From b The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, e Department of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel, a Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, and i Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
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ABSTRACT |
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Transforming growth factor- The transforming growth factor- TGF- The stoichiometry of the signaling complex is not known. T We report here studies on the physical nature of the T Materials and Constructs--
COS7 (CRL 1651), L6 (CRL 1458),
and Mv1Lu (CCL 64) cells were grown as described previously (22, 25).
9E10 ( Binding and Cross-linking--
Radioiodination of TGF- Velocity Centrifugation on Sucrose Gradients--
The velocity
centrifugation technique has been described elsewhere (22). Briefly,
cleared lysates (150 µl in MNT lysis buffer) were mixed with 50 µl
size markers (29-669 kDa; Sigma) and layered over 7.5-30% sucrose
gradients in MNT/1% octyl-POE. Centrifugation was for 8 h, 60,000 rpm, in an SW60 rotor (Beckman) at 4 °C. 250 µl fractions were
removed sequentially from the top of each gradient. After removal of 25 µl for SDS-PAGE and Coomassie staining (to analyze migration of
markers for each individual gradient), fractions were
immunoprecipitated with Immunofluorescence Co-patching--
The method used has been
described elsewhere (21, 22). In the protocol used (detailed in Ref.
21), COS7 cells co-transfected with T GST-Smad3 Kinase Assay--
The construction of cell line
Mv1Lu-Flag-N-Smad3, Mv1Lu in which an N-terminally Flag-tagged Smad3
gene was stably expressed, was described elsewhere (28). GST-Smad3
(construct provided by Ying Zhang and Rik Derynck) (29) protein was
prepared from bacterial lysate as described (30). Mv1Lu cells (Fig.
4A) were incubated in KRH/BSA for 30 min at 37 °C,
followed by 5 min with or without 2 mM DTT. They were
washed three times with KRH/BSA, and incubated with 100 pM
unlabeled TGF- T
L6 rat myoblasts, TGF- T
Fig. 2 shows the results of co-patching
experiments performed on COS7 cells co-transfected with T
Ligand binding and cross-linking to T DTT Increases the Detergent Sensitivity of the Complex--
In
contrast to the persistence of T DTT Pretreatment Prevents TGF- Our major findings are: 1) T We previously used velocity centrifugation to demonstrate that T The tetrameric nature of the complex is also supported by data
indicating that a dimer of T We provide the first evidence in live cells that the formation of the
T The ligand-independent association of a fraction of T To further investigate the relationship between TGF- Our data agree only in part with a previously published report that DTT
pretreatment does not affect type I receptor phosphorylation and does
not measurably alter the amount of ligand-bound and cross-linked T These results raise interesting questions about the stoichiometry of
TGF- The number of ligand molecules in the complex is not known. The most
likely possibilities are one TGF- (TGF-
) binds to
and signals via two serine-threonine kinase receptors, the type I
(T
RI) and type II (T
RII) receptors. We have used different and
complementary techniques to study the physical nature and ligand
dependence of the complex formed by T
RI and T
RII. Velocity
centrifugation of endogenous receptors suggests that ligand-bound
T
RI and T
RII form a heteromeric complex that is most likely a
heterotetramer. Antibody-mediated immunofluorescence co-patching of
epitope-tagged receptors provides the first evidence in live cells that
T
RI·T
RII complex formation occurs at a low but measurable
degree in the absence of ligand, increasing significantly after TGF-
binding. In addition, we demonstrate that pretreatment of cells with
dithiothreitol, which inhibits the binding of TGF-
to T
RI, does
not prevent formation of the T
RI·T
RII complex, but increases
its sensitivity to detergent and prevents TGF-
-activated T
RI from
phosphorylating Smad3 in vitro. This indicates that either
a specific conformation of the T
RI·T
RII complex, disrupted by
dithiothreitol, or direct binding of TGF-
to T
RI is required for signaling.
INTRODUCTION
Top
Abstract
Introduction
References
(TGF-
)1 ligands are
members of a large superfamily of cystine knot growth factors, which includes decapentaplegic (dpp) from Drosophila melanogaster
as well as the Müllerian-inhibiting substance and the activins
and bone morphogenetic proteins from mammals. The TGF-
s are
important modulators of development, the extracellular matrix, and the
immune response; they are potent growth inhibitors in many cell types, and their receptors and some downstream signaling elements are tumor
suppressors (1-5).
signals through the sequential activation of two
serine-threonine kinase cell surface receptors (6-10), termed type I
and type II (T
RI and T
RII). These two receptors physically associate to form a stable complex (8, 11). Several chimeric receptor
systems have established that this complex is required for signaling
(12-16). Whether it is preformed or ligand-induced is controversial.
Isolation of a T
RI·T
RII complex from detergent lysates was
possible only after pretreatment with TGF-
(9). Ligand-independent
interactions between receptor cytoplasmic domains, however, have been
detected in transfected COS cells and in the yeast two-hybrid
system,2 and work with
TGF-
2 (which requires both receptors to bind) suggests that at least
a small percentage of the cell surface receptor population is in
preformed complexes (9, 17-19). Data from experiments on the effect of
DTT also raise questions about the role of ligand in the complex.
Treatment of cells with DTT prevents TGF-
binding to T
RI but not
to T
RII (8, 20) and, in vitro, prevents formation of the
T
RI·T
RII
complex.3
RI and
T
RII form ligand-independent homodimers in the endoplasmic reticulum
and on the cell surface (21, 22). The simplest model is that two
homodimers form a heterotetrameric signaling complex induced or
activated by TGF-
. This view is supported by studies based on
functionally complementary type I receptor mutants and on chimeric
TGF-
/erythropoietin receptors demonstrating that type I dimers are
required for signaling (13, 23). Yamashita et al. (24) used
nonreducing/reducing two-dimensional SDS-PAGE to isolate ligand-bound
T
RI and T
RII homo- and heterodimers. They speculate that these
are derived from heterotetramers, although their data are also
consistent with smaller complexes.
RI·T
RII
complex. We demonstrate that the complex is most likely a stable
heterotetramer. Our studies show conclusively that some T
RI·T
RII complexes exist at the surface of live cells in the absence of ligand, and that TGF-
significantly enhances
heterocomplex formation. DTT treatment, which prevents TGF-
binding
to T
RI, does not inhibit complex formation but does result in the
failure of T
RI from DTT-treated cells to phosphorylate Smad3
in vitro. We suggest that ligand binding to the two
receptors may have different functions, and that complex formation
itself is not sufficient for signal initiation.
EXPERIMENTAL PROCEDURES
-Myc) mouse ascites was from Harvard Monoclonals and 12CA5
(
-HA) from BabCO. Fluorophore-labeled affinity-purified antibodies,
Cy3-streptavidin, and biotinylated F(ab')2 of G
M (goat
anti-mouse F(ab')2 were from Jackson Immunoresearch
Laboratories. IgG fractions and monovalent F(ab') fragments were
prepared as described (22, 26, 27). Untagged T
RI, T
RII, and
N-terminally HA- and Myc-tagged receptors were as described previously
(6, 10, 21, 22).
1
(Celtrix Laboratories and R&D Systems) and binding and cross-linking of
subconfluent cells were as described (25). Cells were preincubated (30 min, 37 °C) in KRH (50 mM HEPES, pH 7.5, 128 mM NaCl, 1.3 mM CaCl2, 5 mM MgSO4, 5 mM KCl) containing
0.5% fatty acid free BSA (KRH/BSA; Sigma). DTT-treated cells were
incubated with 2 mM DTT (5 min, 37 °C) and then rinsed
three times with warm KRH/BSA. Cells were then incubated (1-4 h,
4 °C) in fresh KRH/BSA with 100 pM
125I-TGF-
1. Cross-linking was performed with 0.5 mg/ml
disuccinimidyl suberate (Pierce) for 15 min, followed by quenching with
20 mM glycine. Cells to be used for gradients (Fig. 1) were
then rinsed, incubated in 0.2 mM iodoacetamide in KRH (15 min, 4 °C), and lysed in 150 µl of MNT lysis buffer (20 mM MES, pH 6.0, 30 mM Tris, pH 7.4, 100 mM NaCl, with 2% n-octyl-polyoxyethylene
(octyl-POE; Bachem Bioscience)). For co-immunoprecipitation (Fig. 3),
cells were lysed in 1 ml of various lysis buffers (see Fig. 3 legend). After clearing the lysates, 100 µl were analyzed directly by
SDS-PAGE. The remainder was split in half and immunoprecipitated
(overnight, 4 °C) with either 10 µl/ml of antibody
-IIC, a
polyclonal rabbit antiserum raised against the C-terminal 16 amino
acids of the type II receptor (11), or 14 µl/ml of antibody VPN,
raised against the juxtamembrane region of the human type I receptor
(6). After an additional 30 min of incubation with 50 µl 1:1 protein A-Sepharose in PBS, bound beads were rinsed twice with the original lysis buffer then once with PBS. Protein was eluted into SDS-sample buffer and analyzed by SDS-PAGE.
-IIC or VPN. Samples were analyzed by 7.5%
SDS-PAGE. Autoradiographs were quantified with a LaCie Silverscanner II
and MacBAS (Fuji) software.
RI-Myc and T
RII-HA were
preincubated (30 min, 37 °C) in serum-free Dulbecco's modified
Eagle's medium, washed twice with cold Hanks' balanced salt solution
with 20 mM HEPES, pH 7.4, containing 1% fatty acid-free
BSA, and incubated successively with (a) normal goat IgG
(200 µg/ml) to block nonspecific binding; (b)
-Myc
F(ab') (50 µg/ml); (c) F(ab') of biotinylated G
M (5 µg/ml); (d) F(ab') of unlabeled G
M (200 µg/ml), used
to block all free epitopes on the
-Myc F(ab'); (e)
-HA
IgG (20 µg/ml); (f) fluorescein isothiocyanate-labeled
G
M (20 µg/ml). After fixation (3.2% para-formaldehyde in PBS, pH
7.4, with 1.1% lysine and 0.24% NaIO4) and quenching with
50 mM glycine in PBS (21), the cells were incubated with 0.5 µg/ml Cy3 streptavidin, and mounted with mowiol (Hoechst) containing 29 mM n-propyl gallate (Sigma). Note that
membrane proteins retain some lateral mobility after paraformaldehyde
fixation, and can therefore be patched by streptavidin because of its
super-high binding affinity and multivalent nature. Fluorescence
microscopy and digital image acquisition (CC/CE 200 CCD camera,
Photometrics) were described (21). For each field, fluorescein and Cy3
images were taken separately using highly selective filter sets; the two images were superimposed, exported to Photoshop (Adobe) and printed
(21).
1 (10 min, 37 °C). Cells were then washed and lysed
in 1 ml of lysis buffer (150 mM NaCl, 1% Nonidet P-40, 50 mM Tris-HCl, pH 7.5, 50 mM NaF, 50 mM
-glycerophosphate, 1 mM
Na3VO3, 1 mM DTT, 5 mM
EDTA, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml
leupeptin, 10% glycerol). Supernatants were equalized for protein
content and incubated with 2 µg of GST-Smad3 (3 h, 4 °C). The GST
fusion protein was bound to 10 µl of glutathione beads that were
washed three times with lysis buffer, then washed again with kinase
buffer (50 mM NaCl, 20 mM Tris, pH 7.5, 12 mM MgCl2, 5 mM DTT) and subjected
to an in vitro kinase reaction with 0.2 mM
unlabeled ATP and 50 µCi [
-32P]ATP (20 µl,
30 °C, 30 min). For Fig. 4B, lysates from
Mv1Lu-Flag-N-Smad3 cells treated similarly were immunoprecipitated with
the anti-T
RI antibody VPN (6), with or without 3 µg/ml competing
peptide, before the incubation with GST-Smad3 and
[
-32P]ATP under the above conditions. SDS-containing
sample buffer was added, and the samples boiled to terminate the kinase
reaction. Samples were analyzed by 8% SDS-PAGE.
RESULTS
RI and T
RII Form Stable, Heterotrimeric, or Heterotetrameric
Complexes--
We studied the size of the cell surface ligand-bound
and cross-linked T
RI·T
RII complex using sucrose gradient
velocity centrifugation, a technique we used previously to show that
both receptors form homodimer-sized complexes in the endoplasmic
reticulum (22). Cells were lysed with octyl-POE, a nonionic detergent
with a density of 1 and a high CMC that enables analysis of the
migration of detergent-solubilized proteins rather than of micelles
(31). 125I-TGF-
1-bound and cross-linked T
RII from
COS7 cells transfected with T
RII alone migrated with a peak centered
at fraction 7, the position of a 150-kDa marker protein (Fig.
1A, top and
bottom panels). Although absolute size determinations can be
inaccurate for detergent-solubilized membrane proteins, this is
consistent with a homodimer bound to one or more molecules of TGF-
1,
as expected from previous gradient analysis and immunofluorescence (21,
22). T
RII from COS7 cells cotransfected with both T
RI and T
RII
(Fig. 1A, middle and bottom panels)
and immunoprecipitated with an antibody against T
RI, thus in a
T
RI·T
RII complex, migrated more quickly with a peak centered
around fraction 8, indicative of a heterotrimeric or tetrameric
complex.
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Fig. 1.
Type I and II receptors form stable
heterotetrameric or trimeric complexes in COS7 and L6 cells. Cells
were cross-linked to 125I-TGF- 1, lysed, and subjected to
velocity centrifugation over sucrose gradients. A, COS7
cells were transfected with T
RII alone (top panel) or
together with T
RI (middle panel). Fractions from the
sucrose gradients were immunoprecipitated with anti-T
RII
(top) or anti-T
RI (middle) and analyzed by
SDS-PAGE. Migration of the type II receptor on each gradient was
quantified and normalized to the peak value and is shown in the
bottom panel. Size estimates (in kDa) are from size markers
included in each gradient tube. Receptors from doubly transfected cells
(middle) were immunoprecipitated with antibody against
T
RI, ensuring that all T
RII measured is part of the complex and
not from singly transfected cells. Quantification of the
immunoprecipitated ligand-labeled type I receptor results in a nearly
identical curve (data not shown). The broad nature of the peak for
cells transfected with T
RII alone may reflect some association of
T
RII with the small number of endogenous T
RI found in COS7 cells.
B, L6 cells were treated as in A, except that
cells in the middle panel were treated with 2 mM
DTT before ligand binding. Fractions were immunoprecipitated with
anti-T
RII. Migration of T
RII (middle panel) or both
receptors (top) on the gradients was quantified
(bottom panel). Note that fraction numbers are comparable
only within a given experiment, and that migration of markers for part
A and part B was slightly different. Control
experiments demonstrate that only minimal dissociation of
receptor-bound but noncross-linked ligand occurred in detergent lysates
over the 8 h required for centrifugation (data not shown).
-responsive cells which lack the type III
TGF-
receptor, show similar receptor complexes. TGF-
1-bound type
I and II receptors immunoprecipitated with an antibody against T
RII
both migrated with a peak centered at fraction 10, correlating with
markers between 230 and 265 kDa (Fig. 1B, top and
bottom panels), and consistent with a heterotetrameric or
trimeric complex. Nearly identical results were obtained with TGF-
2
and with other cell lines, including Mv1Lu, HYB2, THP1, and GH3 (data
not shown). Immunoprecipitation with an antibody against T
RI (data
not shown) gave similar results, indicating the absence of a
significant population of ligand-bound T
RII, which is not in a
complex with T
RI. Pretreatment of cells with DTT (Fig.
1B, middle and bottom panels)
prevented ligand binding and cross-linking to T
RI and slowed the
migration of T
RII to a peak centered around 155 kDa. The DTT-treated
complex is likely a dimer (for comparison, see singly transfected COS
cells in Fig. 1A), indicating disruption of the
T
RI·T
RII complex under these conditions. DTT does not appear to
affect T
RII homodimers, a finding consistent with previous results
(21). The DTT effect appears to be on the cross-linking of ligand to
T
RI rather than on cross-linking between T
RI and T
RII, because
immunoprecipitation of T
RI from DTT-treated cells following ligand
binding and cross-linking showed the absence of ligand-labeled T
RI
(Fig. 3). Analogously, ligand binding and cross-linking after DTT
treatment either to endogenous TGF-
receptors (20) or to COS cells
transiently expressing T
RI and T
RII (not shown) failed to reveal
T
RI labeling.
RI and T
RII Form a Ligand-dependent Complex in
Live Cells--
To study the T
RI·T
RII complex in live cells,
we used T
RI and T
RII carrying HA or Myc epitope tags at their
extracellular termini for immunofluorescence co-patching, a technique
that we developed and have described previously (21, 22). Briefly, a
tagged receptor at the surface of live, unfixed cells (in the cold to
avoid internalization) is forced into patches by a double layer of
bivalent IgGs where the secondary antibody is coupled to one
fluorophore (e.g. fluorescein, which emits green
fluorescence). A second receptor, containing a different tag, is
labeled by antibodies coupled to a second fluorophore (e.g.
Cy3, red fluorescence). The cells are examined by fluorescence
microscopy to determine whether the two receptors are swept into mutual
(yellow) or separate (red or green) micropatches. We have employed this
method successfully to demonstrate that all three TGF-
receptors
form ligand-independent homodimers (21, 22).
RI-Myc and
T
RII-HA. The labeling specificity is high, as shown in a control
experiment on cells transfected with T
RI-Myc alone (Fig.
2A, inset); these cells show only Cy3 labeling.
In the absence of ligand (Fig. 2A), 15-20% of the patches
were mutual (yellow), although the majority were separate (either green
or red). In the presence of 250 pM TGF-
1, the percentage
of mutual patches markedly increased to 40-50% (Fig. 2B),
demonstrating that T
RI and T
RII at the surface of live cells have
an inherently low probability of forming heterocomplexes that is
significantly enhanced by ligand binding. The fraction of a given
receptor type in heterocomplexes is proportional to the number of
yellow patches divided by the sum of yellow and red (for the
red-labeled receptor type) or yellow and green (for green-labeled
receptors). These fractions are similar for T
RI and T
RII
(23-29% and 57-63% in the absence and presence of ligand, respectively), in accord with a 1:1 stoichiometric ratio in the heterocomplex.
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Fig. 2.
Type I/II hetero-oligomers are
TGF- -dependent and DTT-independent
in live cells. COS7 cells were co-transfected with T
RI-Myc and
T
RII-HA (A-D), or with T
RI-Myc alone (panel A,
inset). 48 h after transfection, cells in (C) and
(D) were pretreated with 2 mM DTT (15 min,
37 °C). The live cells were incubated in the cold with (B
and D) or without (A and C) 250 pM TGF-
1 for 2 h, followed by successive
incubations with a series of antibodies to mediate patching and
fluorescent labeling (see "Experimental Procedures"). The labeling
protocol results in T
RI-Myc labeled by Cy3 (red), T
RII-HA labeled
by fluorescein (green), and mutual patches containing both receptors
labeled yellow upon superposition of the two fluorescent images. Cells
transfected with T
RI-Myc alone (panel A,
inset) are labeled exclusively with Cy3, demonstrating the
labeling specificity. The numbers of red, green, and yellow patches
were counted on the computer screen on 20 × 20 µm2
flat cell regions (avoiding the nucleus, which contains more
nonspecific staining and is out of the focal plane) for several
independent experiments. Bars, 20 µm.
RI co-expressed with T
RII is
abrogated by DTT pretreatment of cells (Ref. 20 and Fig. 1B,
middle panel). DTT treatment, however, did not dissociate the ligand-independent T
RI·T
RII complexes (around 20%
co-patching), and did not affect the enhancement of heterocomplex
formation by TGF-
1 (around 50% co-patching) (Fig. 2, C
and D). The fraction of each receptor type in yellow patches
also remained similar.
RI·T
RII heterocomplexes in
DTT-treated live cells (Fig. 2, C and D),
velocity sedimentation experiments showed that receptor heterocomplexes
were disrupted by DTT (Fig. 1B). We suspected that this
disparity resulted from destabilizing effects of the detergent used to
solubilize the receptors for velocity sedimentation. We therefore
examined the effect of various detergents on T
RI·T
RII
co-immunoprecipitation from DTT-pretreated Mv1Lu cells (Fig.
3). Immunoprecipitation with an antibody
against T
RI (Fig. 3, right panel) was used to assess the
integrity of the receptor complex in DTT-treated cells, because T
RII
residing in heterocomplexes would still be labeled by ligand and would
co-precipitate with T
RI even if the latter was unlabeled. Under the
most stringent lysis conditions used (buffers 1-3), there was no
co-immunoprecipitation, indicating the absence of intact heteromeric
complexes after detergent solubilization. These results hold in
different cell lines and with antibodies raised against different type
I receptor epitopes (not shown). The same was also true with buffer 4, which was used for the velocity centrifugation experiment in Fig.
1B. With a fifth lysis buffer, however, we detected a small
amount of intact T
RI·T
RII complex in the presence of DTT (Fig.
3, right panel, buffer 5), suggesting that buffer and
detergent conditions determine the integrity of the complex in
DTT-treated cells. Together with the demonstration of T
RI·T
RII
heterocomplex formation in live cells pretreated with DTT (Fig. 2),
these results suggest that DTT treatment alters the conformation of the
heterocomplex formed; the altered complex is less stable (as reflected
in its increased detergent sensitivity) and most likely has a different
conformation, resulting in the failure of ligand binding and
cross-linking to T
RI.
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Fig. 3.
Detergent susceptibility of
T RI·T
RII receptor
complexes in DTT-treated cells. MV1Lu cells pretreated (+) or not
(
) with 2 mM DTT were subjected to binding and
cross-linking with 100 pM 125I-TGF-
1. Cells
were then lysed in various lysis buffers: lane 1,
0.5% Triton X-100, 0.5% deoxycholic acid, 10 mM EDTA in
PBS; lane 2, 0.75% Triton X-100, 0.5% deoxycholic acid, 10 mM EDTA in PBS; lane 3, 1% Triton X-100, 0.5%
deoxycholic acid, 10 mM EDTA in PBS; lane 4, 2%
octyl-POE in MNT; lane 5, 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH
7.4. One-tenth of each lysate was analyzed by SDS-PAGE without further
treatment (not shown); one-half of the remainder was immunoprecipitated
with anti-T
RII (
-IIC), and the other half with anti-T
RI
(VPN).
-induced Activation of the Type I
Receptor--
In epithelial cells, an essential step in
TGF-
-mediated growth inhibition and PAI-1 promoter activation is
activation of the ability of T
RI to rapidly phosphorylate Smad3 at
its C-terminal SSVS motif (28). Because DTT treatment did not block the
ligand-mediated association of T
RI with T
RII, we examined the
signaling capability of these complexes as reflected by their ability
to phosphorylate Smad3 in vitro. Mv1Lu cells (untreated or
pretreated with DTT) were exposed to TGF-
1 to allow heterocomplex
formation and activation of T
RI. In one study, lysates from these
cells were incubated with recombinant GST-Smad3, and the isolated
complex was subjected to an in vitro kinase reaction. In
untreated cells (Fig. 4A,
lanes 1 and 2), TGF-
mediated a greater than
20-fold increase in Smad3 phosphorylation; two-dimensional tryptic
mapping indicated that this in vitro phophorylation occurred
at the same site as in vivo (data not shown). DTT-pretreated
cells, however, showed no ligand-induced phosphorylation (Fig.
4A, lanes 3 and 4). Similar results
(Fig. 4B) were obtained by first immunoprecipitating lysates
with antibodies against T
RI, then incubating the complexes with a
GST-Smad3 fusion protein in an in vitro kinase reaction.
Although there is background phosphorylation of the GST-Smad3 construct
in this assay, peptide competition during the immunoprecipitation (Fig.
4B, lanes 5-8) eliminates all of the
TGF-
-inducible phosphorylation, indicating that T
RI is
responsible. Thus, the T
RI·T
RII complex formed after DTT
treatment is inactive and cannot mediate the earliest step in TGF-
downstream signaling phosphorylation of Smad3.
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Fig. 4.
DTT pretreatment blocks
TGF- -mediated Smad3 phosphorylation.
A, Mv1Lu cells were treated with (lanes 3 and
4) or without (lanes 1 and 2) 2 mM DTT (5 min, 37 °C). After extensive washing, they
were incubated with (lanes 2 and 4) or without
(lanes 1 and 3) 100 pM TGF-
1 (10 min, 37 °C). Cells were rapidly chilled and lysed. Cleared lysates
were incubated with a GST-Smad3 fusion protein for 3 h. After
retrieval of the fusion protein by glutathione beads, it was subjected
to an in vitro kinase reaction then analyzed by SDS-PAGE.
Control cells were allowed to bind 125I-TGF-
1 under the
same conditions and were then cross-linked, lysed, and analyzed by
SDS-PAGE; there was no binding of ligand to T
RI in DTT-pretreated
cells, confirming that the 10-min incubation period at 37 °C was not
sufficient for arrival of significant amounts of non-DTT-exposed T
RI
at the cell surface (data not shown). B, Mv1Lu-Flag-N-Smad3
cells were treated as above, except lysates were immunoprecipitated
with an anti-type I antibody (6) with (lanes 5-8) or
without (lanes 1-4) 3 µg of competing peptide before
incubation with the GST-Smad3 fusion protein.
DISCUSSION
RI and T
RII form a stable,
heteromeric complex, most likely a heterotetramer; 2) in live cells, the two receptors have an intrinsic affinity for each other that is
markedly increased by TGF-
exposure; and 3) DTT does not prevent the
formation of this complex but increases its detergent sensitivity and
blocks TGF-
-induced activation of the type I receptor, as measured
by its ability to phosphorylate Smad3.
RI
and T
RII each form homodimer-sized complexes in the endoplasmic reticulum (22). We use similar technology here to show that ligand-bound and cross-linked receptors in transfected COS7 cells and
in L6 cells expressing native receptors form detergent-stable complexes
whose size is consistent with heterotrimers or heterotetramers. In L6
cells (Fig. 1B), the migration of the T
RI·T
RII
complex is consistent with a heterotetramer or heterotrimer bound to
one TGF-
molecule. In COS7 cells, the complex migrates significantly faster than a homodimeric complex (Fig. 1A, top
panel) (22); the wide nature of the peak, and the shoulder at
higher fractions, may be because of dissociation of larger complexes
during centrifugation. Furthermore, co-patching results obtained in
live cells (Fig. 2) are most consistent with a heterotetramer, the
fractions of T
RI and T
RII in mutual patches are similar, as
expected for a stoichiometric ratio of 1:1. It should be stressed,
however, that a definite determination of the stoichiometric ratio
depends on an accurate measurement of the surface levels of both
receptors on the cells scored, which is not feasible by the current methods.
RII and more than one T
RI reside in
the heterocomplex. Evidence that there are two T
RII in the complex
comes from the demonstration by velocity centrifugation that treatment
of cells with DTT results in a homodimer-sized complex of T
RII. This
suggests that there were two type II receptors in the original,
heteromeric complex; otherwise, one must assume that the normally
dimeric T
RII (21, 22) is monomeric in the heterocomplex and
reassociates to form dimers after DTT treatment and detergent
solubilization. Type I receptors, which are homodimers when expressed
alone and remain dimeric after DTT treatment, (22) are likely to be
multimeric in the active complex, as indicated by experiments
demonstrating the existence of functionally complementary T
RI
mutants (23). Taken together, the sedimentation velocity, co-patching,
and functional complementation studies imply that the signaling TGF-
receptor complex contains two T
RI and two T
RII polypeptides.
RI·T
RII complex is TGF-
-dependent (Fig. 2,
A and B). Previous studies have demonstrated the
TGF-
dependence of the T
RI·T
RII heterocomplex but used
receptors in detergent lysates, leaving open the possibility that
TGF-
stabilized the complex in detergent but did not induce its
formation (8, 9, 11). Co-patching studies examining heterocomplex
formation in the intact plasma membrane show a marked increase in
mutual aggregates in the presence of TGF-
1. There is, however, an
intrinsic affinity between the two receptors, as demonstrated by the
15-20% that reside in mutual aggregates in the absence of TGF-
(Fig. 2A). Although these experiments used cells
overexpressing the transfected receptors, as required for visualization
by immunofluorescence, there are several indications that the
heterocomplexes observed are not the result of high expression levels.
The co-patching experiments examine single cells under the microscope,
and the use of double labeling by IgG or biotin/streptavidin
enhancement enabled us to analyze cells expressing as few as 15,000 surface receptors (evaluated by photomultiplier-based measurement of
the fluorescence intensity on cells expressing known receptor levels, as described by Henis et al. (21)). These cells, with
receptor levels higher but of the same order of magnitude as
untransfected cells, yielded the same co-patching results as cells
expressing receptor levels 10-fold higher. In addition, in all cases
where the level and percentage of higher complexes formed were high enough to allow detection by ultracentrifugation in untransfected cells
expressing the receptors (including ligand-labeled T
RI·T
RII heterocomplexes in the current work, and T
RI or T
RII homodimers in earlier studies) (22), a good agreement was obtained between the
results on these cells and on transiently expressing COS cells. Furthermore, the results obtained in co-patching experiments depended on the receptor types examined, with low co-patching for type II/type
III TGF-
receptor heterodimers versus high co-patching levels for T
RI and T
RII homodimers (21, 22). This emphasizes the
specificity of the interactions measured. We note, however, that COS
cells overexpressing T
RI and T
RII exhibit some ligand-independent receptor phosphorylation (18), raising the possibility that the number
of T
RI·T
RII complexes is larger in this system than in cells
expressing the receptors endogenously.
RI and T
RII
is consistent with our previous finding that TGF-
2, unlike TGF-
1,
binds to a preformed complex of T
RI and T
RII (19). Such preformed
complexes do not appear to mediate TGF-
-independent signal
transduction,4 raising the
question whether the role of ligand is to increase the number of
complexes or to effect a necessary and stabilizing conformational
change. A recent model of the T
RI ectodomain (based on certain
cysteine motifs shared with protectin (CD59)) proposes that its surface
has two distinct binding sites, one each for ligand and T
RII (32).
This model is in agreement with the finding shown in Fig. 2 that there
is an intrinsic affinity between T
RI and T
RII, which is increased
by TGF-
.
-mediated
heterocomplex formation and signaling, we studied the effects of DTT
treatment on complex stability and its ability to phosphorylate Smad3
in vitro. Pretreatment with DTT prevents TGF-
binding and cross-linking to the type I receptor (8, 20). We demonstrate that it
also increases the detergent susceptibility of the complex (Figs.
1B and 3), and blocks TGF-
-induced activation of the
ability of T
RI to phosphorylate Smad3 in vitro (Fig. 4)
but does not change the percentage of T
RI and T
RII in mutual
complexes with or without TGF-
1 (Fig. 2). The observation that
multiple cross-linkers, including difluorodinitrobenzene (with a spacer
length of 3Å) can cross-link TGF-
to T
RI suggests that
cross-linking results reflect direct binding of ligand to T
RI rather
than their fortuitous proximity within a complex.4 Our DTT
data therefore suggest that either a specific T
RI·T
RII complex
conformation, destroyed by DTT, or direct ligand binding to T
RI are
required for signal transduction. It is unclear whether ligand binding
to T
RI stabilizes the complex with T
RII or serves a different
function altogether.
RI·T
RII complex in detergent lysates (33). Although we found that TGF-
1 can mediate heterocomplex formation after DTT treatment, especially in intact cells (Fig. 2), we have been able to demonstrate at most a small percentage of T
RI·T
RII complex using the same detergent lysis conditions as these investigators (see Fig. 3, lysis
buffer 5), in accord with a second report (6). In some of the
experiments by Vivien and Wrana (33), the antibody used was against a
C-terminal epitope of T
RI that has significant sequence identity to
other type I receptors (34); however, antibody cross-reactivity could
not explain all the differences between their results and ours.
receptors in the signaling complex. Although our data are most
consistent with a heterotetrameric
(T
RI)2(T
RII)2 receptor complex, and we
have obtained similar results with several cell lines, there are
suggestions in the literature that the stoichiometry of the complex may
vary. For example, the ratio of T
RI to T
RII in microvascular
endothelial cells was significantly higher in three-dimensional
versus two-dimensional cultures, corresponding to increasing
resistance to the anti-proliferative but not the matrix-inducing
effects of TGF-
(35). In other cell types, including bone, the ratio
of type I to type II receptors may also be important, particularly in
differentiating growth inhibition from other effects of TGF-
(36-38). The migration over sucrose gradients of the T
RI·T
RII
complex from nonepithelial cell systems remains to be determined.
homodimer, each subunit bound to a
type I and a type II receptor, or two ligand homodimers, each subunit
bound to one of the four receptors in a presumed heterotetramer. The
sucrose gradient data presented here show a surprisingly small increase
in complex size when T
RI is added to TGF-
-bound T
RII,
suggesting that the addition of T
RI to T
RII is not accompanied by
the recruitment of additional ligand molecules.
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ACKNOWLEDGEMENTS |
---|
TGF-1 was a kind gift of R&D Biosystems.
We are grateful to Ying Zhang and Rik Derynck (UCSF) for the GST-Smad3
construct and to Ralph Lin for comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA63260 (to H. F. L.) and DK02290 (to R. G. W.) and by grants from the Israel Science Foundation administered by the Israel Academy of Arts and Sciences and from the Israel Cancer Research Fund (to Y. I. H.).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.
c These authors contributed equally to this work.
d Current address: Dept. of Medicine, Yale School of Medicine, P. O. Box 208019, New Haven, CT 06520.
f Recipient of a fellowship from the Clore Scholars Programme.
g Supported by a postdoctoral fellowship from the Robert Steel Foundation for Pediatric Cancer Research.
h Supported by a National Institutes of Health postdoctoral fellowship.
j To whom correspondence should be addressed: The Whitehead Institute for Biomedical Research, 9 Cambridge Ctr., Cambridge, MA 02142. Tel.: 617-258-5216; Fax: 617-258-6768; E-mail: lodish{at}wi.mit.edu.
2 R. Perlman and R. A. Weinberg, personal communication.
3 C. Rodriguez, R. Lin, R. G. Wells, P. Scherer, and H. F. Lodish, manuscript in preparation.
4 R. G. Wells and H. F. Lodish, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
T
RI, T
RII, T
RIII, types I, II,
and III TGF-
receptors;
octyl-POE, n-octylpolyoxyethylene;
DTT, dithiothreitol;
PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered saline;
MES, 4-morpholineethanesulfonic acid;
GST, glutathione
S-transferase;
HA, hemagglutinin.
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REFERENCES |
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