(Received for publication, July 28, 1994; and in revised form, December 7, 1994)
From the
The transforming growth factor (TGF)- type II receptor is a
transmembrane serine/threonine kinase which is essential for all
TGF-
-induced signals. In several cell types TGF-
2 is as
potent as TGF-
1 or TGF-
3 in inducing cellular responses, yet
TGF-
2 does not bind to the majority of expressed type II
receptors. Here we characterized the properties of the soluble
extracellular domain of the human TGF-
type II receptor
synthesized in COS-7 cells. Like the membrane-attached type II
receptor, the soluble receptor contains complex N-linked
oligosaccharides as well as additional sialic acid residues that cause
it to migrate heterogenously upon SDS-polyacrylamide gel
electrophoresis.
I-TGF-
1 binds to and is chemically
cross-linked to this protein. Unlabeled TGF-
1 inhibits the binding
of
I-TGF-
1 with an apparent dissociation constant (K
) of
200 pM, similar to
the apparent K
(
50 pM) of
the cell-surface type II receptor. TGF-
3 inhibits the binding of
I-TGF-
1 to the soluble type II receptor with a
similar dissociation constant,
500 pM. In contrast,
I-TGF-
2 cannot bind and be chemically cross-linked
to the soluble type II receptor, nor does as much as a 125-fold excess
of unlabeled TGF-
2 inhibit the binding of
I-TGF-
1 to the soluble receptor. This is the first
demonstration of the binding affinities of the type II receptor in the
absence of the other cell-surface molecules known to bind TGF-
.
Expressed alone in COS-7 cells the type II receptor also cannot bind
TGF-
2; co-expression of type III receptor enables the type II
receptor to bind TGF-
2. Thus, the type III receptor or some other
component is required for transmission of TGF-
2-induced signals by
the type II receptor.
The transforming growth factor (TGF)-
family of
hormones has important functions in growth, development, and
differentiation(1, 2) . The three mammalian TGF-
isoforms, TGF-
1, -
2, and -
3, share 71-76% amino
acid identities. Although homodimers are the predominant species, the
heterodimers TGF-
1.2 and TGF-
2.3 have been
described(3, 4) . 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.
Most mammalian cells express three abundant high
affinity receptors which can bind and be cross-linked to TGF-: 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(5, 6) . While TGF-
1 binds with
high affinity (50-300 pM) to the types I, II, and III
receptors, TGF-
2 binds with high affinity only to the type III
receptor, binding poorly to the majority of the type I and II receptors
expressed on most mammalian cells. TGF-
3 closely resembles
TGF-
1 in its binding characteristics to cell surface receptors,
although TGF-
3 may bind slightly less well than TGF-
1 to the
type I and II receptors.
The TGF- type III receptor is a
membrane bound proteoglycan with a short cytoplasmic tail that has no
apparent signaling motif(7, 8) . It binds TGF-
2
(apparent K
100 pM) with
slightly greater affinity than TGF-
1 or TGF-
3 (apparent K
300
pM)(9, 10, 11) . The type II
receptor is a type I transmembrane protein with a cytosolic domain
containing a serine/threonine kinase homologous to that in the activin
and several other
receptors(6, 12, 13, 14) . The type
II receptor is essential for all known TGF-
initiated
signals(15, 16, 17) . The type II and type
III receptors interact, as demonstrated by the enhanced ability of the
type II receptor to bind
I-TGF-
1 when co-expressed
in the presence of the type III receptor(8) . After
cross-linking to
I-TGF-
1 or TGF-
2, a fraction
of the type II and type III receptors can be co-immunoprecipitated with
antisera specific to either receptor type(9, 10) .
Homo-oligomers, probably homodimers, of the types II and III 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
minor and probably transient species, most likely intermediates in the
transfer of a TGF-
ligand from a type III to a type II
receptor(18) .
The type I receptors for TGF-s and
activin-A are also transmembrane serine-threonine kinases. The type II
receptors require their corresponding type I receptors for signaling,
while binding of TGF-
s or activin-A to the respective type I
receptors requires co-expression of the corresponding type II receptor.
Heteromeric complexes of the type II with type I receptors are found on
the surface of many cells after ligand binding and may be important for
signal
transduction(15, 19, 20, 21, 22, 23, 24, 25, 26) .
If the type I and II receptors indeed mediate TGF--induced
signals, then it is puzzling that TGF-
2 is equally potent as
TGF-
1 and TGF-
3 in its ability to arrest the growth of cells
(ED
5-20
pM)(27, 28) , since TGF-
2 does not bind
well to either the type I or the type II receptors. One possibility is
that there exists a subpopulation of type I and II receptors which
binds TGF-
2 with high affinity (apparent K
25-50 pM)(29) . Another
possibility is that the type III receptor, which binds TGF-
2 with
relatively high affinity (apparent K
100 pM), could present TGF-
2 to the type
I and/or type II receptors, increasing their affinity for TGF-
2.
Alternatively, another as yet undescribed cell surface component may
interact with the type I and/or the type II receptors and increase
their affinity for TGF-
2. Finally, TGF-
2 may bind to an
undescribed receptor which cannot be chemically cross-linked to
I-TGF-
2 and therefore has escaped identification.
However, 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 potently growth inhibited by TGF-
2(15) .
To
study the binding properties of the type II receptor in isolation, we
constructed a truncated TGF- type II receptor cDNA which encodes
the entire extracellular domain. Transfected COS-7 cells secreted the
soluble receptor, a heterogenously glycosylated protein which binds to
TGF-
1 and TGF-
3 with high affinity (estimated K
200 pM and
500
pM, respectively). In contrast, as much as 10 nM unlabeled TGF-
2 could not inhibit binding of
I-TGF-
1, nor could
I-TGF-
2 be
bound and chemically cross-linked to the soluble type II receptor.
These results are consistent with the ability of TGF-
1 and
TGF-
3 to bind directly to and signal through the high affinity
type II receptor and suggest that at least one additional component is
essential for binding and/or signal transduction by TGF-
2. This
additional component may be the type III receptor, as we show that
co-expression of the type III receptor in COS-7 cells enables the cell
surface type II receptor to bind to TGF-
2.
Figure 1:
Strategy
for construction of a truncated cDNA encoding the extracellular domain
of the human TGF- type II receptor (hTGF-
RII).
Synthetic PCR primers (half-arrows, A and B)
(see ``Materials and Methods'') were used to amplify a
portion of the human TGF-
RII extracellular domain from the
full-length cDNA clone, H2-3FF(12) . The amplified PCR product
was digested with the restriction enzymes PstI and XbaI, and the digested fragment was ligated into the PstI site (bp 545-550) in the full-length cDNA clone and
the XbaI site in the 3` poly-linker region of the vector
(pcDNA-I, InVitrogen). The resulting truncated cDNA encoded a protein
representing the entire extracellular domain of the human TGF-
type II receptor. The last encoded residue of this soluble TGF-
type II receptor (sTGF-
RII) is Asp
. Signal,
hydrophobic signal sequence; TM, transmembrane domain; NH
, N terminus; COOH, C terminus; UT, untranslated region. (Drawings are not to
scale.)
Indicated samples were treated with glycopeptidase F (New England Biolabs) by adding 2000 units of enzyme/50 µl of reaction volume in a buffer containing (final concentrations) 0.5% SDS, 1% Nonidet P-40, and 50 mM sodium phosphate, pH 7.5. The samples were incubated at 37 °C overnight. Samples treated with neuraminidase were incubated in glycopeptidase F reaction buffer in the presence of 0.05 unit of neuraminidase (Genzyme) for 1 h at 37 °C, at which point glycopeptidase F was added to indicated samples, and the incubation was continued overnight. Samples were brought up in SDS-PAGE sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and 100 µg/ml bromphenol blue) and analyzed on 15% polyacrylamide, 1.2% bisacrylamide gels.
Figure 8:
Cross-linking of I-TGF-
2 to COS-7 cells co-transfected with the type
II and III receptors. COS-7 cells were transfected with the type II
receptor (lanes 1-4), the type II and type III receptors (lanes 5-7), or the type III receptor alone (lanes
8-12). Forty-eight hours post-transfection, cells were
incubated with the following concentrations of
I-TGF-
2: 50 pM, lanes 1, 5,
AND 8; 100 PM, lane 9; 200 PM, lanes 2, 6, AND 10; 500 PM, lanes 3, 7, AND 11; 1.0 NM, lanes 4AND 12. AFTER CHEMICAL CROSS-LINKING WITH
DSS, THE SAMPLES WERE SOLUBILIZED AND ANALYZED BY SDS-PAGE. THE TYPE II
RECEPTOR, TYPE III RECEPTOR,
I-TGF-
2, AND MOLECULAR
MASS MARKERS ARE INDICATED ON THE left. UNCHARACTERIZED
SMALLER MOLECULAR MASS SPECIES (U) ARE INDICATED BY THE brackets.
Figure 2:
Metabolic labeling of soluble TGF-
type II receptor expressed in COS cells. COS cells were transfected
with pcDNA-1 expressing either the truncated type II receptor (amino
acids 1-159, lanes 1 and 2) or the full-length
type II receptor cDNA (amino acids 1-567, lanes 3 and 4). Forty-eight hours after transfection, cells were labeled
with 0.5 mCi/ml of a mixture of [
S]cysteine and
[
S]methionine as described under
``Materials and Methods.'' Labeled media were
immunoprecipitated with an antipeptide antibody specific for the
N-terminal extracellular domain of the human type II receptor in the
absence(-) or presence (+) of equimolar concentrations of
immunogenic peptide (Peptide). After an overnight incubation
at 4 °C, immunoprecipitated samples were analyzed by SDS-PAGE on a
11% gel, which was subjected to fluorography. The bracket shows the immunoprecipitated heterogeneous 25-35-kDa soluble
type II receptor (lane 2), which is specifically competed by
the presence of the immunogenic peptide in the immunoprecipitation
reaction (lane 1). The arrow shows a nonspecific
background protein species of
35 kDa which is present even in
control samples (lanes 3 and 4) and is not competed
by immunogenic peptide. The arrowhead shows two protein
species of
46 kDa that are present only in control media, and only
when immunogenic peptide is included in the immunoprecipitation
reaction. Thus, these two are also background
species.
The
antipeptide antibodies we are using are highly specific. -IIN, but
not
-IIC, specific for the C terminus of the full-length type II
receptor, precipitates the soluble receptor secreted by COS cells (Fig. 3, lanes 7 and 11); neither antibody
immunoprecipitates material corresponding to the soluble form of the
type II receptor from mock-transfected cells (lanes
1-6).
-IIN (Fig. 3, lanes 13-16),
but not
-IIC (data not shown), immunoprecipitates the soluble type
II receptor synthesized in a cell-free system in the presence of
microsomes.
Figure 3:
Deglycosylation of soluble TGF- type
II receptor metabolically labeled in transfected cells and synthesized
in a cell-free system. COS cells were transfected with pcDNA-1 alone (Mock, lanes 1-6) or pcDNA-1 expressing the
truncated type II receptor (amino acid residues 1-159, lanes
7-12). As described under ``Materials and
Methods,'' cells were labeled for 4 h with 0.5 mCi/ml of a mixture
of [
S]cysteine and
[
S]methionine. Medium from the labeled cells was
immunoprecipitated with antipeptide antibodies specific for either the
N-terminal extracellular domain (
-II N; lanes
1-4 and 7-10) or the C-terminal intracellular
domain (
-II C; lanes 5, 6, 11,
and 12). Immunoprecipitated samples were deglycosylated with
glycopeptidase F alone (lanes 3, 6, 9, and 12), neuraminidase alone (lanes 2 and 8), or
both (lanes 4 and 10), and then resolved by SDS-PAGE
on a 15% polyacrylamide, 1.2% bisacrylamide gel. In vitro synthesized mRNA encoding the soluble receptor (lanes 13 and 14) or water (lane 15 and 16)
underwent in vitro translation in the presence of microsomes;
microsomal fractions were immunoprecipitated with the
-II N
antibody. The samples in lanes 14 and 16 were treated
with glycopeptidase F. Lanes 13-16 are from a longer
exposure of the same gel as for lanes 1-12. The line (lane 7) indicates the smear representing the mature,
complex glycosylated secreted form of the soluble receptor, which is
reduced in size slightly by neuraminidase treatment (lane 8);
the triangle between lanes 12 and 13 indicates a background protein of
32 kDa seen in
mock-transfected samples. The small arrow in lane 7 shows a minor form of the soluble type II receptor with complex
oligosaccharides. The small arrowhead in lane 9 represents a neuraminidase-sensitive form of the soluble receptor
(most likely O-glycosylated) which is formed by digestion with
glycopeptidase F; the large arrowheads in lanes 10 and 14 show the core receptor without signal peptide or
carbohydrate; it is seen in media treated with neuraminidase and
glycopeptidase F (lanes 9 and 10) and in the in
vitro translation product treated with glycopeptidase F (lane
14). The large arrow represents a form of the receptor
containing high mannose N-linked oligosaccharides that is
generated in the in vitro translation reaction containing
microsomes. The size (in kilodaltons) of molecular mass markers is
noted in the left-hand margin.
Fig. 3shows that the soluble form of the type II
receptor is heterogenously glycosylated, just as is the full-length
type II receptor. ()In vitro translation of mRNA
encoding the soluble type II receptor in a cell-free system containing
microsomes generated a single predominant species of molecular size
28 kDa (lane 13). Potentially this species contains three N-linked oligosaccharides; digestion with glycopeptidase F (lane 14) or endoglycosidase H (data not shown) generated a
single species of molecular size
19 kDa, corresponding to the
soluble type II receptor without signal peptide or carbohydrate.
Treatment of the heterogeneous form of the soluble type II receptor
secreted by COS cells (lane 7) with glycopeptidase F yields
the same
19-kDa species (large arrowhead) as well as a
slower migrating protein (small arrowhead, lane 9).
This latter species contains some form of sialic acid since digestion
with glycopeptidase F together with neuraminidase yields only the
19-kDa species (lane 10). Clearly the secreted soluble
type II receptor contains much sialic acid, since digestion with
neuraminidase alone (lane 8) reduces the average apparent
molecular mass of the heterogeneous protein by
5 kDa. Possibly the
secreted soluble type II receptor contains sialic acid attached to O- as well as N-linked oligosaccharides. However, no
change in migration of the secreted soluble type II receptor was seen
following digestion with O-glycanase, whether or not the
samples were first treated with glycopeptidase F and/or neuraminidase
(data not shown). Thus, there appear to be two major classes of the
secreted soluble type II receptor. One has a heterogeneous set of N-linked oligosaccharides that contain sialic acid, and that
is converted to the 19-kDa ``core'' by digestion with
glycopeptidase F. The other contains this heterogeneous set of N-linked oligosaccharides and also has additional sialic acids
that are found either in O-linked oligosaccharides that are
resistant to O-glycanase or in some unusual type of N-linked oligosaccharide. A similar, complex, pattern of
glycosylation was seen for the full-length type II receptor. (
)
Figure 4:
Chemical cross-linking of the soluble
TGF- type II receptor to
I-TGF-
1. COS-7 cells
were transfected with pcDNA-1 containing either the truncated type II
receptor (lanes 1-3) or the full-length type II receptor (lanes 4-6). Forty-eight hours after transfection, cells
were rinsed twice with PBS and incubated overnight in serum-free medium
(4 ml of Dulbecco's modified Eagle's medium/100-mm tissue
culture dish). Conditioned media were collected and incubated with 200
pM
I-TGF-
1 overnight at 4 °C with
rotation before samples were chemically cross-linked with DSS for 15
min at 4 °C. The samples were then divided into aliquots and
subjected to immunoprecipitation overnight at 4 °C with an
antipeptide antibody specific for the N-terminal extracellular domain
of the human type II receptor in the absence (-, lanes 2 and 5) or presence (+, lanes 1 and 4) of an equimolar concentration of immunogenic peptide (Peptide). Equivalent aliquots were allowed to incubate with
concanavalin A-Sepharose (Con A +, lanes 3 and 6) overnight at 4 °C. Immunoprecipitates and concanavalin
A-bound proteins were analyzed by SDS-PAGE on a 10% gel, which was
dried and exposed to pre-flashed XAR-5 film. The bracket shows the
soluble type II receptor chemically cross-linked to
I-TGF-
1, which migrates as a heterogeneous
37-46-kDa species (lane 2). The arrows point to
additional protein species in the concanavalin A sample (lane
3). Since these species are also present in media from control
transfected COS-7 cells (lane 6), they are likely to be
background species.
Figure 5:
Cross-linking of the soluble TGF-
type II receptor to
I-TGF-
1 or
I-TGF-
2. COS-7 cells were transfected with pcDNA-1
expressing the truncated type II receptor. Forty-eight hours after
transfection, cells were rinsed twice with PBS and incubated overnight
in serum-free medium (5 ml of Dulbecco's modified Eagle's
medium/100-mm tissue culture dish). Conditioned media were collected
and incubated overnight with 100 pM
I-TGF-
1
in the absence (lanes 1 and 2) or presence of 1
nM TGF-
1 (lane 3,
1), 10 nM TGF-
2 (lane 4,
2), or 1 nM TGF-
3 (lane 5,
3). An equivalent
aliquot was incubated with 500 pM
I-TGF-
2 (lane 6). After chemical cross-linking with DSS for 15 min at
4 °C, the samples were subjected to immunoprecipitation overnight
at 4 °C with an antipeptide antibody specific for the N-terminal
extracellular domain of the human type II receptor in the absence (lanes 2-6) or presence (lane 1, P) of
equimolar concentrations of immunogenic peptide. Immunoprecipitated
samples were analyzed by SDS-PAGE on a 10% gel, which was dried and
exposed to pre-flashed XAR-5 film.
The isoform specificity of the soluble receptor is remarkably
similar to that of cell-surface type II receptors expressed in
transfected H216 cells (compare lanes 2 through 5 of Fig. 5and 6). This is a cell line derived from the stable
transfection of the full-length human type II receptor into SW480 colon
adenocarcinoma cells (see ``Materials and Methods''), which
express very low levels of the type II receptor(36) . H216
cells, like SW480, express little if any type III receptor (Fig. 6, lane 2). Note that expression of the type II
receptor in SW480 cells also leads to an increase in the amount of cell
surface type I receptor (Fig. 6, lanes 1 and 2). The ability of the different TGF- isoforms to inhibit
binding of
I-TGF-
1 to the cell surface type I
receptor is the same as for the type II receptor (Fig. 6, lanes 2-5). In particular TGF-
2 does not inhibit
binding of TGF-
1 to either the type II or type I receptor,
indicating that it cannot bind directly to the type II (or type I)
receptor.
Figure 6:
Cross-linking of I-TGF-
1 to SW480 and H216 cells and competition by
unlabeled TGF-
isoforms. SW480 cells (lane 1) and H216
cells (lanes 2-5) were grown on 60-mm tissue culture
dishes until confluent and allowed to bind 100 pM
I-TGF-
1 AT 4 °C IN THE ABSENCE (lanes 1AND 2) OR PRESENCE OF UNLABELED 1 NMTGF-
1 (lane 3,
1), 5 NMTGF-
2 (lane
4,
2), OR 1 NMTGF-
3 (lane 5,
3). CELLS WERE CHEMICALLY CROSS-LINKED WITH 60 µG/ML
DSS BEFORE LYSIS WITH BUFFER CONTAINING DETERGENT AND PROTEASE
INHIBITORS. CELL LYSATES WERE ANALYZED BY SDS-PAGE ON AN 8%GEL, WHICH
WAS DRIED AND EXPOSED TO PRE-FLASHED XAR-5 FILM. THE TYPE II RECEPTOR
IS INDICATED AS A HETEROGENEOUS 90-110-KDA SPECIES (lane
2) WHICH IS NOT ABUNDANT IN SW480 CELLS (lane 1). THE
TGF-
TYPE I RECEPTOR IS INDICATED AS A
69-KDA
SPECIES.
Figure 7:
Cross-linking of the soluble type II
TGF- receptor to
I-TGF-
1 and competition by
unlabeled TGF-
isoforms. A, aliquots of medium from COS-7
cells transfected with pcDNA-1 encoding the soluble type II receptor
were incubated with
I-TGF-
1 (80 pM) in the
absence (lanes 1-3) or presence of increasing amounts of
unlabeled TGF-
1 (lanes 4-8), -
2 (lanes
9-13), or -
3 (lanes 14-18), as
indicated. After chemical cross-linking with DSS, the samples were
subjected to immunoprecipitation with an antipeptide antibody specific
for the N-terminal extracellular region of the human type II receptor (lanes 2-18) in the absence (lanes 3-18)
or presence (lane 2) of equimolar amounts of the immunizing
N-terminal peptide as competitor. For the sample in lane 1,
the antipeptide serum specific for the C terminus of the type II
receptor was used; as expected, no soluble receptor was
immunoprecipitated. B, the autoradiogram was scanned and
densitometric values were normalized to 100 for the highest values. Circles, TGF-
1; squares, TGF-
2; triangles, TGF-
3. The 100 pM values were not
plotted.
It is difficult to determine the binding affinity and
specificity of type II TGF- receptors expressed on the plasma
membrane, since there are no cells that express cell-surface TGF-
type II receptors without either the type I or III receptors. Nor can
we use cell lines to determine whether homodimers of the type II
receptor, heterodimers of type I and II or of type II and III
receptors, or multimers with other receptors, are involved in TGF-
binding and signaling. To this end, we have generated a cDNA encoding a
soluble, secreted form of the human TGF-
type II receptor
containing the entire exoplasmic domain and have expressed it in
transfected COS-7 cells (Fig. 2).
This species migrates
heterogenously on SDS-PAGE because it is heterogenously glycosylated.
Most of the soluble, secreted receptors contain only a heterogeneous
set of N-linked oligosaccharides; these are converted by
digestion with glycopeptidase F to a 19-kDa core species that
lacks the signal peptide and any apparent carbohydrate. The remainder
of the soluble receptor contains these heterogeneous N-linked
oligosaccharides as well as additional sialic acid residues that are
either found in O-linked oligosaccharides that are resistant
to O-glycanase or in some unusual type of N-linked
oligosaccharide; conversion of this group of soluble receptors to the
19-kDa core species requires digestion both with glycopeptidase F
and neuraminidase (Fig. 3).
Our most important result is that
this soluble, secreted form of the human TGF- type II receptor
shows selectivity for TGF-
ligands. It binds to and can be
cross-linked to
I-TGF-
1 (Fig. 4). The
apparent affinities of the soluble form of the receptor for TGF-
1
and -
3 are
200 and
500 pM, respectively, and
TGF-
2 does not bind to the soluble type II receptor (K
> 10 nM) ( Fig. 5and Fig. 7). These properties are qualitatively similar to those of
the human TGF-
type II receptor expressed on the surface of H216
cells (Fig. 6). This is a line of transfected SW480 cells that
express type I and II receptors, but little or no type III receptors.
Similar results were obtained in mink lung epithelial
cells(2) , where the cell surface type II receptor binds
TGF-
1 and TGF-
3 with high affinity (K
25 and
50 pM, respectively) and TGF-
2
with a 10-fold lower affinity. While we can detect no cross-linking of
I-TGF-
2 to the soluble type II receptor (Fig. 5), we can detect a small amount of cross-linking to
cell-surface type II TGF-
receptors in transfected SW480 (H216)
cells (not shown); most likely this is due to a small amount of the
type III receptor or other TGF-
-binding protein expressed in these
cells.
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 which 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 also restores
binding to cell surface type I receptors(15, 16) .
Thus, either the type I receptor in these cells cannot bind TGF-
1
in the absence of the type II receptor or cannot accumulate on the
plasma membrane. 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 co-expressed
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
co-expressed 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 I and II receptors are apparently able to
transduce TGF-
1 or activin signals (19, 20, 21, 22, 23, 24, 25, 26) .
Several cell lines lack the type III receptor, such as fetal bovine
heart endothelial (FBHE) cells (37) and L6
myoblasts(8, 10, 38) , yet express cell
surface type I and II receptors that can bind TGF-1. Thus,
expression of the type III receptor is not necessary for the type II
receptor to bind TGF-
1 in these cells. The type II receptor is
also likely to be the primary binding subunit for TGF-
3, since
unlabeled TGF-
3 can inhibit the binding of
I-TGF-
1 to both the soluble ( Fig. 5and Fig. 7) and cell surface (Fig. 6) type II receptors; a
caveat is that we have not performed binding studies using
I-TGF-
3. Most likely the binding sites on the type
II receptor for TGF-
3 and TGF-
1 overlap.
Our results
suggest that the type II receptor is not the primary binding subunit
for TGF-2. This notion is consistent with the observation that
some cells, such as FBHE cells, which express the type I and II
receptors but lack type III receptors, can bind and respond to
TGF-
1, but not to TGF-
2(37) . Interestingly, these
cells also do not express a subset of type I and type II receptors
which can bind TGF-
2 with high affinity, suggesting that some
other subunit is necessary for the type I and II receptors on these
cells to interact with TGF-
2.
The primary binding subunit for
TGF-2 could be the type III receptor. Several observations support
this notion. First, the type III receptor can bind all three TGF-
isoforms with relatively high affinities (K
100-500 pM). Second, soluble secreted forms of the type
III receptor bind all three TGF-
isoforms with a similar high
affinity (K
500 pM), demonstrating
that the type III receptor alone has the ability to bind
TGF-
2(39) . Finally, there is accumulating evidence that
the type III receptor may interact with a subset of the type II (and
possibly the type I) receptors. For example, expression of the type III
receptor in L6 myoblasts enhances the amount of cell-surface type II
receptor bound and cross-linked to
I-TGF-
1 (8) . More recently, López-Casillas et al.(10) showed that in L6 cells expression of the
type III receptor leads to increased binding of TGF-
2 to the type
II receptor. Here we showed that in COS-7 cells, co-transfection of the
type III receptors with type II receptors enabled the type II receptor
to bind TGF-
2 (Fig. 8).
While our study indicates that
the type II receptor is the primary binding subunit for TGF-1 and
TGF-
3, the soluble type II receptor has
10-fold lower binding
affinity for TGF-
1 or TGF-
3 than does the cell-surface type
II receptor. The soluble type II receptor is monomeric, as judged by
the sedimentation velocity of
S-labeled protein in sucrose
density gradients.
In contrast, the type II receptors found
on the cell surface are primarily homo-oligomers, probably
homodimers(18) , and the TGF-
ligand is a disulfide-linked
dimer. As revealed by x-ray crystallography, TGF-
2 has a novel
monomer fold and dimer association(40, 41) ; a very
similar structure was proposed for TGF-
1 on the basis of
heteronuclear NMR studies(42) . The symmetry of the dimer
suggests that 1 molecule of TGF-
2, and presumably of the other
TGF-
isoforms, could interact simultaneously with two cell-surface
receptor polypeptides. Thus, each of the two receptor subunits in a
homodimer could bind one TGF-
monomer, increasing the binding
energy and thus lowering the dissociation constant for binding of
TGF-
.
The type II receptor may also interact with other
proteins in order to generate a higher affinity receptor complex. Two
likely candidates that may interact with the type II receptor to form
such high affinity complexes are the receptor types I and III. However,
there are many other TGF- binding proteins on the cell surface
besides the types I, II, and III receptors(6) . These other
proteins may also help to stabilize the binding of TGF-
ligands to
the type I and II receptors that generate the intracellular signals.
Soluble forms of these receptors could be used to directly study their
binding properties and their interactions with other receptor subunits.