetal calf serum and 0.3 mg/ml G418. All
cells were grown in 5% CO2 at 37 °C in a humidified atmosphere.
TGF- Phosphorylation Assays in Vitro--
293T cells were transfected
with the type II receptor and harvested by immunoprecipitation with
anti-T Phosphorylation Assays in Vivo--
COS-7 and 293T cells were
transfected with the type II receptors or the type II receptor-KD
(kinase-dead), and the HA epitope-tagged type III receptor. After
48 h, the cells were washed in phosphate-free medium and labeled
with 1.0 mCi/ml 32Pi for 4 h. Cells were
washed with phosphate-buffered saline, lysed with RIPA lysis buffer,
immunoprecipitated with [3H]Thymidine Incorporation Assay--
Cells were
plated in 24-well plates at 2 × 104 cells/ml and
treated with 0-200 pM TGF- Interaction of the Cytoplasmic Domains of the Type III Receptor and
the Type II Receptor--
The types I, II, and III TGF-
We have developed a polyclonal antibody to a small peptide (amino
acids 833-847) in the cytoplasmic domain of the type III receptor
(
To establish the mechanism and relevance of this phenomenon, we
analyzed the ability of the
To determine whether this effect was specific to the Specificity for the Type II TGF- Role of the Cytoplasmic Domain of the Type II Receptor on Type
II-Type III Interactions--
The Role of the Cytoplasmic Domain of the Type III Receptor--
We
have demonstrated previously that a series of deletion mutants of the
extracellular region of the type III receptor which contain the
membrane proximal region retains the ability to bind TGF-
We initially investigated whether the type II receptor interacted with
the cytoplasmic domains of these mutant type III receptors. As
expected, expression of mutants 44-303, 44-382, 44-402, and 44-420,
which all retain GAG attachment sites, migrate as diffuse bands with
progressively lower molecular masses (Fig.
3). In contrast, mutants 44-564 and
44-575 lack the GAG attachment sites and migrate as more discrete
bands. In addition, we are able to detect not only the monomer but also
oligomeric forms of these mutants (Fig. 3). In all cases, these mutants
interacted with the type II receptor, but not with the type II-cyto60
(type II( Role of the Kinase Activity of the Type II Receptor--
The
cytoplasmic domain of the type II receptor contains a serine/threonine
protein kinase that functions by autophosphorylating the type II
receptor and by phosphorylating and activating the type I receptor. The
inability of the type II-cyto60 or the type III receptor in homodimers
to block immunoprecipitation suggests that the type II receptor
interacts with the cytoplasmic domain of the type III receptor in a
nonsteric manner. To determine whether the kinase activity of the type
II receptor mediates the ability of the type II receptor to interact
with the type III receptor, we utilized a kinase-dead version of the
type II receptor (type II-KD). The type II-KD was effectively expressed
and bound TGF- Phosphorylation of the Type III Receptor by the Type II
Receptor--
Two mechanisms can be proposed for why the kinase
activity of the type II receptor is essential for its interaction with
the cytoplasmic domain of the type III receptor. Either phosphorylation of the type III receptor by the type II receptor directly prevents the
The cytoplasmic domain of the type III receptor is rich in serine and
threonine residues and is phosphorylated (21, 22); however, the
kinase(s) responsible for phosphorylating the type III receptor
in vivo has not been elucidated. To determine whether the
type II receptor was the protein kinase responsible for phosphorylating the type III receptor, we initially investigated whether the
cytoplasmic domain of the type III receptor could be a substrate for
the type II receptor. For these studies, a GST fusion of the
cytoplasmic domain of the type III receptor was utilized. A GST fusion
of the cytoplasmic domain of the closely related receptor, endoglin, was utilized as a control substrate, and the type II-KD receptor was
utilized as a control kinase source. As expected, the type II receptor
was able to autophosphorylate, whereas type II-KD was not (data not
shown). The type II receptor was also able to phosphorylate the
GST-type III cytoplasmic domain significantly, with a 3-fold induction
relative to type II-KD (data not shown). The GST-endoglin cytoplasmic
domain was also phosphorylated significantly by the type II receptor
relative to type II-KD (with a 2-fold induction) but not to the same
extent as GST-type III cytoplasmic domain. These studies demonstrate
that the type II receptor can phosphorylate the cytoplasmic domain of
the type III receptor in vitro.
To analyze whether this phosphorylation occurred in vivo,
the type III receptor was expressed in the presence and absence of
either the type II receptor or the type II-KD receptor, the cells were
labeled with 32Pi, and the phosphorylation
state of the type III receptor was analyzed by immunoprecipitation. As
shown in Fig. 4, the phosphorylation state of the type III receptor was increased 7-fold by co-expression of
the type II receptor but not by the type II-KD receptor or in the
absence of an expressed type II receptor. The ability of the type II
receptor to phosphorylate the type III receptor directly in
vitro, together with its ability to enhance the phosphorylation of
the type III receptor in vivo, demonstrates that the type
III receptor is a physiological substrate for the type II receptor.
To determine whether phosphorylation of the type III receptor by the
type II receptor was the mechanism by which the type II receptor
interacts with the type III receptor to block immunoprecipitation by
the
We then investigated whether autophosphorylation of the type II
receptor was responsible for the dependence on type II kinase activity
by testing autophosphorylation site mutants. The type II receptor is
autophosphorylated on at least three serine residues in the cytoplasmic
domain, Ser-213, Ser-409, and Ser-416, with Ser-213 being the major
site (23). Mutational analysis of these autophosphorylation sites (from
serine to alanine) demonstrated that Ser-213 was important for maximal
kinase activity, whereas Ser-409 and Ser-416 were not, and that Ser-409
was important for TGF- Functional Role of the Cytoplasmic Domain of the Type III Receptor
in TGF- Mechanism of Action of the Type III Receptor Cytoplasmic
Domain--
The effects of the cytoplasmic domain of the type III
receptor on TGF- TGF- Here we establish strong evidence for an essential and biologically
significant role of the type III receptor in TGF- Role of the Type III Receptor in TGF-
Recent results have begun to challenge this model. Many of the cells
and cell lines that do not express the type III receptor, including
hematopoietic and endothelial cells, express the closely related
receptor, endoglin, which shares highest homology (70%) to the type
III receptor in the cytoplasmic domain. These cells do respond to
TGF-
The present results demonstrating an interaction of the cytoplasmic
domain of the type III receptor with the type II receptor, the
phosphorylation of the type III receptor by the type II receptor, and
the essential role of the cytoplasmic domain of the type III receptor
in mediating TGF- Roles for the Cytoplasmic Domain of the Type III
Receptor--
Although the cytoplasmic domain of the type III receptor
is highly conserved across species and with the related receptor, endoglin, no function has been described for this domain. Indeed, deletion of the cytoplasmic domain was reported to have no effect on
the ability of the type III receptor to bind TGF-
The association of the cytoplasmic domain of the type III receptor with
the type II receptor may either be direct or via an adaptor protein
that binds to the autophosphorylated type II receptor and/or the
phosphorylated type III receptor cytoplasmic domain. We have been
unable to demonstrate a direct interaction of the cytoplasmic domain of
the type III receptor with the type II receptor in vitro
using GST pull-down assays.3
In addition, yeast two-hybrid screens with the cytoplasmic domain of
the type III receptor did not yield any clones encoding the type II
receptor.3
In other investigations, we have established a protein that does bind
the cytoplasmic domain of the type III receptor. GIPC, a PDZ
domain-containing protein, binds to a class I PDZ binding motif in the
cytoplasmic domain of the type III
receptor.4 Mutating the Class
I PDZ binding motif of the type III receptor abolishes binding of GIPC
to the type III receptor, but does not effect the interaction between
the type III receptor and the type II receptor studied here,
establishing that this protein is not an adaptor protein in this
interaction. GIPC does regulate the expression of the type III receptor
and the response of cells to TGF- Oligomeric Structure of TGF- Implications for Endoglin Signaling--
The high degree of
homology between the cytoplasmic domain of the type III receptor and
the related receptor, endoglin, points to a conserved role for the
cytoplasmic domain of these receptors. The present results suggest that
one of these roles is to interact either directly or indirectly with
the cytoplasmic domain of the autophosphorylated type II receptor. The
epitope recognized by the (TGF-
)
signals through three high affinity cell surface receptors, TGF-
type I, type II, and type III receptors. The type III receptor, also
known as betaglycan, binds to the type II receptor and is thought to
act solely by "presenting" the TGF-
ligand to the type II
receptor. The short cytoplasmic domain of the type III receptor is
thought to have no role in TGF-
signaling because deletion of this
domain has no effect on association with the type II receptor, or with
the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of
the type II receptor and the ability of the type II receptor to
autophosphorylate. This interaction results in the phosphorylation of
the cytoplasmic domain of the type III receptor by the type II
receptor. The type III receptor with the cytoplasmic domain deleted is
able to bind TGF-
, to bind the type II receptor, and to enhance
TGF-
binding to the type II receptor but is unable to enhance
TGF-
2 signaling, determining that the cytoplasmic domain is
essential for some functions of the type III receptor. The type III
receptor functions by selectively binding the autophosphorylated type
II receptor via its cytoplasmic domain, thus promoting the preferential
formation of a complex between the autophosphorylated type II
receptor and the type I receptor and then dissociating from this active
signaling complex. These studies, for the first time, elucidate
important functional roles of the cytoplasmic domain of the type III
receptor and demonstrate that these roles are essential for regulating
TGF-
signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
)1 is a member of a
family of dimeric polypeptide growth factors which, in addition to the TGF-
ligands, includes the bone morphogenetic proteins (BMPs) and
the activins (1). TGF-
regulates cellular proliferation and
differentiation as well as the processes of embryonic development, wound healing, and angiogenesis in a tissue-specific manner. Mutations in TGF-
receptors or their intracellular signaling molecules have
been described, particularly in association with the development of
cancer and hereditary hemorrhagic telangiectasia. Alterations in the
production of TGF-
ligand have also been linked to numerous disease
states, including osteoporosis, hypertension, atherosclerosis, and
fibrotic disease of the kidney, liver, and lung (2).
regulates cellular processes by binding to three high affinity
cell surface receptors, the TGF-
type I, type II, and type III
receptors. Where expressed, the type III receptor, also known as
betaglycan, is the most abundant TGF-
receptor and is traditionally
thought to function by binding TGF-
and then transferring it to its
signaling receptor, the type II receptor. This is particularly important for the TGF-
2 isoform, which cannot bind the type II receptor independently. The type I and II receptors contain
serine/threonine protein kinases in their cytoplasmic domains which
initiate intracellular signaling by phosphorylating members of the
Smad family of transcription factors.
signaling has been established. TGF-
either binds to type III receptors, which then present TGF-
to type
II receptors, or to type II receptors directly. Once activated by
TGF-
, type II receptors recruit, bind, and transphosphorylate type I
receptors, thereby stimulating their protein kinase activity. The
activated type I receptors phosphorylate Smad2 or Smad3 that then bind
to Smad4. The resulting Smad heterocomplex then translocates into the
nucleus where it interacts in a cell-specific manner with numerous
transcription factors to regulate the transcription of many
TGF-
-responsive genes.
signaling at the receptor level as well as
transcription factors, coactivators, and corepressors that interact
with Smads to regulate TGF-
signaling at the transcriptional level
(3). Diversity may also be generated by the formation of discrete
receptor complexes that would then utilize distinct TGF-
pathways.
Indeed, some studies have suggested that the type I receptor mediates
predominantly the effects of TGF-
on the extracellular matrix,
whereas the type II receptor mediates the effects of TGF-
on the
cell cycle/proliferation (4, 5). In addition, Smad-independent
signaling, and signaling through mitogen-activated protein kinase and
other cellular signaling pathways has been reported (6-10).
receptor complexes in TGF-
signaling, we examined the complexes formed by the type III receptor
and the role of the type III receptor in TGF-
signaling. The type
III receptor forms complexes individually with either the type II
receptor or the type I receptor, as well as complexes with the type II
and type I receptors together. Current evidence suggests that the
interaction of the type III receptor with the type II receptor occurs
through their respective extracellular domains because deletion of the
cytoplasmic domain of the type III receptor does not alter the ability
of these receptors to interact (11). Indeed, no functional role for the
cytoplasmic domain of the type III receptor has been established. The
studies described herein establish a role for a specific, functional, and biologically significant interaction between the cytoplasmic domains of the type II and type III receptors. The protein kinase activity of the type II receptor and autophosphorylation of the type II
receptor are both shown to be essential for this interaction, which
results in the phosphorylation of the cytoplasmic domain of the type
III receptor by the type II receptor. The type III receptor with its
cytoplasmic domain deleted is able to bind TGF-
, the type II
receptor, and enhance TGF-
binding to the type II receptor but is
unable to enhance TGF-
2 signaling. The type III receptor functions
by selectively binding autophosphorylated type II receptor via its
cytoplasmic domain, preferentially promoting the formation of a complex
between the autophosphorylated type II receptor and the type I
receptor, and then dissociating from this active signaling complex.
These studies elucidate important functions for the cytoplasmic domain
of the type III receptor and demonstrate that these functional roles
are essential for regulating TGF-
signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Binding and Cross-linking--
Radioligand binding and
cross-linking of 125I-TGF-
1 to L6, L6-III,
L6-III-cyto, or transfected COS-7 cells were performed by incubating
subconfluent cells with KRH buffer (50 mM Hepes, pH 7.5, 130 mM NaCl, 5 mM MgSO4, 1 mM CaCl2, and 5 mM KCl) containing 0.5% bovine serum albumin for 30 min at 37 °C, then with 100 pM 125I-TGF-
1 for 3 h at 4 °C.
125I-TGF-
1 was cross-linked with 0.5-mg/ml
disuccinimidyl suberate for 15 min and quenched with 20 mM
glycine. Cells were then washed with KRH buffer, lysed in RIPA lysis
buffer, immunoprecipitated with the indicated antibodies, and analyzed
by SDS-polyacrylamide gel electrophoresis and phosphorimaging analysis
of dried gels. Quantitation of bands was performed using ImageQuant
version 1.2 software from Molecular Dynamics.
R-II antibodies and protein A-Sepharose 48 h after
transfection. The resulting immunocomplexes were washed and utilized to
phosphorylate recombinant GST-TypeIIIcyto or GST-Endocyto in 25 mM
-glycerophosphate, pH 7.3, 0.5 mM
dithiothreitol, 1.25 mM EGTA, 50 µM sodium
vanadate, 10 mM MgCl2, and 100 µM
ATP ([
-32P]ATP, ~2,000 cpm/pmol), and 5 µg of
recombinant GST-TypeIIIcyto of GST-Endocyto for 30 min at 30 °C. The
reactions were analyzed directly or immunoprecipitated and analyzed on
10% SDS-polyacrylamide gels and detected by phosphorimaging analysis
of the dried gels.
HA antibody and protein G-Sepharose,
analyzed on 10% SDS-polyacrylamide gels, and detected by
phosphorimaging analysis of the dried gels.
1. After 48 h of
incubation, cells were treated with 10 mCi of
[3H]thymidine (Amersham Pharmacia Biotech) for 4 h.
Cells were washed with phosphate-buffered saline and 5%
trichloroacetic acid before harvesting cells with 0.1 N
NaOH. The amount of [3H]thymidine incorporated was
analyzed by liquid scintillation counting. Growth inhibition was
calculated as the ratio of radioactivity with TGF-
treatment/radioactivity in the absence of TGF-
treatment.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptors
have been demonstrated to associate in various complexes through both
co-immunoprecipitation studies (12-16) and in live cells using
co-patching experiments (17, 18). Antibodies to either the type II or
type III receptor are able to co-immunoprecipitate both receptors,
indicating that they form a complex in the presence of ligand.
277). In contrast to other antibodies to the type III receptor, we
have observed that the
277 antibody is unable to co-immunoprecipitate the type II receptor (14). In addition, while
examining the expression of the type III receptor in endothelial cell
lines, we have observed that the
277 antibody was increasingly unable to immunoprecipitate the type III receptor in cell lines with
higher levels of expression of the type II receptor relative to the
type III receptor (19). These observations suggested that the
cytoplasmic domains of the receptors interact with one another, with
the type II receptor preventing immunoprecipitation of the type III
receptor by the
277 antibody.
277 antibody to immunoprecipitate or
co-immunoprecipitate wild type or HA epitope-tagged type II and type
III receptors expressed in COS-7 cells by transient transfection. COS-7
cells were utilized because they express low levels of endogenous receptors that could confound analysis. Expression of these receptors was detected by binding and cross-linking the receptors with iodinated TGF-
1. As expected, the affinity-labeled type III receptor appears as a diffuse band from 180 to 300 kDa, and the type II receptor appears as a band at 95 kDa (Fig. 1). The
277 antibody was able to immunoprecipitate the type III receptor,
however, co-expression of the type II receptor with the type III
receptor abolished the ability of the
277 antibody to
immunoprecipitate the type III receptor (Fig. 1A). Identical
results were obtained when nontagged versions of the receptor were
utilized (data not shown). This effect was dose-dependent,
as increasing the expression of the type II receptor relative to the
expression of the type III receptor was able to inhibit progressively
the ability of the
277 antibody to immunoprecipitate the type III
receptor (Fig. 1B). We were also unable to detect increased
expression of the type II receptor because this was not
co-immunoprecipitated by the
277 antibody. These findings
demonstrate that the
277 antibody immunoprecipitates only the type
III receptors that are not complexed with type II receptors and suggest
that the effect of the type II receptor is the result of an interaction
of its cytoplasmic domain with the cytoplasmic domain of the type III
receptor.
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Fig. 1.
Interaction of the cytoplasmic domain of the
type II TGF- receptor with the cytoplasmic
domain of the type III TGF-
receptor.
Panel A, COS-7 cells were transiently transfected with the
HA-tagged type III TGF-
receptor with (+) or without (
) the type
II TGF-
receptor. Cells were affinity labeled with
125I-TGF-
1, immunoprecipitated (IP) with the
indicated antibodies, and analyzed on 10% SDS-polyacrylamide gels. The
bracket delineates the high molecular mass band from
180-300 kDa characteristic of the type III receptor. The
arrow indicates the position of the type II receptor.
Molecular mass standards (in kDa) are indicated on the
right. Panel B, dose dependence of the type II
receptor interaction with the type III receptor. COS-7 cells were
transiently transfected with 2 µg of the HA-tagged type III TGF-
receptor and increasing amounts (0.5-8 µg) of the type II TGF-
receptor or 0.5-8 µg of the kinase-dead type II TGF-
receptor
(type II-KD). Cells were affinity labeled with
125I-TGF-
1, immunoprecipitated with the indicated
antibodies, and analyzed on 10% SDS-polyacrylamide gels. The positions
of the type III and type II receptors and molecular mass standards (in
kDa) are indicated on the right.
277 antibody we
performed a number of control experiments. First, the ability of the
type II receptor to affect immunoprecipitation of the HA-tagged type
III receptors with the
HA antibody was analyzed. The
HA antibody
was able to immunoprecipitate the affinity-labeled type III receptor,
and co-expression of the type II receptor did not block this ability
(Fig. 1A). In addition, when increasing the expression of
the type II receptor relative to the type III receptor, the
HA
antibody was able to co-immunoprecipitate the type II receptor and to
detect the increased expression of the type II receptor (Fig.
1B). Second, an antibody to the intracellular domain of the
type II receptor (
260) was able to immunoprecipitate the type II
receptor and co-immunoprecipitate the type III receptor and, as
expected, did not co-immunoprecipitate the type III receptor unless the
type II receptor was expressed (Fig. 1A). Finally, the
ability of an antibody to the entire cytoplasmic domain of the type III
receptor was analyzed (
820). This antibody was able to
immunoprecipitate the type III receptor, and expression of the type II
receptor had no effect on this ability (Fig. 1A). In
addition, the type II receptor was co-immunoprecipitated by this
antibody (Fig. 1A). These studies confirm that the ability of the type II receptor to interfere with the immunoprecipitation of
the type III receptor is specific to the
277 antibody, establishing this
277 antibody immunoprecipitation assay as a measure of a specific interaction between the cytoplasmic domains of the type II
receptor and the type III receptor.
Receptor--
To determine
whether the ability of the cytoplasmic domain of the type II TGF-
receptor to interact with the cytoplasmic domain of the type III
receptor was specific to the type II TGF-
receptor, we analyzed the
effect of expression of the type I TGF-
receptor as well as the type
II receptors for activin and BMP, respectively. As shown in Fig.
2, the type I TGF-
receptor, the KD or
constitutively active (T204D) type I receptor, the type II activin
receptor, and the type II BMP receptor were unable to interact with the
cytoplasmic domain of the type III receptor. These results demonstrate
that the ability of the type II TGF-
receptor to interact with the
cytoplasmic domain of the type III receptor is specific to the type II
TGF-
receptor.
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Fig. 2.
Specificity of the type II
TGF- receptor interaction with the type III
receptor and role of the cytoplasmic domain of the type II
receptor. COS-7 cells were transiently transfected with the
HA-tagged type III TGF-
receptor and the type II TGF-
receptor
(RII-TGF-
), the type II activin receptor
(RII-Activin), the type II BMP receptor
(RII-BMP), the type I TGF-
receptor (RI-WT),
the kinase-dead type I TGF-
receptor (RI-KD), the
constitutively active type I TGF-
receptor (RI-T204D),
the type II TGF-
receptor with only 9 amino acids of the cytoplasmic
domain (RII-cyto9), the type II TGF-
receptor with 60 amino acids of the cytoplasmic domain (RII-cyto60), or the
kinase-dead type II TGF-
receptor (RII-KD). Cells were
affinity labeled with 125I-TGF-
1, immunoprecipitated
with the
277 antibody, and analyzed on 10% SDS-polyacrylamide gels.
The bracket delineates the type III receptor, and molecular
mass standards (in kDa) are indicated on the right.
277 antibody immunoprecipitation
assay predicts that the cytoplasmic domain of the type II receptor is
essential for the effect of the type II receptor. To determine whether
this was the case, we utilized two deletion mutants of the type II receptor cytoplasmic domain. When the type III receptor was
co-expressed with a mutant of the type II receptor which lacks all but
the first 9 amino acids of the cytoplasmic domain (type II-cyto9), no interaction with the cytoplasmic domain of the type III receptor was
observed (Fig. 2). Type II-cyto9 may be nonfunctional because of its
inability to sterically hinder access to the epitope of the
277
antibody or because it lacks kinase activity. To evaluate the first
possibility, a deletion mutant of the type II receptor encoding 60 amino acids of the cytoplasmic domain (type II-cyto60) was utilized.
Type II-cyto60 was also unable to interact with the cytoplasmic domain
of the type III receptor (Fig. 2). These studies confirm that an intact
cytoplasmic domain of the type II receptor is necessary for the
interaction with the cytoplasmic domain of the type III receptor and
suggest that the type II receptor interacts with the type III receptor
to exclude the
277 antibody by a nonsteric mechanism.
(20). Some
of these mutants migrate as discrete bands because they lack GAG sites
for proteoglycan attachment. This property allows detection and
analysis of oligomers of the type III receptor, thus allowing us to
determine the effect of the cytoplasmic domains of type III receptors
interacting with each other using the
277 antibody
immunoprecipitation assay.
cyto)) mutant (Fig. 3). These results confirm that, as
expected, these regions of the extracellular domain of the type III
receptor were not necessary for the interactions of the cytoplasmic
domains of these receptors. In addition, we were able to detect the
oligomers of the type III receptor with the
277 antibody,
demonstrating that the interaction of the cytoplasmic domain of the
type III receptor with itself does not interfere with access of the
277 antibody epitope, in contrast to the situation with the
heterocomplex of the type II and type III receptors.
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Fig. 3.
Role of the cytoplasmic domain of the type
III receptor in homo-oligomeric complexes. COS-1 cells were
transiently transfected with the HA-tagged type III TGF- receptor or
mutants lacking the indicated amino acids from the extracellular
domains (
44-303,
44-382,
44-402,
44-420,
44-564,
44-575) and either the type II TGF-
receptor (type II) or the type II TGF-
receptor without
the cytoplasmic domain (type II
cyto). Cells
were affinity labeled with 125I-TGF-
1,
immunoprecipitated with the
277 antibody, and analyzed on 10%
SDS-polyacrylamide gels. The bracket delineates the type III
receptor, the arrows delineate the type III receptor
oligomeric (oligo) and monomeric (mono)
complexes, the type III receptor core, and the TGF-
ligand as
indicated. The molecular mass standards (in kDa) are indicated on the
right.
; however, it did not effectively interact with the
cytoplasmic domain of the type III receptor (Fig. 2), even with
increasing expression of the type II-KD mutant relative to the type III
receptor (Fig. 1B). In contrast with the type II receptor,
the type II-KD mutant was effectively co-immunoprecipitated by the
277 antibody, indicating that the
277 antibody could detect the
type III receptor complexed with the type II-KD mutant receptor (Fig.
1B). These studies demonstrate that the kinase activity of
the type II receptor is essential for the ability of the type II
receptor to interact with the cytoplasmic domain of the type III receptor.
277 antibody from binding its epitope on the type III receptor, or
autophosphorylation of the type II receptor indirectly blocks the
epitope of the
277 antibody either by inducing a close association between the type III and type II receptors or by recruiting an adaptor
protein that binds to the autophosphorylated type II receptor and/or
the phosphorylated type III receptor cytoplasmic domain.
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Fig. 4.
The type II receptor phosphorylates the type
III receptor in vivo. COS-7 cells were
transiently transfected with the HA-tagged type III TGF- receptor
and either the type II TGF-
receptor (Type II-WT) or the
kinase-dead type II TGF-
receptor (Type II-KD), labeled
with Pi, and immunoprecipitated (IP) with the
HA antibody. Panel A, the products were analyzed on 10%
SDS-polyacrylamide gels. The bracket delineates the type III
receptor, and molecular mass standards (in kDa) are indicated on the
right. Panel B, the phosphorimaging data were
quantified utilizing Image Gauge version 3.0, and the data are
expressed in arbitrary units. Panel C, the
277 antibody
is able to immunoprecipitate the phosphorylated type III receptor. The
GST fusion protein of the cytoplasmic domain of the type III receptor
(GST-III) was phosphorylated by the type II receptor
in vitro. This reaction was then immunoprecipitated with the
277 antibody and analyzed on 10% SDS-polyacrylamide gel.
277 antibody, we investigated whether phosphorylation of the
cytoplasmic domain of the type III receptor by the type II receptor
altered the ability of the
277 antibody to immunoprecipitate the
type III receptor. When the GST-type III cytoplasmic domain was
phosphorylated by the type II receptor in vitro, the
277 antibody was able to immunoprecipitate and detect this phosphorylation (Fig. 4C). In addition, when the type III receptor was
expressed in the presence and absence of the type II receptor,
immunoprecipitated by the
HA antibody and analyzed by Western blot
with the
277 antibody, the
277 antibody was able to detect
the type III receptor that had been expressed alone or with the type II
receptor (data not shown). These results demonstrate that
phosphorylation of the type III receptor by the type II receptor is not
responsible for the inability of the
277 antibody to detect the type
III receptor in the presence of the type II receptor.
signaling. As shown in Fig.
5A, when either Ser-213 or
Ser-416 was mutated to alanine, there was no effect on the ability of the type II receptor to interact with the cytoplasmic domain of the
type III receptor. In contrast, mutation of Ser-409 to alanine either
alone, or in combination with Ser-416, completely blocked the ability
of the type II receptor to interact with the cytoplasmic domain of the
type III receptor. To investigate the ability of these
autophosphorylation mutants of the type II receptor to phosphorylate the type III receptor and autophosphorylate in vivo, the
type III receptor was expressed in the presence of either the type II
receptor or the autophosphorylation site mutants of the type II
receptor, the cells were labeled with 32Pi, and
the phosphorylation state of the type III receptor and type II receptor
was analyzed by immunoprecipitation. As shown in Fig.
5B, all of the type II receptors with the exception
of the type II-KD were able to phosphorylate the type III receptor with
varying ability (S416A > S213A > wild type > S409A).
However, only the S213A and S416A mutants of the type II receptor were able to autophosphorylate and their ability to autophosphorylate (S416A > S213A > wild type) correlated directly with their
ability to interact with the cytoplasmic domain of the type III
receptor (Fig. 5). These results demonstrate that kinase activity of
the type II receptor is necessary but not sufficient for its
interaction with the type III receptor and that autophosphorylation of
the type II receptor is required as well. As the cytoplasmic domain of
type III receptor interacted selectively with the autophosphorylated form of the type II receptor and autophosphorylation of the type II
receptor is required for type II receptor activation and subsequent downstream TGF-
signaling, these results suggest a mechanism by
which this interaction could functionally regulate TGF-
signaling.
View larger version (36K):
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Fig. 5.
Requirement for autophosphorylation of the
type II receptor. COS-7 cells were transiently transfected with
the HA-tagged type III TGF- receptor and the type II TGF-
receptor (RII-WT), the kinase-dead type II TGF-
receptor
(RII-KD), the type II TGF-
receptor with 60 amino acids
of the cytoplasmic domain (RII-cyto60), the type II TGF-
receptor with serine 213 mutated to alanine (RII-S213A),
with serine 409 mutated to alanine (RII-S409A), with serine
416 mutated to alanine (RII-S416A), or the double mutant
RII-S409A/S416A. Panel A, cells were affinity labeled with
125I-TGF-
1, immunoprecipitated with the
277 antibody,
and analyzed on 10% SDS-polyacrylamide gels. Panel B, cells
were labeled with Pi and immunoprecipitated with the
HA
antibody and analyzed on 10% SDS-polyacrylamide gel. The
brackets delineate the type III and type II receptors, and
molecular mass standards (in kDa) are indicated on the
right.
Signaling--
Although previous studies have suggested
that the cytoplasmic domain of the type III receptor is not essential
for formation of a complex between the type II and type III receptors
or for the presentation role of the type III receptor, the present
studies suggested that the cytoplasmic domain of the type III receptor plays an important role in regulating TGF-
signaling. To investigate the role of the cytoplasmic domain of the type III receptor in TGF-
signaling, we analyzed the ability of the type III receptor and mutants
of the type III receptor lacking the cytoplasmic domain to bind
TGF-
, bind the type II receptor, present TGF-
to the type II
receptor, and mediate TGF-
signaling. The type III receptor and a
mutant of the type III receptor which lacks the entire cytoplasmic domain (III-cyto) were transfected into the L6 myoblast cell line, which normally lacks expression of the type III receptor, and selected
for stable expression. As shown in Fig.
6A, the type III receptor and
the type III-cyto receptor in these stable cell lines were both
expressed at the cell surface and bound TGF-
to a similar degree. In
addition, both receptors bound the type II receptor as demonstrated by
the ability of both receptors to co-immunoprecipitate the type II
receptor using the
HA antibody and the ability of both receptors to
be co-immunoprecipitated with the type II receptor using the
260
antibody for the type II receptor (Fig. 6A). Both the type
III receptor and the type III-cyto receptor also increased binding of
TGF-
to the type II receptor relative to the parental L6 cell line
to a similar degree (Fig. 6B). The stable cell lines
expressing the type III receptor and the type III-cyto receptor were
then utilized in [3H]thymidine incorporation assays to
investigate the effect of these type III receptors on TGF-
signaling. The TGF-
2 isoform was utilized because this isoform
cannot bind the type II receptor directly and thus depends on the
presence of the type III receptor to signal. As shown in Fig.
6C, the parental L6 myoblast cell line is largely
insensitive to the TGF-
2 isoform; however, expression of the
full-length type III receptor restores sensitivity to TGF-
2. In
contrast, expression of the type III-cyto receptor failed to restore
sensitivity of L6 cells to TGF-
2. To our knowledge, this is the
first demonstration that the cytoplasmic domain of the type III
receptor is essential for mediating TGF-
2 signaling and that the
type III receptor does more than present TGF-
ligand to the type II
receptor.
View larger version (48K):
[in a new window]
Fig. 6.
Requirement for the cytoplasmic domain of the
type III receptor for type III receptor-mediated signaling. Stable
cell lines of the L6 myoblast cell line that express the HA-tagged type
III receptor (L6-III) or the HA-tagged type III receptor
lacking the cytoplasmic domain (L6-III-cyto) were made.
Panel A, full-length type III receptor and the type III
receptor lacking the cytoplasmic domain are expressed and bind the type
II receptor to a similar degree. The L6-III and L6-III-cyto cell lines
were affinity labeled with 125I-TGF- 1,
immunoprecipitated (IP) with the
HA antibody or the
260 (type II receptor) antibody, and analyzed on 10%
SDS-polyacrylamide gels. The bracket delineates the type III
receptor, and the arrow indicates the type II receptor. The
data were quantified using ImageQuant software, and the data are
expressed in arbitrary units. Panel B, full-length type III
receptor and the type III receptor lacking the cytoplasmic domain
enhance TGF-
1 binding to the type II receptor to a similar degree.
The L6, L6-III, and L6-III-cyto cell lines were affinity labeled with
12.5-100 pM 125I-TGF-
1 and analyzed
directly on 10% SDS-polyacrylamide gels (upper image). The
L6-III and L6-III-cyto cell lines were treated with 12.5-100
pM TGF-
1, and extracts were resolved on 10%
SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with
antibody to the type II TGF-
receptor (lower
image). The data were quantified using ImageQuant software, and
the data are expressed arbitrary units, normalized to the expression of
the type II TGF-
receptor. Panel C, the L6-III and
L6-III-cyto cell lines were utilized in thymidine incorporation assays
in the presence of 200 pM TGF-
2. Results are the average
of four experiments done in triplicate, with S.E. indicated.
signaling occur downstream of the type III receptor binding TGF-
, associating with the type II receptor and presenting the TGF-
ligand to the type II receptor. The results with the autophosphorylation site mutants determined further that the
cytoplasmic domain of the type III receptor selectively interacts with
and regulates autophosphorylated/activated type II receptor. After delivery of the TGF-
ligand to the type II receptor, the type III
receptor delivers the type II receptor to the type I receptor; however,
the fate of the type III receptor in these complexes is unknown. Either
the type III receptor remains associated with the type II receptor and
the type I receptor, or the type III receptor dissociates from the
active "signaling complex" between the type II receptor and the
type I receptor. To determine which of these possibilities was the
case, the effect of the cytoplasmic domain of the type III receptor on
interactions among the type III receptor, the type II receptor, and the
type I receptor was investigated. To approximate most closely the
physiological situation where the type II receptor and the type III
receptor form a complex with each other and then the type I receptor is
recruited to this complex, increasing amounts of the type I receptor
were added to a fixed quantity of the type II receptor and the
HA-tagged type III receptor or the HA-tagged type III-cyto receptor.
The complexes among the type III receptor, the type II receptor, and the type I receptor were then detected by immunoprecipitation with the
HA antibody, and type III receptor not complexed with the type II,
or type I receptor was detected by immunoprecipitation with the
277
antibody. In the presence of the type III receptor, increasing the
expression of the type I receptor increased the amount of the type III
receptor not bound to the type II receptor (Fig.
7A) and decreased the amount
of the type II receptor and type I receptor bound to the type III
receptor (Fig. 7, B and C). These results suggest
that the type III receptor normally dissociates from the activated
complex of the type II receptor and the type I receptor. In contrast,
when the type III-cyto receptor was expressed, increasing the
expression of the type I receptor did not decrease the amount of the
type II receptor bound to the type III receptor but led to a stable
complex of the type III receptor bound to the type II receptor and the
type I receptor (Fig. 7, B and C). These results
suggest that the cytoplasmic domain of the type III receptor functions
not only to bring autophosphorylated/activated type II receptor into
the complex, but also to dissociate the type III receptor from the
activated signaling complex between the type II receptor and type I
receptor. As the type II receptor phosphorylates the type III receptor
on the cytoplasmic domain, this phosphorylation represents a potential
mechanism by which the type III receptor dissociates from the active
signaling complex between the type II receptor and the type I receptor
(Fig. 8).
View larger version (40K):
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Fig. 7.
Mechanism of action for the cytoplasmic
domain of the type III receptor. COS-7 cells were transiently
transfected with 2 µg of the HA-tagged type III TGF- receptor with
(III) or without (III-cyto) the cytoplasmic
domain, 2 µg of the type II TGF-
receptor, and increasing amounts
(0.5-8 µg) of the type I TGF-
receptor. Cells were affinity
labeled with 125I-TGF-
1, immunoprecipitated
(IP) with the
277 antibody (panel A) or the
HA antibody (panel B), and analyzed on 10%
SDS-polyacrylamide gels. The brackets delineate the type III
receptor, the type II receptor, and the type I receptor. Molecular mass
standards (in kDa) are indicated on the right. Panel
C, the data were quantified using ImageQuant software, and the
data are expressed as a percent of control.
View larger version (28K):
[in a new window]
Fig. 8.
Model for role of the type III
receptor in TGF- signaling. A,
TGF-
binds to the type III receptor
(TGF
RIII). B,
TGF
RIII preferentially presents TGF-
to the autophosphorylated
type II receptor (TGF
RII) via a specific
interaction of the cytoplasmic domain of TGF
RIII with the
cytoplasmic domain of autophosphorylated TGF
RII. C, the
complex of TGF-
, TGF
RIII, and TGF
RII recruits and binds the
type I receptor (TGF
RI). D, TGF
RII transphosphorylates
and activates TGF
RI, and it transphosphorylates the cytoplasmic
domain of TGF
RIII, releasing it from the active signaling complex of
TGF
RI and TGF
RII (E). F, the
active signaling complex phosphorylates Smad2 or Smad3 to propagate
further downstream TGF-
signaling. Phosphorylation sites/events are
signified by the black circles.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mediates a vast array of biology through an apparently
simplistic signaling pathway. A number of factors that interact with
the TGF-
signaling pathway, including the type I and type II
receptor-interacting proteins, TRIP-1, STRAP, FKBP-12, PP2A, and SARA,
and transcription factors that interact with the Smad transcription factors including NF-1, Sp-1, AP-1, CREB, TFE3, and
FAST1/2, have been described. These proteins increase the complexity of
the pathway and may serve to regulate the TGF-
pathway (1).
Alternative mechanisms for the generation of complexity are through
interactions among the numerous cell surface receptors for TGF-
as
well as through potentially novel downstream signaling pathways from
these receptors.
signaling mediated
by a functional interaction of the cytoplasmic domains of the type III
receptor with the type II receptor. This interaction is demonstrated by
1) the ability of cytoplasmic domain of the type II receptor to
interact with the cytoplasmic domain of the type III receptor as
measured by the
277 antibody immunoprecipitation assay
(i.e. binding of the type III receptor to the
277
antibody and the type II receptor are mutually exclusive); 2) the
requirement for kinase activity of the type II receptor for this
interaction; 3) the ability of the type II receptor to phosphorylate
the cytoplasmic domain of the type III receptor; and 4) the specificity
of the cytoplasmic domain of the type III receptor for
autophosphorylated type II receptor. The significance of this
interaction is demonstrated by the inability of mutants of the type III
receptor lacking the cytoplasmic domain to carry out a type III
receptor function, namely enhancing TGF-
2 signaling. The mechanism
of action of the type III receptor in mediating signaling involves the
specific ability of the cytoplasmic domain of the type III receptor to bind autophosphorylated/activated type II receptor and the ability of
the type III receptor to dissociate from the active signaling complex
comprised of the type II receptor and the type I receptor.
Superfamily
Signaling--
The type III receptor has previously been thought to
have a nonessential role in TGF-
signaling, acting only to
"present" ligand to the signaling type I and type II receptors. In
this presentation role, the type III receptor binds TGF-
and then the type II receptor and presents TGF-
to the type II receptor. The
type II receptor is activated by binding TGF-
and recruits the type
I receptor into a complex and phosphorylates it to activate further
downstream signaling (Fig. 8). The presentation role for the type III
receptor was suggested by the somewhat lower affinity of the type III
receptor for TGF-
ligands (30-300 pM for the type III
receptor versus 25-50 pM for the type II
receptor), the lack of an obvious signaling motif in the short
cytoplasmic domain of the type III receptor, and the ability of cells
to respond to TGF-
in the absence of type III receptor expression.
1 but are unresponsive to TGF-
2, as endoglin does not bind
TGF-
2. Sensitivity to TGF-
2 can be restored by ectopic expression
of the type III receptor, supporting an essential role for the type III
receptor in TGF-
2 signaling (24). The type III receptor has also
been shown to have an essential nonredundant role in TGF-
signaling,
mediating the effects of TGF-
(TGF-
1 or TGF-
2) on mesenchymal
transformation in chick embryonic heart development (25). In addition,
the loss of functional type III receptor expression on intestinal
goblet cells is sufficient to mediate resistance to TGF-
(26).
Finally, the type III receptor was shown to bind and modulate signaling
by another TGF-
superfamily member, inhibin (27).
signaling establish that the type III receptor is
essential for mediating the effects of TGF-
, particularly for the
TGF-
2 isoform. The type III receptor without the cytoplasmic domain
can bind the type II receptor and increase TGF-
binding to the type
II receptor, but this is insufficient to enhance signaling. Thus, the
type III receptor does more than simply present ligand. We propose a
model depicted in Fig. 8 in which the type III receptor selectively
enhances the formation of the active signaling complex between the
autophosphorylated type II receptor and the type I receptor. The type
III receptor carries out this function by selectively binding the
autophosphorylated, activated type II receptor via its cytoplasmic
domain, mediating the specific interaction of the autophosphorylated
type II receptor with the type I receptor, and then dissociating itself
from the activated signaling complex between the type II receptor and
the type I receptor. This dissociation may involve the phosphorylation of the type III receptor by the type II receptor as discussed below.
, to bind the type
II receptor, or to enhance TGF-
binding to the type II receptor
(11). Although we find that the type III receptor lacking its
cytoplasmic domain can bind TGF-
, associate with the type II
receptor, and present TGF-
to the type II receptor, its ability
to mediate TGF-
2 signaling in a biological assay is affected. The
previous observations regarding the role of the cytoplasmic domain of
the type III receptor were based on experiments done in transiently
transfected COS cells. In addition, although the ability of the type
III receptor without the cytoplasmic domain to bind the type II
receptor and enhance TGF-
binding was investigated, the ability to
enhance TGF-
signaling was not (11). The functions ascribed here to
the cytoplasmic domain of the type III receptor, namely phosphorylation
by the type II receptor, associating specifically with the cytoplasmic
domain of the autophosphorylated type II receptor and dissociating the
type III receptor from the complex of the type II receptor and the type
I receptor, appear to be essential for the role of the type III
receptor in TGF-
signaling. Indeed, phosphorylation of the type III
receptor by the type II receptor may be the mechanism by which the type
III receptor is released from the active signaling complex. The vital
role of the cytoplasmic domain is supported further by evidence that
the extracellular domains of the type II and type III receptors do not
bind TGF-
2 in an cooperative manner and that the binding of the
extracellular domain of the type III receptor to TGF-
2 does not
promote binding of the extracellular domain of the type II receptor to
TGF-
2.2
, further establishing the
importance of the cytoplasmic domain of the type III receptor in
TGF-
signaling.
Receptors on the Cell
Surface--
The type III receptor exists as homodimers and oligomers
in vivo. We were able to detect these homodimers and
oligomers with the
277 antibody, indicating that the
277 epitope
is accessible when the type III receptor is complexed with other type
III receptors. The accessibility of the type III receptor cytoplasmic
domain suggests that these oligomeric complexes may be able to interact with the type II receptor. This is supported further by the finding that type II receptor expression inhibits immunoprecipitation of the
homo-oligomers of the 44-564 and 44-575 mutants of the type III
receptor by the
277 antibody (Fig. 5). Although the precise
stoichiometry of TGF-
receptor complexes at the cell surface has not
been elucidated, these results support a model in which oligomers of
the type III receptor form complexes with one or more type II
receptors. The type II receptor has been found in complexes with either
the type I receptor or the type III receptor, as well as in complexes
with both the type I and III receptors. We had observed previously that
only a minority of the type II receptor is complexed with a minority of
the type III receptor in vivo (17). The present results,
which demonstrate that the type III receptor binds preferentially to
autophosphorylated and activated type II receptor and dissociates from
the type II receptor complex when the type I receptor is recruited, are
consistent with this finding.
277 antibody is critical for this
interaction, which is consistent with the high degree of conservation
of this region. The region of high homology between the type III
receptor and endoglin can be divided into three domains: box 1 (94%),
which includes part of the transmembrane domain and the first five
amino acids in the cytoplasmic domain, and box 2 (88%), which is
composed of the last 17 amino acids of the cytoplasmic domain
(including all of the epitope for the
277 antibody). The
22-24-amino acid sequence linking these two regions is much less
conserved (30%). In contrast to the
277 antibody, the polyclonal
antibody to the entire cytoplasmic domain of the type III receptor
(
820) and a polyclonal antibody to the linker region of endoglin are
both able to immunoprecipitate the type II receptor along with either
endoglin or the type III receptor, respectively (16, 28). Thus, it
appears that within the type II-type III receptor complex, box 2 is
shielded from antibody interaction, whereas the other regions remain
accessible for antibody binding. Interestingly, box 2, which associates
with the type II receptor, is absent in a splice variant of endoglin which is truncated after box 1 (29). This presents the intriguing possibility that one difference between these splice variants will be
in their ability to interact with the type II receptor and the TGF-
signaling pathway. Indeed, in the one report in which the role of these
splice variants was investigated, it was shown that full-length
endoglin was able to antagonize TGF-
signaling, whereas the
truncated version was not (30). The role the type II receptor
interactions with endoglin and the relevance of this to TGF-
signaling will require further evaluation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Josee Plamondon for expert technical
assistance, Dr. Joan Massague for the generous supply of the 820
antibody, Dr. Kunxin Luo for the type II receptor autophosphorylation
site mutant constructs, and R & D Systems, Inc. for generous supply of
TGF-
1.
![]() |
FOOTNOTES |
---|
* This work was supported by a Howard Hughes Medical Institute postdoctoral research fellowship for physicians (to G. C. B.) and by Grants CA73161-01 from the NCI, National Institutes of Health (to W. P. S.) and CA63260 from the National Institutes of Health (to H. F. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Medicine, Box 2631, Duke University Medical Center, Durham, NC 27710. Tel.: 919-668-1352; Fax: 919-668-2458; E-mail: blobe001@mc.duke.edu.
Present address: Dept. of Pediatrics, National Jewish Medical
and Research Center, Denver, CO 80206.
Published, JBC Papers in Press, April 25, 2001, DOI 10.1074/jbc.M100188200
2 De Crescenzo, G., Grothe, S., Zwaagstra, J., Tsang, M., and O'Connor-McCourt, M. D. (May 29, 2001) J. Biol. Chem. 10.1074/jbc.M009765200
3 G. C. Blobe and H. F. Lodish, unpublished observations.
4 G. C. Blobe, X. Liu, and H. F. Lodish, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor
;
BMP, bone morphogenetic protein;
GST, glutathione S-transferase;
KD, kinase-dead;
HA, hemagglutinin.
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