From the
Transforming growth factor-
Transforming growth factor
The type II receptor is a
transmembrane serine/threonine kinase, the cytoplasmic domain of which
can be autophosphorylated when expressed in Escherichia
coli(12) . This receptor is constitutively
autophosphorylated when transfected into mammalian cells
(13) ,
which is consistent with its homodimeric complex
formation
(14, 15, 16) . The type I TGF-
Previous studies suggest
that heteromeric complex formation between type II and type I receptors
is required for ligand binding to the type I receptor and may play a
role in TGF-
To make the deletion
mutants of the type II receptor cytoplasmic domain and the
kinase-defective mutants of the type I and type II receptor cytoplasmic
domains, the two cytoplasmic domains were cloned into M13mp19 vector.
Deletions or point mutations were generated using the Sculptor in
vitro mutagenesis system (Amersham Corp.) and oligonucleotide
primers to introduce the desired mutations. The mutated type II
cytoplasmic domains containing the different deletions were subcloned
into pJG4-5
(28) . The point-mutated kinase-defective
cytoplasmic domains were subcloned into pAS1-CYH2
(22) for the
type II mutant and into pACTII
(29) for the type I mutant.
The deletion mutants of type I receptor cytoplasmic domain were
constructed as follows. Plasmids pJGcRI-(147-235) and
pJGcRI-(147-414) were generated by removing the
EcoRI/XmnI and EcoRI/MscI fragments from the
type I receptor cytoplasmic domain in pJGcRI and then inserting the
mutated cDNA into pJG4-53, respectively. pJG4-53 was
constructed by inserting two annealed oligonucleotides
(5`-AATTCATCCCGGGTACTCGAGTAAGTAAGTAA and
5`-TCGATTACTTACTTACTCGAGTACCCGGGATG) between the EcoRI and
XhoI sites of plasmid pJG4-5, thereby inserting stop
codons in all three frames. To construct pJGcRI-(147-360), an
EcoRI/NsiI fragment derived from pJGcRI was first
inserted into pSP72 (Promega) to be converted to
EcoRI/XhoI fragment and then cloned into
pJG4-53. To generate pJGcRI-(237-509), an
XmnI/XhoI fragment was removed from pJGcRI, converted
to EcoRI/XhoI fragment by inserting into and
subsequently excising from pSP72, and cloned into pJG4-53.
Plasmids pJGcRI-(361-509) and pJGcRI-(416-509) were
constructed by removing the NsiI/XhoI and
MscI/XhoI fragments from pJGcRI, converting them into
EcoRI/XhoI fragments using strategy described above,
and cloning them into pJG4-53.
We next examined whether this association of the two types
of cytoplasmic domains also occurs in vivo. Cells were
cotransfected with the cytoplasmic portions of type I and type II
receptors, preceded by an initiator methionine and C-terminally tagged
with myc and Flag epitopes, respectively. The lysates of metabolically
labeled, cotransfected cells were subjected to immunoprecipitations
using tag-specific antibodies. As shown in Fig. 1, the anti-Flag
antibody precipitated not only the type II receptor, but also a band
with the same mobility as the type I receptor (lane 2). Such
coimmunoprecipitation was not detected from cells transfected with only
the type II cytoplasmic domain (Fig. 1, lane 1).
Similarly, the anti-myc antibody precipitated not only the myc-tagged
type I cytoplasmic domain but also a band with a mobility identical to
the Flag-tagged type II receptor cytoplasmic domain (lane 3).
To confirm the identity of the coprecipitated band, we performed a
double immunoprecipitation analysis, in which low stringency
immunoprecipitates using an anti-myc antibody were solubilized and
reprecipitated under high stringency conditions using an anti-Flag
antibody. Such double immunoprecipitations revealed that the
coprecipitated band was recognized by the anti-Flag antibody and thus
corresponds to the type II receptor cytoplasmic domain (Fig. 1,
lane 4), thus indicating the association of the two
cytoplasmic domains. As expected, the two sequential
immunoprecipitation steps resulted in a considerably lower intensity of
the two receptor bands. Taken together, these results indicate that the
two cytoplasmic domains can interact independent of the
extracellular/transmembrane domains and can be involved in the
formation of the heteromeric complex between the type I and type II
receptors.
Expression plasmids for HA-epitope-tagged type I Tsk 7L
and myc-tagged type II receptors
(16) were constructed and
cotransfected into 293 cells. The lysates of metabolically labeled,
cotransfected cells were subjected to immunoprecipitations using
tag-specific antibodies. These analyses demonstrated a diffuse band of
the size of the type I receptor in the type II receptor
immunoprecipitates which was absent in cells expressing only the type
II receptor (Fig. 3, lanes 2-4). Unfortunately,
the expression level of the type II receptor was consistently much
higher than that of the type I receptor. However, in reciprocal
experiments, bands of the size of the type II receptor were clearly
apparent in type I receptor immunoprecipitations when both receptor
types were coexpressed, but not when only the type I receptor was
expressed (Fig. 3, lanes 7-9). These data thus
suggest association of both receptor types. To further evaluate the
identity of the coprecipitated bands, we performed double
immunoprecipitations using first anti-myc and then anti-HA antibodies.
These analyses revealed that the HA-tagged type I receptor
coprecipitated and thus was associated with the myc-tagged type II
receptor (Fig. 3, lanes 12 and 13). In control
experiments, no coprecipitation could be detected in untransfected
cells, nor in 293 cells transfected with only the myc-tagged type II
receptor or the HA-tagged type I receptor (Fig. 3, lanes 5,
10, 14, and 15). The reciprocal immunoprecipitation using
first the HA- and then the myc-specific antibodies further documented
this association (Fig. 3, lane 16). This double
immunoprecipitation revealed a high level of myc-tagged type II
receptor coprecipitated with the HA-tagged type I receptor using the HA
antibody, since a comparable level of type I receptor was recovered
from two sequential immunoprecipitations with HA antibody
(Fig. 3, compare lanes 16 and 17). Furthermore,
the association of the two receptors was also detected when lysates of
cotransfected cells were first immunoprecipitated using the myc or HA
antibody and then subjected to Western blotting using the HA or myc
antibody, respectively (data not shown). Interestingly, the amounts of
coprecipitated HA-tagged type I receptor did not differ when the
cotransfected cells were treated with TGF-
We next examined whether the ability of both types of
full-size receptors to associate with each other was solely based on
the possible direct interaction of the cytoplasmic domains. We
therefore generated expression plasmids for a Flag-tagged truncated
type I
(23) and a myc-tagged truncated type II receptor
(16) lacking most of their cytoplasmic domains. Double
immunoprecipitation analyses showed that the Flag-tagged truncated type
I receptor was coimmunoprecipitated with the myc-tagged truncated type
II receptor using the anti-myc antibody (Fig. 6, lanes 11 and 12) and vice versa (Fig. 6, lanes
9 and 10). No coprecipitated truncated type I or type II
receptor was detected in cells transfected with only the truncated type
II or type I receptor respectively (Fig. 6, lanes 13 and
14). Treatment of cells with TGF-
We have shown that the cytoplasmic domains of the type I and
type II receptors are able to interact directly with each other, as is
apparent from yeast two-hybrid assays and double immunoprecipitation
analyses. If the expression of
The
constitutive autophosphorylation of the type II receptor cytoplasmic
domain
(13) and the direct association of the kinase-active
cytoplasmic domain of the type II receptor with the cytoplasmic domain
of the type I receptor suggest that both receptor types may have an
inherent affinity for each other. This possibility was evaluated by
double immunoprecipitation analyses using metabolically labeled cells
cotransfected with differentially tagged type I and type II receptors.
Using our experimental conditions, we demonstrated that both receptor
types can already associate with each other in the absence of TGF-
Whereas the direct association of the cytoplasmic
domains is likely to play a role in the formation and stabilization of
the heteromeric complex, our results also indicate that this
cytoplasmic association is not absolutely required for the heteromeric
interaction. Indeed, truncated receptors lacking the cytoplasmic
domains still have the ability to associate. This is reminiscent of
tyrosine kinase receptors, which dimerize through interaction of their
extracellular/transmembrane
segments
(36, 37, 38) . Thus, multiple contact
points are involved in the formation and stabilization of the
heteromeric complex of the type I and type II receptors.
In the case
of tyrosine kinase receptors, overexpression of truncated receptors
lacking most of their cytoplasmic kinase domains abrogates receptor
function
(39, 40, 41) . Similar dominant-negative
inhibition of receptor functions by overexpressing cytoplasmically
truncated receptors has also been demonstrated in the case of TGF-
The
characteristics of the heteromeric TGF-
Y190 yeast was transformed with various plasmids as indicated, and
We thank Drs. R. Brent and S. Elledge for providing
the yeast two-hybrid reagents and Drs E. Filvaroff and S. Lawler for
stimulating discussions.
Note Added in Proof-Chen and
Weinberg (Chen, R.-H., and Weinberg, R. A.(1995) Proc. Natl. Acad.
Sci. U. S. A.
92, 1565-1569) recently reported the
ligand-independent association of the type I and type II TGF-
(TGF-
) transduces signals
through its type I and type II receptors. Both receptor types have
previously been shown to interact in a heteromeric complex in the
presence of TGF-
. We have now characterized these interactions
between both receptor types using a combination of yeast two-hybrid
interaction assays and coimmunoprecipitation analyses. Our results
indicate a direct association between the cytoplasmic domains of the
two receptor types. Mutation analysis of these cytoplasmic domains
reveals that this direct interaction requires kinase activity and,
thus, depends on phosphorylation, probably via a transphosphorylation
mechanism. Furthermore, the two receptor types already have an inherent
affinity for each other in the absence of TGF-
, and the
heteromeric complex can be detected in coimmunoprecipitations under
these conditions. Taken together, our results reveal a novel mechanism
of receptor complex formation, whereby two different cytoplasmic
domains directly associate with each other. This interaction may play a
major role in activation of serine/threonine kinase receptors.
(TGF-
)
(
)
belongs to a family of multifunctional proteins which
regulate cell proliferation, differentiation, and formation of the
extracellular matrix
(1, 2) . TGF-
exerts its
biological activities through binding to specific cell surface
receptors. Most cells express three classes of TGF-
receptors,
defined by their sizes and ability to bind and cross-link
I-TGF-
(3, 4) . The type III TGF-
receptor is a proteoglycan
(5, 6) and may be involved in
presenting ligand to type I and type II receptors, but does not
directly mediate TGF-
signaling
(7, 8) . In
contrast, both type II and type I receptors are required for
TGF-
-induced signaling
(9, 10) . Dominant negative
inhibition of the type II receptor signaling suggests the existence of
two receptor-mediated signaling pathways, one of which requires a
functional type II receptor, presumably in combination with the type I
receptor, and leads to the antiproliferative effect of TGF-
. The
other pathway is associated with the type I receptor without the
necessity of type II receptor signaling and directs TGF-
-induced
expression of several genes
(11) .
receptor was identified using chemical cross-linking techniques as a
53-kDa glycoprotein
(17) , and several closely related type I
TGF-
receptors have been
cloned
(18, 19, 20, 21, 22) .
They contain an intracellular serine/threonine kinase domain, but do
not bind ligand unless coexpressed with the type II receptor. The
specificity of ligand binding to the type I receptor is determined by
the nature of the type II
receptors
(19, 22, 23) . Although only one of
them, ALK-5/R4, restores TGF-
responsiveness in a mutant cell line
lacking type I receptors
(20, 21) , another type I
receptor, Tsk 7L, is involved in TGF-
-induced transdifferentiation
of epithelial cells
(24) . The physiological importance of the
different type I receptors in the various biological activities of
TGF-
still remains to be determined.
signaling. A physical interaction of the two receptor
types has been shown by coimmunoprecipitation experiments, in which
type II or type I receptor-specific antibodies precipitated both
ligand-bound type II and type I receptors. This interaction with the
type II TGF-
receptor was apparent with all type I
receptors
(8, 19, 22, 23, 25, 26) .
Furthermore, it has been suggested that ligand cooccupation is required
for the physical association of the two receptor types
(13) . In
the present studies, we characterized the interaction of both receptor
types. As a model, we studied the association of type II receptor with
the type I receptor Tsk 7L. The cytoplasmic domains of the two
receptors can undergo a direct physical association with each other as
determined by coimmunoprecipitations and yeast two-hybrid assays. This
direct association depends on the kinase activity of the receptors. In
addition, coimmunoprecipitation studies demonstrated that both receptor
types interact in the absence of TGF-
and thus have an inherent
affinity for each other. The phosphorylation-dependent interaction of
two cytoplasmic domains represents a novel mechanism for dimerization
and/or oligomerization of receptors, which may be required for
signaling of TGF-
-related factors through their transmembrane
serine/threonine kinase receptors.
Yeast Expression Plasmids
To construct the
plasmid pEGcRII encoding the entire cytoplasmic domain of type II
receptor inserted in frame to the DNA-binding domain of LexA, a
3.5-kilobase pair HpaI/XhoI fragment containing the
cytoplasmic domain of the type II receptor (amino acids 192-567)
was removed from pH2-3FF
(12) and ligated into pEG202
(28) that was digested by BamHI, filled in with Klenow
DNA polymerase I, and digested with XhoI. pJGcRI allows the
expression of a fusion protein between the B42 acidic activation domain
and the cytoplasmic domain of type I receptor Tsk 7L. To make this
plasmid, the entire cytoplasmic sequences (amino acids 147-509)
of the Tsk 7L was PCR-amplified and cloned into
pJG4-5
(28) . pJGcRII, encoding the B42-type II cytoplasmic
domain fusion, was constructed by excising the EcoRI fragment
from pEGcRII and then cloned into pJG4-5. pEGcRI, containing the
LexA DNA binding domain-type I receptor cytoplasmic domain fusion, was
made by excising the EcoRI/XhoI fragment from
pJG4-5 and then cloned into pEG202. To construct the plasmid
pAScRII containing the cytoplasmic domain of the type II receptor fused
to the 3` end of the GAL4 DNA binding domain, a 2.3-kilobase pair
SmaI/BamHI fragment was excised from pEGcRII and
cloned into the SmaI/BglII sites of the
pAS1-CYH2
(29) . Plasmid pACTcRI allows expression of the fusion
protein of the GAL4 activation domain and the type I receptor Tsk 7L
cytoplasmic domain. To construct this, the entire cytoplasmic domain
(amino acids 147-509) of the type I receptor Tsk 7L was generated
by PCR and cloned into pACTII
(29) .
Two-hybrid Assays
Yeast transformation was
performed as described
(32) . Y190 transformants were plated on
Sc media lacking Trp, Leu, His, but including 25 mM
3-aminotriazole (Sigma). EGY48 transformants were isolated on
glucose-Ura His
Trp
plates and assayed as described (28). Interaction was determined
by screening for
-galactosidase activity using a filter lift
assay
(33) . Quantitation of
-galactosidase activity in
yeast was performed using
o-nitrophenyl-
-D-galactoside as
substrate
(33) .
Mammalian Expression Plasmids
Plasmid pIR-HA
drives expression of the full-length type I receptor Tsk 7L linked to
an HA epitope tag (GLYDVPDYASLG) at its C terminus. The entire coding
region of Tsk 7L
(18) was amplified by PCR and cloned into the
expression plasmid, pRK5
(27) , together with a synthetic adaptor
encoding the HA epitope tag. pIRC-myc is an expression plasmid for the
entire cytoplasmic domain (amino acids 153-509) of the type I
receptor Tsk 7L with an N-terminal myc epitope tag. An EcoRI
fragment for the cytoplasmic domain of the Tsk 7L was generated by PCR
and ligated to the 3` of a single-stranded synthetic adaptor encoding
the myc epitope tag (GEQKLISEEDLN) preceded by an initiator methionine.
The ligated product was subcloned into the expression plasmid
pRK7
(27) . The construction of pIIRC-Flag, which allows
expression of the cytoplasmic domain of the type II receptor with an
N-terminal Flag tag, has been described before
(16) .
Transient Transfections, Cell Surface Biotinylations, and
Double Immunoprecipitations
293 cells were transfected using the
calcium-phosphate precipitation method
(30) . The conditions for
treatment with TGF- or its neutralizing antibody have been
described previously
(16) . Metabolic labeling of cells was
initiated at 36-48 h after transfection using
S-protein-labeling mix (DuPont NEN). Labeled cells were
lysed and subjected to double immunoprecipitations as
described
(16) . To detect the receptors located at cell surface,
cells were labeled using sulfosuccinimdyl 6-(biotinamino) hexanoate
(Pierce) at 48 h after transfection using procedures described
(31) and then lysed for immunoprecipitation. The biotinylated
proteins were detected with the enhanced chemiluminescence system
(Amersham Corp.) using streptavidin-conjugated horseradish peroxidase
as probe.
RESULTS
Direct Interaction of the Cytoplasmic Domains of the
Type I and Type II Receptors
Since the type I and type II
receptors have the ability to interact in a heteromeric complex, we
evaluated the involvement of different receptor domains in this
interaction. In this context, we examined whether the cytoplasmic
domains of the two receptor types undergo a direct physical
interaction. For this purpose, we used the yeast two-hybrid system,
which lacks endogenous type I and type II receptors and scores direct
physical interactions between proteins, based on transcriptional
activation of reporter genes
(34) . Two expression plasmids were
constructed by fusion of the cytoplasmic domains of type II and type I
receptors to the LexA DNA-binding domain and the nuclear localized B42
acidic activation domain, respectively. Cotransformation of the two
plasmids in yeast strain EGY48
(28) resulted in transactivation
of the Leu and lacZ reporter genes, as detected by its ability to grow
in the absence of leucine and its blue color when stained for
-galactosidase activity (). No transactivation was
found when yeast was transformed with only one of the plasmids. Similar
transactivation was observed when yeast was cotransformed with a pair
of the reciprocal plasmids, in which the type I cytoplasmic domain was
fused to the LexA DNA-binding domain, and the type II cytoplasmic
domain was made as a hybrid with the B42 activation domain
(). The specificity of this interaction was further
confirmed using a different two-hybrid system
(29) , in which the
coexpression of a hybrid GAL4 DNA-binding domain-type II cytoplasmic
domain and a fusion protein of the GAL4 activation domain and the type
I cytoplasmic domain resulted in transactivation of the His and lacZ
reporter genes in the yeast strain Y190. Taken together, these results
indicate a direct and specific association between the cytoplasmic
domains of the type I and type II receptors. In contrast, no direct
interaction was detected in the same two-hybrid assays between the type
II receptor cytoplasmic domains, the type I receptor cytoplasmic
domains, and between the type II receptor cytoplasmic domain
() and the cytoplasmic domain of TGF-
as a control
protein.
Figure 1:
The cytoplasmic domains of type I and
type II receptors can interact with each other. 293 cells transfected
with pIIRC-Flag, an expression plasmid encoding the Flag-tagged type II
cytoplasmic domain (20) (II) or cotransfected with pIIRC-Flag
and pIRC-myc, which expresses a myc-tagged cytoplasmic domain of the
Tsk 7L type I receptor (D), were metabolically labeled. Cell
lysates were subjected to single (lane 1) or double (lanes
2-4) immunoprecipitations using antibodies as indicated
(M, anti-myc; F, anti-Flag). cRI and
cRII indicate the cytoplasmic domains of the type I and type
II receptors, respectively. Molecular weight markers are shown to the
right. The cytoplasmic domain of the type I receptor
coimmunoprecipitates with the cytoplasmic domain of the type II
receptor (compare lane 2 with lane 1) and vice
versa (lane 3). Double immunoprecipitations show
coprecipitation of type II receptor cytoplasmic domain in type I
receptor immunoprecipitates as well as associated type I receptor
cytoplasmic domain (lane 4).
The Interaction of the Two Cytoplasmic Domains Depends on
the Kinase Activity
Because the interaction between the two
cytoplasmic domains represents a novel mechanism of receptor
interactions that may be a feature unique to the TGF- receptor
family, we investigated whether specific regions in these domains are
involved in their association. A panel of deletion mutants that span
the length of the type II cytoplasmic region was constructed and
individually fused with the B42 activation domain
(Fig. 2A). Their capacity to directly and specifically
interact with the cytoplasmic domain of the type I receptor was tested
again using the two-hybrid system. As shown in Fig. 2A,
deletion of the spacer region upstream from the kinase domain or the
C-terminal tail region immediately downstream from the kinase sequences
did not affect association with the type I cytoplasmic portion.
However, deletion of any part of the type II receptor kinase domain
tested abolished its ability to interact with the type I receptor
cytoplasmic portion. Using a similar approach, the subregion in the
type I cytoplasmic domain involved in the interaction with the type II
cytoplasmic domain was also determined. A panel of deletion mutants of
the type I cytoplasmic domain, each lacking a portion of the kinase
domain, was constructed and individually fused to the B42
transactivation domain (Fig. 2B). None of these mutants
were able to interact with the type II cytoplasmic portion in the same
yeast two-hybrid assays. Thus, our data indicate that an intact kinase
domain, but not the flanking sequences, is required for the association
between the cytoplasmic domains of the type I and type II receptors.
Figure 2:
Kinase domains are required for the
association between the cytoplasmic portions of type I and type II
receptors. Deletion mutants of the cytoplasmic domains of the type I
(A) and type II (B) receptors were used to define the
interacting domains and are shown schematically. Transmembrane domains
(TM) of the two receptors are indicated. The numbers in parentheses indicate the amino acid number of the
cytoplasmic sequence present in the fusion proteins; when preceded by a
``d,'' the numbers indicate the deleted segments.
The deletion mutants of the cytoplasmic domains of the type II and type
I receptors were expressed as fusion proteins with the B42 activation
domain and these plasmids were named JG-cRII and JG-cRI, respectively.
EGY48 yeast cells (21) were cotransformed with the indicated panel of
plasmids for type II deletion mutants together with the expression
plasmid pEG-cRI which fuses the LexA domain and the cytoplasmic domain
of type I receptor (A) or with plasmids for the panel of
pJG-cRI deletion mutants and pEG-cRII containing the type II receptor
cytoplasmic domain (B). Binding was determined by the dark
blue staining following a filter lift assay for -galactosidase
activity (25).
The results obtained with the deletion mutants suggest that the
overall structure of the kinase domains per se may be critical
for association between the two cytoplasmic domains and that deletion
of any portion of the kinase domains may alter such structure, thus
disrupting the association. However, it was equally possible that the
kinase activities of the two receptors play a role in the association,
since any deletion within the kinase domains tested is likely to
inactivate the kinase activity. To test the latter possibility in yeast
that lacks endogenous TGF- receptor kinase activity,
kinase-defective type I and type II receptor cytoplasmic domains were
constructed by replacing the lysine in the ATP-binding site with an
arginine, and the mutated cytoplasmic domains were then fused to the
GAL4 activation and DNA-binding domains, respectively. The strengths of
the interactions between the wild type and mutant as well as between
the two mutants were tested in the yeast two-hybrid system. As shown in
, inactivation of the kinase activity in both receptors
abolished the direct association as assessed by the lack of
-galactosidase activity. Combination of the type II wild type and
type I kinase-defective mutant showed a somewhat weaker interaction
compared with the kinase-active pair. Furthermore, the type II
kinase-negative mutant showed only a very weak interaction with the
wild type type I receptor. These data indicated that the kinase
activity of the two receptors, especially the type II receptor, is
required for the direct association of both cytoplasmic domains,
probably via a transphosphorylation mechanism. To examine the role of
the kinase activity in the heteromerization of these two domains in
mammalian cells, we generated expression plasmids for the cytoplasmic
domains identical to the ones used in Fig. 1, except that, as in
the yeast two-hybrid expression plasmids, we replaced the lysine in the
ATP-binding site of each cytoplasmic domain with an arginine.
Unfortunately, the kinase-inactive versions of the cytoplasmic domains
had a much lower intracellular stability than the kinase-active
versions, thereby precluding an evaluation of their association in
transfected cells using coimmunoprecipitation analysis (data not
shown).
Interaction of Full-size Type I and Type II
Receptors
The experiments outlined above demonstrated that the
kinase-active cytoplasmic domains of the type I and type II receptors
have an inherent affinity for each other which results in their
association. Furthermore, they indicate that the kinase activity of the
type II receptor plays a major role in this association, which could be
due to an autophosphorylation of the cytoplasmic domains. In mammalian
cells, the type II receptor forms a constitutive
homodimer
(14, 15, 16) which is
autophosphorylated both in the presence and absence of
TGF-
(13) . Based on these results, we evaluated whether the
intact, full-size receptors also had an inherent affinity for each
other, which could result in an association in the absence of
TGF-
.
or a neutralizing
antibody that interferes with TGF-
binding to its receptors
(35) (Fig. 3, compare lanes 13 and 14).
Figure 3:
Heteromeric complex formation between the
type I and type II receptors in metabolically labeled, transiently
transfected cells. 293 cells cotransfected with equal amounts of
expression plasmids for the HA-epitope-tagged Tsk 7L type I receptor,
pIR-HA, and the myc-tagged type II receptor, pIIR-myc (20)
(D), or transfected with pIIR-myc alone (II), pIR-HA
alone (I), or untransfected (-) were metabolically
labeled and treated with TGF- (+) or the neutralizing
antibody (-) as indicated. In each transfection, 25 µg of
plasmid DNA was used. Cell lysates were subjected to single (lanes
1-11) or double (lanes 12-17)
immunoprecipitations using conditions as described (20) and antibodies
as indicated (M, anti-myc; H, anti-HA) and analyzed
by denaturing gel electrophoresis under reducing conditions. The
molecular markers (111, 74, and 45 kDa) are indicated as dots on both
sides. The type I (RI) and type II (RII) receptors
are indicated. Lane 1 is a shorter exposure of a pIIR-myc
transfection, similar to lane 2, albeit from different
experiments (hence the differences in the degradation products of the
type II receptor). In contrast to lane 2 (transfection with
pIIR-myc only), the type I receptor was coprecipitated in type II
receptor immunoprecipitates, when pIR-HA was cotransfected with
pIIR-myc (lanes 3 and 4). In immunoprecipitations of
the type I receptor, the type II receptor coprecipitated with the type
I receptor when pIIR-myc was cotransfected with pIR-HA (lanes 8 and 9), but not in the absence of pIIR-myc (lane
7). Double immunoprecipitation using first the myc and then the HA
antibody showed the presence of the HA-tagged type I receptor (most
intense band) and some myc-tagged type II receptor (lanes 12 and 13). Furthermore, the myc-tagged type II receptor was
detected in double immunoprecipitations using first the HA and then the
myc antibody (lane 16).
In principle, the coprecipitation of both receptor types could have
been due to overcrowding of these receptors at the surface of
cotransfected cells, thereby leading to an increased and, to some
extent, nonspecific level of oligomerization or aggregation. However,
no coimmunoprecipitation was detected in cells overexpressing the
myc-tagged type II receptor and a control transmembrane protein,
TGF-, using the same cotransfection and immunoprecipitation
methods (16). In addition, to address whether this constitutive
interaction occurs under physiological conditions, i.e. at
receptor expression levels comparable with receptor levels in
untransfected cells, the epitope-tagged type II and type I receptors
were expressed in 293 cells at low levels, which based on cell surface
cross-linking with
I-TGF-
, were comparable with the
endogenous receptor levels of C2C12 cells (data not shown). The
ligand-independent association of the two receptor types could be
demonstrated by double immunoprecipitation analysis under these
conditions as well (Fig. 4). Finally, we performed
coimmunoprecipitation analysis of the receptors at the cell surface,
that were labeled using surface biotinylation, thus excluding the
intracellular receptor pool (Fig. 5). Also under these conditions
was the type II receptor expression considerably higher than the type I
receptor levels, thereby requiring sequential immunoprecipitation
analysis, as in Fig. 3and Fig. 4. As is apparent
especially from lanes 9-12, both receptor types
associate with each other at the cell surface in the absence of ligand
binding, consistent with the results from the
S-labeled
receptors in total cell lysates.
Figure 4:
Formation of the heteromeric type I-type
II receptor complex in cells expressing a low level of transfected
receptors, comparable to endogenous receptor levels. 293 cells were
cotransfected with 2 µg of pIR-HA and 2 µg of pIIR-myc in the
presence of 21 µg of carrier DNA. Cells were treated with TGF-
(+) or neutralizing antibody (-), metabolically labeled, and
subjected to single (lanes 1 and 2) or double
immunoprecipitations (lanes 3 and 4) using antibodies
as indicated. The molecular weight markers and the type I and II
receptors are indicated. The double immunoprecipitations using first
the myc and then the HA antibody showed the presence of the HA-tagged
type I receptor (most intense band) and some myc-tagged type II
receptor (lanes 3 and 4).
Figure 5:
Formation of the heteromeric receptor
complex at the surface of cotransfected cells. 293 cells were
cotransfected with pIR-HA and pIIR-myc (D) or transfected with
pIIR-myc alone (II), pIR-HA alone (I), or
untransfected (-). This experiment was carried out in the absence
of TGF- and the presence of a neutralizing antibody (16), and
immunoprecipitations were carried out using antibodies as indicated.
The molecular markers (111, 74, and 45 kDa) are indicated as dots to the right. The type I (RI) and type II
(RII) receptors and a frequently observed degradation product
of the type II receptor (asterisk) are indicated. Single
immunoprecipitations show the type II receptor as a double band
(because of the degradation product) and the type I receptor as a
single band. Double immunoprecipitations of cells expressing both
receptor types show the presence of the HA-tagged type I receptor in
type II receptor immunoprecipitates (lane 9) and myc-tagged
type II receptor in type I receptor immunoprecipitates (lane
11). Lanes 10 and 12 show control double
immunoprecipitations in which the type II receptor and type I receptor
were subjected to two sequential
immunoprecipitations.
Taken together, these results
indicate that both receptor types have already an affinity for each
other in the absence of TGF-. Under our immunoprecipitation
conditions, a similar degree of receptor association was apparent in
both the presence and absence of TGF-
, implicating the presence of
a pre-existing heteromeric receptor complex independent of ligand
occupation.
did not stimulate their
association (Fig. 6, compare lanes 9 and 10; 11 and 12), similar to the results obtained using the two
full-length receptors. Thus, as in the case of the type I and type II
receptors, the extracellular and transmembrane domains display already
an affinity toward each other. This affinity is high enough to allow
the detection of the associated heteromeric complex by
coimmunoprecipitation analysis.
Figure 6:
Interaction between the truncated type I
and type II receptors lacking most of their cytoplasmic domains. 293
cells cotransfected with pIRDN-Flag, an expression plasmid for the
Flag-epitope tagged, cytoplasmically truncated Tsk 7L type I receptor
(15) and pIIRDN-myc, which expresses the myc-tagged, cytoplasmically
truncated type II receptor (20) (D), with pIIRDN-myc alone
(II), or with pIRDN-Flag alone (I), or untransfected
(-) were metabolically labeled and treated with TGF-
(+) or with a neutralizing antibody (-). Single (lanes
1-8) or double (lanes 9-14)
immunoprecipitations were performed using antibodies as indicated
(M, anti-myc; F, anti-Flag). tRII and tRI indicate
the truncated type II and type I receptors, respectively. Molecular
weight markers are shown to the right. Both the truncated type
II and I receptors migrated as double bands reflecting differences in
glycosylation. The highest band of the type I receptor (lanes
1-3) comigrates with a band of the type II receptor
(lanes 5-7). The lowest band in the type II receptor
lanes, which is most apparent in lanes 9-12, corresponds
to a frequently observed degradation product of the receptor. Double
immunoprecipitations using first the Flag and then the myc antibody
showed the presence of the myc-tagged type II receptor in the type I
receptor immunoprecipitates (lanes 9 and 10). Double
immunoprecipitations using antibodies in a reversed order showed the
presence of the Flag-tagged type I receptor in the type II receptor
immunoprecipitates (lanes 11 and
12).
DISCUSSION
Receptor dimerization is essential for kinase activation of
growth factor receptors that are themselves transmembrane tyrosine
kinases or are associated with cytoplasmic tyrosine
kinases
(36, 37) . Typically, occupation of the receptors
by ligands induces dimerization of adjacent extracellular domains and
leads to interactions between the cytoplasmic domains and their rapid
transphosphorylation. As a result, a cascade of signaling events is
activated and leads to the various growth factor-stimulated biological
responses. In the case of TGF- receptors, which could be
considered as prototypes for the recently discovered transmembrane
serine/threonine kinase receptors, formation of heteromeric complexes
between type I and type II receptors in the presence of TGF-
has
been demonstrated following cross-linking with
I-TGF-
(8, 19, 22, 23, 25, 26) .
In addition, Wrana et al.(13) proposed, based on
coimmunoprecipitation analysis of cotransfected cells, that ligand
induces the interaction of the type II and type I receptors. Analyses
of the ligand-bound heteromeric
complex
(8, 13, 19, 22, 23, 25, 26) and the constitutive homodimerization of the type II
receptor independent of ligand binding (14-16) strongly suggest
that the heteromeric TGF-
receptor complex may be a tetramer which
combines two type II and two type I receptors. In the current study we
further characterized the structural basis of the heteromeric
interaction between the type I and type II receptors using a
combination of double immunoprecipitation analyses and yeast two-hybrid
assays.
-galactosidase in the yeast
two-hybrid system is a measure of the strength of the interaction, we
conclude that the association between both types of cytoplasmic domains
is comparable with the reported interactions between pRB and
phosphatase 1
2
(33) or cdk2 and the cdk-inhibitor
p21
(29) . This specific association between two different
cytoplasmic domains represents a novel mechanism of receptor
interactions and may contribute to the stability of the heteromeric
receptor complex. Analyses of cytoplasmic domain mutants in the
two-hybrid system indicate that the kinase activity is essential for
direct association. Furthermore, the two kinase-active cytoplasmic
domains can undergo a heteromeric interaction in vivo. Whether
this physical interaction of the cytoplasmic domains is mediated
through phosphorylated amino acids or results from conformational
changes induced by cytoplasmic domain phosphorylation remains to be
determined. In this context, it is possible that specific sequences in
the cytoplasmic domains bind to phosphorylated serines and/or
threonines in the opposing receptor domain, similar to the interactions
of SH2 domains with phosphotyrosine residues
(36) . Our results
also suggest the existence of transphosphorylation of adjacent
cytoplasmic kinase domains in the heteromeric TGF-
receptor
complex. Thus, transphosphorylation of both receptors may induce the
interaction between the cytoplasmic domains and play a major role in
the stabilization of the receptor complex. The observation that
elimination of the type II receptor kinase activity diminishes the
association more dramatically than inactivation of the type I receptor
kinase suggests that transphosphorylation of the type I receptor by the
type II receptor kinase is more important for this interaction than the
reciprocal phosphorylation event. This is consistent with the recent
conclusion that the type II receptor phosphorylates the type I receptor
in the heteromeric complex
(13) . Thus, in a tetrameric receptor
complex of two type II and two type I receptors
(15) , the
phosphorylation of the type II receptor cytoplasmic domains may result
from ligand-independent autophosphorylation
(13) and mediate the
direct interaction with the type I receptor cytoplasmic domain.
and in the presence of a neutralizing anti-TGF-
antibody that
interferes with receptor binding to endogenous TGF-
. Thus, our
results indicate the presence of a pre-existing heteromeric type I-type
II receptor complex and show that ligand binding is not required for
formation of this complex. Furthermore, we did not detect a difference
in the amount of heteromeric complex in cells treated with or without
TGF-
. The association of both receptor types in the absence of
TGF-
was not a result of overexpression of these receptors at the
cell surface, since heteromerization was also apparent at physiological
expression levels and no association with an unrelated transmembrane
protein, e.g. transmembrane TGF-
, was detected using
overexpression conditions. The ability of both receptor types to
associate in the absence of TGF-
may seem inconsistent with the
previously reported conclusion that ligand binding induces formation of
the heteromeric complex
(13) . However, our results are not
necessarily in contradiction. In the case of tyrosine kinase receptors,
two receptors can undergo a low affinity dimerization in the absence of
ligand, and ligand binding stabilizes this interaction thus resulting
in a dimeric complex which can be immunoprecipitated as
such
(38) . Similarly, ligand occupation may not be required for
low affinity heteromerization of type I and type II receptors as
suggested
(13) , but may increase the stability of this complex.
However, in contrast with receptor tyrosine kinases, the interaction
between both receptor types in the absence of ligand occupation may be
of sufficiently high affinity to allow their coimmunoprecipitation.
Thus, whether or not ligand induces complex formation may be a semantic
issue and a reflection of the sensitivity of the experimental
conditions.
receptor family, such as activin
(42) and TGF-
type II
receptors (11, 43, 44), suggesting formation of receptor complexes via
interactions between their extracellular/transmembrane domains. In
accordance, we have now provided direct evidence for the involvement of
this domain in the formation of the receptor complex.
receptor complex,
determined in the present study, are somewhat different from typical
tyrosine kinase receptors, in which the extracellular/transmembrane
domains mediate ligand-induced
dimerization
(36, 37, 38) . However, this complex
may resemble to some extent the receptors for insulin and insulin
growth factor-1, which are more stable homodimers in the absence of
ligand
(45) . Similarly, transmembrane guanylyl cyclase receptors
exist as homomers and/or heteromers and in fact may form a tetrameric
protein complex in the absence of ligand
(46) . How ligand
binding induces receptor activation is largely unknown for any of these
receptors, but is likely a consequence of ligand-induced conformational
changes. In the case of the TGF-
receptor family, ligand binding
may stabilize a pre-existing heteromeric complex, thereby inducing a
conformational alteration and, as a consequence, exposing critical
amino acids in the cytoplasmic domains. These substrate sites could
then undergo transphosphorylation by the adjacent cytoplasmic kinase
domain in the receptor complex, as documented for the type I receptor
phosphorylation by the type II receptor
(13) . Phosphorylation of
these critical residues presumably plays a key role in the recruitment
of downstream signaling proteins and/or receptor activation. Future
studies will be conducted to evaluate this model and to determine the
contributions of the homomeric and heteromeric receptor complexes in
TGF-
-mediated signal transduction.
Table:
Interaction between the cytoplasmic domains of
type II receptor and type I receptor Tsk 7L
Table:
Effect of kinase activity of the type I and
type II receptors on the association between their cytoplasmic domains
-galactosidase activity was determined by the colony lift method
and quantitated using
o-nitrophenyl-
-D-galactoside as substrate (33).
The expression levels of the type II and type I cytoplasmic domain
fusion proteins were verified using Western blot analyses and were very
similar in the yeast transformed with the different sets of plasmids
(data not shown).
,
transforming growth factor-
; PCR, polymerase chain reaction.
receptors in COS-1 cells, which is consistent with our results.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.