©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Soluble Exoplasmic Domain of the Type II Transforming Growth Factor (TGF)- Receptor
A HETEROGENEOUSLY GLYCOSYLATED PROTEIN WITH HIGH AFFINITY AND SELECTIVITY FOR TGF-beta LIGANDS (*)

(Received for publication, July 28, 1994; and in revised form, December 7, 1994)

Herbert Y. Lin (1) (2)(§) Aristidis Moustakas (1)(¶) Petra Knaus (1)(**) Rebecca G. Wells (1) (4)(§§) Yoav I. Henis (1) (3) Harvey F. Lodish (1) (2)(¶¶)

From the  (1)Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, (2)Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, (3)Department of Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel, and (4)Division of Gastroenterology, Brigham and Women's Hospital, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The transforming growth factor (TGF)-beta type II receptor is a transmembrane serine/threonine kinase which is essential for all TGF-beta-induced signals. In several cell types TGF-beta2 is as potent as TGF-beta1 or TGF-beta3 in inducing cellular responses, yet TGF-beta2 does not bind to the majority of expressed type II receptors. Here we characterized the properties of the soluble extracellular domain of the human TGF-beta type II receptor synthesized in COS-7 cells. Like the membrane-attached type II receptor, the soluble receptor contains complex N-linked oligosaccharides as well as additional sialic acid residues that cause it to migrate heterogenously upon SDS-polyacrylamide gel electrophoresis. I-TGF-beta1 binds to and is chemically cross-linked to this protein. Unlabeled TGF-beta1 inhibits the binding of I-TGF-beta1 with an apparent dissociation constant (K) of 200 pM, similar to the apparent K ( 50 pM) of the cell-surface type II receptor. TGF-beta3 inhibits the binding of I-TGF-beta1 to the soluble type II receptor with a similar dissociation constant, 500 pM. In contrast, I-TGF-beta2 cannot bind and be chemically cross-linked to the soluble type II receptor, nor does as much as a 125-fold excess of unlabeled TGF-beta2 inhibit the binding of I-TGF-beta1 to the soluble receptor. This is the first demonstration of the binding affinities of the type II receptor in the absence of the other cell-surface molecules known to bind TGF-beta. Expressed alone in COS-7 cells the type II receptor also cannot bind TGF-beta2; co-expression of type III receptor enables the type II receptor to bind TGF-beta2. Thus, the type III receptor or some other component is required for transmission of TGF-beta2-induced signals by the type II receptor.


INTRODUCTION

The transforming growth factor (TGF)^1-beta family of hormones has important functions in growth, development, and differentiation(1, 2) . The three mammalian TGF-beta isoforms, TGF-beta1, -beta2, and -beta3, share 71-76% amino acid identities. Although homodimers are the predominant species, the heterodimers TGF-beta1.2 and TGF-beta2.3 have been described(3, 4) . The sequences of the mature, proteolytically processed forms of each TGF-beta family member are almost entirely conserved across species, and thus there has been evolutionary pressure to retain both the similarities and differences in these isoforms.

Most mammalian cells express three abundant high affinity receptors which can bind and be cross-linked to TGF-beta: the type I (53 kDa), type II (65 kDa), and type III (100-280 kDa) receptors, based upon the molecular mass of the cross-linked products analyzed by gel electrophoresis(5, 6) . While TGF-beta1 binds with high affinity (50-300 pM) to the types I, II, and III receptors, TGF-beta2 binds with high affinity only to the type III receptor, binding poorly to the majority of the type I and II receptors expressed on most mammalian cells. TGF-beta3 closely resembles TGF-beta1 in its binding characteristics to cell surface receptors, although TGF-beta3 may bind slightly less well than TGF-beta1 to the type I and II receptors.

The TGF-beta type III receptor is a membrane bound proteoglycan with a short cytoplasmic tail that has no apparent signaling motif(7, 8) . It binds TGF-beta2 (apparent K 100 pM) with slightly greater affinity than TGF-beta1 or TGF-beta3 (apparent K 300 pM)(9, 10, 11) . The type II receptor is a type I transmembrane protein with a cytosolic domain containing a serine/threonine kinase homologous to that in the activin and several other receptors(6, 12, 13, 14) . The type II receptor is essential for all known TGF-beta initiated signals(15, 16, 17) . The type II and type III receptors interact, as demonstrated by the enhanced ability of the type II receptor to bind I-TGF-beta1 when co-expressed in the presence of the type III receptor(8) . After cross-linking to I-TGF-beta1 or TGF-beta2, a fraction of the type II and type III receptors can be co-immunoprecipitated with antisera specific to either receptor type(9, 10) . Homo-oligomers, probably homodimers, of the types II and III receptors exist on the cell surface in the absence or presence of TGF-beta1 or TGF-beta2. Hetero-oligomers of the types II and III receptors are minor and probably transient species, most likely intermediates in the transfer of a TGF-beta ligand from a type III to a type II receptor(18) .

The type I receptors for TGF-betas and activin-A are also transmembrane serine-threonine kinases. The type II receptors require their corresponding type I receptors for signaling, while binding of TGF-betas or activin-A to the respective type I receptors requires co-expression of the corresponding type II receptor. Heteromeric complexes of the type II with type I receptors are found on the surface of many cells after ligand binding and may be important for signal transduction(15, 19, 20, 21, 22, 23, 24, 25, 26) .

If the type I and II receptors indeed mediate TGF-beta-induced signals, then it is puzzling that TGF-beta2 is equally potent as TGF-beta1 and TGF-beta3 in its ability to arrest the growth of cells (ED 5-20 pM)(27, 28) , since TGF-beta2 does not bind well to either the type I or the type II receptors. One possibility is that there exists a subpopulation of type I and II receptors which binds TGF-beta2 with high affinity (apparent K 25-50 pM)(29) . Another possibility is that the type III receptor, which binds TGF-beta2 with relatively high affinity (apparent K 100 pM), could present TGF-beta2 to the type I and/or type II receptors, increasing their affinity for TGF-beta2. Alternatively, another as yet undescribed cell surface component may interact with the type I and/or the type II receptors and increase their affinity for TGF-beta2. Finally, TGF-beta2 may bind to an undescribed receptor which cannot be chemically cross-linked to I-TGF-beta2 and therefore has escaped identification. However, TGF-beta2 does interact with the type II receptor, since reintroduction of the cloned type II receptor into mink lung cell mutants lacking the type II receptor restored the ability of these cells to be potently growth inhibited by TGF-beta2(15) .

To study the binding properties of the type II receptor in isolation, we constructed a truncated TGF-beta type II receptor cDNA which encodes the entire extracellular domain. Transfected COS-7 cells secreted the soluble receptor, a heterogenously glycosylated protein which binds to TGF-beta1 and TGF-beta3 with high affinity (estimated K 200 pM and 500 pM, respectively). In contrast, as much as 10 nM unlabeled TGF-beta2 could not inhibit binding of I-TGF-beta1, nor could I-TGF-beta2 be bound and chemically cross-linked to the soluble type II receptor. These results are consistent with the ability of TGF-beta1 and TGF-beta3 to bind directly to and signal through the high affinity type II receptor and suggest that at least one additional component is essential for binding and/or signal transduction by TGF-beta2. This additional component may be the type III receptor, as we show that co-expression of the type III receptor in COS-7 cells enables the cell surface type II receptor to bind to TGF-beta2.


MATERIALS AND METHODS

Construction of the Truncated Human TGF-beta Type II Receptor cDNA Encoding the Entire Extracellular Domain

A portion of the extracellular domain of the human TGF-beta type II receptor was amplified by PCR from a full-length human TGF-beta type II receptor cDNA, H2-3FF, in the vector pcDNA-1 (12) (see Fig. 1). The 5` sense primer was: 5`-AATCCTGCATGAGCAAC-3`, corresponding to bp 529-545 of the cDNA (encoding residues Lys to Asn). This immediately precedes a PstI site (bp 545-550) in the cDNA. The 3` antisense primer had the sequence 5`-TCTAGATCTAGACTAGTCAGGATTGCTGG-3`, representing bp 797-810, which encoded residues Thr to Asp, immediately preceded by an antisense stop codon (CTA, underlined) and two tandem XbaI sites (double underlined). TGF-beta type II receptor cDNA (1 µg) was mixed with 2 µM final concentration of each primer, 1.25 mM each of dATP, dCTP, dGTP, and dTTP, 2.5 units of Taq polymerase, and 10 µl of 10 times buffer provided by the PCR kit (Perkin-Elmer) in a total volume of 100 µl. Amplification was performed for 30 cycles (1 min, 94 °C; 2 min 55 °C; 1 min 72 °C) with a final extension at 72 °C for 5 min(30) . The amplified PCR product was digested with PstI and XbaI and ligated into the PstI site (bp 545-550) in the full-length cDNA clone and the XbaI site in the polylinker of the vector (pcDNA-1, InVitrogen, San Diego, CA), thus replacing the C terminus and 3`-untranslated region of the full-length type II receptor cDNA. The resulting truncated cDNA encoded a protein representing the entire extracellular domain of the human TGF-beta type II receptor. The last encoded residue is Asp. The portion of the truncated cDNA which was derived from the PCR reaction was sequenced (31) to confirm the fidelity of the PCR reaction.


Figure 1: Strategy for construction of a truncated cDNA encoding the extracellular domain of the human TGF-beta type II receptor (hTGF-betaRII). Synthetic PCR primers (half-arrows, A and B) (see ``Materials and Methods'') were used to amplify a portion of the human TGF-betaRII extracellular domain from the full-length cDNA clone, H2-3FF(12) . The amplified PCR product was digested with the restriction enzymes PstI and XbaI, and the digested fragment was ligated into the PstI site (bp 545-550) in the full-length cDNA clone and the XbaI site in the 3` poly-linker region of the vector (pcDNA-I, InVitrogen). The resulting truncated cDNA encoded a protein representing the entire extracellular domain of the human TGF-beta type II receptor. The last encoded residue of this soluble TGF-beta type II receptor (sTGF-betaRII) is Asp. Signal, hydrophobic signal sequence; TM, transmembrane domain; NH(2), N terminus; COOH, C terminus; UT, untranslated region. (Drawings are not to scale.)



Generation of Antipeptide Antibodies

Polyclonal rabbit antisera were raised against peptides specific for the N-terminal region (Lys to Ala, KSVNNDMIVTDNNGAC, with an additional cysteine residue not encoded by the receptor cDNA after Ala) and the C terminus (Cys to Lys, CSEEKIPEDGSLNTTK) of the human TGF-beta type II receptor. The peptides were coupled to keyhole limpet hemocyanin via the cysteine, and rabbits were injected and boosted five to seven times with 1 mg of keyhole limpet hemocyanin-peptide conjugate. The resultant antipeptide antibodies are referred to as ``-IIN'' and ``-IIC,'' specific for the N terminus and C terminus of the type II receptor, respectively. Immunoglobulins (IgGs) were prepared from the crude rabbit serum by ammonium sulfate precipitation followed by DEAE-cellulose chromatography (32) prior to use in immunoprecipitation reactions.

Transient Transfection of COS-7 Cells

COS-7 cells (American Type Culture Collection CRL 1651) were transfected with the plasmid pcDNA-1 or pcDNA-1-neo containing either the full-length human TGF-beta type II receptor cDNA (H2-3FF), the full-length rat TGF-beta type III receptor (R3-0FF), or the truncated TGF-beta type II receptor cDNA encoding the soluble extracellular domain of the type II receptor, using the DEAE-dextran/chloroquine method described previously(12) .

Metabolic Labeling of Soluble Type II Receptor

COS7 cells were transfected with plasmid DNA and labeled approximately 48 h post transfection. They were rinsed once with PBS and placed in Dulbecco's modified Eagle's medium minus cysteine and methionine with 0.5 mCi/ml of a mixture of [S]cysteine and [S]methionine (Express, DuPont-NEN) for 4 h at 37 °C. The labeling medium was collected, chilled on ice and filtered (pore size 0.22 mM) to remove loose cells and debris. Phenylmethylsulfonyl fluoride was added to 1 mM. The medium was then concentrated in a Centricon 10 column (Amicon), divided into equal aliquots for immunoprecipitation, and brought up in lysis buffer (0.5% deoxycholate, 1% Triton X-100, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride in PBS).

In Vitro Translation

The cDNA encoding the soluble type II receptor was cloned into pcDNA-1 and was transcribed in vitro into mRNA, using the T7 RNA polymerase system (Stratagene) and the protocols detailed by the manufacturer. It was translated in a rabbit reticulocyte lysate (Promega) in the presence of [S]methionine (Amersham Corp.) and canine pancreatic microsomes(33) . The microsomal fraction was isolated by centrifugation through a sucrose cushion (0.5 M sucrose, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl) in a TL100 centrifuge (Beckman) at 100,000 rpm for 25 min. The microsomal pellet was lysed in lysis buffer in preparation for immunoprecipitation.

Immunoprecipitation of S-Labeled Soluble Type II Receptor and Digestion with Glycosidases

Samples in lysis buffer were incubated at 4 °C overnight with 30-50 µg/ml IgG fraction from alpha-IIN or alpha-IIC antipeptide antibodies. 50 µl of protein A-Sepharose (Sigma; a 1:1 v/v mixture with PBS) was added per ml of immunoprecipitation reaction; the samples were incubated for an additional 30 min. The beads were pelleted by brief centrifugation, washed three times with lysis buffer containing SDS (0.2% for alpha-IIN, 0.5% for alpha-IIC), then twice with PBS. Samples were eluted by boiling in 0.5% SDS.

Indicated samples were treated with glycopeptidase F (New England Biolabs) by adding 2000 units of enzyme/50 µl of reaction volume in a buffer containing (final concentrations) 0.5% SDS, 1% Nonidet P-40, and 50 mM sodium phosphate, pH 7.5. The samples were incubated at 37 °C overnight. Samples treated with neuraminidase were incubated in glycopeptidase F reaction buffer in the presence of 0.05 unit of neuraminidase (Genzyme) for 1 h at 37 °C, at which point glycopeptidase F was added to indicated samples, and the incubation was continued overnight. Samples were brought up in SDS-PAGE sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and 100 µg/ml bromphenol blue) and analyzed on 15% polyacrylamide, 1.2% bisacrylamide gels.

Chemical Cross-linking of 125I-TGF-beta1 to Soluble Receptors

TGF-beta1 was iodinated by the chloramine T method as described(34) . Forty-eight hours post-transfection COS-7 cells were rinsed twice with PBS and placed in serum-free medium (4-5 ml of Dulbecco's modified Eagle's medium/100-mm dish) overnight at 37 °C. This conditioned medium was collected and filtered (pore size: 0.22 µm) to remove loose cells and debris before being chilled on ice and equilibrated with Hepes (20 mM, pH 7.4) and phenylmethylsulfonyl fluoride (1 mM). The conditioned medium was divided into equal aliquots and I-TGF-beta1 or I-TGF-beta2 was added in the presence of various concentrations of different unlabeled TGF-beta isoforms as noted in the figure legends. After incubation overnight at 4 °C with constant rotation, soluble receptors in the conditioned medium were chemically cross-linked with 60 µg/ml DSS for 15 min at 4 °C with rotation; the reaction was stopped by addition of 2 mM ethanolamine. Samples were then either immunoprecipitated or incubated with concanavalin A.

Immunoprecipitation of Affinity-labeled Soluble Type II Receptors

Cross-linked samples were immunoprecipitated with alpha-IIN (100 µg of IgG/ml). After incubation overnight at 4 °C with rotation, 50 µl of protein A-Sepharose CL-4B (Sigma) (1:1, v/v with PBS) were added, and incubation was continued for an additional 30 min. Protein A-Sepharose beads were then pelleted by a brief centrifugation and washed three times with PBS, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.2% SDS, and then two times with PBS, 1% Triton X-100, 0.5% sodium deoxycholate without SDS. Proteins attached to the beads were solubilized in SDS-PAGE sample buffer before being boiled for 5 min and resolved by SDS-PAGE on 10% gels under reducing conditions.

Binding to Concanavalin A-Sepharose

Fifty µl of concanavalin A-Sepharose CL-4B (1:1, v/v in PBS) (Pharmacia) were added to 1 ml of cross-linked sample in the presence of 2 mM manganese chloride, and allowed to incubate overnight with rotation at 4 °C. Concanavalin A-Sepharose beads were then pelleted by a brief centrifugation and treated exactly as described above for protein A-Sepharose.

Generation of a Stably Transfected SW480 Cell Line Expressing the Human Type II Receptor

SW480 human colon adenocarcinoma cells (ATCC-CCL 228) were transfected using the calcium phosphate method (35) with the human TGF-beta type II receptor cloned into the EcoRI site in the polylinker region of the vector pcDNA-1-neo (InVitrogen). Individual G418-resistant colonies were selected and amplified; one clone, H216, was selected for further study.

Chemical Cross-linking of I-TGF-beta1 and I-TGF-beta2 to Cells

SW480 and H216 cells were grown to confluence on 60-mm tissue culture plates. COS-7 cells were used 48 h after transfection. Cells were rinsed twice with PBS, pH 7.4, and preincubated with binding buffer consisting of KRH buffer (50 mM Hepes (pH 7.5), 128 mM NaCl, 1.3 mM CaCl(2), 5 mM MgSO(4), 5 mM KCl) with 0.5% bovine serum albumin for 30 min at 37 °C. Cells were washed five times in ice-cold binding buffer, then incubated in binding buffer at 4 °C for 3.5 h with rotation in the presence of 100 pMI-TGF-beta1 and with various concentrations of unlabeled TGF-beta isoforms or (see Fig. 8) in the presence of different concentrations of I-TGF-beta2. Cells were then washed five times with ice-cold KRH buffer, followed by cross-linking with 60 µg/ml DSS in KRH buffer under constant rotation for 15 min at 4 °C. Cells were scraped into 1 ml of KRH buffer and were pelleted by a brief microcentrifugation (1,500 rpm, 5 min). Cell pellets were dissolved in 100 µl of lysis buffer containing detergent and protease inhibitors (PBS, 1% Triton X-100, 0.5% deoxycholate, 1 mM EDTA (pH 8.0), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, 10 µg/ml leupeptin, 10 µg/ml antipain, 10 µg/ml benzamidine hydrochloride, 10 µg/ml soybean trypsin inhibitor, and 10 µg/ml aprotonin), and membrane proteins were solubilized by rotation at 4 °C for 40 min. Insoluble material was pelleted by microcentrifugation at 14,000 rpm for 10 min at 4 °C and discarded. Samples were then analyzed by SDS-PAGE on a 8% gel under reducing conditions.


Figure 8: Cross-linking of I-TGF-beta2 to COS-7 cells co-transfected with the type II and III receptors. COS-7 cells were transfected with the type II receptor (lanes 1-4), the type II and type III receptors (lanes 5-7), or the type III receptor alone (lanes 8-12). Forty-eight hours post-transfection, cells were incubated with the following concentrations of I-TGF-beta2: 50 pM, lanes 1, 5, AND 8; 100 PM, lane 9; 200 PM, lanes 2, 6, AND 10; 500 PM, lanes 3, 7, AND 11; 1.0 NM, lanes 4AND 12. AFTER CHEMICAL CROSS-LINKING WITH DSS, THE SAMPLES WERE SOLUBILIZED AND ANALYZED BY SDS-PAGE. THE TYPE II RECEPTOR, TYPE III RECEPTOR, I-TGF-beta2, AND MOLECULAR MASS MARKERS ARE INDICATED ON THE left. UNCHARACTERIZED SMALLER MOLECULAR MASS SPECIES (U) ARE INDICATED BY THE brackets.



Processing and Quantification of Gels

Gels containing S-labeled proteins were fixed, soaked in 2,5-diphenyloxazole, dried, and exposed to pre-flashed Kodak XAR-5 film. Gels containing I-labeled proteins were dried, then exposed to pre-flashed Kodak XAR-5 film. Autoradiograms were scanned and quantitated on a ImageQuant densitometer (Molecular Dynamics).


RESULTS

A Soluble Type II Receptor is Secreted by COS Cells

COS cells were transfected with the expression plasmid pcDNA-1 containing a truncated TGF-beta type II receptor (amino acid residues 1-159; predicted molecular mass, 18 kDa) (Fig. 1). After cleavage of the hydrophobic leader sequence, the length of this truncated receptor is 136 amino acid residues, and the predicted molecular mass is 15.5 kDa. However, Fig. 2, lane 2, shows that transfected COS cells secrete little, if any, [S]methionine- and [S]cysteine-labeled proteins immunoreactive with the type II receptor antibody (alpha-IIN) that migrate on these reducing gels between 15 and 20 kDa. Instead, they secrete a heterogeneous species of 25-35 kDa, almost twice the expected size of the predicted protein core. This material is immunoprecipitated by an antipeptide antibody specific for the N-terminal region of the type II receptor, but not when immunogenic peptide is added to the immunoprecipitation reaction (compare lanes 2 and 1). No material resembling the soluble, secreted receptor is found in the medium of cells transfected with the full-length receptor (lanes 3 and 4). All of the other proteins seen in Fig. 2are nonspecific, background species (see legend).


Figure 2: Metabolic labeling of soluble TGF-beta type II receptor expressed in COS cells. COS cells were transfected with pcDNA-1 expressing either the truncated type II receptor (amino acids 1-159, lanes 1 and 2) or the full-length type II receptor cDNA (amino acids 1-567, lanes 3 and 4). Forty-eight hours after transfection, cells were labeled with 0.5 mCi/ml of a mixture of [S]cysteine and [S]methionine as described under ``Materials and Methods.'' Labeled media were immunoprecipitated with an antipeptide antibody specific for the N-terminal extracellular domain of the human type II receptor in the absence(-) or presence (+) of equimolar concentrations of immunogenic peptide (Peptide). After an overnight incubation at 4 °C, immunoprecipitated samples were analyzed by SDS-PAGE on a 11% gel, which was subjected to fluorography. The bracket shows the immunoprecipitated heterogeneous 25-35-kDa soluble type II receptor (lane 2), which is specifically competed by the presence of the immunogenic peptide in the immunoprecipitation reaction (lane 1). The arrow shows a nonspecific background protein species of 35 kDa which is present even in control samples (lanes 3 and 4) and is not competed by immunogenic peptide. The arrowhead shows two protein species of 46 kDa that are present only in control media, and only when immunogenic peptide is included in the immunoprecipitation reaction. Thus, these two are also background species.



The antipeptide antibodies we are using are highly specific. alpha-IIN, but not alpha-IIC, specific for the C terminus of the full-length type II receptor, precipitates the soluble receptor secreted by COS cells (Fig. 3, lanes 7 and 11); neither antibody immunoprecipitates material corresponding to the soluble form of the type II receptor from mock-transfected cells (lanes 1-6). alpha-IIN (Fig. 3, lanes 13-16), but not alpha-IIC (data not shown), immunoprecipitates the soluble type II receptor synthesized in a cell-free system in the presence of microsomes.


Figure 3: Deglycosylation of soluble TGF-beta type II receptor metabolically labeled in transfected cells and synthesized in a cell-free system. COS cells were transfected with pcDNA-1 alone (Mock, lanes 1-6) or pcDNA-1 expressing the truncated type II receptor (amino acid residues 1-159, lanes 7-12). As described under ``Materials and Methods,'' cells were labeled for 4 h with 0.5 mCi/ml of a mixture of [S]cysteine and [S]methionine. Medium from the labeled cells was immunoprecipitated with antipeptide antibodies specific for either the N-terminal extracellular domain (alpha-II N; lanes 1-4 and 7-10) or the C-terminal intracellular domain (alpha-II C; lanes 5, 6, 11, and 12). Immunoprecipitated samples were deglycosylated with glycopeptidase F alone (lanes 3, 6, 9, and 12), neuraminidase alone (lanes 2 and 8), or both (lanes 4 and 10), and then resolved by SDS-PAGE on a 15% polyacrylamide, 1.2% bisacrylamide gel. In vitro synthesized mRNA encoding the soluble receptor (lanes 13 and 14) or water (lane 15 and 16) underwent in vitro translation in the presence of microsomes; microsomal fractions were immunoprecipitated with the alpha-II N antibody. The samples in lanes 14 and 16 were treated with glycopeptidase F. Lanes 13-16 are from a longer exposure of the same gel as for lanes 1-12. The line (lane 7) indicates the smear representing the mature, complex glycosylated secreted form of the soluble receptor, which is reduced in size slightly by neuraminidase treatment (lane 8); the triangle between lanes 12 and 13 indicates a background protein of 32 kDa seen in mock-transfected samples. The small arrow in lane 7 shows a minor form of the soluble type II receptor with complex oligosaccharides. The small arrowhead in lane 9 represents a neuraminidase-sensitive form of the soluble receptor (most likely O-glycosylated) which is formed by digestion with glycopeptidase F; the large arrowheads in lanes 10 and 14 show the core receptor without signal peptide or carbohydrate; it is seen in media treated with neuraminidase and glycopeptidase F (lanes 9 and 10) and in the in vitro translation product treated with glycopeptidase F (lane 14). The large arrow represents a form of the receptor containing high mannose N-linked oligosaccharides that is generated in the in vitro translation reaction containing microsomes. The size (in kilodaltons) of molecular mass markers is noted in the left-hand margin.



Fig. 3shows that the soluble form of the type II receptor is heterogenously glycosylated, just as is the full-length type II receptor. (^2)In vitro translation of mRNA encoding the soluble type II receptor in a cell-free system containing microsomes generated a single predominant species of molecular size 28 kDa (lane 13). Potentially this species contains three N-linked oligosaccharides; digestion with glycopeptidase F (lane 14) or endoglycosidase H (data not shown) generated a single species of molecular size 19 kDa, corresponding to the soluble type II receptor without signal peptide or carbohydrate. Treatment of the heterogeneous form of the soluble type II receptor secreted by COS cells (lane 7) with glycopeptidase F yields the same 19-kDa species (large arrowhead) as well as a slower migrating protein (small arrowhead, lane 9). This latter species contains some form of sialic acid since digestion with glycopeptidase F together with neuraminidase yields only the 19-kDa species (lane 10). Clearly the secreted soluble type II receptor contains much sialic acid, since digestion with neuraminidase alone (lane 8) reduces the average apparent molecular mass of the heterogeneous protein by 5 kDa. Possibly the secreted soluble type II receptor contains sialic acid attached to O- as well as N-linked oligosaccharides. However, no change in migration of the secreted soluble type II receptor was seen following digestion with O-glycanase, whether or not the samples were first treated with glycopeptidase F and/or neuraminidase (data not shown). Thus, there appear to be two major classes of the secreted soluble type II receptor. One has a heterogeneous set of N-linked oligosaccharides that contain sialic acid, and that is converted to the 19-kDa ``core'' by digestion with glycopeptidase F. The other contains this heterogeneous set of N-linked oligosaccharides and also has additional sialic acids that are found either in O-linked oligosaccharides that are resistant to O-glycanase or in some unusual type of N-linked oligosaccharide. A similar, complex, pattern of glycosylation was seen for the full-length type II receptor. (^3)

The Heterogenously Glycosylated Soluble Type II Receptor Secreted by COS-7 Cells Can Bind and Become Chemically Cross-linked to ITGF-beta1

Fig. 4shows that the soluble secreted receptor is able to bind and be chemically cross-linked to 200 pMI-TGF-beta1. Serum-free conditioned medium from COS-7 cells transfected with pcDNA-1 expressing the soluble type II receptor was incubated with 200 pMI-TGF-beta1, chemically cross-linked with DSS, and immunoprecipitated with alpha-IIN. A heterogeneous species of 35-46 kDa is chemically cross-linked to I-TGF-beta1 (lane 2). This cross-linked species is specific, since it was not seen in an immunoprecipitation reaction containing the immunogenic peptide (lane 1), and was not present in conditioned media from COS-7 cells transfected with the full-length receptor (lane 5). The size of this species is consistent with a 1:1 complex of a monomer of TGF-beta1 (molecular mass, 12.5 kDa) and a single molecule of the soluble type II receptor (25-35 kDa, Fig. 2, lane 2). Fig. 4, lane 3, shows that this chemically cross-linked species can be precipitated by concanavalin A-Sepharose, indicating that the species has exposed mannose residues. The mature form of TGF-beta1 is not glycosylated, and thus the heterogeneity of the soluble type II receptor-TGF-beta1 complex can be ascribed to the heterogeneity of the oligosaccharides attached to the soluble type II receptor (Fig. 3). As expected, this TGF-beta1-binding, concanavalin A-reactive, species is not secreted by COS-7 cells expressing the full-length type II receptor (lane 6). Another chemically cross-linked species of 46 kDa is precipitated by concanavalin A-Sepharose, but as this species is also precipitated from the conditioned medium of mock-transfected COS-7 cells or cells transfected with the full-length receptor (lane 6), it is likely a nonspecific background species. Thus, the extracellular domain of the type II receptor can bind TGF-beta1.


Figure 4: Chemical cross-linking of the soluble TGF-beta type II receptor to I-TGF-beta1. COS-7 cells were transfected with pcDNA-1 containing either the truncated type II receptor (lanes 1-3) or the full-length type II receptor (lanes 4-6). Forty-eight hours after transfection, cells were rinsed twice with PBS and incubated overnight in serum-free medium (4 ml of Dulbecco's modified Eagle's medium/100-mm tissue culture dish). Conditioned media were collected and incubated with 200 pMI-TGF-beta1 overnight at 4 °C with rotation before samples were chemically cross-linked with DSS for 15 min at 4 °C. The samples were then divided into aliquots and subjected to immunoprecipitation overnight at 4 °C with an antipeptide antibody specific for the N-terminal extracellular domain of the human type II receptor in the absence (-, lanes 2 and 5) or presence (+, lanes 1 and 4) of an equimolar concentration of immunogenic peptide (Peptide). Equivalent aliquots were allowed to incubate with concanavalin A-Sepharose (Con A +, lanes 3 and 6) overnight at 4 °C. Immunoprecipitates and concanavalin A-bound proteins were analyzed by SDS-PAGE on a 10% gel, which was dried and exposed to pre-flashed XAR-5 film. The bracket shows the soluble type II receptor chemically cross-linked to I-TGF-beta1, which migrates as a heterogeneous 37-46-kDa species (lane 2). The arrows point to additional protein species in the concanavalin A sample (lane 3). Since these species are also present in media from control transfected COS-7 cells (lane 6), they are likely to be background species.



The Soluble Receptor Binds TGF-beta1 and TGF-beta3 but Cannot Bind TGF-beta2

Fig. 5shows that binding and chemical cross-linking of 100 pMI-TGF-beta1 to the soluble type II receptor, forming the heterogeneous 35-45 kDa species (lane 2), is competed effectively by a 10-fold excess (1 nM) of unlabeled TGF-beta1 (lane 3) and to a slightly lesser extent by a 10-fold excess of unlabeled TGF-beta3 (lane 5), but not at all by a 100-fold excess (10 nM) of unlabeled TGF-beta2 (lane 4). Furthermore, incubation of an equivalent aliquot of conditioned medium with 500 pMI-TGF-beta2, followed by chemical cross-linking with DSS, did not generate an immunoprecipitable, chemically cross-linked species (lane 6), even after prolonged exposure of the autoradiogram. The I-TGF-beta2 was functional, since it could be bound and cross-linked efficiently to the types I, II, and III receptors expressed on the surface of Rat-1 (9) and transfected COS-7 cells (see Fig. 8). Thus, we conclude that TGF-beta2 cannot bind to the soluble type II receptor.


Figure 5: Cross-linking of the soluble TGF-beta type II receptor to I-TGF-beta1 or I-TGF-beta2. COS-7 cells were transfected with pcDNA-1 expressing the truncated type II receptor. Forty-eight hours after transfection, cells were rinsed twice with PBS and incubated overnight in serum-free medium (5 ml of Dulbecco's modified Eagle's medium/100-mm tissue culture dish). Conditioned media were collected and incubated overnight with 100 pMI-TGF-beta1 in the absence (lanes 1 and 2) or presence of 1 nM TGF-beta1 (lane 3, beta1), 10 nM TGF-beta2 (lane 4, beta2), or 1 nM TGF-beta3 (lane 5, beta3). An equivalent aliquot was incubated with 500 pMI-TGF-beta2 (lane 6). After chemical cross-linking with DSS for 15 min at 4 °C, the samples were subjected to immunoprecipitation overnight at 4 °C with an antipeptide antibody specific for the N-terminal extracellular domain of the human type II receptor in the absence (lanes 2-6) or presence (lane 1, P) of equimolar concentrations of immunogenic peptide. Immunoprecipitated samples were analyzed by SDS-PAGE on a 10% gel, which was dried and exposed to pre-flashed XAR-5 film.



The isoform specificity of the soluble receptor is remarkably similar to that of cell-surface type II receptors expressed in transfected H216 cells (compare lanes 2 through 5 of Fig. 5and 6). This is a cell line derived from the stable transfection of the full-length human type II receptor into SW480 colon adenocarcinoma cells (see ``Materials and Methods''), which express very low levels of the type II receptor(36) . H216 cells, like SW480, express little if any type III receptor (Fig. 6, lane 2). Note that expression of the type II receptor in SW480 cells also leads to an increase in the amount of cell surface type I receptor (Fig. 6, lanes 1 and 2). The ability of the different TGF-beta isoforms to inhibit binding of I-TGF-beta1 to the cell surface type I receptor is the same as for the type II receptor (Fig. 6, lanes 2-5). In particular TGF-beta2 does not inhibit binding of TGF-beta1 to either the type II or type I receptor, indicating that it cannot bind directly to the type II (or type I) receptor.


Figure 6: Cross-linking of I-TGF-beta1 to SW480 and H216 cells and competition by unlabeled TGF-beta isoforms. SW480 cells (lane 1) and H216 cells (lanes 2-5) were grown on 60-mm tissue culture dishes until confluent and allowed to bind 100 pM I-TGF-beta1 AT 4 °C IN THE ABSENCE (lanes 1AND 2) OR PRESENCE OF UNLABELED 1 NMTGF-beta1 (lane 3, beta1), 5 NMTGF-beta2 (lane 4, beta2), OR 1 NMTGF-beta3 (lane 5, beta3). CELLS WERE CHEMICALLY CROSS-LINKED WITH 60 µG/ML DSS BEFORE LYSIS WITH BUFFER CONTAINING DETERGENT AND PROTEASE INHIBITORS. CELL LYSATES WERE ANALYZED BY SDS-PAGE ON AN 8%GEL, WHICH WAS DRIED AND EXPOSED TO PRE-FLASHED XAR-5 FILM. THE TYPE II RECEPTOR IS INDICATED AS A HETEROGENEOUS 90-110-KDA SPECIES (lane 2) WHICH IS NOT ABUNDANT IN SW480 CELLS (lane 1). THE TGF-betaTYPE I RECEPTOR IS INDICATED AS A 69-KDA SPECIES.



Determination of the Binding Affinity of the Soluble Type II Receptor for Different TGF-beta Isoforms

Conditioned medium from COS-7 cells transfected with pcDNA-1 expressing the soluble type II receptor was incubated with 80 pMI-TGF-beta1 in the presence of increasing concentrations of unlabeled TGF-beta1, TGF-beta2, or TGF-beta3 (Fig. 7A). From the densitometric scan of the autoradiogram (Fig. 7B), we calculated the apparent K(d) for the soluble type II receptor to be 200 pM for TGF-beta1 and 500 pM TGF-beta3. These dissociation constants, which were reproducible in repeats of this experiment, are 5-10-fold higher than those for binding of TGF-beta1 and TGF-beta3 to the type II receptor on the surface of mink lung cells, 25 and 50 pM, respectively (2) . Up to 10 nM TGF-beta2 did not inhibit binding of I-TGF-beta1 to the soluble type II receptor, and thus we estimate that its dissociation constant for the receptor must be greater than 10M. Similarly, TGF-beta2 is unable to block binding of I-TGF-beta1 to the full-length human type II receptor expressed on the surface of transfected SW480 cells (Fig. 6, lane 4).


Figure 7: Cross-linking of the soluble type II TGF-beta receptor to I-TGF-beta1 and competition by unlabeled TGF-beta isoforms. A, aliquots of medium from COS-7 cells transfected with pcDNA-1 encoding the soluble type II receptor were incubated with I-TGF-beta1 (80 pM) in the absence (lanes 1-3) or presence of increasing amounts of unlabeled TGF-beta1 (lanes 4-8), -beta2 (lanes 9-13), or -beta3 (lanes 14-18), as indicated. After chemical cross-linking with DSS, the samples were subjected to immunoprecipitation with an antipeptide antibody specific for the N-terminal extracellular region of the human type II receptor (lanes 2-18) in the absence (lanes 3-18) or presence (lane 2) of equimolar amounts of the immunizing N-terminal peptide as competitor. For the sample in lane 1, the antipeptide serum specific for the C terminus of the type II receptor was used; as expected, no soluble receptor was immunoprecipitated. B, the autoradiogram was scanned and densitometric values were normalized to 100 for the highest values. Circles, TGF-beta1; squares, TGF-beta2; triangles, TGF-beta3. The 100 pM values were not plotted.



The Type III Receptor Enhances the Ability of the Type II Receptor to Bind TGF-beta2

COS-7 cells transfected with pcDNA-1 expressing the human type II receptor bind little, if any, radioiodinated TGF-beta2 (Fig. 8, lanes 1-4) but do bind I-TGF-beta1 efficiently(12) . Importantly, co-expression of the rat type III receptor greatly enhances the ability of the type II receptor to bind TGF-beta2 (compare lanes 5-7 with lanes 1-3). Using 500 pM of I-TGF-beta2, the amount cross-linked to the type II receptor is enhanced >10-fold by co-expression of the type III receptor (compare lanes 5 and 1). Expression of the type III receptor alone does not lead to enhanced binding of I-TGF-beta2 to the endogenous type II receptors (lanes 8-12). The smaller molecular mass species seen in lanes 5-7 are nonspecific and have been observed in other cells overexpressing the type III receptor(9, 16) .


DISCUSSION

It is difficult to determine the binding affinity and specificity of type II TGF-beta receptors expressed on the plasma membrane, since there are no cells that express cell-surface TGF-beta type II receptors without either the type I or III receptors. Nor can we use cell lines to determine whether homodimers of the type II receptor, heterodimers of type I and II or of type II and III receptors, or multimers with other receptors, are involved in TGF-beta binding and signaling. To this end, we have generated a cDNA encoding a soluble, secreted form of the human TGF-beta type II receptor containing the entire exoplasmic domain and have expressed it in transfected COS-7 cells (Fig. 2).

This species migrates heterogenously on SDS-PAGE because it is heterogenously glycosylated. Most of the soluble, secreted receptors contain only a heterogeneous set of N-linked oligosaccharides; these are converted by digestion with glycopeptidase F to a 19-kDa core species that lacks the signal peptide and any apparent carbohydrate. The remainder of the soluble receptor contains these heterogeneous N-linked oligosaccharides as well as additional sialic acid residues that are either found in O-linked oligosaccharides that are resistant to O-glycanase or in some unusual type of N-linked oligosaccharide; conversion of this group of soluble receptors to the 19-kDa core species requires digestion both with glycopeptidase F and neuraminidase (Fig. 3).

Our most important result is that this soluble, secreted form of the human TGF-beta type II receptor shows selectivity for TGF-beta ligands. It binds to and can be cross-linked to I-TGF-beta1 (Fig. 4). The apparent affinities of the soluble form of the receptor for TGF-beta1 and -beta3 are 200 and 500 pM, respectively, and TGF-beta2 does not bind to the soluble type II receptor (K(d) > 10 nM) ( Fig. 5and Fig. 7). These properties are qualitatively similar to those of the human TGF-beta type II receptor expressed on the surface of H216 cells (Fig. 6). This is a line of transfected SW480 cells that express type I and II receptors, but little or no type III receptors. Similar results were obtained in mink lung epithelial cells(2) , where the cell surface type II receptor binds TGF-beta1 and TGF-beta3 with high affinity (K(d) 25 and 50 pM, respectively) and TGF-beta2 with a 10-fold lower affinity. While we can detect no cross-linking of I-TGF-beta2 to the soluble type II receptor (Fig. 5), we can detect a small amount of cross-linking to cell-surface type II TGF-beta receptors in transfected SW480 (H216) cells (not shown); most likely this is due to a small amount of the type III receptor or other TGF-beta-binding protein expressed in these cells.

The ability of I-TGF-beta1 to bind directly to the extracellular domain of the human TGF-beta type II receptor suggests that the type II receptor may be the primary binding subunit for TGF-beta1. Several facts support this notion. First, mutant cell lines which lack cell-surface type II receptors and are resistant to growth inhibition by TGF-beta1, such as DR mink lung cells and Hep 3B-TR cells, also lack type I receptors that are able to bind ligand, and expression of the type II receptor in these cells also restores binding to cell surface type I receptors(15, 16) . Thus, either the type I receptor in these cells cannot bind TGF-beta1 in the absence of the type II receptor or cannot accumulate on the plasma membrane. Several type I receptors for TGF-beta and activin have been cloned. When expressed alone in transfected cells none are able to bind TGF-beta1 or any other ligand tested. When co-expressed in COS cells with the type II TGF-beta receptor all of these species are able to bind TGF-beta1, and when the type II activin receptor is co-expressed all are able to bind and be cross-linked to activin. Thus, the nature of the type II receptor determines the nature of the ligand that is bound to the type I receptor, even though only certain combinations of type I and II receptors are apparently able to transduce TGF-beta1 or activin signals (19, 20, 21, 22, 23, 24, 25, 26) .

Several cell lines lack the type III receptor, such as fetal bovine heart endothelial (FBHE) cells (37) and L6 myoblasts(8, 10, 38) , yet express cell surface type I and II receptors that can bind TGF-beta1. Thus, expression of the type III receptor is not necessary for the type II receptor to bind TGF-beta1 in these cells. The type II receptor is also likely to be the primary binding subunit for TGF-beta3, since unlabeled TGF-beta3 can inhibit the binding of I-TGF-beta1 to both the soluble ( Fig. 5and Fig. 7) and cell surface (Fig. 6) type II receptors; a caveat is that we have not performed binding studies using I-TGF-beta3. Most likely the binding sites on the type II receptor for TGF-beta3 and TGF-beta1 overlap.

Our results suggest that the type II receptor is not the primary binding subunit for TGF-beta2. This notion is consistent with the observation that some cells, such as FBHE cells, which express the type I and II receptors but lack type III receptors, can bind and respond to TGF-beta1, but not to TGF-beta2(37) . Interestingly, these cells also do not express a subset of type I and type II receptors which can bind TGF-beta2 with high affinity, suggesting that some other subunit is necessary for the type I and II receptors on these cells to interact with TGF-beta2.

The primary binding subunit for TGF-beta2 could be the type III receptor. Several observations support this notion. First, the type III receptor can bind all three TGF-beta isoforms with relatively high affinities (K(d) 100-500 pM). Second, soluble secreted forms of the type III receptor bind all three TGF-beta isoforms with a similar high affinity (K(d) 500 pM), demonstrating that the type III receptor alone has the ability to bind TGF-beta2(39) . Finally, there is accumulating evidence that the type III receptor may interact with a subset of the type II (and possibly the type I) receptors. For example, expression of the type III receptor in L6 myoblasts enhances the amount of cell-surface type II receptor bound and cross-linked to I-TGF-beta1 (8) . More recently, López-Casillas et al.(10) showed that in L6 cells expression of the type III receptor leads to increased binding of TGF-beta2 to the type II receptor. Here we showed that in COS-7 cells, co-transfection of the type III receptors with type II receptors enabled the type II receptor to bind TGF-beta2 (Fig. 8).

While our study indicates that the type II receptor is the primary binding subunit for TGF-beta1 and TGF-beta3, the soluble type II receptor has 10-fold lower binding affinity for TGF-beta1 or TGF-beta3 than does the cell-surface type II receptor. The soluble type II receptor is monomeric, as judged by the sedimentation velocity of S-labeled protein in sucrose density gradients.^3 In contrast, the type II receptors found on the cell surface are primarily homo-oligomers, probably homodimers(18) , and the TGF-beta ligand is a disulfide-linked dimer. As revealed by x-ray crystallography, TGF-beta2 has a novel monomer fold and dimer association(40, 41) ; a very similar structure was proposed for TGF-beta1 on the basis of heteronuclear NMR studies(42) . The symmetry of the dimer suggests that 1 molecule of TGF-beta2, and presumably of the other TGF-beta isoforms, could interact simultaneously with two cell-surface receptor polypeptides. Thus, each of the two receptor subunits in a homodimer could bind one TGF-beta monomer, increasing the binding energy and thus lowering the dissociation constant for binding of TGF-beta.

The type II receptor may also interact with other proteins in order to generate a higher affinity receptor complex. Two likely candidates that may interact with the type II receptor to form such high affinity complexes are the receptor types I and III. However, there are many other TGF-beta binding proteins on the cell surface besides the types I, II, and III receptors(6) . These other proteins may also help to stabilize the binding of TGF-beta ligands to the type I and II receptors that generate the intracellular signals. Soluble forms of these receptors could be used to directly study their binding properties and their interactions with other receptor subunits.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant CA-63260 (to H. F. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Program in Membrane Biology, Renal Unit, Massachusetts General Hospital, Boston, MA 02114 and Department of Medicine, Harvard Medical School, Boston, MA 02115.

Postdoctoral fellow of the Anna Fuller Fund (grant 719).

**
Recipient of an AIDS Research Fund Postdoctoral Fellowship from the German Cancer Institute.

§§
Physician Postdoctoral Fellow of the Howard Hughes Medical Institute.

¶¶
To whom correspondence should be addressed: Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA, 02142. Tel.: 617-258-5216; Fax: 617-258-9872; lodish{at}wi.mit.edu.

(^1)
The abbreviations used are: TGF, transforming growth factor; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; DSS, disuccinimidyl suberate; FBHE, fetal bovine heart endothelial; PCR, polymerase chain reaction.

(^2)
H. Y. Lin, unpublished observations.

(^3)
R. G. Wells, unpublished observations.


ACKNOWLEDGEMENTS

We thank A. G. Geiser for providing SW480 cells, and Celtrix Laboratories, Inc., Palo Alto, CA, and R& Systems, Minneapolis, MN, for their generous gifts of TGF-beta1, -beta2, and -beta3.


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