(Received for publication, November 8, 1994)
From the
Transforming growth factor- (TGF-
) signaling in Mv1Lu
lung epithelial cells requires coexpression of TGF-
receptors I
(T
R-I) and II (T
R-II), two distantly related transmembrane
serine/threonine kinases that form a heteromeric complex upon ligand
binding. Here, we examine the formation of TGF-
receptor
homooligomers and their possible contribution to signaling. T
R-I
can contact ligand bound to T
R-II, but not ligand free in the
medium, and thus cannot form ligandinduced homo-oligomers. T
R-II,
which binds ligand on its own, formed oligomeric complexes when
overexpressed in transfected COS cells. However, these complexes were
largely ligand-independent and involved immature receptor protein.
Since ligand-induced homo-oligomers could not be obtained with the
wild-type TGF-
receptors, we studied receptor cytoplasmic domain
homo-oligomerization by using receptor chimeras. The extracellular
domain of T
R-II was fused to the transmembrane and cytoplasmic
domains of T
R-I, yielding T
R-II/I, and the extracellular
domain of T
R-I was fused to the transmembrane and cytoplasmic
domains of T
R-II, yielding T
R-I/II. When cotransfected with
wild-type receptors and exposed to ligand, T
R-II/I formed a
complex with T
R-I, and T
R-I/II formed a complex with
T
R-II, thus yielding complexes with homologous cytoplasmic
domains. T
R-II/I transfected alone or with T
R-I did not
restore TGF-
responsiveness in T
R-II-defective cell mutants.
Furthermore, T
R-II/I acted in a dominant negative fashion,
inhibiting restoration of TGF-
responsiveness by a cotransfected
T
R-II in T
R-II-defective cells and by a cotransfected
T
R-I in T
R-I-defective cells. Similarly, T
R-I/II
transfected alone or with T
R-II did not restore TGF-
responsiveness and acted in a dominant negative fashion against
T
R-I. Together with previous genetic and biochemical evidence,
these results suggest that TGF-
mediates transcriptional and
antiproliferative responses through the heteromeric
T
R-I
T
R-II complex and not through homo-oligomeric
T
R-I or T
R-II complexes.
Transforming growth factor- (TGF-
) (
)is a
multifunctional cytokine that binds to various membrane
proteins(1, 2) . Genetic and biochemical evidence
indicates that two of these proteins, known as receptor types I and II,
are involved in
signaling(3, 4, 5, 6, 7, 8) .
Both receptors are transmembrane serine/threonine kinases and consist
of a short extracellular domain, a single transmembrane region, a
kinase domain, and, in the type II receptors, a serine/threonine-rich
tail. Despite their similar domain structure, TGF-
receptors I and
II show only
40% amino acid sequence identity in the kinase domain
and even lower similarity outside this domain. The same holds true for
receptors that bind other TGF-
-related factors such as activin and
bone morphogenetic proteins (BMPs). The type I receptors for these
various factors are more similar to each other than they are to their
corresponding type II receptors, generating two distinct subfamilies of
transmembrane serine/threonine kinases(1) .
Evidence that
receptors I and II are essential for signaling derived originally from
chemically induced cell mutants selected for resistance to TGF-
action(3, 9, 10) . Some of the mutants lack
binding to receptor I, and others lack binding to receptors I and II.
Hybrids created by fusion of cells from one group with cells from the
other recovered receptors and full responsiveness to
TGF-
(3) . These observations suggested a model in which
receptor I requires receptor II for ligand binding and both receptors
act in concert to elicit transcriptional and antiproliferative
responses(3) . This model was recently confirmed in studies
with the cloned receptors. The type II receptors identified to date can
bind ligand on their
own(5, 11, 12, 13, 14) ,
but cannot signal independently of type I
receptors(4, 6, 7, 8) . The type I
receptors for TGF-
and activin can contact ligand bound to
receptor II, but not ligand that is free in the
medium(6, 7, 15, 16, 17) .
This interaction leads to formation of a tight trimolecular complex
between ligand, receptor I, and receptor II. Certain BMP type I
receptors can bind ligand when transfected alone into test cells;
however, they associate with cotransfected certain type II receptors
and bind ligand more effectively in their presence (18, 19, 20, 21) . (
)
The type II receptor for a given ligand may interact
with different type I receptor isoforms, forming combinations of
different signaling capacity(8, 22) .
Antiproliferative and extracellular matrix transcriptional responses to
TGF- in Mv1Lu mink lung epithelial cells are mediated by the
TGF-
type II receptor (T
R-II) in concert with the TGF-
type I receptor (T
R-I)(6, 7, 8) . No
TGF-
responses are observed when TGF-
binds to T
R-II
expressed in the absence of T
R-I (4, 6, 7, 8) or to T
R-I via a
T
R-II construct lacking the cytoplasmic domain (23) or
when T
R-I and T
R-II are expressed in the same cell, but one
of the two harbors a mutation that disrupts kinase
activity(4, 7, 8) .
The interaction
between TR-I and T
R-II suggests two alternative models for
TGF-
signal transduction. In one model, T
R-I and T
R-II
might signal separately, initiating distinct pathways whose cooperation
produces TGF-
responses(15) . After contacting the ligand,
T
R-I and T
R-II could each signal as monomers or, by analogy
with the tyrosine kinase receptors(24) , as ligand-induced
homodimers. Alternatively, the signaling entity could be the
heteromeric T
R-I
T
R-II complex. Formation of this
complex might be necessary to allow each receptor access to its
substrates or to allow one receptor kinase to stimulate the other,
which then propagates the signal to downstream substrates. Support for
the latter model comes from the recent demonstration that T
R-II is
a constitutively active kinase that, upon ligand binding, recruits and
phosphorylates T
R-I as a necessary step for signal propagation via
the T
R-I kinase(25) . However, the evidence to date does
not exclude the possibility that ligand-induced homologous receptor
interactions might participate in TGF-
signaling.
To
investigate this possibility, we have sought evidence for
ligand-induced homo-oligomeric TR-I or T
R-II complexes. In
addition, we forced homo-oligomerization of receptor cytoplasmic
domains by using chimeras that contain the extracellular domain of one
receptor and the transmembrane and cytoplasmic domains of the other.
The results of studies using these constructs argue that homologous
receptor kinase interactions do not mediate TGF-
signals.
For metabolic labeling, transiently transfected
cells were incubated in methionine-free minimal essential medium
containing 50 µCi/ml [S]methionine
(Tran
S-label, ICN) for 2.5 h at 37 °C followed by 1 h
at 4 °C in the presence or absence of TGF-
(1 nM).
Cell monolayers were then washed once in ice-cold phosphate-buffered
saline and lysed in buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% (v/v) Triton X-100, and protease
inhibitor mixture).
To determine the
relative levels of functional TR-II/HA and T
R-II/His
expressed on the surface of cotransfected COS cells, receptors were
affinity-labeled by cross-linking to bound
I-TGF-
1
and precipitated from cell lysates with anti-HA antibody or
Ni-NTA-agarose beads, respectively (Fig. 1A). The yield
of labeled T
R-II/HA was
3-fold less than the yield of labeled
T
R-II/His; however, this difference is attributable to different
recoveries in the two isolation procedures. Previous work indicated
that precipitation with anti-HA antibody recovers only
30% of the
total affinity-labeled T
R-II/HA, whereas precipitation with
Ni-NTA-agarose recovers T
R-II/His almost
quantitatively(16, 25) . Taking this difference into
account, the results indicated that functional T
R-II/HA and
T
R-II/His are expressed at equivalent levels on the surface of COS
cells.
Figure 1:
TGF- type II receptors do not form
ligand-dependent homo-oligomers. A, COS cells were transiently
transfected with HA-tagged T
R-II alone, His-tagged T
R-II
alone, both receptor vectors, or empty pCMV5 vector. Lysates from
transfected cells affinity-labeled with
I-TGF-
1 were
precipitated with anti-HA antibody or Ni-NTA-agarose beads as
indicated. Another aliquot was incubated with Ni-NTA-agarose, and the
eluate from these beads was precipitated with anti-HA antibody.
Precipitated material was visualized by SDS-PAGE and autoradiography. B, COS cells, Mv1Lu cells, and L
E
cells were transiently transfected with HA-tagged T
R-II and
metabolically labeled with [
S]methionine in
methionine-free medium for 30 (COS) or 10 (L
E
and Mv1Lu) min, and the label was chased with regular medium for
the indicated periods. Cell lysates were precipitated with HA antibody,
and the precipitate was subjected to SDS-PAGE and fluorography. The
immature T
R-II core and the mature T
R-II product are
indicated. C, COS cells transfected with the indicated
T
R-II constructs or empty vector were metabolically labeled with
[
S]methionine for 2.5 h and then incubated for 1
h at 4 °C in the presence or absence of 1 nM TGF-
1.
Cell lysates were precipitated with anti-HA antibody either directly or
after one cycle of binding and elution from
Ni-NTA-agarose.
More important, when these cell lysates were bound to
Ni-NTA-agarose and eluted with imidazole, and the eluates were
precipitated with anti-HA antibody, little or no ligand-labeled
TR-II was recovered (Fig. 1A). This two-step
precipitation method can readily recover ligand-induced complexes of
T
R-II/His and T
R-I/HA from COS cells (see Fig. 3)(25) . The present results suggested that
T
R-II did not form stable ligand-induced homo-oligomers in COS
cells.
Figure 3:
Cell-surface expression of TR-II/I
and T
R-I/II and association with wild-type receptors. Confluent
monolayers of COS-1 cells (A and B) or R-1B cells (C and D) transfected with the indicated receptor
combinations or empty pCMV5 vector were affinity-labeled by sequential
incubation with
I-TGF-
1. Cell lysates were subjected
to SDS-PAGE and autoradiography either directly (A and C) or after precipitation with a monoclonal antibody against
the HA epitope tagged onto T
R-I or with Ni-NTA-agarose beads that
bind hexahistidine sequences tagged onto T
R-II or T
R-I/II (B and D). Labeled material was visualized by PAGE
and autoradiography.
Since COS cells have been used in many TGF- receptor
studies, we examined the interaction of transfected T
R-II in more
detail by immunoprecipitation of metabolically labeled receptor.
Pulse-chase labeling with [
S]methionine revealed
that T
R-II was synthesized as a 70-kDa product that was converted
with time into a heterogeneous product of
80 kDa (Fig. 1B). Based on previous studies(30) ,
these products are identified as the newly synthesized T
R-II core
before N-linked glycan chain maturation and the mature
T
R-II glycoprotein, respectively. Metabolically labeled receptor
began to disappear 2 h after the start of the chase (Fig. 1B), at which time only about half of the
receptor population had reached maturity. Surface labeling of
T
R-II-transfected COS cells indicated that immature T
R-II did
not reach the cell surface (data not shown; see (25) ).
Continued labeling with [
S]methionine confirmed
that only about half of the steady-state receptor population was the
mature form (Fig. 1C). COS cells support episomal
replication of vectors that contain an SV40 origin of
replication(29) , resulting in vast overexpression of products
encoded by these vectors. In contrast to COS cells, Mv1Lu mink lung
epithelial cells and L
E
rat skeletal myoblasts,
which expressed stably transfected T
R-II at modest levels,
completely processed this receptor within 60 min after synthesis (Fig. 1B). Collectively, these results argue that
transiently transfected COS cells express more T
R-II protein than
they can fully process.
To search for homo-oligomeric TR-II
interactions, COS cells transfected with T
R-II/HA, T
R-II/His,
or the two receptors were metabolically labeled and subjected to the
two-step precipitation protocol described above to recover complexes
containing both tagged receptor forms. As expected, lysates from singly
transfected cells yielded no labeled products with this protocol (Fig. 1C). Two-step precipitation of lysates from
doubly transfected cells yielded a large amount of immature T
R-II
core and very little mature receptor form (Fig. 1C).
The amount of immature T
R-II form recovered in this process was
approximately half of the total amount, as determined from the
autoradiographic signals (Fig. 1C, compare the second and fifthlanes). The receptor yield
was only marginally increased by incubation of cells with a high
concentration (1 nM) of TGF-
1 (Fig. 1C).
These results indicate that in COS cells overexpressing T
R-II, a
large proportion of excess immature T
R-II and a small proportion
of mature T
R-II form ligand-independent complexes and that the
addition of ligand has little effect on the yield of these complexes.
Similar experiments performed with Mv1Lu cells did not yield
ligand-independent or -dependent T
R-II complexes (data not shown).
Figure 2:
Schematic representation of TGF-
receptor chimeras. Boxes represent the receptor kinase
domains. The amino acid positions of each receptor portion at the
chimera break points are indicated by single letter
code.
When
transfected into COS cells, TR-II/I had the ligand binding and
receptor interaction properties of wild-type T
R-II, as determined
by affinity labeling with
I-TGF-
. T
R-II/I bound
ligand when expressed alone (Fig. 3A), supported ligand
binding to a cotransfected T
R-I (Fig. 3A), and
formed a coprecipitating complex with T
R-I (Fig. 3B). Thus, T
R-II/I was able to bind ligand
in concert with T
R-I, generating oligomeric receptor complexes
with multiple T
R-I cytoplasmic domains. Similar results were
obtained by transfection of T
R-II/I and T
R-I into R-1B cells (Fig. 3, C and D). R-1B cells are a chemically
induced mutant derivative of the Mv1Lu cell line and lack TGF-
responses because of a defect in endogenous T
R-I(9) .
Untransfected R-1B cells express endogenous T
R-II at levels much
lower than those of transfected T
R-II, hence the lack of
T
R-II signal in the autoradiographs shown here.
Like TR-I,
the chimera T
R-I/II did not bind TGF-
when transfected alone
into COS or R-1B cells (Fig. 3, A and C).
Cotransfection of a hexahistidine-tagged T
R-I/II with T
R-II,
followed by affinity labeling and precipitation with Ni-NTA-agarose,
showed coprecipitation of T
R-II with T
R-I/II
(Fig. 3, B and D). This complex was
ligand-dependent as demonstrated by two-step precipitation of
metabolically labeled receptors from cells expressing T
R-II/HA and
hexahistidine-tagged T
R-I/II (Fig. 4A). Under
these conditions, T
R-I/II did not become efficiently labeled by
cross-linking to bound
I-TGF-
(Fig. 3).
However, T
R-I/II labeling was detected when a higher concentration
of
I-TGF-
(1 nM instead of 250 pM)
was used in the assays (Fig. 4B). The low labeling
efficiency could be due to formation of anomalous contacts between
TGF-
and T
R-I/II, diminishing the efficiency of the
cross-linking reaction. Nevertheless, T
R-I/II was clearly capable
of forming ligand-induced complexes with T
R-II, generating
oligomers containing multiple T
R-II cytoplasmic domains.
Figure 4:
Ligand-dependent interaction of
TR-I/II with T
R-II. A, R-1B cells cotransfected with
hexahistidine-tagged T
R-I/II and HA epitope-tagged T
R-II were
metabolically labeled with [
S]methionine and
then incubated for 1 h at 4 °C in the presence or absence of 1
nM TGF-
1. Cell lysates were precipitated with
Ni-NTA-agarose beads, the beads were eluted with imidazole, and the
eluate was precipitated with HA antibody. Immunoprecipitates were
subjected to SDS-PAGE and autoradiography, which demonstrated a
ligand-dependent interaction between the two receptors. B,
R-1B cells cotransfected with T
R-I/II and T
R-II were
affinity-labeled with the indicated concentrations of
I-TGF-
1, revealing labeling of T
R-I/II only at
the high
I-TGF-
1
concentration.
TGF- receptors, singly or in relevant combinations, were
cotransfected with the TGF-
-inducible construct p3TP-Lux, which
contains a luciferase reporter gene(4) . Transfection of
T
R-I alone or in combination with T
R-II restored TGF-
responsiveness in R-1B cells, whereas transfection of T
R-II/I did
not (Fig. 5A). Cotransfection of T
R-II/I prevented
T
R-I from restoring this TGF-
response (Fig. 5A). Similar results were obtained with the
antimitogenic effect of TGF-
under conditions (16) in
which
60% of the R-1B cell population takes up transfected
T
R-I cDNA and becomes inhibited in response to TGF-
(Fig. 6). As determined by
[
I]iododeoxyuridine incorporation into DNA, the
ability of R-1B cells to respond to TGF-
was not diminished by
cotransfection of T
R-II, but was blocked by cotransfection of
T
R-II/I (Fig. 6).
Figure 5:
Dominant negative effect of TR-II/I
on TGF-
transcriptional responses. R-1B cells (A) or
DR-26 cells (B) were transiently cotransfected with the
indicated receptor vectors and/or empty pCMV5 to equalize the amount of
DNA in the transfections. A fixed amount of the reporter vector
p3TP-Lux was included in all transfections. TGF-
1 (250
pM) was added 36 h later. Cell extracts were prepared 20 h
later, and luciferase activity was determined in a
luminometer.
Figure 6:
Dominant negative effect of TR-II/I
on TGF-
antiproliferative response. R-1B cells were transfected
with the indicated receptor vectors under conditions that yield
60% transient transfection efficiency. TGF-
1 (250
pM) was added 36 h later for 20 h.
[
I]Iododeoxyuridine (
I-dU) was added during the last 4 h of
incubation, and its incorporation into DNA was determined. Data are
presented as the percent inhibition of
[
I]iododeoxyuridine incorporation relative to
controls that did not receive TGF-
. Results are the means ±
S.D. of triplicate determinations.
These results indicate that
ligand-induced association of TR-I intracellular domains via the
T
R-I and T
R-II/I combination does not generate
transcriptional or antimitogenic responses to TGF-
in R-1B cells.
Instead, T
R-II/I acted in a dominant negative fashion presumably
by competing with endogenous T
R-II for transfected T
R-I or by
titrating out a substrate. T
R-II/I was also unable to signal when
transfected alone or with T
R-I into DR-26 cells (Fig. 5B) and inhibited the rescue of TGF-
responsiveness by T
R-II in these cells (Fig. 5B).
This effect was probably due to competition for the limited amount of
endogenous T
R-I. These results provide further evidence that
complexes of T
R-II/I with either endogenous or cotransfected
T
R-I lack the signaling capacity of heteromeric
T
R-I
T
R-II complexes.
We determined the signaling
capacity of TR-I/II by transfection of this construct into R-1B
cells. In these transfectants, TGF-
concentrations up to 2 nM did not elicit a transcriptional response (Fig. 7) or an
antiproliferative response (data not shown). Furthermore, the ability
of transfected T
R-I to restore responsiveness in R-1B cells, which
was increased by cotransfection with T
R-II in some experiments,
was decreased by cotransfection with T
R-I/II (Fig. 7). This
dominant negative effect was most evident at the higher TGF-
concentration range (Fig. 7), where the ability of transfected
T
R-I/II to compete for the limited pool of endogenous T
R-II
would be greater. Thus, ligand-induced association of T
R-II
intracellular domains via the T
R-II and T
R-I/II combination
was ineffective as a mediator of TGF-
responses in Mv1Lu cells.
Figure 7:
Effect of TR-I/II on the TGF-
transcriptional response. R-1B cells were transiently transfected with
empty pCMV5 (open circles), T
R-I vector (closed
circles), T
R-I and T
R-II vectors (open
squares), T
R-I/II vector (closed squares), or
T
R-I and T
R-I/II vectors (closed triangles). The
amount of DNA in all transfections was equalized by the addition of an
appropriate amount of empty pCMV5. The p3TP-Lux vector was included in
all transfections. 36 h after transfection, TGF-
1 was added at the
indicated concentrations. Cell extracts were prepared after 20 h, and
luciferase activity was determined in a luminometer. Data are the
average of triplicate determinations, with standard deviations smaller
than the size of the symbols.
Cotransfection of TR-I/II and T
R-II/I into R-1B cells and
incubation with up to 1 nM
I-TGF-
yielded a
very low level of receptor complex, as determined by the two-step
precipitation assay (data not shown). The untagged versions of these
two chimeras did not restore TGF-
responsiveness when
cotransfected into R-1B cells (data not shown). However, the weak
interaction of these two constructs precluded interpretation of these
results.
Tyrosine kinase receptors are usually activated by
ligand-induced homodimerization(24) . In contrast, the
serine/threonine kinase receptors for TGF- and related factors
form ligand-induced heterodimers. The type II receptors for TGF-
and activin bind ligand when expressed alone, whereas the type I
receptors contact ligand only when coexpressed with the corresponding
type II receptors, with both receptors forming a tight heteromeric
complex(1, 4, 6, 7, 25) .
Certain type I receptors for BMP deviate from this rule since they bind
ligand when transfected alone into COS
cells(19, 20, 21) . However, they still form
ligand-induced complexes with a coexpressed BMP-type II receptor (19, 20, 21) .
The existence of
these heterologous interactions, together with genetic evidence that
TGF-
signaling requires the presence of receptor types I and II in
the same cell(3) , led to the recent demonstration that
T
R-II is a constitutively active kinase that can phosphorylate an
associated T
R-I, which then propagates the signal to downstream
substrates(25) .
Although the evidence for TGF--induced
heteromeric receptor complexes is extensive, the homodimeric nature of
TGF-
raises the possibility that its receptors might also form
ligand-induced homodimers. In the case of T
R-II, this possibility
is in principle compatible with the ability of this receptor to bind
ligand on its own. However, the present results argue against the
existence of ligand-induced T
R-II homo-oligomers. We searched for
such receptor complexes using a two-step precipitation method that
readily reveals ligand-induced association of T
R-I with
T
R-II(25) . This method relies on the retrieval of
receptors via sequences tagged onto their C termini. This modification
is not likely to interfere with T
R-II complex formation since the
C-terminal tail of T
R-II is dispensable for
signaling(23) , and its extension with short sequence tags does
not disrupt association with T
R-I or signaling
activity(25) .
Using this approach, a large proportion of
TR-II in transfected COS cells was found as oligomers. However,
these oligomers are ligand-independent and mostly include immature
receptor protein. COS cells constitutively express SV40 large T
antigen, which allows episomal replication of expression vectors
containing an SV40 origin of replication, resulting in vast
overexpression of the encoded protein (29) . Our results show
that the amount of T
R-II expressed by this method in COS cells
saturates the cell's capacity to properly process the protein and
forward it to the plasma membrane. As a result, a large proportion of
T
R-II in transfected COS cells accumulates as immature core. In
contrast, Mv1Lu mink lung epithelial cells and L
E
rat skeletal myoblasts, expressing transfected T
R-II at
lower levels than COS cells, showed less accumulation of the immature
T
R-II form or formation of ligand-independent complexes.
Collectively, the evidence suggests that formation of TR-II
homo-oligomers in COS cells is ligand-independent and results largely
from receptor overexpression. In agreement with recent observations (32, 33) , we found that a proportion of mature
T
R-II protein forms ligand-independent complexes. The proportion
of mature T
R-II that we detected in complexes was very small, a
result that is in contrast with the more substantial levels reported by
others(32, 33) . This discrepancy could be due to
differences in receptor expression levels or in stability of the
receptor complex under the different precipitation conditions.
Although TR-I contains a ligand-binding domain, the possibility
of ligand-induced T
R-I homo-oligomerization is precluded by the
fact that this receptor requires the presence of T
R-II in order to
bind
ligand(4, 6, 7, 15, 25) .
At variance with this notion, it has been reported that human kidney
293 cells appear to bind TGF-
solely through the type I receptor
in receptor affinity labeling assays(31) . However,
immunoprecipitation of affinity-labeled cell lysates with
anti-T
R-II antibodies has demonstrated the presence of T
R-II
in 293 cells and its association with T
R-I. (
)
The
stoichiometry of the TR-I
T
R-II complex has not been
established yet. However, since the ligand is itself a disulfide-linked
homodimer, the stoichiometry of the receptor complex could be higher
than 1:1. Evidence supporting this notion has been provided by recent
receptor cross-linking experiments(34) . This raises the
possibility of interactions between homologous kinase domains within
the ligand-induced heteromeric receptor complex. Therefore, we probed
the signaling capacity of ligand-induced homologous kinase complexes by
using chimeras containing the extracellular domain of one receptor and
the transmembrane and cytoplasmic domains of the other. The binding
properties of these chimeras are those expected from the extracellular
domains they contain. The chimera T
R-II/I containing the
extracellular domain of T
R-II and the transmembrane and
cytoplasmic domains of T
R-I binds TGF-
when transfected alone
and recruits a cotransfected T
R-I into a complex with ligand. This
complex contains T
R-I cytoplasmic domains contributed by both
T
R-I and T
R-II/I. However, this complex does not generate
TGF-
responses when expressed in receptor-defective cells. In
fact, T
R-II/I acts in a dominant negative fashion, preventing
wild-type receptors from signaling. This dominant negative effect
presumably results from competition of T
R-II/I with endogenous
T
R-II for transfected T
R-I or from competition with T
R-I
for a rate-limiting substrate. These results indicate that
homo-oligomerization of the T
R-I cytoplasmic domain is not
sufficient to generate TGF-
responses. This phenomenon is of
particular interest since the T
R-I kinase domain is the downstream
signaling element in the heteromeric receptor
complex(25, 35) .
An analogous conclusion can be
drawn from our results with the TR-I/II chimera. TGF-
readily
induces association of T
R-I/II with T
R-II, yielding a complex
that contains T
R-II cytoplasmic domains contributed by both
T
R-II and T
R-I/II. Formation of this complex does not lead to
signaling activity, but rather interferes with T
R-I in a dominant
negative fashion. The limited ability of T
R-I/II and T
R-II/I
to form a complex in the presence of ligand did not allow us to
determine if their intracellular domains can generate a response when
brought together by ligand via heterologous ectodomains (but see note
added in proof).
In conclusion, wild-type TR-I and T
R-II
do not appear to form stable ligand-induced homo-oligomers even in
cells overexpressing this receptor. Furthermore, when ligand-induced
homo-oligomerization of TGF-
receptor cytoplasmic domains is
enforced with the use of receptor chimeras, it is not sufficient to
generate a signal. These observations are consistent with the general
conclusion that TGF-
signaling is the result of an interaction
between T
R-I and T
R-II. Together with the observation that
T
R-II is a constitutively active kinase(25) , these
results argue that the ligand-induced homodimerization/activation
phenomenon characteristic of tyrosine kinase receptors (24) is
not shared by the TGF-
receptors. The main role of the ligand
appears to be the induction or stabilization of a complex between
T
R-II and its substrate, T
R-I, allowing phosphorylation and
activation of T
R-I as a requirement for signal propagation.
Note Added in Proof-After submission of
this manuscript, Okadome et al.(36) reported an
experimental strategy, results, and conclusions similar to those
reported here, and additionally reported signaling by TR-I/II
cotransfected with T
R-II/I.