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Communication
Vol.
267,
No.
27,
Issue
of
September 25, pp. 19027-19030,1992
THE JOURNAL
OF
BIOLOGICAL CHEMISTRY
0
1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed
in
U.S.A.
Endoglin
Is
a
Component
of
the
Transforming Growth Factor-@
Receptor System in Human
Endothelial Cells*
(Received for publication, May 18, 1992)
Sela CheifetzS, Teresa BellonQ, Carmela CalesQ,
Sonia Vera(, Carmelo BernabeuQ,
Joan
Massague$II**, and Michelle Letartell$$
From the $Cell Biology and Genetics Program
and
I(
Howard Hughes Medical Institute, Memorial Sloan-
Kettering Cancer Center, New
York,
New York
10021,
CCentro de Investigaciones Bioldgicas, Consejo Superior de
Inuestigaciones Cientificas,
28006
Madrid, Spain, and
the
TDivision
of
Immunology and Cancer Research,
The
Hospital
for
Sick Children and the University of Toronto,
Toronto, M5G
LX8
Ontario, Canada
Endoglin, a dimeric membrane glycoprotein ex-
pressed at high levels on human vascular endothelial
cells, shares regions
of
sequence identity with beta-
glycan, a major binding protein for transforming
growth factor-j3 (TGF-#I) that co-exists with TGF-j3
receptors I and I1 in
a
variety of cell lines but
is
low or
absent in endothelial cells.
We
have examined whether
endoglin also binds TGF-j3 and demonstrate here that
the major TGF-81-binding protein co-existing with
TGF-j3 receptors I and I1 on human umbilical vein
endothelial cells is endoglin,
as
determined by specific
immunoprecipitation of endoglin affinity-labeled with
12’I-TGF-j3. Furthermore, endoglin ectopically ex-
pressed in
COS
cells binds TGF-j31. Competition affin-
ity-labeling experiments showed that endoglin binds
TGF-B1
(KO
-
50
p~)
and TGF-j33 with high affinity
but fails to bind TGF-DS. This difference in affinity of
endoglin for the TGF-j3 isoforms is in contrast to beta-
glycan which recognizes all three isoforms. TGF-j3
however is binding with high affinity to only a small
fraction of the available endoglin molecules, suggest-
ing that some rate-limiting event is required to sustain
TGF-j3 binding to endoglin.
Endoglin is
a
homodimeric membrane glycoprotein com-
posed of disulfide-linked subunits
of
95 kDa
(1,
2). It is
expressed on human pre-erythroblasts, macrophages, leu-
kemic cells
of
the lymphoid and myeloid lineages, and at
higher levels on syncytiotrophoblasts of term placenta and
vascular endothelial cells
(1,
3-5).
A
relationship between
*
This work was supported by grants from the National Institutes
of
Health (to
J.
M.), Medical Research Council-Canada (to M.
L.),
National Cancer Institute-Canada
(to
M.
L.),
and CICYT-Spain (to
C. B.). The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with
18
U.S.C. Section 1734
solely to indicate this fact.
**
Howard Hughes Medical Institute Investigator. To whom cor-
respondence should be addressed: Cell Biology and Genetics Program,
Box 116, Memorial Sloan-Kettering Cancer Center, 1275 York Ave.,
New York,
NY
10021.
$$
Terry Fox Scientist of the National Cancer Institute-Canada.
Endoglin
Betaglycan
N
c
FIG.
1.
Domain structures of betaglycan and endoglin.
Sche-
matic representation highlighting regions of similarity between the
linear sequences of betaglycan, an 853-amino acid transmembrane
proteoglycan
(6,
7),
and endoglin, a disulfide-linked transmembrane
protein composed of subunits
of
633 amino acids
(1).
The transmem-
brane and short cytoplasmic regions (dark shaded
box)
of endoglin
have
a
high level
of
sequence similarity to the corresponding regions
of betaglycan. Two regions of weaker similarity are detected in the
ectodomains of these proteins (light shaded boxes). Numbers represent
the percent amino acid sequence similarity between the indicated
domains of betaglycan and endoglin. Closed ovals represent position
of
cysteine residues. Two putative sites for glycosaminoglycan chain
attachment in betaglycan (6) are indicated.
human endoglin and TGF-0’ receptor system was discovered
with the molecular cloning of the rat TGF-@-binding proteo-
glycan, betaglycan (also known as the type
I11
TGF-P recep-
tor), which revealed that the transmembrane domain and the
relatively short (43 amino acids) cytoplasmic tail
of
this
protein (6, 7) were remarkably similar
(71%
amino acid se-
quence similarity with 63% identity) to the corresponding
regions in endoglin
(1)
(Fig.
1).
The extracellular domains of
these two proteins show limited amino acid sequence homol-
ogy
(1, 6-8), and although endoglin contains O-linked oligo-
saccharides, it is not a proteoglycan (9). Endoglin contains an
RGD sequence and
so
is potentially involved in RGD-me-
diated cellular adhesion (5), whereas betaglycan does not
contain this sequence.
In addition to betaglycan, the TGF-P receptor system (10)
in most mesenchymal and epithelial cells consists of the type
I receptor,
a
53-kDa glycoprotein whose structure has not
been determined yet (lo), and the type
I1
receptor
(11),
which
belongs to the protein serinelthreonine kinase receptor family
(10-14).
Additional cell surface TGF-@-binding proteins, some
of which have a more restricted distribution, have also been
described (15-17). In particular, endothelial cells which ex-
press the TGF-@ receptors
I
and
I1
but have little or
no
betaglycan (17-20) have been shown to express a disulfide-
linked protein dimer
of
95-kDa subunits, which binds
TGF-
Pl
but not TGF-@2
(17).
The size and restricted distribution
of
this TGF-@-binding protein is remarkably similar to that
of endoglin. These observations in conjunction with the struc-
The abbreviations used are: TGF-0, transforming growth factor-
p;
DTT, dithiothreitol; HUVEC, human umbilical cord endothelial
cell(s); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis; mAb, monoclonal antibody.
19027
19028
Endoglin Binds
TGF-p
tural relationship between endoglin and betaglycan prompted
an examination of whether endoglin binds TGF-/3. We have
used monoclonal antibodies specific to human endoglin and
an endoglin expression vector to demonstrate that endoglin
is
a
major TGF-p-binding protein in human vascular endo-
thelial cells.
EXPERIMENTAL PROCEDURES
Cell Culture
and
Transfections-Human umbilical vein endothelial
cells (HUVEC, CRL 1730, ATCC) were maintained in &-minimal
essential media supplemented according to supplier's instructions or
prepared from umbilical veins as previously described (2). Similar
results were obtained using cells from either source. COS-M6 cells,
maintained in Dulbecco's modified Eagle's medium supplemented
with 10% bovine serum, were transfected with a cDNA encoding full-
length endoglin'
(1)
ligated into the EcoRI site of the mammalian
expression vector pcEXV (21), or with a control vector without cDNA
insert (pcMV5; Ref. 6) by the DEAE-dextran-chloroquine procedure
(22). 24 h post-transfection, cells were trypsinized and reseeded into
multicluster dishes and allowed to grow an additional 48 h before
being affinity-labeled with 1251-TGF-/31
as
described below.
Receptor Affinity Labeling
and
Immunoprecipitations-TGF-01
and TGF-j32 were purchased from R
&
D Systems (Minneapolis,
MN), and TGF-03 was obtained from Oncogene Science. "'1-TGF-
e1
used in these studies was prepared by the chloramine-?' method
as
previously described (23) or purchased from Amersham Corp.; both
preparations gave identical results. The conditions for affinity label-
ing cell monolayers with '251-TGF-f11 and disuccinimidyl suberate
(Pierce Chemical Co.) have been described previously (24). The
concentrations of lZ5I-TGF-fll and competing unlabeled ligands used
for each experiment are indicated in the figure legends. Triton X-100
extracts of the affinity-labeled cells were either analyzed directly on
sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) or first
incubated with monoclonal antibody (mAb) 44G4 directed against
human endoglin (25) or with control antibody (see below). For im-
munoprecipitations, detergent extracts were diluted with an equal
volume of phosphate-buffered saline containing 1% Triton X-100 and
precleared by incubation for 20 min at 4 "C with protein G-Sepharose
(Pharmacia LKB Biotechnology Inc.) prior to overnight incubation
at
4 "C with mAb 44G4. Immune complexes were collected by incu-
bation with protein G-Sepharose for
1
h at 4 "C. For some experi-
ments, mAb 44G4 was used coupled to Sepharose (25). The immu-
noprecipitate? were washed three times (saline with 1% Triton X-
100) and then resolved by SDS-PAGE in the presence or absence of
dithiothreitol (DTT) and visualized by autoradiography. Irrelevant
mAb (44D7) (25) used in control experiments to monitor specificity
of
the immunoprecipitations did not immunoprecipitate any affinity-
labeled bands (data not shown).
RESULTS
Analysis of the affinity-labeled profile of HUVEC revealed
that like vascular endothelial cells from other sources, these
cells have little or no betaglycan, which characteristically
migrates as a diffuse band between 200 and 400 kDa on
reducing SDS-PAGE (Fig.
24).
Instead, HUVEC expressed
a
disulfide-linked cell surface protein that, together with TGF-
/3
receptors
I
and 11, was affinity-labeled by cross-linking with
'"I-TGF-@l. Receptors I and I1 were detected in HUVEC as
labeled complexes of approximately 65 and 100 kDa, which is
similar to the size of these labeled receptors reported for other
human cell lines (26). The disulfide-linked TGF-pl-binding
protein migrated
as
a
labeled complex
at
180 kDa and above
on nonreducing SDS-PAGE gels while on reducing gels it
migrated as
a
labeled 110-kDa species that overlapped the
labeled type I1 receptor band (Fig.
2A).
Resolution of these disulfide-linked TGF-pl-binding pro-
teins on two-dimensional gels (Fig. 2B) confirmed that the
disulfide-linked complexes (probably dimers and higher order
oligomers) contained subunits of approximately 95 kDa (value
estimated by subtracting the cross-linked TGF-p1 monomer
'
T.
Bell6n and C. Bernabeu, manuscript in preparation.
A
.DTT
[
R
NR
C
q-
%MI
MrW)
-P
-180
200-
RII
-
*[
-105
-100
'00
-
97.4-
.
*.
RI
-
-
70
69
-
46
-
4
FIG.
2. Cell
surface TGF-f31-binding proteins expressed by
HUVEC.
Near confluent cultures of HUVECs were affinity-labeled
by incubation with 100 p~ '2sI-TGF-f11 alone or in the presence of 4
nM unlabeled TGF-fl1 followed by chemical cross-linking with 0.16
mM disuccinimidyl suberate. A, Triton X-100 extracts
of
affinity-
labeled HUVEC were resolved on SDS-PAGE gels under reducing
(R)
or nonreducing
(NR)
conditions.
Lane
C contains extract from
cells affinity-labeled in presence of excess unlabeled TGF-01. The
migration position of TGF-0 receptors
I
(RI)
and
I1
(RII)
are indi-
cated. Arrow, the major affinity-labeled proteins of 180 kDa and
higher molecular mass apparent on nonreducing gels. Arrowhead, the
affinity-labeled proteins of 110-120 kDa seen on reducing gels.
B,
detergent extracts of affinity-labeled HUVEC were resolved under
nonreducing conditions on
a
first gel that was then resolved under
reducing conditions in the second dimension as previously described
(17). The 110-120-kDa labeled species migrating off-the-diagonal are
indicated (arrowheads).
mass, 12.5 kDa from the reduced 110-kDa affinity-labeled
complex). Together with the type I1 receptor, the disulfide-
linked TGF-61-binding proteins are the major affinity-labeled
species expressed by HUVEC.
Given:
1)
the similarity in subunit composition between
this TGF-@-binding protein and endoglin, 2) the presence of
endoglin on endothelial cells, and
3)
the structural relation-
ship between endoglin and betaglycan, we sought to determine
whether this disulfide-linked TGF-/3-binding protein on endo-
thelial cells is indeed endoglin. To this end, affinity-labeled
HUVEC extracts were immunoprecipitated with monoclonal
antibody 4464, which is specific for human endoglin
(1,
2,
25). Electrophoretic analysis of these immunoprecipitates re-
vealed
a
labeled protein complex whose subunit structure was
similar to that of endoglin (Fig.
3A).
Thus under reducing
conditions, a major affinity-labeled band of approximately
110 kDa was seen which migrated as complexes of 180 kDa
and greater than
200
kDa when analyzed under nonreducing
conditions. The higher order oligomers might contain multiple
endoglin molecules cross-linked by TGF-p1, itself
a
disulfide-
linked dimer (27). Repeated immunoprecipitation with 4464-
IgG-Sepharose completely depleted these labeled species from
cell extracts (Fig.
3B).
No
affinity-labeled bands were im-
munoprecipitated from three other human cell lines (A549,
Hep G2, MCF-7), which lack endoglin and were used as
negative-controls for these experiments (data not shown).
The identity of this dimeric TGF-P-binding protein of
HUVEC with endoglin was confirmed by ectopically express-
ing the endoglin cDNA in COS monkey kidney cells. After
affinity-labeling with '251-TGF-pl,
a
labeled species with the
characteristics of endoglin could be specifically precipitated
by mAb 44G4 only from detergent extracts of endoglin-trans-
fectants (Fig. 4). Differences in glycosylation likely account
for the smaller size of endoglin expressed in COS cells relative
to endogenous endoglin of HUVEC.
The TGF-/3 isoform binding specificity of endoglin was
assessed by testing the ability of TGF-Dl, $2, and
-83
to
Endoglin
Binds
TGF-p
19029
200
-
r)
-
Dimer
x
97.4
-
m
-
Mommerl-
69
-
RII-
46
-
"
FIG.
3.
Specific immunoprecipitation of TGF-B1-endoglin
complexes.
HUVECs were affinity-labeled with 100 p~ '251-TGF-/31
as
described in Fig. 2.
A,
detergent extracts of affinity-labeled cells
were incubated with mAb 44G4 and immune complexes were collected
on protein G-Sepharose. After washes, equal aliquots of the samples
were analyzed under reducing
(R)
or
nonreducing
(NR)
conditions
by SDS-PAGE (5-8% polyacrylamide gradient gels).
B,
affinity-
labeled HUVEC lysates were maximally depleted of endoglin by two
successive 45 min incubations at 4 "C with 100
pl
of 44G4-IgG-
Sepharose.
S,
supernatant after second immunoprecipitation.
I,
the
first 44G4 immunoprecipitate which contained 83% of the endoglin.
T,
corresponding amount of total extract used for the depletion
experiment. All samples were analyzed under nonreducing conditions
on SDS-PAGE with the exception of
IR,
which was run under reducing
conditions. The migration positions of TGF-0 receptor
I1
(RII),
and
endoglin monomer, dimer, and oligomer are indicated.
I
NR
Mr
(K)
200
-
97.4
-
100
-
69
-
46
-
FIG.
4.
Endoglin transiently expressed in
COS-M6
cells
binds
TGF-j31.
COS-M6 cells were transfected with
a
cDNA encod-
ing full-length endoglin
(Endoglin)
or
control vector
(C).
Cells were
affinity-labeled with 150 p~ '251-TGF-fi1 and the detergent extracts
incubated with mAb 44G4 followed by protein G-Sepharose. Immu-
noprecipitated proteins were analyzed by SDS-PAGE under reducing
(R)
and nonreducing
(NR)
conditions and visualized by autoradiog-
raphy.
compete with the binding of '251-TGF-P1 in endoglin trans-
fectants and in HUVEC. Endoglin from transfected COS cells
(Fig.
5A)
and from HUVEC (Fig.
5B
and data not shown)
A
0
COS-MG/Endoglin
HUMC
FIG.
5.
The specificity of endoglin for TGF-/3 isoforms
as-
sessed
in
COS
cell
transfectants
and
in
HUVEC.
A,
COS-M6
cells transfected with endoglin vector were affinity-labeled with 150
pM '2sI-TGF-01 alone
or
in the presence of
1
or 10 nM unlabeled
TGF-01, $2
or
$33.
B,
HUVEC were affinity-labeled with 100 pM
"'II-TGF-Pl alone
or
in the presence of 5 nM unlabeled TGF-01
or
TGF-02. Lysates from these cells were immunoprecipitated with mAb
44G4. Immunoprecipitates were fractionated under reducing condi-
tions on SDS-PAGE gels. The region of the gels containing mono-
meric endoglin is shown along with the migration position of 100-
kDa marker.
binds TGF-01 and
43
but fails to bind TGF-P2. A
KO
-
50
PM
for TGF-01 was estimated for HUVEC endoglin from
expanded competition assays performed at lower concentra-
tions of radiolabeled tracer (data not shown). The difference
in affinity of endoglin for TGF-P2 relative to TGF-Pl and
TGF-03 is in contrast to betaglycan, which binds all three
isoforms with high affinity
(6,
23, 27).
Scatchard analysis of '251-TGF-/31 equilibrium binding data
from HUVEC indicated
a
single high affinity TGF-Pl binding
site (average
KO
=
60
PM) and approximately 2
X
lo4
TGF-
Dl
binding sites/cell (data not shown), which is comparable
to the values obtained for endothelial cells from other sources
(19). From the affinity-labeling profile, at least three proteins
are contributing to these binding sites. Since saturation analy-
sis with mAb 44G4 indicated that HUVEC express of the
order of
IO6
endoglin molecules/cell(2,4), it is clear that some
rate-limiting event is required to sustain high affinity TGF-/3
binding to endoglin.
DISCUSSION
The present studies demonstrate that one function of en-
doglin is to bind TGF-P in an isoform-restricted manner.
Thus, endoglin is identified here as
a
major TGF-/3 binding
glycoprotein in human umbilical vein endothelial cells. The
disulfide-linked dimeric TGF-@binding protein previously
identified on fetal bovine heart endothelial cells (17) is likely
to be bovine endoglin, although this point could not be proven
in the present studies because of the lack of appropriate
antibodies. Given the presence of endoglin in human pre-
erythroblasts, macrophages, lymphoid, and myeloid leukemic
cells, and placental syncytiotrophoblasts, it will be of interest
to determine if endoglin participates in TGF-P binding in
these cell types as well. Likewise, it will be of interest to
determine whether various previously described TGF-&bind-
ing proteins of size and TGF-P binding properties similar to
those of endoglin (28, 29) might indeed correspond to this
molecule.
It is important to note that the molecular mass and electro-
phoretic migration of the reduced endoglin monomer and the
human TGF-6 receptor
11,
as well as their ability to bind
TGF-Pl and
83
better than p2, are similar. However, the
primary structure and presumed functional role of endoglin
and the TGF-8 receptor I1 are very different. Therefore,
conventional receptor affinity-labeling procedures alone are
insufficient to distinguish between endoglin and the TGF-/3
receptor I1 in studies of the expression, function, and regula-
tion of these two molecules. Additional reagents, such as
antibodies to TGF-0 receptor 11, will be essential for such
studies.
The results also indicate that only
a
portion of the endoglin
19030
Endoglin
Binds
TGF-/i’
molecules expressed in HUVEC bind TGF-P with high affin-
ity. The rate-limiting event required to sustain high affinity
TGF-/3 binding to endoglin is unknown
at
present. It is
possible that
a
post-translational modification of endoglin, a
conformational change
or
an interaction with another cellular
component may be required to generate the high affinity TGF-
p
binding site in endoglin. The nature
of
this event is currently
under investigation.
The specific role of endoglin
as
a mediator of cell interaction
with TGF-/3 remains to be determined. The homology between
the transmembrane and cytoplasmic portions of endoglin and
betaglycan suggests that they might associate through these
regions with similar molecules in order to fulfill their func-
tion(s).
As
no signal transducing structure is discernible in
the cytoplasmic domain of either endoglin
or
betaglycan, for
these proteins to be involved in signaling, they must interact
with specific signaling components. In the TGF-/3 system, the
evidence suggests that TGF-fl signaling is primarily mediated
by TGF-P receptors I and I1
(IO),
and as previously proposed
for betaglycan
(6,
7),
endoglin may be involved in presenting
TGF-P to these signaling receptors
or
participate as an acces-
sory molecule in the TGF-P receptor signaling complexes. We
note that the TGF-/3 isoform binding specificity of endoglin
is
remarkably similar to the profile of TGF-P responsiveness
in endothelial cells; TGF-Pl and
P3
are strong inhibitors of
proliferation of endothelial cells from various sources
(23,30,
31),
whereas TGF-P2
is
not
(20, 23, 31).
Endoglin, and its
relative betaglycan, might act as modulators of TGF-P inter-
action with the signaling receptors, I and/or 11, thus affecting
the ability of cells to respond to the TGF-P isoforms.
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