From the
TGF-
The TGF-
Most mammalian cells express three abundant high affinity receptors,
which can bind and be cross-linked to TGF-
Studies
in chemically mutagenized cell lines showed that TGF-
Oligomerization of TGF-
If the type II and type I receptors
indeed mediate TGF-
To study the binding properties of TGF-
The secreted exoplasmic domain of the human TGF-
TGF-
However, growth inhibition by TGF-
The current model for binding of members
of the TGF-
Our data indicate that
interaction of TGF-
We thank Drs. Petra Knaus, Kunxin Luo, Riki Perlman,
and Rebecca Wells for helpful discussions of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 binds with high affinity (K
= 25-50 pM) directly to the TGF-
type II receptor serine-threonine kinase (T
-RII) in the absence of
expression of the TGF-
type I or III receptors (T
-RI and
T
-RIII). The serine-threonine kinase T
-RI is essential for
TGF-
1 signaling but not for binding to T
-RII. TGF-
2, in
contrast, does not bind directly to T
-RII, although coexpression
of T
-RIII does allow binding and cross-linking of TGF-
2 to
T
-RII. Here we show that in transfected COS cells binding and
cross-linking of
I-TGF-
2 to T
-RI or T
-RII
requires expression of both receptors. In cells transfected with the c-myc-tagged human T
-RII cDNA, only low amounts
of
I-TGF-
2 cross-linked to T
-RI and T
-RII
were detected even with high concentrations (700 pM) of
ligand. Cotransfection of the influenza-hemagglutinin-tagged human
T
-RI cDNA dramatically increased the binding of TGF-
2 to
T
-RII; the concentration of
I-TGF-
2 required
for half-maximal binding and cross-linking to T
-RI and T
-RII
was
40 pM. Coimmunoprecipitation studies showed that the
high affinity receptor for TGF-
2 is composed of a hetero-oligomer
of T
-RI and T
-RII. Thus TGF-
1 and -
2 bind to
TGF-
receptors in different ways; TGF-
1 binds directly to
T
-RII, while binding of TGF-
2 to T
-RII requires
coexpression of T
-RI or T
-RIII.
(transforming growth factor
)
(
)family of cytokines has important functions in
growth, development, and differentiation(1) . The three
mammalian TGF-
isoforms, TGF-
1, -
2, and -
3, share
71-76% amino acid identities. Although disulfide-linked
homodimers of 25 kDa are the predominant species, the heterodimers
TGF-
1.2 and TGF-
2.3 have been described(2) . The
sequences of the mature, proteolytically processed forms of each
TGF-
family member are almost entirely conserved across species,
and thus there has been evolutionary pressure to retain both the
similarities and differences in these isoforms. In most cell types, all
three forms share very similar biological activities(3) .
: the type I (
53
kDa), type II (
65 kDa), and type III (
100-280 kDa)
receptors, based upon the molecular mass of the cross-linked products
analyzed by gel electrophoresis(4) . T
-RI and T
-RII,
the type I and II receptors, are type I transmembrane proteins with
cytosolic domains containing a serine-threonine
kinase(5, 6, 7, 8, 9) . Both
receptors are essential for intracellular signaling. The TGF-
type
III receptor, or betaglycan, is a membrane-bound proteoglycan with a
short cytoplasmic tail that has no apparent signaling
motif(10, 11) . It binds TGF-
2 (apparent K
100 pM) with slightly
greater affinity than TGF-
1 or TGF-
3 (apparent K
300
pM)(12, 13) . The main role of betaglycan seems
to be in binding and then presenting TGF-
ligand to the signaling
receptors T
-RI and T
-RII(14) ; overexpression of
T
-RIII in L6 myoblasts leads to a dramatic increase in TGF-
2
binding to T
-RI and T
-RII(10, 15) .
1 binds
T
-RII with high affinity (mass
25-50 pM) in
the absence of T
-RI and that binding of TGF-
1 to T
-RI
requires the presence of T
-RII(16, 17) . TGF-
1
binds with relatively high affinity to the soluble secreted exoplasmic
domain of T
-RII(18) . Heteromeric complexes of the type II
and type I receptors are found on the surface of many cells after
ligand binding, and are important for signal
transduction(15, 19, 20) . Addition of
TGF-
1 allows the cytosolic domains of T
-RI and T
-RII to
interact such that the cytoplasmic domain of T
-RI is
transphosphorylated by the constitutively active T
-RII kinase.
Phosphorylation of the cytoplasmic domain of T
-RI is believed to
activate its kinase activity and to allow its phosphorylation of and/or
its association with (unknown) downstream substrates(21) .
receptors plays important roles both in
ligand binding and signal transduction. Homo-oligomers, probably
homodimers, of the types II (22, 23) and III (22) receptors exist on the cell surface in the absence or
presence of TGF-
1 or TGF-
2. Hetero-oligomers of the types II
and III receptors are probably transient species, most likely
intermediates in the transfer of a TGF-
ligand from a type III to
a type II receptor(15) . Heteromeric complexes of T
-RI and
T
-RII, and of T
-RII and T
-RIII, have been detected after
addition of TGF-
1 (15, 19, 20) but it is
not known whether the T
-RI
T
-RII complexes exist in the
absence of TGF-
1 ligand.
-induced signals, then it is puzzling that
TGF-
2 is as potent as TGF-
1 and TGF-
3 in its ability to
arrest the growth of cells (ED
5-20
pM)(24) , since TGF-
2 does not bind well to either
the type I or the type II receptors. TGF-
2 does interact with the
type II receptor, since reintroduction of the cloned type II receptor
into mink lung cell mutants lacking the type II receptor restored the
ability of these cells to be growth-inhibited by
TGF-
2(19) . Depending on the cell line TGF-
2 binds to
T
-RII and T
-RI with an affinity
10- to
100-fold
lower than does
TGF-
1(24, 25, 26, 27) . TGF-
2
does not bind at all to the soluble secreted exoplasmic domain of
T
-RII, and in transfected COS cells, binding of TGF-
2 to
T
-RII requires coexpression of T
-RIII (18).
Epitope-tagged Receptors
The expression vectors
for the epitope-tagged TGF- receptors have been described
previously(22, 29) .
Transient Transfection of COS-7 Cells
COS-7 cells
(American Type Culture Collection CRL 1651) were grown in
Dulbecco's modified Eagle's medium supplemented with
penicillin, streptomycin, L-glutamine, and 10% fetal bovine
serum (Life Technologies, Inc.). Approximately 30% confluent cells in a
10-cm diameter dish were transfected with different combinations of the
plasmids pcDNA-1 containing the N-terminal Myc-tagged human T-RII
receptor (22) and CMV7 containing the C-terminal HA-tagged
T
-RI receptor(28, 29) , using the
DEAE-dextran/chloroquine method described previously(5) .
Receptor Cross-linking and
Immunoprecipitation
TGF-1 and TGF-
2 (R& Systems,
Minneapolis, MN) were iodinated by the chloramine-T method as
described(30) . COS cells were used in binding studies 2 days
after transfection, as detailed in Ref. 22. Affinity labeling of
transfected cells was done in the presence of
I-TGF-
1 or
I-TGF-
2 and the
cross-linking agent disuccinimidyl suberate (DSS; Pierce) as described
previously(31) . After binding and cross-linking, portions of
the cell lysates were analyzed directly by electrophoresis through a
10% SDS-polyacrylamide gel, exposed overnight to a Fuji BAS III screen,
and quantified with the BAS-2000 bio-imaging analyzer (Fuji).
Subsequently the gel was exposed for 3 weeks to a preflashed Kodak
XAR-5 film. The remainder of the lysate was immunoprecipitated with
either the anti-HA monoclonal antibody 12CA5 (32) (Harvard
Monoclonals, Boston, MA) or the monoclonal antibody 9E10 that
recognizes a human c-myc epitope (33) (BAbCO, Berkeley,
CA) as described previously(22) . The immunoprecipitates were
also analyzed by SDS-PAGE, autoradiography, and bio-imaging
quantification.
2 to T
-RI
and T
-RII, we transfected the full-length human receptors into COS
cells. TGF-
1 binds efficiently to T
-RII expressed in COS
cells, and binding is not enhanced by coexpression of T
-RI
(6).
(
)The cotransfection experiment in Fig. 1confirms this point, and serves as an essential control for
the experiments with TGF-
2. Almost undetectable binding of
I-TGF-
1 to either T
-RI or endogenous T
-RII
occurred when cells were transfected with T
-RI alone (Fig. 1, lane1). In contrast, substantial
binding of
I-TGF-
1 to T
-RII but not to
endogenous T
-TI occurred after transfection of T
-RII alone (lane2). This confirms that binding of TGF-
1 to
T
-RII does not require expression of T
-RI(19) . In
addition, and as previously reported(6) , after cotransfection
of T
-RI and T
-RII
I-TGF-
1 is bound and
cross-linked to both receptors (Fig. 1, lane3). We do not know why less
I-TGF-
1
was cross-linked to T
-II when T
-RI was coexpressed; one
possibility is that cotransfection of both receptors decreased surface
expression of T
-RII.
Figure 1:
Binding and
cross-linking of I-TGF-
1 to transfected COS cells
expressing T
-RI or/and T
-RII. COS cells were transfected with
1 µg of T
-RI-HA (lane1), 1 µg of
T
-RII-Myc cDNA (lane2), or 1 µg of each
cDNA (lane3). Forty-eight hours after transfection,
the cells were incubated 2 h at 4 °C with 50 pM
I-TGF-
1. The cells were then incubated for 10 min at
4 °C in the presence of 50 mM of the cross-linking reagent
DSS. The cell lysates were resolved by electrophoresis through a 10%
SDS-polyacrylamide gel and autoradiographed using Kodak XAR
film.
Consistent with our previous
results(18) , Fig. 2A (lanes 1-4)
show that, in COS cells transfected with T-RII cDNA, little
I-TGF-
2 is bound and cross-linked to T
-RII or
endogenous T
-RI or T
-RIII, even at high concentrations (700
pM) of TGF-
2. Importantly, cotransfection of T
-RI
dramatically increased the amount of
I-TGF-
2
cross-linked to T
-RII and T
-RI (lanes 5-8).
Quantification (Fig. 2B) showed that, at concentrations
of TGF-
2 less than 300 pM, over 10 times more
I-TGF-
2 was cross-linked to T
-RII when
T
-RI was coexpressed. After coexpression of both T
-RII and
T
-RI, the concentration of
I-TGF-
2 required for
half-maximal binding and cross-linking to cell surface T
-RI and
T
-RII was
40 pM. In the cotransfection experiments,
the amount of
I-TGF-
2 cross-linked to T
-RI was
higher than that to T
-RII. This may reflect a real difference in
the proportion of both receptors in the binding complex or differences
in the efficiency of ligand-receptor cross-linking.
Figure 2:
Binding
and cross-linking of I-TGF-
2 to transfected COS
cells expressing T
-RI and T
-RII. COS cells were transfected
using the dextran-chloroquine technique with 1 µg of TB-RII-Myc
cDNA without (lanes 1-4) or together with (lanes
5-8) 1 µg of T
-RI-HA cDNA. Forty-eight hours after
transfection, the cells were incubated at 4 °C with increasing
concentrations of
I-TGF-
2: 6 pM (lanes1 and 5), 30 pM (lanes2 and 6), 150 pM (lanes3 and 7), or 750 pM (lanes4 and 8). After cross-linking with DSS, as detailed in the legend to
Fig. 1, the cell lysates were resolved by electrophoresis through a 10%
SDS-polyacrylamide gel and autoradiographed using Kodak XAR film. PanelA shows the autoradiogram. In PanelB, the gel was exposed for a short period of time to a
Fuji BAS III screen; the individual protein bands were quantified using
a Fuji BAS-2000 Bio-imaging analyzer. The amount of radioactivity in
each protein species is expressed in arbitrary units. Closedsquares,
I-TGF-
2 cross-linked to
T
-RI; closedtriangles,
I-TGF-
2 cross-linked to T
-RII; solidlines, cells transfected with both T
-RI-HA and
T
-RII-Myc; dashedlines, cells transfected only
with T
-RII-Myc.
To confirm that
the cross-linked receptor species correspond to the transfected
TGF- receptors and not to endogenous COS cell receptors, the cell
lysates from the experiment in Fig. 2were immunoprecipitated
with either anti-HA or anti-Myc antibodies (Fig. 3). When the
cells were transfected only with T
-RII-Myc and cross-linked to
I-TGF-
2, the anti-Myc antibody precipitated no
I-TGF-
2 cross-linked to T
-RII (Fig. 3, lanes 11-14). Similarly, when cells were transfected
only with T
-RI-HA and cross-linked to
I-TGF-
2,
only trace amounts of labeled receptors could be detected (Fig. 4, lanes 1-4) and the anti-HA antibody
precipitated no
I-TGF-
2 cross-linked to T
-RI
(data not shown). Thus,
ITGF-
2 cannot bind either to
T
-RII-Myc or T
-RI-HA when expressed alone in COS cells; the
low level of binding of
I-TGF-
2 to type I and type
II receptors singly expressed in COS cells (Fig. 2A, lanes 1-4) most likely involves endogenous type I and II
COS cell receptors since these are not precipitated by the anti-epitope
antibodies. However, when COS cells were transfected both with
T
-RII-Myc and T
-RI-HA and cross-linked to
I-TGF-
2, both anti-Myc and anti-HA antibodies
immunoprecipitated
I-TGF-
2 cross-linked to both
T
-RII-Myc and T
-RI-HA (Fig. 3, lanes 5-8 and 15-18). Thus, both T
-RII and T
-RI are
associated in a tight complex after ligand binding. The total amount of
both receptors immunoprecipitated with anti-Myc antibodies is lower
than that with anti-HA, most likely reflecting a lower efficiency of
immunoprecipitation by the anti-Myc antibody.
(
)
Figure 3:
Binding and cross-linking of I-TGF-
2 to transfected COS cells expressing
T
-RI and T
-RII: analysis by immunoprecipitation. The cell
lysates from the experiment in Fig. 2 were immunoprecipitated overnight
in the presence of 1% Triton X-100 using 10 mg/ml anti-HA or anti-Myc
(9E10) antibody. Lanes 1-8 represent the samples in lanes 1-8 of Fig. 1A immunoprecipitated with
the anti-HA antibody, respectively, and lanes 11-18 represent the samples in lanes 1-8 of Fig. 1A immunoprecipitated with the anti-Myc antibody. The
immunoprecipitates were resolved by electrophoresis through a 10%
SDS-polyacrylamide gel and autoradiographed using Kodak XAR film.
Figure 4:
Binding and
cross-linking of I-TGF-
2 to transfected COS cells
expressing increasing amounts of T
-RI in the presence or absence
of T
-RII. COS cells were transfected with 0 µg (lanes1 and 5), 0.1 µg (lanes2 and 6), 0.3 µg (lanes3 and 7), or 1 µg (lanes4 and 8) of
T
-RI-HA cDNA without (lanes 1-4) or together with (lanes 5-8) 1 µg of T
-RII-Myc cDNA. Two days
after transfection, the cells were incubated at 4 °C for 2 h with
150 pM
I-TGF-
2, then for 10 min at 4 °C
in the presence of 50 µM DSS. The cell lysates were
analyzed as in Fig. 1.
The increased amount of type II receptor
cross-linked to I-TGF-
2 after T
-RI
overexpression indicates that T
-RI increases the affinity of
TGF-
2 for T
-RII. On the other hand, the amount of type I
receptor cross-linked to
I-TGF-
2 dramatically
increases when this receptor is coexpressed with T
-RII, suggesting
that T
-RII increases the affinity of T
-RI for TGF-
2. Fig. 4demonstrates directly that T
-RII increases the
affinity of TGF-
2 for T
-RI. In this study, different
concentrations of T
-RI-HA cDNA were transfected alone or
cotransfected with a fixed amount of T
-RII-Myc cDNA. Transfected
cells were incubated at 4 °C for 2 h with a fixed amount of
TGF-
2 (150 pM) and then subjected to cross-linking with
DSS. Transfection of T
-RI-HA in the absence of transfected
T
-RII does allow a small but significant binding of
I-TGF-
2 to T
-RI-HA (Fig. 4, lanes
1-4); presumably this is due to endogenous T
-RII in the
COS cell clone used in this study interacting with the transfected
T
-RI. Binding of
I-TGF-
2 to T
-RI-HA was
dramatically increased when T
-RII was cotransfected (Fig. 4, lanes 5-8).
type II
receptor binds to and can be cross-linked to
I-TGF-
1
with an apparent affinity of
200 pM; in contrast,
TGF-
2 does not bind to this receptor domain (K
> 10 nM)(18) . The ability of
I-TGF-
1 to bind directly to the extracellular domain
of the human TGF-
type II receptor suggests that the type II
receptor may be the primary binding subunit for TGF-
1. Several
facts support this notion. First, mutant cell lines that lack
cell-surface type II receptors and are resistant to growth inhibition
by TGF-
1, such as DR mink lung cells and Hep 3B-TR cells, also
lack type I receptors that are able to bind ligand, and expression of
the type II receptor in these cells restores binding and cross-linking
of TGF-
1 to cell surface type I receptors(19) . Second,
several type I receptors for TGF-
and activin have been cloned.
When expressed alone in transfected cells none are able to bind
TGF-
1 or any other ligand tested. When coexpressed in COS cells
with the type II TGF-
receptor all of these species are able to
bind TGF-
1, and when the type II activin receptor is coexpressed
all are able to bind and be cross-linked to activin. Thus, the nature
of the type II receptor determines the nature of the ligand that is
bound to the type I receptor, even though only certain combinations of
type II and I receptors are apparently able to transduce TGF-
1 or
activin signals(9, 34) .
2 does not bind at
all to the soluble secreted extracellular domain of T
-RII (18) or to T
-RII expressed in transfected COS cells (Ref.
18 and this work), yet T
-RII is essential for TGF-
2
signaling(16) . Thus, binding of TGF-
2 to T
-RII might
require other receptor subunits on the cell surface. One of these could
be T
-RIII, since expression of T
-RIII enhances binding of
TGF-
2 to T
-RII in transfected COS cells and also in L6
myoblasts(15, 18) . Furthermore, the affinity of
TGF-
2 for T
-RI and T
-RII is higher in cells that
endogenously express high numbers of T
-RIII than in cells that do
not(24) .
2 does not
require expression of T
-RIII(15) . The data presented here
strongly indicate the existence of a high affinity TGF-
2 receptor
that is a hetero-oligomer of the two serine-threonine kinase receptors,
T
-RI and T
-RII. In COS cells overexpressing T
-RI or
T
-RII alone, the amount of receptors cross-linked to iodinated
TGF-
2 was very low, even at high concentrations of ligand.
Cotransfection of T
-RI with T
-RII dramatically increased the
ability of both T
-RI and T
-RII to be cross-linked to
I-TGF-
2.
family of cytokines to cell surface receptors is based
mainly on studies using TGF-
1. High affinity binding of TGF-
1
to T
-RII does not require the presence of T
-RI since mutant
cells lacking functional T
-RI display a normal pattern of binding
of TGF-
1 to T
-RII (16, 19). Binding and cross-linking of
TGF-
1 to T
-RI is dependent on prior binding of TGF-
1 to
T
-RII(19) . This interaction presumably involves the
extracellular and/or transmembrane domains of both receptors since a
mutant truncated T
-RII, lacking a complete cytoplasmic domain,
restores binding of TGF-
1 to T
-RI in a cell line that lacks a
functional T
-RII(35) .
2 with TGF-
receptors follows a different
sequence of events. Since expression of both T
-RI and T
-RII
are required for TGF-
2 to bind with high affinity to either
receptor, cell surface T
-RI and T
-RII probably interact in
the absence of ligand. A weak ligand-independent interaction between
T
-RI and T
-RII has been observed when the proteins are
overexpressed in insect (36) and COS cells(29) . Since
most of the cell surface T
-RII is in homodimers(22) ,
heteromers of T
-RI and T
-RII could represent a small
proportion of the total receptors on the cell surface. We cannot say
whether these presumed heteromers would contain one or two subunits of
T
-RI or T
-RII. We hypothesize that the small number of high
affinity receptors for TGF-
2 in Mv1Lu epithelial cells are
preformed T
-RI-T
-RII complexes. In the Mv1Lu cell line,
TGF-
2 apparently binds only to a small subset of the T
-RI and
T
-RII receptors that can bind TGF-
1(24) . However, the
affinity of binding of TGF-
2 to these high affinity receptors is
the same (
20 pM TGF-
2 required for half-saturation)
as for binding of TGF-
1 to a larger number of cell surface
receptors, and the concentrations of TGF-
1 and TGF-
2 required
for growth inhibition are also the same (24). Thus, these presumably
preformed T
-RI and T
-RII heteromers could be the preferred
receptors for signaling by TGF-
2.
,
transforming growth factor
; T
-RI, T
-RII, and
T
-RIII, type I, II, and III TGF-
receptors; PAGE,
polyacrylamide gel electrophoresis; DSS, disuccinimidyl suberate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.