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INTRODUCTION |
Transforming growth factor-
(TGF-
)1 is the prototype
of a superfamily of growth factors involved in the regulation of cell proliferation, differentiation, and development (1, 2). TGF-
signals
through a complex of transmembrane serine/threonine kinase receptors,
the TGF-
type I and type II receptors. Ligand binding promotes the
association between the type I and II receptors. In this complex,
phosphorylation of the type I receptor kinase by the constitutively
active type II receptor kinase results in its activation. Active type I
receptor phosphorylates members of a novel family of transcriptional
regulators, the Smads, which transduce the TGF-
signal into the cell
nucleus (3, 4).
TGF-
has two known co-receptors, betaglycan and endoglin,
which are transmembrane glycoproteins with large extracellular regions
that bind TGF-
and small cytoplasmatic regions without any clearly
identifiable signaling motif (5-8). Betaglycan is a membrane
proteoglycan containing heparan and chondroitin sulfate chains whose
core protein binds all three TGF-
isoforms (9-11). Betaglycan is
capable of fine tuning the availability of TGF-
to the signaling
receptors, thereby determining the outcome of the TGF-
stimulation
(12, 13). This regulation is both positive and negative. Although
the membrane-bound form of betaglycan increases the binding of TGF-
to the signaling complex, the soluble form of betaglycan prevents this
binding and therefore blocks the actions of TGF-
(14). These effects
are more dramatic for TGF-
2, the isoform for which betaglycan has
higher affinity (15-17). Expression of membrane betaglycan in cells
that normally do not express this co-receptor increases their binding
to TGF-
2 and corrects for their low sensitivity to this TGF-
isoform (18, 19). Presumably, this effect is mediated by a
TGF-
-induced "presentation complex" formed between
membrane-bound betaglycan and the TGF-
type II receptor (18, 20).
However, the presentation function is insufficient to account for the
betaglycan strict requirement for the epithelial-mesenchymal transition
leading to the heart valve formation (21). This latter work has raised
the possibility of a more direct, albeit unknown, role of
betaglycan in the TGF-
signaling.
Betaglycan also interacts with type II receptors of another TGF-
superfamily member. Lewis et al. (22) shows that betaglycan also binds inhibin A and participates in a ternary complex composed of
the activin type II receptor and inhibin. This complex mediates the
inhibin antagonism of activin by a simple and elegant mechanism: the
routing the activin type II receptor into an inactive complex with
betaglycan instead of the signaling complex with activin and the
activin type I receptor (22). These findings indicate that betaglycan
is a versatile co-receptor for at least two distinct members of the
TGF-
superfamily and open the question of the nature of the
structural determinants that make this versatility possible.
The TGF-
binding function of betaglycan is a property of its
ectodomain core protein. Although betaglycan GAG chains are capable of
binding basic fibroblast growth factor, they are dispensable for
TGF-
binding and TGF-
2 presentation function (8, 10, 14, 23).
Amino acid sequence comparisons have disclosed regions in betaglycan
ectodomain with similarity to other receptors or extracellular
proteins. The 260 residues at its amino-terminal end have 28%
similarity to the corresponding amino-terminal portion of endoglin
ectodomain (7), whereas the 330 residues at its carboxyl-terminal end
have similarity to proteins related to uromodulin (24) (Fig.
1A). Several groups have utilized deletion mutants of the
wild type receptor to map the ligand binding domain of betaglycan (14,
25-29). These studies show the existence of two ligand binding regions
in betaglycan that roughly match to each half of its ectodomain, the
membrane-distal and the membrane-proximal regions (26). In the present
work we have characterized the functional properties of these ligand
binding regions. Our results indicate that betaglycan ligand binding
regions are equivalent for its TGF-
isoform affinities and
TGF-
-enhancing function but differ in their ability to bind inhibin A.
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EXPERIMENTAL PROCEDURES |
Materials--
TGF-
1 and inhibin A were from R&D Systems
(Minneapolis, MN); TGF-
2 was from Ciba-Geigy AG (Basel,
Switzerland). Restriction endonucleases and modifying enzymes were from
Roche Molecular Biochemicals. Buffers, salts, and protease and
phosphatase inhibitors were from Sigma-Aldrich. The expression vectors
for the human TGF-
type II receptor (tagged with the HA1 epitope)
and the human activin type II receptor (tagged with a carboxyl-terminal
hexa-histidine tail) have been described (18, 30).
Betaglycan Deletion Mutants--
Betaglycan mutants were
constructed from the wild type rat betaglycan cDNA engineered with
the human c-Myc epitope recognized by the monoclonal antibody
9E10 (18). Construction of deletion mutants
2,
3,
10, and
gag
has been described (14). Mutant
11 (
45-409)
was constructed from our previously described
8 (
45-282) mutant.
The construction of
8 was done by the insertion at the
StuI/BclI sites of the wild type betaglycan
cDNA of a double-stranded oligonucleotide encoding, in addition to
the
8 intended changes, a novel NaeI site (14). For the
creation of
11,
8 was cut at the EcoRV site and at its
engineered NaeI site, and the resulting large restriction
fragment was self-ligated. Mutant
12 (
44-499) resulted from the
self-ligation of the large fragment produced by digestion of the wild
type c-Myc-tagged betaglycan vector with StuI and XhoI followed by filling-in with the Klenow fragment of the
DNA polymerase. In addition, untagged versions of
10 and
11
betaglycan mutants were created and used in diverse experiments as
indicated in the figure legends. The soluble versions of the
10 and
11 mutants were created by suitable insertion of double-stranded oligonucleotides encoding a stop codon preceded by a hexa-histidine carboxyl-terminal tail. For soluble
10, the oligonucleotide was inserted between the EcoRV and NcoI sites of wild
type c-Myc-tagged betaglycan, and for soluble
11, at the
NcoI and AvrII sites of c-Myc-tagged
11. All
DNA manipulations were done and verified by nucleotide sequencing after
standard techniques (31).
Cell Culture, Transfections, and Generation of Baculoviral
Strains--
COS-1 and L6E9 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% or 20% fetal bovine serum,
respectively. Sf9 cells were grown in Grace's insect
cell culture medium (Life Technologies, Inc.) supplemented with 10%
fetal calf serum, yeastolate, and lactalbumin hydrolysate. High five
cells (H5, Invitrogen, Carlsbad, CA) were grown in spinner flasks at
27 °C at 100 rpm using Express Five serum-free medium (Life
Technologies, Inc.) supplemented with 4 mM glutamine. For
expression in COS-1 cells, the cDNAs encoding the diverse mutant
betaglycan constructs were subcloned in the pCMV5 vector (32). For
stable L6E9 cells transfections, the cDNAs encoding the wild type
betaglycan and the untagged
10 and
11 mutants were subcloned in
the pcDNA3 vector (Invitrogen). Transfer vectors for the generation
of baculoviral strains consisted of the pBlueBac4 vector (Invitrogen)
subclones of the inserts encoding c-Myc and hexa-histidine-tagged
soluble
10 and
11 mutants. Transient transfections were done by
the diethylaminoethyldextran method (33), and assays were done 48 h post-transfection. Stable L6E9 transfection was done by the calcium
phosphate precipitation method (34). Stable transfectants were selected
with 400 µg/ml geneticin (Life Technologies, Inc.) and enriched by
three rounds of magnetic cell sorting using anti-betaglycan antiserum
#822, which is directed against the ectodomain (18), and goat
anti-rabbit IgG magnetic microbeads (Miltenyi Biotec Inc, Auburn, CA).
The generation of recombinant baculoviral strains expressing the
soluble
10 and
11 was done by co-transfection in Sf9 cells
of the pBlueBac4-based constructs with Bac-N-Blue DNA (Invitrogen)
employing the calcium phosphate precipitation method.
Purification of Baculoviral Soluble
10 and
11
Proteins--
High five cells at a density of 2 × 106/ml were infected at a multiplicity of infection of 10 with a high titer stock of the soluble
10 or
11 baculovirus. The
conditioned media were harvested 48 h post-infection and
immediately processed. Conditioned media were supplemented with 1 mM phenylmethylsulfonyl fluoride, spun at 11,000 rpm for 20 min at 4 °C to remove debris, concentrated 10 times using the
Minitan ultrafiltration system (Millipore, Bedford, MA), and subjected
to immobilized metal ion affinity chromatography using fast
flow chelating Sepharose (Amersham Pharmacia Biotech). Chelating
Sepharose (a 60-ml bead column) was loaded with 50 mM
NiCl2 and then equilibrated in washing buffer (25 mM Hepes pH 7.5, 1 M KCl, 20 mM
imidazole, 1 mM phenylmethylsulfonyl fluoride). The
concentrated conditioned media were loaded, and the column was washed
with 6 volumes of washing buffer and eluted with a 20-250
mM linear gradient of imidazole. Identification of the
fractions containing soluble
10 or
11 proteins was done by
SDS-PAGE and silver staining. Positive fractions were pooled and
concentrated in an Amicon ultrafiltration chamber (Millipore, Bedford,
MA). After dialysis against phosphate-buffered saline containing 1%
(v/v) glycerol and 1 mM phenylmethylsulfonyl fluoride, the
recombinant soluble
10 and
11 proteins were stored at
70 °C.
This procedure usually yielded between 3 and 4 mg of homogeneously purified proteins from each liter of conditioned media.
Affinity Labeling and Binding Assays--
TGF-
was
radiolabeled with [125I]iodine using chloramine T (11),
whereas a lactoperoxidase method was employed for inhibin A labeling
(35). Binding assays, affinity labeling, and immunoprecipitations under
native or denaturing/reducing conditions were done as described (18).
TGF-
affinity labeling in solution was done with the purified
soluble
10 or
11 proteins (50 ng/assay) as described before
(14).
Smad2 Phosphorylation Assay--
Determination of phosphorylated
Smad2 was done by Western blot with anti-phospho-Smad2 (Ser465/467)
anti-serum (a generous gift of C.-H. Heldin, Uppsala, Sweden). L6E9
myoblast cells expressing the wild type and betaglycan mutants were
stimulated with 20 pM TGF-
2 or TGF-
1 for 15 min.
Cells were lysed in TTE buffer (0.1% Triton X-100, 10 mM
Tris-HCl, pH 7.4, 1 mM EDTA) supplemented with inhibitors
of proteases (10 µg/ml leupeptin, 10 µg/ml antipain, 100 µg/ml
benzamidine hydrochloride, 50 µg/ml aprotinin, 100 µg/ml soybean
trypsin inhibitor, 10 µg/ml pepstatin, 1 mM
phenylmethylsulfonyl fluoride) and phosphatases (10 mM
sodium pyrophosphate, 50 mM sodium fluoride, 100 µM sodium orthovanadate, 10 mM
-glycerophosphate). Equal amounts of protein from cell lysates were
separated by SDS-PAGE (9% gels), transferred to nitrocellulose
membranes, and probed with anti-phospho-Smad2. Equal loading of Smad2
was verified by probing the membranes again with an anti-Smad2 antibody
(a generous gift of J. L. Wrana). Immunoblots were revealed using
the enhanced chemiluminescence (Amersham Pharmacia Biotech) and autoradiography.
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RESULTS |
Betaglycan Deletion Mutagenesis Has Identified 2 Independent
TGF-
Binding Regions--
At least 20 deletion mutants of rat
betaglycan have been created and assayed in eukaryotic expression
systems with the purpose of mapping its TGF-
binding sites (14,
25-27). These studies identify two binding regions that are
approximately located at the membrane-distal (amino-terminal) half (14,
27) and at the membrane-proximal (carboxyl-terminal) half of betaglycan
ectodomain (25, 26). Because these halves contain the regions of
similarity to endoglin and uromodulin, here we will refer to them as
the E-related and the U-related regions, respectively (Fig.
1A).

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Fig. 1.
Betaglycan mutants and their
TGF- binding activity. A, the
rat betaglycan mutants employed in this study are presented
schematically. The amino acids deleted are indicated in
parentheses. Betaglycan GAG attachment sites at serines 535 and 546 (circles), its signal peptide (black
box), and the position of its cysteines (dots) are
indicated. The cell plasma membrane (stripped bar)
and the c-Myc epitope tag (asterisk) are also indicated.
Untagged versions of the wild type betaglycan and its 10 and 11
mutants were also used in the present work as described in the
corresponding figure legends. Regions of sequence similarity to
endoglin and uromodulin are indicated as shadowed and
cross-hatched boxes, respectively. Flanking the
uromodulin-related region there are sequences (empty boxes)
without similarity to known proteins. Within the uromodulin-related
region, there is an additional short region of similarity to endoglin
that includes its first three cysteines (7). A dashed line
drawn through residues 409-410 indicates the boundaries of the
membrane-distal (E-related) and membrane-proximal (U-related) TGF-
binding regions of betaglycan ectodomain. COS-1 cells were transiently
transfected with the empty expression vector (pCMV5) or the indicated
c-Myc-tagged betaglycan mutants (B) or with the indicated
untagged betaglycan mutants (C). Two days post-transfection
the cultures were affinity-labeled with 200 pM
125I-labeled TGF- 1 (B) or 200 pM
125I-labeled TGF- 2 (C). Cell lysates were
immunoprecipitated with the anti-c-Myc 9E10 monoclonal antibody
(B) or with the anti-betaglycan ectodomain rabbit antiserum
#822 (C). The immunoprecipitates were separated by SDS-PAGE
and visualized by autoradiography (B) or phosphorimager
scanning (C) of the gels. Migration of molecular mass
standards (in kDa) and the position of cross-linked oligomers of some
mutant receptors (arrowheads) are indicated.
W.T., wild type betaglycan.
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To determine the functional properties of betaglycan TGF-
binding
regions, we decided to test their ligand binding affinities, TGF-
2
presentation, and TGF-
2-enhancing functions. For that purpose we
constructed betaglycan mutants analogous to those described by Pepin
et al. (25), i.e. lacking the amino-terminal half
of betaglycan ectodomain. Our new truncated mutants
11 and
12
lack residues 45-409 (
45-409) and 44-499 (
44-499) of wild
type rat betaglycan, respectively (Fig. 1A). Mutants
11
and
12 are complementary to the set that we have described before
(14). In particular
11, which lacks the entire amino-terminal half,
is the complement of our previous
10 (
410-781) mutant, which
lacks the entire carboxyl-terminal half of the ectodomain. Mutants
11 and
12 along with the wild type betaglycan and our previously
characterized
2 (
200-500),
3 (
499-783),
10, and
gag
mutants (14), were tested for TGF-
binding
activity. For that purpose, COS-1 cells transiently transfected with
these mutant vectors were affinity-labeled with
125I-labeled TGF-
1 or 125I-labeled TGF-
2.
Betaglycan-specific labeled products were revealed by
immunoprecipitation of the cell lysates followed by SDS-PAGE and
autoradiography (Fig. 1, B and C). As reported
before, the wild type betaglycan appeared as a "part time"
proteoglycan, that is, a GAG-containing product of smeared mobility
above 200 kDa plus the 130-kDa core protein (Fig. 1, B and
C, lanes 2). Also as before, the
gag
mutant showed exclusively as a core protein, devoid
of GAG chains (Fig. 1B, lane 3). The
gag
betaglycan is a double point mutant of the serines
that have been shown to be the GAG chain acceptor amino acids (S535,
546A), and therefore, it is not processed as a proteoglycan; however, it displays the wild type receptor TGF-
binding activity (14). Mutants
10 and
3, which do not contain the serines 535 and 546, also appeared as core proteins with relative mobilities of ~85 and 97 kDa, respectively. The high molecular weight affinity-labeled proteins
exhibited by gag
,
10, and
3 mutants (Fig. 1,
B and C, arrowheads), result from cross-linked dimeric and oligomeric forms of these mutant receptors (14, 25). In agreement with Pepin et al. (25), our new
11 mutant, which includes all the 10 cysteines residues that define the
uromodulin similarity (24), also bound TGF-
1 and TGF-
2 (Figs. 1,
B, lane 6, and C, lane 4).
The protein expressed by
11 was affinity-labeled both as a core
protein of ~95 kDa as well as a proteoglycan (smear between 120-194
kDa), as could be expected since it contains the serines 535 and 546. Mutant
12, which lacks residues 44-499 of betaglycan core protein,
including the first two cysteines of the U-related region, did not bind any of the three TGF-
isoforms (Fig. 1B, lane
8, and data not shown). In addition, the empty pCMV5 vector and
our previously reported
2 mutant were included in these experiments
as negative controls. The TGF-
binding incapacity of
2 and
12
was not due to lack of expression. All the deletion mutants shown in
Fig. 1A were verified for cell surface expression by
fluorescence-activated cell sorter analysis using the appropriate
antibody (data not shown). In summary, the analysis of our new mutants
along with those previously described confirmed the existence of two
discrete regions of betaglycan ectodomain with TGF-
binding
activity. The functional characterization of these regions, which are
located between residues 45-410 and 410-781 and are present in our
10 and in
11 mutants, respectively, will be presented in the
following sections.
Both Betaglycan Ligand Binding Regions Bind TGF-
1 and TGF-
2
with Relative Affinities Similar to the Wild Type Receptor--
As a
first step to characterize the betaglycan TGF-
binding regions, we
assayed the
10 and
11 mutants in binding competitions with the
1 and
2 isoforms. As a reference, we also tested the wild type
betaglycan, which has a characteristic higher affinity for TGF-
2
that is revealed by a very weak TGF-
2 binding competition by the
other isoforms (15-17). For this purpose, COS-1 cells were transfected
with the wild type betaglycan, the
10, or the
11, pCMV5-based constructs and subjected to a 125I-labeled
TGF-
2 binding assay in the presence of increasing concentrations of
competing unlabeled TGF-
1 or TGF-
2. As expected for the wild type
betaglycan, 50% of its binding of 50 pM
125I-labeled TGF-
2 was prevented by ~3 nM
cold TGF-
2, whereas it was not affected by concentrations as high as
10 nM cold TGF-
1 (Fig.
2A, WT). The
10
and
11 mutants exhibited 125I-labeled TGF-
2 total
binding and competition patterns that are similar to those observed in
the intact receptor, indicating that they have the same TGF-
isoform
relative affinities as the wild type betaglycan (Fig.
2A,
10 and
11).

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Fig. 2.
TGF- isoform
specific binding competition of betaglycan mutants.
A, COS-1 cells were transiently transfected with the empty
expression vector (pCMV5 (circles)) or the indicated
untagged betaglycan vectors (squares). One day after
transfection, the cells were split into 24-well plates for assay. On
the next day, cells were labeled with 50 pM
125I-labeled TGF- 2 in the presence of the indicated
concentrations (0, 1, 3, or 10 nM) of cold TGF- 1
(open symbols) or TGF- 2 (closed symbols). The
bound iodinated ligand is plotted against the concentration of the
unlabeled competitor. B, purified soluble 10
(Sol 10) or soluble 11 (Sol
11) proteins (50 ng/assay) were subjected to
affinity-labeling in solution with 100 pM
125I-labeled TGF- 2 in the absence (C) or
presence of the indicated concentration of competing unlabeled TGF- 1
or TGF- 2. After cross-linking with disuccinimidyl suberate, the
labeling reactions were quenched with Tris-Cl and immunoprecipitated
with anti-betaglycan antiserum #822, and the immunoprecipitates were
revealed by SDS-PAGE and phosphorimager scanning (left
panels). The percentage of competition was estimated from
densitometric analysis of the labeled soluble 10 and soluble 11
using the ImageQuant Software and plotted against the competitor
TGF- concentration (right panels). WT, wild
type betaglycan.
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To demonstrate that these TGF-
binding properties are intrinsic to
these regions, we did a similar 125I-labeled TGF-
2
binding competition analysis with the purified soluble forms of the
10 and
11 mutants (Fig. 2B). For this purpose we
created baculoviral strains of soluble
10 and
11 whose protein products could be secreted into the conditioned media of infected insect cells. Since the soluble
10 and soluble
11 mutants were engineered with a hexa-histidine carboxyl-terminal tail, they could be
purified to homogeneity by immobilized metal ion affinity chromatography as described under "Experimental Procedures."
Purified soluble
10 and soluble
11 proteins were subjected to
affinity labeling in solution with a constant amount of
125I-labeled TGF-
2 and increasing amounts (from 0-2
nM) of competing unlabeled TGF-
1, or TGF-
2. Purified
soluble
10 and soluble
11 are glycoproteins with a molecular mass
of 52 and 63 kDa, respectively (data not shown), which after
125I-labeled TGF-
2 cross-linking, migrate just below and
above and 64-kDa marker (Fig. 2B). The amount of labeled
soluble
10 and soluble
11 was quantified from the
PhosphorImager (Molecular Dynamics) scans of the SDS-PAGE
gels and plotted as the percent of affinity-labeling competition
against the cold competitor concentration (Fig. 2B,
right panels). Similar to what was observed for their membrane counterparts, the half-maximal homologous competition of the
TGF-
2 labeling was at least one order of magnitude higher than the
TGF-
1 heterologous competition. Taken together, the data suggested
that the relative affinities of both membrane-bound and soluble
10
and
11 mutants for TGF-
1 and TGF-
2 are very similar to each
other and to the wild type betaglycan.
TGF-
2 Presentation Function of Betaglycan Ligand Binding
Regions--
Since both betaglycan TGF-
binding regions have
TGF-
2 binding affinities that are comparable with those of the
intact receptor, we decided to test whether or not they conserved the
TGF-
2 presentation function of the wild type betaglycan (6, 18, 20).
This presentation function refers to the capacity of the membrane-bound betaglycan to increase the TGF-
2 labeling of the TGF-
type II receptor. Presumably, this function accounts for the enhanced potency
of the TGF-
2 isoform in the presence of betaglycan (18). To measure
the TGF-
2 presentation function of betaglycan TGF-
binding
regions, we co-expressed a few of our c-Myc-tagged mutants along with
the human TGF-
type II receptor by transient transfection of COS-1
cells. The type II receptor cDNA used in this experiment has been
tagged with the influenza virus hemagglutinin HA1 epitope that is
recognized by the monoclonal antibody 12CA5 (36). After co-transfection, these cells were affinity-labeled with
125I-labeled TGF-
2, and the cell lysates were subjected
to denaturation and reduction and then to immunoprecipitation with
either the anti-c-Myc antibody 9E10 (37) or the anti-HA1 antibody. The immunoprecipitates were resolved in SDS-PAGE and revealed by
autoradiography. The denaturation and reduction step abrogates any
noncovalent complex formed between the two receptors during the
affinity labeling, thus preventing their co-immunoprecipitation. The
anti-c-Myc immunoprecipitates revealed that the TGF-
2-labeled
c-Myc-betaglycan mutants (Fig. 3A) exhibit gel migration
patterns similar to those observed with TGF-
1 (Fig. 1B).
This suggests that the co-expression of the TGF-
type II receptor
did not alter the binding properties of the betaglycan mutants;
however, the labeling of the former was regulated by the
expression of the latter. The anti-HA1 immunoprecipitates revealed the
extent to which the co-transfection partner enhanced the labeling of
the type II receptor (Fig. 3B). Co-transfection with the
wild type betaglycan, either in its proteoglycan (W.T.) or
core protein form (gag
) resulted in an increased labeling
of the type II receptor (Fig. 3B, lanes 5 and
6). Co-transfection with the empty pCMV5 vector resulted in
negligible labeling of the type II receptor, revealing its endogenous
poor TGF-
2 binding activity (lane 1, Fig. 3B). Similarly, co-transfection with
2 or
12, mutants without TGF-
2 binding, resulted in a barely detectable increase in the labeling of
type II receptor (when compared with the empty vector, lanes 2 and 3, Fig. 3B), which may be regarded as
nonspecific "background" of this kind of experiments.
Co-transfection with
11, which had a level of TGF-
2 labeling
similar to the wild type betaglycan (lanes 4 and
5, Fig. 3A), resulted only in a slight increase
over the background level of labeling of the type II receptor (compare lane 4 against lanes 2 and 3 in Fig.
3B). On the other hand, co-transfection with
3 or
10
resulted in an approximate 2- or 3-fold increase, respectively, over
the level of type II receptor labeling promoted by the wild type
betaglycan (compare lanes 7 and 8 against
lanes 5 and 6 in Fig. 3B). Thus,
despite sharing the property of binding TGF-
2, the
3,
10, and
11 betaglycan mutants are not equal in their ability to
"present" this isoform to the TGF-
type II receptor.

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Fig. 3.
TGF- 2 presentation
activity of betaglycan mutants. A and B,
COS-1 cells were transiently co-transfected with the HA1 epitope-tagged
TGF- type II receptor (T RII/HA) vector and
the indicated c-Myc-tagged betaglycan mutants or the empty expression
vector (pCMV5). Two days post-transfection the cultures were
affinity-labeled with 100 pM 125I-labeled
TGF- 2, and the cell lysates were denatured by boiling in the
presence of SDS and dithiothreitol. Then, one-half of the cell lysate
was immunoprecipitated with the 9E10 monoclonal anti-c-Myc antibody
(A) and the other half with the 12CA5 monoclonal anti-HA
antibody (B). The immunoprecipitates were separated by
SDS-PAGE and visualized by autoradiography of the gels. C,
cultures of L6E9 cells, stably transfected with indicated
untagged betaglycan (BG) mutants or the empty
expression vector (pcDNA3), were affinity-labeled with 150 pM 125I-labeled TGF- 2. Equivalent aliquots
of their cell lysates were immunoprecipitated with the anti-betaglycan
antiserum (#822) or with the anti-TGF- type II receptor antibody
(L21, Santa Cruz). The immunoprecipitates as well as an aliquot of the
total cell lysates (T) were separated by SDS-PAGE and
visualized by phosphorimager scanning of the gels. WT, wildtype
betglycan.
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To confirm that this effect also could be observed with the
endogenous type II receptor and that it did not depend on any of the
epitope tags present in the tested receptors, we analyzed the TGF-
2
presentation function of the untagged betaglycan mutants in rat L6E9
myoblasts. For that purpose we transfected L6E9 cells, which normally
do not express betaglycan (6, 18), with the wild type and the
10 and
11 betaglycan mutants and used magnetic bead immunoabsorption to
obtain enriched pools of stable transfectants. The enriched pools were
subjected to affinity labeling with 150 pM
125I-labeled TGF-
2, and the identity of the labeled
products was revealed by specific immunoprecipitation under
nondenaturing conditions (Fig. 3C). At this TGF-
2
concentration, the level of labeling of control-transfected L6E9 cells
was practically negligible (Fig. 3C, lanes 4-6).
On the other hand, expression of the wild type betaglycan resulted in a
significant increase in the labeling of the endogenous type II receptor
(Fig. 3C, compare lanes 3 and 6). This
effect, which has been observed before, is due to an increase in
TGF-
2 relative affinity of the endogenous type II receptor in the
presence of betaglycan rather than to an increase in the amount of the
receptor (6, 18). Also, as has been observed before (18), in the
presence TGF-
2 the endogenous type II receptor forms a complex with
betaglycan that can be demonstrated by co-immunoprecipitation with
specific receptor antibodies (Fig. 3C, lanes
2-3). In this kind of assay, the betaglycan mutant
10 was
capable of increasing the TGF-
2 labeling of the endogenous type II
receptor and of forming an immunoprecipitable complex with it (Fig.
3C, lanes 10-12). However, and similar to what
was observed in co-transfected COS-1 cells, the
11 mutant
efficiently bound TGF-
2 but fails to increase the labeling of the
type II receptor (Fig. 3C, lanes 7-9).
Both Betaglycan Ligand Binding Regions Enhance the
TGF-
2-dependent Smad2 Phosphorylation--
The data
presented in Fig. 3 indicated that the TGF-
2 presentation function
of betaglycan is a property of the E-related region and, therefore,
strongly suggested that this ligand binding region would preferentially
enhance the TGF-
2 signals. To evaluate this possibility, we decided
to determine how the expression of the
10 and
11 mutants affected
the TGF-
2-induced Smad2 phosphorylation in L6E9 cells. We chose to
measure Smad2 phosphorylation because it is one of the earliest steps
in TGF-
signaling and could most directly reveal if the TGF-
2
presentation function of betaglycan had any effect in the TGF-
pathway (38). Also, to rule out any possible idiosyncratic behavior of
a single clones, we preferred to employ for this experiment the pool of
stable L6E9 transfectants analyzed in Fig. 3C and to include
as an additional negative controls, the untransfected L6E9 cells. These
cells were treated with 20 pM TGF-
2 for 15 min, and
their lysates were separated in SDS-PAGE and blotted for
immunodetection with an antiserum that recognizes Smad2 phosphorylated
at serines 465 and 467 (39). It has been demonstrated that these Smad2
serines are the phosphorylation target of the activated TGF-
type I
receptor kinase (40, 41). The phopho-Smad2 signal was
normalized using as a standard the amount of total Smad2, as detected
by probing the stripped blot with an antiserum that recognizes all
forms of Smad2 (42). Control untransfected L6E9 or
pcDNA3-transfected cells did not significantly increase basal
phosphorylation of Smad2. As it was expected for the wild type
betaglycan, its expression caused a 1.9-fold increase in the Smad2
phosphorylation. Surprisingly, expression of the
10 or
11
betaglycan mutants caused similar increases of Smad2 phosphorylation,
3.0- and 3.2-fold, respectively (Fig. 4).
The experiment shown in Fig. 4 is representative of a total of three in
which the same result was observed, namely, that both betaglycan ligand
binding regions were equally capable of enhancing the TGF-
2-induced Smad2 phosphorylation. Importantly, this short term exposure and limiting concentration of TGF-
2 was required to demonstrate any betaglycan dependence of the Smad2 phosphorylation. Similar treatment with TGF-
1 or longer treatment with higher concentrations of TGF-
2 (50 pM) caused equivalent increases of the Smad2
phosphorylation in the tested cell pools (data not shown). Overall,
these experiments indicate that the E- and U-related regions, despite
their contrasting TGF-
presentation ability, both enhance the
TGF-
signal as well as the wild type betaglycan. In addition, this
enhancement occurs in a TGF-
isoform-specific manner and only under
limiting concentrations of the factor.

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Fig. 4.
TGF- 2-enhancing
activity of betaglycan mutants. Cultures of untransfected L6E9
cells or pools of L6E9 cells stably transfected with indicated untagged
betaglycan mutants or the empty expression vector (pcDNA3) were
treated with 20 pM TGF- 2 (+) or with the plain medium
( ; DMEM plus 0.2% FBS) for 15 at 37 °C. After treatment the
cultures were quickly chilled and lysed in the presence of phosphatase
and protease inhibitors. Equivalent amounts of cell lysates were
separated SDS-PAGE and transferred to nitrocellulose membranes for
immunoblotting with antibodies that recognize Smad2 (Total
Smad2) or, specifically, its phosphorylated form
(PO4-Smad2). WT, wild type
betaglycan.
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Betaglycan Ability to Bind Inhibin A Resides in Its U-related
Ligand Binding Region--
Recently it has been described that
betaglycan binds inhibin A, enhances inhibin A binding to the activin
type II receptor, and establishes a receptor complex that mediates the
inhibin antagonism of activin (22). Therefore, we determined whether or
not the inhibin A binding function of betaglycan could be assigned to any of its 2 TGF-
binding regions. For that purpose, we transfected COS-1 cells with the wild type betaglycan or its mutants alone or in
the presence of the activin type II receptor and measured their
125I-labeled inhibin A binding. Table
I shows the total counts bound to these
cells in one representative experiment of the three performed with
similar results. Cells transfected with the wild type or the
gag
betaglycan vector had higher inhibin A binding than
the pCMV5-transfected control cells. These results are in agreement
with those of Lewis et al. (22) and indicate that
betaglycan GAG chains are dispensable for the inhibin A binding. In all
the experiments performed, the
11 mutant exhibited inhibin A binding
that was comparable with that of the cells expressing the wild type or
the gag
betaglycan. On the other hand, the
10 mutant
consistently showed inhibin A binding that was similar to
pCMV5-transfected control cells. COS-1 cells transfected with the
activin type II receptor also exhibited inhibin A binding above the
control cells. As expected, the inhibin A binding of cells expressing
the activin type II receptor could be further increased by
co-transfection with the wild type, the gag
betaglycan,
or the
11 vectors. However, the
10 mutant could only marginally
increase the inhibin A binding of the type II activin receptor. These
results suggest that the intrinsic inhibin A binding capacity of
betaglycan resides exclusively in the U-related ligand binding
region.
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Table I
Betaglycan mutants and their inhibin A binding activity
COS-1 cells were transiently co-transfected with the indicated untagged
betaglycan vector and the empty expression vector (pCMV5) or the type
II activin receptor (ActRII) vector. Two days after transfection the
cells were labeled with 125 pM 125I-labeled inhibin
A, and the bound ligand was counted. The bound counts were normalized
using as the unit the counts bound by the pCMV5 vector alone or in the
presence of the ActRII, and the normalized values are shown within
parenthesis.
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To probe the relative affinities of this region for its TGF-
superfamily ligands, the binding of 50 pM
125I-labeled inhibin A was competed with a 10- and 100-fold
excess of unlabeled TGF-
1, TGF-
2, or inhibin A. This experiment,
shown in Fig. 5, confirms that the
U-related region, but not the E-related region, binds inhibin A as
efficiently as the wild type and gag
betaglycan. A
practically complete homologous competition of this binding could be
obtained with 0.5 nM unlabeled ligand. However, in the
heterologous competition, even at 100-fold excess, TGF-
1 could only
partially compete the inhibin A binding. On the other hand, TGF-
2
was a strong competitor of the inhibin A binding, exhibiting degrees of
competition that are indistinguishable from those observed with the
homologous ligand. Interestingly, the binding of 100 pM
125I-labeled TGF-
2 to wild type betaglycan or its
gag
,
10, and
11 mutants could not be competed at
all by 7 nM inhibin A (data not shown). All together, these
experiments suggest that, although the E-related ligand binding region
of betaglycan binds only TGF-
, the U-related region also binds
inhibin A with the following relative affinities: TGF-
2 > inhibin A > TGF-
1.

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Fig. 5.
Ligand binding competition of inhibin A
binding of the betaglycan mutants. COS-1 cells were transiently
transfected with the empty expression vector (pCMV5) or the indicated
untagged betaglycan vectors. One day after transfection, the cells were
split into 24-well plates for assay. On the next day, cells were
labeled with 50 pM 125I-labeled inhibin A in
the absence (C (closed bars)) or in the presence
of the indicated concentrations (0.5 or 5.0 nM) of cold
TGF- 1 (cross-hatched bars), TGF- 2 (dotted
bars), or inhibin A (empty bars). The bound iodinated
ligand is plotted against the indicated unlabeled competitor.
WT, wild type betaglycan.
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DISCUSSION |
Betaglycan belongs to a class of cell surface receptor molecules
that regulate the access of ligands to the signaling receptors. The
functional relevance of this growing class of co-receptors is
exemplified by cell surface heparan sulfates, which are necessary for
the high affinity binding and signaling of basic fibroblast growth
factor (43, 44). Betaglycan is a particularly interesting and versatile
co-receptor because it modulates the effects of at least three members
of the TGF-
superfamily. In one hand, membrane-bound betaglycan
positively regulates TGF-
, whereas in the other, it mediates the
inhibin antagonism of the activin signal (18, 22). Furthermore, a
soluble form of betaglycan capable of binding TGF-
with high
affinity has been found in serum and extracellular matrix (45). A
recombinant version of soluble betaglycan binds, sequesters, and
thereby antagonizes TGF-
, playing an opposite role to the
membrane-bound counterpart (14). Because of these dual actions, it has
been proposed that betaglycan could be one of TGF-
major in
vivo regulators (46). To fully understand and take advantage of
betaglycan properties, it is necessary to characterize its interactions
with its different ligands and signaling receptors. Betaglycan binds
inhibin A and all three mammalian TGF-
isoforms through its
ectodomain core protein, which is disproportionally large when compared
with the ectodomains of the corresponding signaling receptors. The
regional similarities that betaglycan ectodomain exhibits with endoglin (7) and with a class of extracellular proteins related to uromodulin (24) suggest a modular design with separate domains. Interestingly, this modular design seems to adjust well to the identified regions of
TGF-
binding activity. Several groups have used deletion mutants of
rat betaglycan to map betaglycan TGF-
binding regions (14, 25-29).
This experimental approach has revealed two ample portions of
betaglycan with TGF-
binding activity that approximately correspond to each half of its ectodomain, the membrane-distal (E-related) and the
membrane-proximal (U-related) regions (Fig. 1A). In the present work we report the creation of additional betaglycan mutants that contain the U-related regions (
11 and
12) and, thus,
complete our previously described set of mutants (14). The availability of these new mutants has provided us with the materials to study the
functional properties of these regions.
The TGF-
affinity labeling of our new and a few selected old mutants
(Figs. 1, B and C, and 3A) confirmed
the presence of TGF-
binding activity in the E-related region,
present in mutants
3,
10, and in the U-related region, present
intact in mutant
11. In addition, we have found that these regions
have TGF-
binding affinities that are very similar to the intact
receptor. Ligand binding competitions (Fig. 2) indicate that
10 and
11 exhibit the same relative TGF-
affinities, approximately one order of magnitude higher for TGF-
2 than for TGF-
1. This TGF-
isoform selectivity has been shown before for the intact wild type
receptor (15-17). Interestingly, when the E-related and the U-related
regions are expressed as soluble receptors, they have the same relative
TGF-
affinities of their membrane counterparts and the wild type
receptor. These results further support the possibility that the
residues in the E- and U-related regions may constitute bona
fide independent structural domains of the betaglycan
extracellular region.
Nonetheless, the precise determination of the sites or residues
involved in the ligand binding activity in these domains will be
difficult to obtain by further analysis of betaglycan-truncated mutants, an approach that has met with puzzling results. As an example
is the case of our previously published
1 (
45-199),
5
(
200-285),
8 (
45-282), and
9 (
287-409) mutants, which despite having complete the U-related region, do not bind TGF-
(14).
A plausible explanation for this inability is that the portions of the
E-related region that were not deleted in
1,
5,
8, and
9
indirectly affect the TGF-
binding activity of the U-related region
by a "downstream folding effect." Presumably, the leftover segments
of the upstream E-related region in these mutants would cause an
improper folding of the downstream U-related region and thereby loss of
its TGF-
binding activity. The incomplete maturation of the mutant
receptor, which would be another consequence of its putative improper
folding, has been ruled out for all our mutants (old and new), since
each one of them has been shown to reach the cell surface. Downstream
folding effects may also explain the discrepancies in TGF-
binding
activity shown by mutants that look alike. A case in point is our
inactive
12 (
44-499) mutant (Figs. 1 and 3), which is very
similar to the active
44-564 and
44-575 mutants published by
the group of O'Connor-McCourt (25). The removal of the first two
cysteines that define the uromodulin similarity of the U-related
region, as in mutant
12, renders this TGF-
binding region
inactive. Surprisingly, O'Connor's group has found that the
additional removal of the third or the third and fourth of these
cysteines, as in their mutants
44-564 and
44-575, do not
affect the binding activity of the U-related region (25). This would
suggest that downstream folding effects impair the major determinants
of the TGF-
binding activity of the U-related region in a very
unpredictable manner. Also, these results would indicate that the last
213 residues of the U-related region are the most relevant for its
TGF-
binding activity; unfortunately, further deletion of residues
within this region has been uninformative. The inactivity of their
44-596 mutant has been attributed to improper maturation of the mutant
receptor, making it difficult to assess its intrinsic TGF-
binding
activity (26). In view of the shortcomings of the analysis by deletion
mutants, it is likely that the precise determination of the residues
directly involved in the TGF-
-binding activity of betaglycan E- and
U-related regions will require the use of biophysical techniques. These techniques, such as x-ray diffraction or nuclear magnetic resonance could be employed with our purified soluble
10 and
11 mutants.
Another issue addressed in this work is the TGF-
2 presentation
activity of the betaglycan TGF-
binding regions. We and others have
demonstrated that the endogenous low affinity that the type II receptor
has for TGF-
2 is compensated by the TGF-
presentation activity of
the membrane-bound wild type betaglycan (18, 20). We evaluated the
TGF-
2 presentation activity of the betaglycan TGF-
binding
regions by determining the extent of TGF-
2 labeling that they confer
to the type II receptor. For that we performed two kinds of
experiments; in one we used epitope-tagged receptors in transiently
transfected COS-1 cells (Fig. 3, A and B), and in
another we used stably expressed untagged receptors in L6E9 cells (Fig.
3C). Both experiments led, essentially, to the same conclusion; the TGF-
binding regions of betaglycan have opposite ability to present this TGF-
isoform to the type II receptor. In
both experiments the basal level of TGF-
2 binding of type II
receptor, either the one endogenously present in the L6E9 cells or the
one transiently transfected in COS-1 cells, is negligible in the
absence of betaglycan (Fig. 3, B and C). In COS-1
cells, co-transfection with the wild type betaglycan or its
gag
mutant greatly increased the TGF-
2 labeling of the
type II receptor. Co-expression of
11, the betaglycan mutant
encoding the complete U-related region, did not promote the same level
of binding. At best,
11 improved the levels obtained with mutants
devoid of TGF-
binding activity, which may be regarded as
background. Similarly, when the
11 mutant was expressed in L6E9
cells, it did not increase the TGF-
2 binding of the type II
receptor. On the other hand, in COS-1 cells, co-expression of
10 or
3 betaglycan mutants encoding the complete E-related region promoted
the type II receptor TGF-
2 binding at levels even higher than those
observed with the wild type betaglycan. In the L6E9 cells, the
expression of the
10 mutant also increased the labeling of the type
II receptor; however, in this case the wild type betaglycan was a
better TGF-
2 presenter than the
10 mutant. The slightly better
labeling of the L6E9 endogenous TGF-
type II receptor in the
presence of the wild type betaglycan than in the presence of the
10
mutant contrasts with the opposite situation in the COS-1 cells
experiment (compare the
10 and WT
lanes in Figs. 3, B and C). However,
very little can be said about the structure or nature of the
interacting receptors based on this result. The experiments shown in
Figs. 3, B and C correspond to different type of
assays. In one we used a transiently expressed, tagged human type II
receptor, whereas in the other, an untagged endogenous rat receptor was
evaluated. These facts, added to unknown TGF-
cross-linking
efficiencies for these receptors, would make any quantitative
conclusion derived from these results unreliable. Nonetheless, the
finding that the E-related region confers better TGF-
2 labeling of
the type II receptor than the U-related region is evidenced by the two
types of assays shown in Fig. 3. Based on these observations, it was reasonable to guess that the E-related region would be responsible of
the TGF-
2 functional enhancement property of the wild type betaglycan. Contrary to this expectation, our experiments measuring the
TGF-
2-dependent Smad2 phosphorylation indicated that
both ligand binding regions were equally capable of performing that function (Fig. 4). This fact raises a paradox which indicates that the
so-called TGF-
presentation function is, very likely, an artifact of
the cross-linking step during the affinity labeling. These results also
indicate that without sustaining functional experiments, affinity
labeling data should be interpreted very cautiously.
The last issue addressed in this work is related to the betaglycan
inhibin A binding ability. Here we have shown that the intrinsic
inhibin A binding capacity of betaglycan is confined to the U-related
ligand binding region and that the GAG chains are dispensable for this
capacity. Additionally, these findings suggest that the U-related
region is the one that creates with the type II activin receptor the
high affinity inhibin binding site, and that is the most likely
candidate to mediate the inhibin antagonism of activin. This raises
another question of why two polypeptide sequences so distinct
from each other, as is the case for the E- and U-related
regions, have such peculiar ligand binding properties. Their similar
TGF-
binding and functional properties, their different inhibin A
binding abilities, plus the fact that they are confined to discrete
portions of the co-receptor would support the hypothesis that the E-
and U-related regions reflect two independently folded domains of
betaglycan. These facts also would favor the hypothesis that the
TGF-
2-enhancing effect of betaglycan results from a simple
ligand-concentrating mechanism; that is, that wild type receptor or its
independently expressed ligand binding regions simply increase the
local TGF-
2 concentration in the neighborhood of the TGF-
type II
receptor. However, with the currently available evidence, the argument
for a specific more-favorable TGF-
2 conformation, imposed by
betaglycan or any of its ligand binding regions, cannot be ruled out.
This last possibility is especially enticing due to the fact that only
one of the regions is capable of binding inhibin A, which would suggest that very specific ligand-receptor interactions occur for each region.
A more conclusive answer to these questions will have to wait for
biophysical structural studies of betaglycan ligand binding regions.