From the Ludwig Institute for Cancer Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia
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ABSTRACT |
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Transforming growth factor- Transforming growth factor- Recently, receptors for TGF- While the overwhelming majority of studies are consistent with the
above model of TGF- Most recent studies on chimeric granulocyte-macrophage
colony-stimulating factor (GM-CSF)/TGF- Given that the type I and II receptors form signaling-incompetent
receptor complexes in the absence of ligand, we need to understand what
change occurs in the receptor complex following ligand binding. The
observations that (i) T Construction of C-terminal Tagged Wild-type, Transmembrane
Chimeric, and Transmembrane Mutant TGF-
The above primers were designed to exchange the type I receptor
transmembrane domain (22 amino acids, positions 126-147) (11) with the
type II receptor transmembrane domain (30 amino acids, positions
160-189) (12). PCR products of extracellular, transmembrane, and
cytoplasmic domains of type I and II were obtained using a Perkin-Elmer
DNA thermal cycler with Taq DNA polymerase (BIOTECH), ALK-5
(11), or H2-3FF (12) as templates and primers as indicated in Fig.
1. The PCR products were first ligated to
a linearized pCRII vector (Invitrogen). Deletion of about
20 nucleotides in the signal sequence region of T
Similar PCR strategy has been used to generate the type II
transmembrane mutant receptors (Fig. 1). Ligation of the extracellular fragment generated using primers RII-1s and RII Cell Culture and Transient Transfection--
COS-1 cells were
obtained from the American Type Culture Collection. Mutant mink lung
epithelial (Mv1Lu) cells R1B and DR26 (10) were gifts from A. B. Roberts (National Institutes of Health). The cells were grown in a 5%
CO2 atmosphere at 37 °C in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) containing 10% fetal bovine serum
(CSL, Melbourne, Australia), 60 µg/ml penicillin, and 100 µg/ml
streptomycin. Transient transfections were performed using a
DEAE-dextran protocol (29), and transfected cells were assayed 48 or
72 h later.
Binding and Affinity Cross-linking--
125I-TGF- Luciferase Assay--
The p3TP-Lux (10) TGF- Expression of C-terminal Tagged TGF- T T Expression of both the Wild-type T Expression of T Receptor Complex Formation Parallels Receptor Complex
Activation--
To investigate the correlation between TGF- T Expression of T Previous studies on TGF- Expression of the wild-type T The type I receptor consists of 22 amino acids in its transmembrane
region (Leu126-Ile147) (11), and the type II
transmembrane region contains 30 amino acids
(Leu160-Tyr189) (12). Given that the
transmembrane domain folds in a predicted
(TGF-
) delivers diverse growth and differentiation signals by
binding two distantly related transmembrane serine/threonine kinase
receptors: the type I receptor (T
RI) and the type II receptor
(T
RII). In an attempt to establish the role of the transmembrane
domain in receptor signaling, two chimeric TGF-
receptors,
T
RI-II-I and T
RII-I-II, containing the opposite transmembrane
domain were generated. When transfected into a mutant mink lung
epithelial cell line R1B, which lacks functional T
RI, T
RI-II-I
restored TGF-
1-induced transcriptional activation of a TGF-
reporter p3TP-Lux to ~25% of the levels restored by wild-type
T
RI. In the mutant mink lung epithelial cell line DR26, which
contains a truncated, nonfunctional T
RII, wild-type receptor T
RII
restored the TGF-
responsiveness, while the T
RII-I-II cDNA
was inactive. When both T
RI and T
RII were transfected into R1B,
DR26, or Mv1Lu cells, a low level of constitutive p3TP-Lux activity was
observed. However, cotransfection of both transmembrane chimeric
receptors, T
RI-II-I and T
RII-I-II, or the wild-type T
RI with
the transmembrane chimeric T
RII-I-II resulted in high levels of
ligand-independent receptor activation. These results suggest that the
transmembrane domains of both TGF-
receptors are essential and play
a pivotal role in receptor activation. To investigate the role of the
transmembrane domain further, four type II transmembrane mutants were
generated: T
RII
-1, T
RII
-2, T
RII
-3, and T
RII
-4,
which have one, two, three, or four amino acids deleted at the N
terminus of the transmembrane domain, respectively. Interestingly,
co-expression of T
RII
-1 with the wild-type T
RI in DR26 cells
resulted in high levels of constitutive activation, while only low
levels of the activation were observed when T
RII
-2, T
RII
-3,
or T
RII
-4 were co-expressed with the wild-type T
RI. However,
T
RII
-1 restored very little the TGF-
responsiveness in
DR26cells. Expression of T
RII
-2, T
RII
-3, and T
RII
-4
resulted in a progressive increase in TGF-
responsiveness, with
T
RII
-4 reaching the level of activity of the wild-type T
RII.
Furthermore, like T
RII-I-II, co-expression of T
RII
-1 with
T
RI-II-I also resulted in high levels of constitutive activation.
These results are consistent with an important role for the
transmembrane region of the receptors. We further propose a model of
receptor activation in which receptor activation occurs via relative
orientational rotation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
)1 is a member of a
superfamily of secreted signaling molecules that play important roles in intercellular communication (1-3). TGF-
superfamily members regulate proliferation, differentiation, migration, and adhesion of
many cell types and affect a wide range of biological functions, including embryonic development, hematopoiesis, and immune and inflammatory cell responses (4). Alterations in the activity of these
growth factors or their receptors have been implicated in fibrosis,
immunosuppression, cancer, and other disorders (4-6).
superfamily members have been
identified and cloned (1, 3, 7-9). In mammalian cells, most responses
to TGF-
are mediated by the type I and II cell surface receptors,
which are expressed in most cell types and tissues (2, 8-10).
Molecular cloning of T
RI and T
RII (11-13) has shown that they
are members of a family of proteins with a small extracellular region,
a single transmembrane domain, and a cytoplasmic region with a
serine/threonine kinase domain. There is 40% homology between the
T
RI and T
RII kinase domains (3). Genetic evidence from mutant
cells resistant to the action of TGF-
suggests that both type I and
type II receptors are required for TGF-
signaling (10, 14).
Furthermore, loss of functional T
RII by deletion or mutation results
in abnormal cell overproliferation and is thought to contribute to the
malignant phenotype of certain types of cancers (5, 15). T
RII is a
constitutively active kinase and is autophosphorylated (16). While
TGF-
binds directly to T
RII, it is currently believed that
TGF-
signaling occurs through T
RI (17). Early studies have shown
that TGF-
binds directly to T
RII; T
RI is then recruited into
the complex and becomes phosphorylated by T
RII. T
RI then
propagates a signal to downstream substrates (17-21).
receptor activation and signaling (17-21), more
recent studies (22-25) suggest that some fundamental questions
concerning the molecular mechanism of receptor activation have yet to
be answered. 125I-TGF-
affinity labeling and
cross-linking followed by immunoprecipitation using T
RI- and
T
RII-specific antisera and analyses by two-dimensional gel
electrophoresis under nonreducing and reducing conditions, demonstrates
that TGF-
induces the formation of heteromeric receptor complexes
(22). This complex is most likely a heterotetramer containing two
molecules each of T
RI and T
RII (22). Further double
immunoprecipitation analyses using lysates from metabolically labeled
cells cotransfected with differentially epitope-tagged type II
receptors have demonstrated that type II receptors form a homomeric
complex both in the presence and absence of TGF-
(23). It has also
shown that both the extracellular and intracellular domains of type II
receptor can interact with each other and form homomeric associations
(23, 24). These results indicate that both type II extracellular and
intracellular domains have an inherent affinity for each other in the
absence of TGF-
. In addition, experiments using a yeast two-hybrid
interaction assay and double immunoprecipitation analyses (25) have
also shown that type I and type II receptors form heteromeric complexes
even in the absence of TGF-
. Also, the type I and type II receptor
extracellular domains alone or the intracellular domains alone form
heteromeric complexes, again in the absence of ligand. Taken together,
these results suggest that both the extracellular and intracellular type I and type II receptors domains have inherent affinities for each
other in the absence of TGF-
and form preexisting, heterooligomeric receptor complexes. More recently, it has been shown that after ligand
binding, T
RII formed a heteromeric complex with T
R-2.1 (26), a
chimeric receptor containing the extracellular and transmembrane domains of type II receptor and the intracellular domain of type I
receptor. The T
RII·T
R-2.1 failed to signal any TGF-
response in R1B cells, which lack a functional type I receptor. These results suggest that the receptor heterooligomerization may be necessary but
not sufficient for TGF-
signaling.
receptors (27) and chimeric erythropoietin (Epo)/TGF-
receptors (28) have shown some surprising results. GM-CSF induces the TGF-
signaling through chimeric
receptors
I and
II or
II and
I, which consist of the
extracellular domain of GM-CSF
or
receptor fused to the
transmembrane and cytoplasmic domain of TGF-
receptor type I or II,
but chimeric receptors
I and
I or
II and
II are
signaling-incompetent. Similarly, Epo signals the TGF-
activity by
binding to chimeric Epo/TGF-
receptors E-RI and E-RII, consisting of
the extracellular domain of Epo receptor and the cytoplasmic TGF-
receptor type I and II, respectively. These studies confirmed that
homodimerization of the receptor cytoplasmic domains of either type I
or type II receptors does not activate the receptor complex and that
heterooligomerization of the receptor cytoplasmic domains is required
for receptor activation. However, given that the extracellular domain
of the type II TGF-
receptor may also homodimerize (23-25), it is
surprising that the T
RII·T
R-2.1 complex failed to signal (26),
yet the Epo or GM-CSF chimeric receptors were signaling-competent. In
addition, the intensity of E-RI and E-RII signaling depends on the
combination of transmembrane domains in the chimeric receptors (28). In cells expressing E-RI and E-RII chimeras where the transmembrane domains are Epo receptor in one construct and TGF-
receptor in the
other, the maximum extent of growth inhibition was lower, compared with
cells expressing E-RI and E-RII chimeras with either Epo receptor's
transmembrane domains or TGF-
receptor's transmembrane domains
(28). Taking together the observations in these studies (26-28), we
propose that the transmembrane domain may play a role in receptor activation.
R-2.1 fails to reconstitute signaling despite
heterodimerization with the type II receptor (26), (ii)
heterodimerization of the chimeric GM-CSF/TGF-
or Epo/TGF-
receptors results in active signaling (27, 28), and (iii) the
transmembrane domain plays a role in E-RI and E-RII signaling (28)
indicate that more work is required to define the role of the
transmembrane domain in receptor activation. In particular, the role of
the transmembrane domain in transmitting a signal following ligand
binding is not clear. In order to investigate these questions, we have
generated two TGF-
transmembrane chimeric receptors, in which the
transmembrane domains of the type I and II receptors were interchanged,
and four type II transmembrane mutants with a series of deletions in
the transmembrane domain. The signaling activity of these chimeric and
mutant receptors in mutant mink lung cells has been examined, and our
results identify an important role played by the transmembrane domain
in receptor activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Receptors--
Polymerase
chain reaction (PCR) and human cDNAs ALK-5 (T
RI) (11) and
H2-3FF (T
RII) (12) were used to generate transmembrane chimeric and
mutant TGF-
receptors. The following primers were used (where a
single underline indicates the cDNAs of the type I receptor, a
double underline indicates the type II receptor, lowercase type
indicates restriction sites, boldface type indicates stop and
start codes, and italic type indicates the M2-flag coding sequence): RI-1s,
GGTaagcttGCCACCATGGAGGCGGCGGTCGCTGCTCCGCGT; RI-Ss, ACTgcatgcCTGCTcccgggGGCGACGGCGTT; RI-2s,
GTGGAgctagcAGCTGTCATTGCTGGACCA; RI-3s,
CACAttcgaaCTGTCATTCACCATCGAGTG; RI-1a,
AATGactagtAGCAGTTCCACAGGACCAAGGCCAGGTGATGA; RI-2a,
AGatcgatTAACGCGGATATAGACCATCAACATGAG;
RI-3a,
gcatgcTCACTTGTCATCGTCGTCCTTGTAGTCCATTTTGATGCCTTCCTGTTG; RII-1s,
GCAaagcttGCCACCATGGGTCGGGGGCTGCTCAGGGGCCTGT;
RII-2s, TTGTTactagtCATATTTCAAGTGACAGGC; RII-3s,
GTTAggcgccAGCAGAAGCTGAGTTCAACC; RII-1a,
TAGCtctagaTCAGGATTGCTGGTGTTATATTCTTC;
RII-2a,
CTatcgatTGTGGCAGTAGCAGTAGAAGATGATGAT; RII-3a, gaattcTTTGGTAGTGTTTAGGGAGCC; RII
-1a,
GactagtAAGTCAGGATTGCTGGTGTT; RII
-3a,
gacgtcAGGATTGCTGGTGTT; RII
-3s,
gacgtcATATTTCAAGTGACAGGC; RII
-4a,
gatatcAGGATTGCTGGTGTTATA; RII
-4s,
gatatcTTTCAAGTGACAGGCATC.
RI was observed
frequently in the resulting PCR product, T
RI-E. A similar deletion
has been reported in other work (26). A
NcoI-XhoI fragment of ALK-5 (nucleotides 1-236)
was used to replace the deleted portion of T
RI-E-pCRII
by ligating at NcoI and XhoI sites. Three
fragments, T
RI-E, T
RII-T, and T
RI-C, were ligated to pcDNA
I/Amp (Invitrogen) at HindIII and SphI sites to
form the T
RI-II-I(-M2) cDNA construct. To create
HA3-tagged transmembrane chimeric type II receptor
T
RII-I-II(-HA3), a EcoRI-XbaI
fragment consisting of three repeats of HA coding sequence was ligated
to pcDNA I/Amp at corresponding sites; then, at its
HindIII and EcoRI sites, fragments T
RII-E,
T
RI-T, and T
RII-C were ligated to the pcDNA I/Amp vector
containing HA3. By replacing the
XhoI-BamHI portion in T
RI-II-I(-M2) with a
corresponding fragment in ALK-5, tagged wild-type T
RI(-M2) was
created. Similarly, by replacing MluI-BglII
cDNAs in T
RII-I-II(-HA3) with those in H2-3FF,
T
RII(-HA3) was constructed.
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Fig. 1.
Schematic illustration of construction of
TGF- wild-type, chimeric, and type II
transmembrane residue deletion receptors. T
RI-E, T
RI-T, and
T
RI-C are type I receptor extracellular, transmembrane, and
cytoplasmic domains; T
RII-E, T
RII-T, and T
RII-C are the
corresponding type II receptor domains. All of the six domains were
generated by PCR, and primers used are indicated with
arrows. T
RI(-M2) is the wild-type type I receptor with a
C-terminal M2-FLAG tag; T
RII(-HA3) is type II with a
C-terminal three repeats of hemagglutinin epitope HA3 tag.
T
RI-II-I(-M2) contains the extracellular and cytoplasmic domains of
type I and the transmembrane domain of type II, and
T
RII-I-II(HA3) is a wild-type type II with type I
transmembrane domain. T
RII
-1 contains a deletion of one
transmembrane residue Leu160 in T
RII. T
RII
-2
deletes Leu160 and Leu161, T
RII
-3 deletes
Leu160, Leu161, and Leu162; and
T
RII
-4 deletes Leu160, Leu161,
Leu162, and Val163. All four deletion mutants
are C-tagged with HA3.
-1a with the
transmembrane-cytoplasmic fragment generated by primers RII-2s and
RII-3a at their SpeI sites into the pcDNA I/Amp vector
containing HA3 forms C-terminal HA3-tagged
T
RII
-1. This resulted in T
RII
-1 containing a
Leu160 deletion in the transmembrane domain of the type II
receptor. Replacing RII
-1a by RII-1a and ligating the two fragments
at their XbaI/SpeI sites, T
RII
-2 was
generated, which results in a deletion of two residues
(Leu160 and Leu161). To construct T
RII
-3,
which contains a deletion of three residues (Leu160,
Leu161, and Leu162), the extracellular fragment
generated by primers RII-1s and RII
-3a and the
transmembrane-cytoplasmic fragment generated by primers RII
-3s and
RII-3a were ligated at their AatII sites. Primers RII
-4a
and RII
-4s were used to produce corresponding fragments, which were
then ligated at their EcoRV sites to generate T
RII
-4.
It resulted in a deletion of four residues (Leu160,
Leu161, Leu162, and Val163) in the
transmembrane domain of the type II receptor (Fig. 1).
was purchased from Amersham Corp. Binding and affinity cross-linking
assays using bis(sulfosuccinimidyl) suberate (Pierce) were performed as
described previously (22). Briefly, 2 days after transient transfection
with TGF-
receptor constructs, COS-1 cells in six-well plates were
washed with binding buffer (phosphate-buffered saline containing 0.9 mM CaCl2, 0.49 mM
MgCl2, and 1 mg/ml bovine serum albumin), and incubated on
ice for 3 h with 0.4 µCi of 125I-TGF-
/well in 200 µl of binding buffer. After incubation, the cells were washed with
the binding buffer without bovine serum albumin and cross-linked with
0.5 ml of 0.28 mM bis(sulfosuccinimidyl) suberate (3) (in
binding buffer without bovine serum albumin) for 15 min on ice. The
cells were then washed with phosphate-buffered saline and lysed in 100 µl of lysis buffer consisting of 25 mM Tris-phosphate, pH
7.8, 2 mM dithiothreitol, 2 mM
1,2-diaminocyclohexane-N,N,N',N',-tetraacetic acid, 10%
glycerol, 1% Triton X-100, and 1.5% Trasylol (Bayer). The cell
lysates were immunoprecipitated using anti-M2 FLAG antibody conjugated
beads (IBI; Eastman Kodak Co.) followed by SDS-gel electrophoresis
using 10% polyacrylmide and autoradiography.
-inducible
luciferase reporter construct, containing a region of the human
plasminogen activator inhibitor-1 gene promoter and three repeats of
12-O-tetradecanoylphorbol-13-acetate-responsive elements
upstream of the luciferase gene (10), was obtained from A. B. Roberts. p3TP-Lux (6 µg) was co-transfected into mutant Mv1Lu cells
together with 6 µg TGF-
receptor construct(s). The cells in a
10-cm dish were divided into six wells in six-well plates 24 h
after transfection. At 48 h post-transfection, the media were
changed to Dulbecco's modified Eagle's medium, 0.2% bovine serum
albumin, and three wells of each transfected cell line were stimulated
with TGF-
(2 ng/ml). Thereafter, cells were lysed in 100 µl/well
lysis buffer and assayed for luciferase activity using the luciferase
assay system (Promega). Cell lysates (20 µl/well) were used to
measure the total light emission in 10 s using a luminometer (ML
3000 Microtiter Plate Luminometer). The rest of the cell lysates were
directly analyzed by SDS-gel electrophoresis using 12% polyacrylamide
followed by Western blotting. Anti-M2 monoclonal antibody was purchased
from IBI-Kodak. Anti-HA3 polyclonal antibody was obtained
from D. Bowtell (Peter MacCallum Cancer Institute, Melbourne).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Receptors and
Receptor-Receptor Complex Formation--
In order to identify and
monitor the expression of TGF-
receptors, an octapeptide FLAG marker
M2 was tagged to the C terminus T
RI and T
RI-II-I. A triple
hemagglutinin epitope tag HA3 was fused to the C termini of
T
RII and T
RII-I-II. The M2-tagged T
RI(-M2) and
T
RI-II-I(-M2) receptors were transfected into COS-1 cells, and
Western blot analysis using a monoclonal anti-M2 antibody showed that
both were expressed (Fig. 2A).
The HA3-tagged T
RII, T
RII-I-II, and T
RII
-1,
T
RII
-2, T
RII
-3, and T
RII
-4 receptor cDNAs were
transfected into Mv1Lu cells and analyzed using Western blotting by a
polyclonal anti-HA3 antibody (Fig. 2B). The
receptor cDNA constructs transfected into COS-1 cells were used to
investigate the ligand binding properties of the wild-type and chimeric
receptors. Binding of 125I-TGF-
1 was detected by
affinity cross-linking followed by immunoprecipitation with anti-M2
antibody (Fig. 2C). Both the wild-type T
RI and the transmembrane chimeric T
RI-II-I bind TGF-
in the presence of either the wild type T
RII or the transmembrane chimeric T
RII-I-II (Fig. 2C). The fact that the type II receptors (wild-type,
chimeric, or transmembrane mutant) were present after
immunoprecipitation with anti-M2 (type I receptor) antibody confirmed
the formation of type I-type II complexes. These results indicate that
the tagged wild-type, chimeric, and transmembrane mutated receptors are
transported to the cell surface, bind TGF-
, and form receptor
complexes.
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Fig. 2.
Expression and ligand binding properties of
wild-type, chimeric, and transmembrane deletion TGF-
receptors. A, detection of expression of the
wild-type and the transmembrane chimeric type I receptors in COS-1
cells by Western blotting. T
RI(-M2) and T
RI- II-I(-M2) were
transfected into COS-1 cells using the DEAE-dextran method and lysed
48 h later. The cell lysates were subjected to SDS-gel
electrophoresis, transferred onto nitrocellulose membrane, and Western
blotted (29) using a monoclonal anti-M2 antibody. B,
detection of expression of wild-type, chimeric, and transmembrane
residue de- letion type II receptors in Mv1Lu cells by Western
blotting with a polyclonal anti-HA3 antibody. C,
125I-TGF-
cross-linking to receptor. As described under
"Experimental Procedures," COS-1 cells were transfected with
receptor constructs, and 125I-TGF-
was added and
cross-linked to receptors using bis(sulfosuccinimidyl)
suberate. Cell lysates were immunoprecipitated using anti-M2 FLAG
antibody-conjugated beads followed by SDS-gel electrophoresis using
10% polyacrylmide and autoradiography.
RI(-M2) and T
RII(-HA3) Are Functional TGF-
Receptors--
Activation of the plasminogen activator inhibitor
promoter has been frequently used as a marker for TGF-
signaling
activity (10). A reporter gene construct, p3TP-Lux (16), in which the plasminogen activator inhibitor-1 promoter drives expression of luciferase, is cotransfected with the TGF-
receptor into mutant Mv1Lu cells. The luciferase activity was used as a measure of the
activation of TGF-
receptor. As shown above, T
RI(-M2) and T
RII(-HA3) can be successfully expressed in COS-1 and
Mv1Lu cells and form heteromeric receptor complexes with TGF-
. It
was necessary to first confirm that the M2 and HA3 tags did
not interfere with the receptor functions. When T
RI(-M2) was
transfected into mutant Mv1Lu cells (R1B), which lack functional T
RI
and do not normally respond to TGF-
stimulation, the p3TP-Lux
transcriptional response to TGF-
was restored (Fig.
3A). In DR26 cells, which are
mutant Mv1Lu cells lacking functional T
RII and therefore cannot
transduce TGF-
induced signals, transfection of
T
RII(-HA3) restored TGF-
responsiveness (Fig.
3B). These results demonstrate that the C-terminal tags,
i.e. M2 and HA3, do not interfere the function
of the TGF-
receptors.
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Fig. 3.
Signaling from wild-type and chimeric
TGF- receptors. cDNA constructs for
TGF-
receptors and p3TP-Lux were transiently transfected into mutant
Mv1Lu R1B cells (A), which lack functional T
RI, or DR26
cells (B), which contain a truncated, nonfunctional T
RII.
Luciferase activity was assayed as described under "Experimental
Procedures." The results are representative of three separate
experiments. C, Western blots of the expression levels of
T
RII and T
RII-I-II in DR26 cells using the same cell lysates for
luciferase assay in B.
RI-II-I Restores TGF-
Responsiveness in R1B Cells, but
T
RII-I-II Cannot Functionally Substitute for T
RII in DR26
Cells--
T
RI-II-I expression restored TGF-
responsiveness in
R1B cells. However, the degree of TGF-
-stimulated luciferase
activity was less than that resulting from transfection of the
wild-type receptor T
RI (Fig. 3A). In contrast, expression
of T
RII-I-II in DR26 cells did not restore the TGF-
responsiveness, whereas the expression of the wild-type T
RII did
restore TGF-
responsiveness (Fig. 3B). The expression
level of the transmembrane chimeric T
RII-I-II was similar to the
wild-type T
RII (Fig. 3C) in DR26 cells. Interestingly,
transfection of particularly T
RI, but also T
RII, into DR26 cells
increased the basal level of luciferase activity significantly
(2.6-fold; Fig. 3B).
RI and T
RII Results in a
Low Level of Constitutive Activation, while Expression of both
Transmembrane Chimeras T
RI-II-I and T
RII-I-II Results in a Very
High Level of Constitutive Activity--
Expression of T
RI or
T
RII in DR26 cells resulted in some constitutive activation of the
p3TP-Lux reporter in the absence of ligand (Fig. 3B). To
further evaluate the effect of receptor overexpression on TGF-
receptor activation, T
RI and T
RII were cotransfected into R1B
cells. This contransfection resulted in not only the restoration of
TGF-
responsiveness in the cells but also an elevated basal level of
activity in the absence of TGF-
(Fig.
4A), indicating some level of
constitutive receptor activation following overexpression. To
investigate whether expression of transmembrane chimeric receptors
results in a change in this constititutive activation, T
RI-II-I and
T
RII-I-II were cotransfected into R1B cells. A significantly high
basal level/constitutive activity was observed (Fig. 4A).
The activity marginally increased upon TGF-
stimulation. The level
of the constitutive activity was compatible with TGF-
stimulation of
T
RI-R1B cells (Fig. 3A). Given that T
RI-II-I alone
restored TGF-
responsiveness to only ~25% and T
RII-I-II failed
to restore the TGF-
responsiveness in DR26 cells (Fig.
3A), the high level of ligand-independent p3TP-Lux
activation suggests that the receptor transmembrane domain plays a
significant role in receptor activation. Transfection of both chimeric
receptor constructs into DR26 cells also resulted in a high level of
constitutive activity, confirming the results obtained in R1B cells
(Fig. 4B).
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Fig. 4.
Constitutive activation of wild-type and
chimeric TGF- receptors. cDNA
constructs for TGF-
receptors and p3TP-Lux were transiently
transfected into R1B cells (A) or DR26 cells (B).
Luciferase activities were assayed as described under "Experimental
Procedures." The results are representative of at least three
separate experiments. C, Western blot of the expression
levels of T
RII and T
RII-I-II in DR26 cells using the same cell
lysates for luciferase assay in B.
RI with T
RII-I-II Results in a High Level of
Constitutive Activity, but Expression of T
RI-II-I with T
RII Gives
Low Level Constitutive Activity--
Given that expression of both
wild-type receptors T
RI and T
RII results in a low level receptor
constitutive activity while expression of both transmembrane chimeric
receptors T
RI-II-I and T
RII-I-II gives rise to a very high
constitutive activity (Fig. 4), we undertook further experiments to
clarify the function of transmembrane domain on receptor activation.
Cotransfection of the wild-type T
RI and the transmembrane chimeric
T
RII-I-II in R1B cells gave rise to a relatively high constitutive
activity (Fig. 4A), which was higher than that due to
T
RI/T
RII but lower than that following T
RI-II-I/T
RII-I-II
cotransfection. The level of activation was not further enhanced by the
addition of TGF-
, although the wild-type T
RI was transfected,
perhaps because the constitutive activity was already close to a
maximal level of receptor (type I) activation. The high constitutive
activity resulting from cotransfection of T
RI and T
RII-I-II also
suggests that the transmembrane chimeric receptor T
RII-I-II is as
capable of signaling as the wild-type type II receptor T
RII. When
the transmembrane chimeric T
RI-II-I and the wild-type T
RII were
cotransfected into R1B cells, a low constitutive activity was observed
(compatible with T
RI/T
RII constitutive activity) (Fig.
4A). Little TGF-
stimulation was observed, only to a
level similar to that obtained by ligand stimulation following
T
RI-II-I transfected in R1B cells. Similar results were obtained
when DR26 (Fig. 4B) or the wild-type Mv1Lu (data not shown)
cells were used, except that in Mv1Lu cells the p3TP-Lux activity was
slightly stimulated by TGF-
, probably due to the cells expressing
wild-type receptors. Western blotting analysis (Fig. 4C)
shows that the expression level of type II receptors and chimeras was
similar in all experiments.
receptor complex formation and activation, various combinations of
receptor constructs were transfected into COS cells. Similar levels of
expression of the wild-type or chimeric receptors were achieved (Fig.
5, B and C). In
co-immunoprecipitation experiments, immunoprecipitation with anti-M2
(type I receptor) antibody and the Western blotting with
anti-HA3 (type II receptor) antibody demonstrated that the receptor complex formation exactly paralleled the high level of ligand-independent constitutive receptor activation (Fig.
5A). These results demonstrate that by simply changing
the transmembrane regions, a drastic effect is observed on
receptor complex formation.
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Fig. 5.
Receptor-receptor complex formation.
Indicated cDNA constructs were transfected into COS-1 cells using
the DEAE-dextran method, and the cells were lysed 72 h later. The
cell lysates were then immunoprecipitated using anti-M2 FLAG
antibody-conjugated beads. The precipitates were subjected to SDS-gel
electrophoresis using 10% polyacrylmide, transferred onto
nitrocellulose membrane, and Western blotted using a polyclonal
anti-HA3 antibody (A), without
immunoprecipitation (B), and without immunoprecipitation,
blotted with a monoclonal anti-M2 antibody (C).
RII
-1 Is Almost Inactive, T
RII
-2 and T
RII
-3 Are
Partially Active, and T
RII
-4 Is Fully Active in DR26
Cells--
As demonstrated above, exchanging the transmembrane domains
of the type I and II receptors had a dramatic impact on the activities of the receptor. To further identify the importance of the
transmembrane domain in receptor activation, the signaling activity of
type II transmembrane deletion mutants was examined in DR26 cells. Expression of T
RII
-1, which has a Leu160 deletion in
the transmembrane domain of the type II receptor, restored very little
of the TGF-
responsiveness in DR26 cells (Fig.
6A). This observation is
similar to the expression of T
RII-I-II. However, transfection of the
cDNAs of the type II receptor containing a two- or three-residue
deletion in the transmembrane domain (T
RII
-2 and T
RII
-3)
restored about half of the wild-type T
RII activity (Fig.
5A). Moreover, the expression of the four-residue deletion type II receptor, T
RII
-4, fully restored the activity of the wild-type receptor (Fig. 5A). Western blotting analysis
(Fig. 6B) demonstrates similar expression levels of the
wild-type and the mutant type II receptors, and all were found to bind
TGF-
(Fig. 2C).
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Fig. 6.
Signaling activity of type II receptors with
transmembrane residue deletion in DR26 cells. A,
cDNA constructs for TGF- receptors and p3TP-Lux were transiently
transfected into DR26 cells. Luciferase activities were assayed as
described under "Experimental Procedures." The results are
representative of four separate experiments. B, Western blot
of the expression levels of T
RII and type II transmembrane residue
deletion mutants in DR26 cells using the same cell lysates for
luciferase assay in A.
RII
-1 with T
RI or T
RI-II-I Resulted in a
High Constitutive Activation--
As shown earlier, T
RII-I-II was
inactive in DR26 cells (Fig. 3B) but exhibited high levels
of constitutive activation when co-expressed with T
RI or T
RI-II-I
(Fig. 5, A and B). To investigate whether
T
RII
-1 and T
RII-I-II share other signaling properties, the
transcriptional activation of p3TP-Lux was examined following the
co-expression of T
RII
-1 with either T
RI or T
RI-II-I. In DR26 cells, a very high ligand-independent activation was obtained when
T
RII
-1 was co-expressed with the wild-type T
RI, while only a
moderate activation was exhibited by T
RII
-2, T
RII
-3, or
T
RII
-4 with T
RI (Fig.
7A). Similarly, co-expression
of T
RII
-1 and T
RI-II-I in DR26 cells also resulted in a high
constitutive activation. However, T
RII
-2 with T
RI-II-I showed
little constitutive activation, while T
RII
-3 or T
RII
-4 with
T
RI-II-I resulted in a moderate activation (Fig. 7B).
This trend of constitutive activation was also obtained in R1B cells
(data not shown). These results demonstrated that the relative
constitutive activity among the type II transmembrane residue deletion
mutants differs from their TGF-
-induced activity, and more
importantly, T
RII
-1 and T
RII-I-II share similar signaling
activity.
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Fig. 7.
Constitutive activation of type II receptors
with transmembrane residue deletion in DR26 cells. cDNA
constructs for TGF- type II receptors and p3TP-Lux were transiently
transfected into DR26 cells together with the wild-type T
RI
(A) or the transmembrane chimeric T
RI-II-I
(B). Luciferase activities were assayed as described under
"Experimental Procedures." The results are representative of at
least three separate experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
signaling have suggested a clearly
postulated mechanism of TGF-
receptor activation (17-21). TGF-
binds directly to the type II receptor, which is a constitutively active kinase and is autophosphorylated. The TGF-
/type II receptor then recruits and phosphorylates the type I receptor, which is activated and signals to downstream substrates (21). This mechanism is
consistent with a model of heteromeric association of the type I and II
receptors that follows TGF-
binding. Subsequent double immunoprecipitation and yeast two-hybrid studies have shown that the
type I and II receptors form a heteromeric complex even in the absence
of TGF-
(25). The formation of a homomeric type II-type II complex
has also been detected (23). Interactions between T
RI and T
RII or
between T
RII and T
RII occur between both the extracellular
domains and the intracelluar domains without ligand stimulation (23,
24). Given that the receptors preexist in a heteromeric complex but
only are activated by TGF-
binding, the ligand may perform functions
in addition to simply bringing the receptors together. We hypothesize
that the binding of ligand to the extracellular domains of the type I
and type II receptors also results in specific changes in the
intracellular region of the receptor complex that lead to signaling.
Such changes are transmitted from the extracellular to the
intracellular region of the receptor through the transmembrane domains.
Indeed the fact that T
R2.1, a chimeric receptor containing the
extracellular and the transmembrane domains of the type II receptor and
the intracellular domain of the type I receptor, fails to reconstitute signaling despite the fact that the wild-type type II receptor heterodimerizes with it (26) confirms that receptor
heterooligomerization is not sufficient for TGF-
signaling. The data
presented in our study establish that by simply changing the
hydrophobic transmembrane domain, the signaling ability of the receptor
is radically altered.
RI restores the TGF-
responsiveness
in R1B cells (Ref. 10; Fig. 3A). Changing the transmembrane domain of T
RI from type I to type II reduces the signaling activity of the receptor to ~25% (Fig. 3A). Expression of T
RII
restores the TGF-
responsiveness in DR26 cells (10, Fig.
3B). Simply changing the T
RII transmembrane domain from
type II to type I all but abolishes the signaling activity of the
receptor (Fig. 3B) despite high levels of expression,
complex formation, and ligand binding (Fig. 2, B and
C). Our results further suggest that the transmembrane domains play an important role in receptor activation. Overexpression of T
RI and T
RII in cells results in some ligand-independent constitutive activation of the receptor in our studies (Fig. 4, A and B) and works by others (30). However, by
simply swapping the transmembrane domains of T
RI and T
RII we see
a marked increase in constitutive activity to a maximal level (Fig. 4,
A and B). A slightly lower level of activation is
seen if both receptors have type I transmembrane domains, while if both
receptors have the type II transmembrane domains only a modest
constitutive activation is seen (Fig. 4, A and
B). This suggests that the transmembrane domain may mediate
a change in the T
RI-II-I·T
RII-I-II or T
RI·T
RII-I-II receptor complex resulting in activation and that this change is
similar to that induced by ligand binding to the wild-type receptor.
-helical structure (29),
exchanging the type I and type II transmembrane domains has two
consequences: (i) increasing the distance between the extracellular and
intracellular domains of the type I receptor but decreasing the
distance between these domains in the type II receptor and (ii)
altering the relative orientation between the extracellular and
intracellular domains about 100° in the type I receptor in one
direction but 100° in the other direction in the type II receptor. If
the signaling incompetence of the type II transmembrane chimeric
receptor T
RII-I-II in DR26 cells (Fig. 3B) is due to the
shortening of the distance between the extracellular and the
intracellular domains or if the low activity of the type I
transmembrane chimeric receptor T
RI-II-I in R1B cells (Fig.
3A) is due to the lengthening of the distance between the
extracellular and intracellular domains, then coexpression of
T
RI-II-I and T
RII-I-II would not be expected to have any TGF-
signaling activity. However, coexpression of T
RI-II-I and
T
RII-I-II results in a very high constitutive activation of the
receptors in either R1B or DR26 cells (Fig. 4, A and
B), indicating that the distance between the extracellular
and intracellular domains may not be critical for achieving receptor
activation. Perhaps the relative orientation between the extracellular
and intracellular domains in the receptors and the relative orientation between the T
RI and T
RII receptors in the receptor complexes are
more important. We propose that TGF-
binding to the preexisting receptor complex results in relative rotation between the extracellular domains of the type I and type II receptors, which, through the transmembrane domains, results in a change in orientation between the
T
RI and T
RII intracellular domains. This relative reorientation of the intracellular kinase domains results in a productive interaction between the type I and type II kinase (Fig.
8).
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Fig. 8.
A general model for TGF-
receptor activation via relative rotation. TGF-
receptors
form a receptor complex in the absence of ligand. To the wild-type
receptor complex, TGF-
binds to the receptor extracellular domains,
resulting in relative rotation between receptors and therefore the
receptor activation and signaling. Exchanging the type I and type II
receptor transmembrane domains in the receptors results in relative
rotation between the extracellular and intracellular domains in the
receptor. The interaction between extracellular domains brings the
cytoplasmic domains of the transmembrane chimeric receptors into a
productive interaction orientation, leading to constitutive activation
and signaling in the absence of the ligand.
By changing the transmembrane domains, the resulting reorientation
between the type I and II intracellular domains may be similar to that
normally induced by TGF- resulting in receptor activation. Hence,
coexpression of T
RI-II-I and T
RII-I-II or T
RI and T
RII-I-II
results in constitutive activity in the absence of ligand (Fig. 4,
A and B). These data suggest that a 100-200° (approximately) reorientation between the kinase domains of T
RI and
T
RII results in activation. Since the constitutive activity resulting from coexpression of T
RI with T
RII-I-II is similar to
that induced by ligand when T
RI and T
RII are expressed in cells
(Fig. 4, A and B), we postulate that TGF-
may
induce approximately 100° of relative rotation between the type I and
type II kinase domains in the wild-type receptor complex. The addition
of TGF-
to the T
RI·T
RII-I-II complex has no effect on the
activation level (Fig. 4, A and B) possibly
because the complex has assumed a rotated confirmation and ligand is no
longer capable of inducing further rotation. Introducing the type II
transmembrane domain to T
RI results in 100° (approximately)
rotation in the opposite direction. The kinase domains may then be in a
less favorable orientation in the resulting T
RI-II-I·T
RII
complex, and therefore little constitutive activation is seen (Fig. 4,
A and B). Ligand binding to the
T
RI-II-I·T
RII receptor complex changes the orientation between
the kinase domains to provide some receptor activation but less than
that following ligand binding to the wild-type receptor complex (Fig.
3A).
Since the type I and type II extracellular domains interact with each
other in the absence of ligand, it is possible that they may provide a
constraint preventing the receptor intracellular kinase domains from
interacting in an active conformation in the preexisting wild-type
receptor complex. In support of this possibility, a recent paper
suggests that overexpression of the extracellular domain-truncated type
I and type II receptors results in a high level of ligand-independent
receptor activation and signaling (30). Further consistent with this is
the supposition that the extracellular interactions work against the
intracellular kinase interactions in the receptor complex. Ligand
binding to the receptor complex may change the orientation of the
receptor-receptor interactions in the extracellular domain, which in
turn results in more favorable, or even enhanced, interactions between
the kinase domains (activation). In the TRI-II-I·T
RII-I-II and
T
RI·T
RII-I-II complexes, the extracellular interactions
strengthen the kinase interactions in the absence of ligand, resulting
in constitutive receptor activation. Accordingly, the interactions in
the T
RI-II-I·T
RII-I-II and T
RI·T
RII-I-II complexes are
expected to be stronger than those in the T
RI·T
RII and
T
RI-II-I·T
RII complexes. The immunoprecipitation and Western
blotting results in Fig. 5 confirm the above prediction. Furthermore,
this result demonstrates an important role played by the receptor
transmembrane domains in the formation of receptor complex.
To further elucidate the critical role of the transmembrane domain in
receptor activation, we constructed four TGF- type II receptor
mutants with consecutive residue deletions in the transmembrane domain
(Fig. 1). T
RII
-1 has a deletion of Leu160;
T
RII
-2 has a deletion of Leu160 and
Leu161; T
RII
-3 has a deletion of Leu160,
Leu161, and Leu162; and T
RII
-4 has a
deletion of Leu160, Leu161, Leu162,
and Val163. Since the transmembrane domain is
-helical
and 3.4-3.7 residues constitute one
-helical turn, each residue
deletion in the transmembrane domain may result in approximately 100°
of relative rotation between the extracellular and intracellular
domains. A deletion of four residues in the transmembrane region should
return the receptor close to the original wild-type orientation. As
described before, changing the transmembrane domain of type II receptor
to type I receptor may result in 100° relative rotation. Therefore,
the relative orientations between the extracellular and the
intracellular domains of T
RII-I-II and T
RII
-1 are similar.
Expression of the T
RII one-residue deletion construct T
RII
-1
in DR26 cells failed to restore TGF-
-induced plasminogen activator
inhibitor-1 activation as assayed by luciferase activity using p3TP-Lux
reporter construct (Fig. 5A), similar to the results
obtained by expression of T
RII-I-II (Fig. 3B). However,
expression of a transmembrane two- or three-residue deletion
construct (T
RII
-2 or T
RII
-3) resulted in some
restoration of TGF-
responsiveness in DR26 cells. More importantly,
the four-residue transmembrane deletion receptor T
RII
-4 fully
restored the TGF-
responsiveness (Fig. 6A) in DR26 cells.
This would be predicted, since T
RII
-4 retains a relative
orientation close to that of the wild-type T
RII. These results
demonstrate a pivotal role for the transmembrane domain in receptor
activation, and further support our model (Fig. 8) in which relative
orientations between the extracellular and intracellular receptor
domains and hence of the receptor kinase domains are critical in
activation of the TGF-
receptor complex.
The TGF--induced signaling activity of both T
RII-I-II and
T
RII
-1 has been shown to be severely impaired (Figs.
3B and 6A). The constitutive activation observed
when T
RII-I-II was co-expressed with T
RI or T
RI-II-I (Fig. 4,
A and B) indicates that the transmembrane
chimeric receptor T
RII-I-II can be as potent as the wild-type
T
RII. It is expected that the one-transmembrane residue deletion
mutant T
RII
-1 exhibits similar signaling potency, since the
relative orientations between the extracellular and intracellular
domains of T
RII-I-II and T
RII
-1 are expected to be similar.
The constitutive activation resulting from cotransfection of
T
RII
-1 with T
RI or T
RI-II-I (Fig. 7, A and
B) meets this expectation. It should be noted that the trend
of TGF-
-induced activity exhibited in Fig. 6A among the
type II receptors differs dramatically from the trend of the
constitutive activity observed when cotransfected with the wild-type
T
RI (Fig. 7A). In addition, these trends are different
from the one of constitutive activity exhibited in Fig. 7B
when the transmembrane chimeric T
RI-II-I was contransfected. The
orientation of the type I intracellular domain (relative to the type II
receptors) in each case differs from the other. These results provide
further evidence supporting our conclusion that the relative
orientation between receptors determines whether the receptor complex
is activated or not.
The type I and the type II receptors for the TGF- superfamily are
related, with high conservation in the kinase domain (3). Since the
transmembrane domains play a pivotal role in TGF-
receptor activation as demonstrated above, it is instructive to compare the
length and primary sequence of the domain in the superfamily. Using a
BLAST program (31), the transmembrane sequences of the TGF-
receptor
superfamily were retrieved and aligned (Fig.
9). The length and the primary sequence
of the domain vary between subfamily receptors. However, the length
within each subfamily is strictly conserved, although the primary
sequence may not be. The conservation of the length of the
transmembrane domain within each subfamily may contribute to the
ligand-specific activation of the receptor complex.
|
In conclusion, we have demonstrated that the transmembrane region of
the TGF- receptors is not just a passive hydrophobic membrane anchor
but is critically important in receptor association and activation. We
propose that TGF-
activates the type I and II receptor complex
through induction of relative rotation between the receptors (Fig. 8).
We are currently analyzing various mutants of the receptor
transmembrane regions in order to gain even further insights into the
regulatory role played by the transmembrane region of the TGF-
receptor and indeed how generally this mechanism may be applied in the
activation of other transmembrane receptors.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Ludwig Institute for
Cancer Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia. Tel: 61-3-93413155; Fax: 61-3-93413104; E-mail: Hong.Jian.Zhu{at}ludwid.edu.au.
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
T
R, TGF-
receptor;
GM-CSF, granulocyte/macrophage colony-stimulating factor;
Epo, erythropoietin;
PCR, polymerase chain reaction.
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REFERENCES |
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