Ligand Binding and Functional Properties of Betaglycan, a Co-receptor of the Transforming Growth Factor-beta Superfamily

SPECIALIZED BINDING REGIONS FOR TRANSFORMING GROWTH FACTOR-beta AND INHIBIN A*

José Esparza-LópezDagger , José Luis MontielDagger , M. Magdalena Vilchis-LanderosDagger , Toshihide Okadome§, Kohei Miyazono§, and Fernando López-CasillasDagger

From the Dagger  Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, UNAM Apartado Postal 70-246, México City, D. F., 04510, México and § The Cancer Institute, Tokyo, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan

Received for publication, September 28, 2000, and in revised form, December 28, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Betaglycan, also known as the transforming growth factor-beta (TGF-beta ) type III receptor, is a membrane-anchored proteoglycan that binds TGF-beta via its core protein. Deletion mutagenesis analysis has revealed two regions of betaglycan ectodomain capable of binding TGF-beta : one at the amino-terminal half, the endoglin-related region (López-Casillas, F., Payne, H., Andres, J. L., and Massagué, J. (1994) J. Cell Biol. 124, 557-568), and the other at the carboxyl-terminal half, the uromodulin-related region (Pepin, M.-C., Beauchemin, M., Plamondon, J., and O'Connor-McCourt, M. D. (1994) Proc. Natl. Acad. Sci. U. S. A 91, 6997-7001). In the present work we have functionally characterized these ligand binding regions. Similar to the wild type receptor, both regions bind TGF-beta 2 with higher affinity than TGF-beta 1. However, only the endoglin-related region increases the TGF-beta 2 labeling of the TGF-beta type II receptor, the so-called "TGF-beta -presentation" function of the wild type receptor. Despite this preference, both regions as well as the wild type receptor mediate the TGF-beta 2-dependent Smad2 phosphorylation, indicating that they can function indistinguishably as TGF-beta -enhancing co-receptors. On the other hand, we found that the recently described ability of the wild type betaglycan to bind inhibin A is a property of the core protein that resides in the uromodulin-related region. Binding competition experiments indicate that this region binds inhibin and TGF-beta with the following relative affinities: TGF-beta 2 > inhibin A > TGF-beta 1. All together, the present results suggest that betaglycan ectodomain is endowed with two bona fide independent ligand binding domains that can perform specialized functions as co-receptors of distinct members of the TGF-beta superfamily.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor-beta (TGF-beta )1 is the prototype of a superfamily of growth factors involved in the regulation of cell proliferation, differentiation, and development (1, 2). TGF-beta signals through a complex of transmembrane serine/threonine kinase receptors, the TGF-beta 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-beta signal into the cell nucleus (3, 4).

TGF-beta has two known co-receptors, betaglycan and endoglin, which are transmembrane glycoproteins with large extracellular regions that bind TGF-beta 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-beta isoforms (9-11). Betaglycan is capable of fine tuning the availability of TGF-beta to the signaling receptors, thereby determining the outcome of the TGF-beta stimulation (12, 13). This regulation is both positive and negative. Although the membrane-bound form of betaglycan increases the binding of TGF-beta to the signaling complex, the soluble form of betaglycan prevents this binding and therefore blocks the actions of TGF-beta (14). These effects are more dramatic for TGF-beta 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-beta 2 and corrects for their low sensitivity to this TGF-beta isoform (18, 19). Presumably, this effect is mediated by a TGF-beta -induced "presentation complex" formed between membrane-bound betaglycan and the TGF-beta 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-beta signaling.

Betaglycan also interacts with type II receptors of another TGF-beta 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-beta superfamily and open the question of the nature of the structural determinants that make this versatility possible.

The TGF-beta 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-beta binding and TGF-beta 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-beta isoform affinities and TGF-beta -enhancing function but differ in their ability to bind inhibin A.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- TGF-beta 1 and inhibin A were from R&D Systems (Minneapolis, MN); TGF-beta 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-beta 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 Delta 2, Delta 3, Delta 10, and gag- has been described (14). Mutant Delta 11 (Delta 45-409) was constructed from our previously described Delta 8 (Delta 45-282) mutant. The construction of Delta 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 Delta 8 intended changes, a novel NaeI site (14). For the creation of Delta 11, Delta 8 was cut at the EcoRV site and at its engineered NaeI site, and the resulting large restriction fragment was self-ligated. Mutant Delta 12 (Delta 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 Delta 10 and Delta 11 betaglycan mutants were created and used in diverse experiments as indicated in the figure legends. The soluble versions of the Delta 10 and Delta 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 Delta 10, the oligonucleotide was inserted between the EcoRV and NcoI sites of wild type c-Myc-tagged betaglycan, and for soluble Delta 11, at the NcoI and AvrII sites of c-Myc-tagged Delta 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 Delta 10 and Delta 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 Delta 10 and Delta 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 Delta 10 and Delta 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 Delta 10 and Delta 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 Delta 10 or Delta 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 Delta 10 or Delta 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 Delta 10 and Delta 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-beta 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-beta affinity labeling in solution was done with the purified soluble Delta 10 or Delta 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-beta 2 or TGF-beta 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 beta -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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Betaglycan Deletion Mutagenesis Has Identified 2 Independent TGF-beta 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-beta 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-beta 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 Delta 10 and Delta 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-beta 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-beta 1 (B) or 200 pM 125I-labeled TGF-beta 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.

To determine the functional properties of betaglycan TGF-beta binding regions, we decided to test their ligand binding affinities, TGF-beta 2 presentation, and TGF-beta 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 Delta 11 and Delta 12 lack residues 45-409 (Delta 45-409) and 44-499 (Delta 44-499) of wild type rat betaglycan, respectively (Fig. 1A). Mutants Delta 11 and Delta 12 are complementary to the set that we have described before (14). In particular Delta 11, which lacks the entire amino-terminal half, is the complement of our previous Delta 10 (Delta 410-781) mutant, which lacks the entire carboxyl-terminal half of the ectodomain. Mutants Delta 11 and Delta 12 along with the wild type betaglycan and our previously characterized Delta 2 (Delta 200-500), Delta 3 (Delta 499-783), Delta 10, and gag- mutants (14), were tested for TGF-beta binding activity. For that purpose, COS-1 cells transiently transfected with these mutant vectors were affinity-labeled with 125I-labeled TGF-beta 1 or 125I-labeled TGF-beta 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-beta binding activity (14). Mutants Delta 10 and Delta 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-, Delta 10, and Delta 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 Delta 11 mutant, which includes all the 10 cysteines residues that define the uromodulin similarity (24), also bound TGF-beta 1 and TGF-beta 2 (Figs. 1, B, lane 6, and C, lane 4). The protein expressed by Delta 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 Delta 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-beta isoforms (Fig. 1B, lane 8, and data not shown). In addition, the empty pCMV5 vector and our previously reported Delta 2 mutant were included in these experiments as negative controls. The TGF-beta binding incapacity of Delta 2 and Delta 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-beta binding activity. The functional characterization of these regions, which are located between residues 45-410 and 410-781 and are present in our Delta 10 and in Delta 11 mutants, respectively, will be presented in the following sections.

Both Betaglycan Ligand Binding Regions Bind TGF-beta 1 and TGF-beta 2 with Relative Affinities Similar to the Wild Type Receptor-- As a first step to characterize the betaglycan TGF-beta binding regions, we assayed the Delta 10 and Delta 11 mutants in binding competitions with the beta 1 and beta 2 isoforms. As a reference, we also tested the wild type betaglycan, which has a characteristic higher affinity for TGF-beta 2 that is revealed by a very weak TGF-beta 2 binding competition by the other isoforms (15-17). For this purpose, COS-1 cells were transfected with the wild type betaglycan, the Delta 10, or the Delta 11, pCMV5-based constructs and subjected to a 125I-labeled TGF-beta 2 binding assay in the presence of increasing concentrations of competing unlabeled TGF-beta 1 or TGF-beta 2. As expected for the wild type betaglycan, 50% of its binding of 50 pM 125I-labeled TGF-beta 2 was prevented by ~3 nM cold TGF-beta 2, whereas it was not affected by concentrations as high as 10 nM cold TGF-beta 1 (Fig. 2A, WT). The Delta 10 and Delta 11 mutants exhibited 125I-labeled TGF-beta 2 total binding and competition patterns that are similar to those observed in the intact receptor, indicating that they have the same TGF-beta isoform relative affinities as the wild type betaglycan (Fig. 2A, Delta 10 and Delta 11).


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Fig. 2.   TGF-beta 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-beta 2 in the presence of the indicated concentrations (0, 1, 3, or 10 nM) of cold TGF-beta 1 (open symbols) or TGF-beta 2 (closed symbols). The bound iodinated ligand is plotted against the concentration of the unlabeled competitor. B, purified soluble Delta 10 (Sol Delta 10) or soluble Delta 11 (Sol Delta 11) proteins (50 ng/assay) were subjected to affinity-labeling in solution with 100 pM 125I-labeled TGF-beta 2 in the absence (C) or presence of the indicated concentration of competing unlabeled TGF-beta 1 or TGF-beta 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 Delta 10 and soluble Delta 11 using the ImageQuant Software and plotted against the competitor TGF-beta concentration (right panels). WT, wild type betaglycan.

To demonstrate that these TGF-beta binding properties are intrinsic to these regions, we did a similar 125I-labeled TGF-beta 2 binding competition analysis with the purified soluble forms of the Delta 10 and Delta 11 mutants (Fig. 2B). For this purpose we created baculoviral strains of soluble Delta 10 and Delta 11 whose protein products could be secreted into the conditioned media of infected insect cells. Since the soluble Delta 10 and soluble Delta 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 Delta 10 and soluble Delta 11 proteins were subjected to affinity labeling in solution with a constant amount of 125I-labeled TGF-beta 2 and increasing amounts (from 0-2 nM) of competing unlabeled TGF-beta 1, or TGF-beta 2. Purified soluble Delta 10 and soluble Delta 11 are glycoproteins with a molecular mass of 52 and 63 kDa, respectively (data not shown), which after 125I-labeled TGF-beta 2 cross-linking, migrate just below and above and 64-kDa marker (Fig. 2B). The amount of labeled soluble Delta 10 and soluble Delta 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-beta 2 labeling was at least one order of magnitude higher than the TGF-beta 1 heterologous competition. Taken together, the data suggested that the relative affinities of both membrane-bound and soluble Delta 10 and Delta 11 mutants for TGF-beta 1 and TGF-beta 2 are very similar to each other and to the wild type betaglycan.

TGF-beta 2 Presentation Function of Betaglycan Ligand Binding Regions-- Since both betaglycan TGF-beta binding regions have TGF-beta 2 binding affinities that are comparable with those of the intact receptor, we decided to test whether or not they conserved the TGF-beta 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-beta 2 labeling of the TGF-beta type II receptor. Presumably, this function accounts for the enhanced potency of the TGF-beta 2 isoform in the presence of betaglycan (18). To measure the TGF-beta 2 presentation function of betaglycan TGF-beta binding regions, we co-expressed a few of our c-Myc-tagged mutants along with the human TGF-beta 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-beta 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-beta 2-labeled c-Myc-betaglycan mutants (Fig. 3A) exhibit gel migration patterns similar to those observed with TGF-beta 1 (Fig. 1B). This suggests that the co-expression of the TGF-beta 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-beta 2 binding activity (lane 1, Fig. 3B). Similarly, co-transfection with Delta 2 or Delta 12, mutants without TGF-beta 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 Delta 11, which had a level of TGF-beta 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 Delta 3 or Delta 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-beta 2, the Delta 3, Delta 10, and Delta 11 betaglycan mutants are not equal in their ability to "present" this isoform to the TGF-beta type II receptor.


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Fig. 3.   TGF-beta 2 presentation activity of betaglycan mutants. A and B, COS-1 cells were transiently co-transfected with the HA1 epitope-tagged TGF-beta type II receptor (Tbeta 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-beta 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-beta 2. Equivalent aliquots of their cell lysates were immunoprecipitated with the anti-betaglycan antiserum (#822) or with the anti-TGF-beta 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.

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-beta 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 Delta 10 and Delta 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-beta 2, and the identity of the labeled products was revealed by specific immunoprecipitation under nondenaturing conditions (Fig. 3C). At this TGF-beta 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-beta 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-beta 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 Delta 10 was capable of increasing the TGF-beta 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 Delta 11 mutant efficiently bound TGF-beta 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-beta 2-dependent Smad2 Phosphorylation-- The data presented in Fig. 3 indicated that the TGF-beta 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-beta 2 signals. To evaluate this possibility, we decided to determine how the expression of the Delta 10 and Delta 11 mutants affected the TGF-beta 2-induced Smad2 phosphorylation in L6E9 cells. We chose to measure Smad2 phosphorylation because it is one of the earliest steps in TGF-beta signaling and could most directly reveal if the TGF-beta 2 presentation function of betaglycan had any effect in the TGF-beta 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-beta 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-beta 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 Delta 10 or Delta 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-beta 2-induced Smad2 phosphorylation. Importantly, this short term exposure and limiting concentration of TGF-beta 2 was required to demonstrate any betaglycan dependence of the Smad2 phosphorylation. Similar treatment with TGF-beta 1 or longer treatment with higher concentrations of TGF-beta 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-beta presentation ability, both enhance the TGF-beta signal as well as the wild type betaglycan. In addition, this enhancement occurs in a TGF-beta isoform-specific manner and only under limiting concentrations of the factor.


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Fig. 4.   TGF-beta 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-beta 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.

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-beta 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 Delta 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 Delta 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 Delta 11 vectors. However, the Delta 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.

To probe the relative affinities of this region for its TGF-beta superfamily ligands, the binding of 50 pM 125I-labeled inhibin A was competed with a 10- and 100-fold excess of unlabeled TGF-beta 1, TGF-beta 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-beta 1 could only partially compete the inhibin A binding. On the other hand, TGF-beta 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-beta 2 to wild type betaglycan or its gag-, Delta 10, and Delta 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-beta , the U-related region also binds inhibin A with the following relative affinities: TGF-beta 2 > inhibin A > TGF-beta 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-beta 1 (cross-hatched bars), TGF-beta 2 (dotted bars), or inhibin A (empty bars). The bound iodinated ligand is plotted against the indicated unlabeled competitor. WT, wild type betaglycan.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta superfamily. In one hand, membrane-bound betaglycan positively regulates TGF-beta , 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-beta with high affinity has been found in serum and extracellular matrix (45). A recombinant version of soluble betaglycan binds, sequesters, and thereby antagonizes TGF-beta , 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-beta 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-beta 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-beta binding activity. Several groups have used deletion mutants of rat betaglycan to map betaglycan TGF-beta binding regions (14, 25-29). This experimental approach has revealed two ample portions of betaglycan with TGF-beta 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 (Delta 11 and Delta 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-beta affinity labeling of our new and a few selected old mutants (Figs. 1, B and C, and 3A) confirmed the presence of TGF-beta binding activity in the E-related region, present in mutants Delta 3, Delta 10, and in the U-related region, present intact in mutant Delta 11. In addition, we have found that these regions have TGF-beta binding affinities that are very similar to the intact receptor. Ligand binding competitions (Fig. 2) indicate that Delta 10 and Delta 11 exhibit the same relative TGF-beta affinities, approximately one order of magnitude higher for TGF-beta 2 than for TGF-beta 1. This TGF-beta 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-beta 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 Delta 1 (Delta 45-199), Delta 5 (Delta 200-285), Delta 8 (Delta 45-282), and Delta 9 (Delta 287-409) mutants, which despite having complete the U-related region, do not bind TGF-beta (14). A plausible explanation for this inability is that the portions of the E-related region that were not deleted in Delta 1, Delta 5, Delta 8, and Delta 9 indirectly affect the TGF-beta 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-beta 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-beta binding activity shown by mutants that look alike. A case in point is our inactive Delta 12 (Delta 44-499) mutant (Figs. 1 and 3), which is very similar to the active Delta  44-564 and Delta  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 Delta 12, renders this TGF-beta 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 Delta  44-564 and Delta  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-beta 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-beta binding activity; unfortunately, further deletion of residues within this region has been uninformative. The inactivity of their Delta  44-596 mutant has been attributed to improper maturation of the mutant receptor, making it difficult to assess its intrinsic TGF-beta 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-beta -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 Delta 10 and Delta 11 mutants.

Another issue addressed in this work is the TGF-beta 2 presentation activity of the betaglycan TGF-beta binding regions. We and others have demonstrated that the endogenous low affinity that the type II receptor has for TGF-beta 2 is compensated by the TGF-beta presentation activity of the membrane-bound wild type betaglycan (18, 20). We evaluated the TGF-beta 2 presentation activity of the betaglycan TGF-beta binding regions by determining the extent of TGF-beta 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-beta binding regions of betaglycan have opposite ability to present this TGF-beta isoform to the type II receptor. In both experiments the basal level of TGF-beta 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-beta 2 labeling of the type II receptor. Co-expression of Delta 11, the betaglycan mutant encoding the complete U-related region, did not promote the same level of binding. At best, Delta 11 improved the levels obtained with mutants devoid of TGF-beta binding activity, which may be regarded as background. Similarly, when the Delta 11 mutant was expressed in L6E9 cells, it did not increase the TGF-beta 2 binding of the type II receptor. On the other hand, in COS-1 cells, co-expression of Delta 10 or Delta 3 betaglycan mutants encoding the complete E-related region promoted the type II receptor TGF-beta 2 binding at levels even higher than those observed with the wild type betaglycan. In the L6E9 cells, the expression of the Delta 10 mutant also increased the labeling of the type II receptor; however, in this case the wild type betaglycan was a better TGF-beta 2 presenter than the Delta 10 mutant. The slightly better labeling of the L6E9 endogenous TGF-beta type II receptor in the presence of the wild type betaglycan than in the presence of the Delta 10 mutant contrasts with the opposite situation in the COS-1 cells experiment (compare the Delta 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-beta 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-beta 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-beta 2 functional enhancement property of the wild type betaglycan. Contrary to this expectation, our experiments measuring the TGF-beta 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-beta 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-beta 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-beta 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-beta 2 concentration in the neighborhood of the TGF-beta type II receptor. However, with the currently available evidence, the argument for a specific more-favorable TGF-beta 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.

    ACKNOWLEDGEMENTS

We thank P. ten Dijke, C.-H. Heldin, and J. Massagué and J. A. García-Sainz for helpful suggestions and comments, Valentín Mendoza for superb technical assistance, N. Cerletti (Ciba-Geigy AG) for the generous gift of TGF-beta , and C.-H. Heldin and J. L. Wrana for the generous gift of anti-Smad2 antibodies.

    FOOTNOTES

* This work was supported in part by grants from Consejo Nacional de Ciencia y Tecnología, México, Dirección General de Apoyo al Personal Académico, UNAM, the Howard Hughes Medical Institute, and the International Center for Genetic Engineering and Biotechnology, Italy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

An International Research Scholar of the Howard Hughes Medical Institute. To whom correspondence should be addressed. Tel.: 52-56-22-56-25; Fax: 52-56-22-56-11; E-mail: fcasilla@ifisiol.unam.mx.

Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M008866200

    ABBREVIATIONS

The abbreviations used are: TGF-beta : transforming growth factor-beta , GAG, glycosaminoglycan chains; E-related, endoglin-related; U-related, uromodulin-related; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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