©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Rat Pituitary Tumor Cell Line (GH) Expresses Type I and Type II Receptors and Other Cell Surface Binding Protein(s) for Transforming Growth Factor- (*)

(Received for publication, August 10, 1994; and in revised form, November 2, 1994)

Hidetoshi Yamashita (§) Toshihide Okadome Petra Franzén Peter ten Dijke (¶) Carl-Henrik Heldin Kohei Miyazono (**)

From the Ludwig Institute for Cancer Research, Box 595 Biomedical Center, S-751 24 Uppsala, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A rat pituitary tumor cell line (GH(3)) has been reported to express transforming growth factor-beta (TGF-beta) binding components of 70-74 kDa (ligand included), denoted TGF-beta type IV receptor. We investigated whether the type IV receptor corresponds to any of the recently cloned type I receptors for proteins in the TGF-beta superfamily. TGF-beta type I receptor (TbetaR-I) complexes of 69-72 kDa formed a heteromeric complex with TbetaR-II in GH(3) cells, as detected by immunoprecipitation. In addition, TGF-beta formed complexes of 72-74 kDa, which were different from TbetaR-I and the other known type I receptors, and were not dependent on TbetaR-II for binding. The GH(3) cells were resistant to the growth inhibitory activity of TGF-beta, but a transcriptional response was activated by TGF-beta in this cell line, presumably through the TbetaR-II and TbetaR-I complex. These results indicate that GH(3) cells have TbetaR-I and TbetaR-II and, in addition, other binding protein(s) which form 72-74-kDa complexes with TGF-beta; the function of the latter component(s) remains to be elucidated.


INTRODUCTION

Transforming growth factor-beta (TGF-beta) (^1)is a family of 25-kDa dimeric proteins which regulate cell proliferation and differentiation, accelerate extracellular matrix production, and modulate immune functions(1) . TGF-betas belong to a larger protein family denoted the TGF-beta superfamily which also includes activins and inhibins and bone morphogenic proteins (BMPs)(2) . These proteins have sequence similarities to each other, and in particular 7 cysteine residues are conserved in most of the members in the family. The active forms of most of the members are dimers, which are derived from larger precursor proteins. Inhibins are heterodimers of one alpha chain and one beta chain (beta(A) or beta(B)), whereas activins are homodimers composed of two inhibin beta(A) chains (activin A) or two inhibin beta(B) chains (activin B), or a heterodimer of one beta(A) and one beta(B) chain (activin AB)(3) . Activins stimulate the secretion of follicle-stimulating hormone from the pituitary gland and the differentiation of hematopoietic cells. Activins are also known to induce the mesoderm formation in Xenopus embryo. Inhibins, on the other hand, inhibit the secretion of follicle-stimulating hormone from the pituitary gland.

TGF-betas exert their functions through the interaction with various receptors and binding proteins on the cell surface(4, 5, 6, 7, 8) . These include TGF-beta type I receptor (TbetaR-I, 53 kDa), type II receptor (TbetaR-II, 75 kDa), betaglycan or type III receptor (more than 200 kDa), endoglin (180 kDa), type IV receptor (57-62 kDa), type V receptor (400 kDa), type VI receptor (180 kDa), and phosphatidylinositol-anchored membrane proteins. TbetaR-I and TbetaR-II are widely expressed on many different cell types (9, 10, 11) and form a heteromeric complex on the cell surface after ligand binding(12, 13, 14, 15, 16) . Betaglycan (17, 18) and endoglin (19) are also expressed on several different cell types. They have short intracellular domains, which are highly similar to each other in their amino acid sequences. Betaglycan and endoglin may present the ligands to TbetaR-II and/or TbetaR-I, which are the receptors primarily involved in signaling(18, 20, 21, 22) . The functions for the other receptors and binding proteins remain to be elucidated.

cDNA cloning of TbetaR-II revealed that it has an intracellular serine/threonine kinase domain(9) . Type II receptors for activin (ActR-II and ActR-IIB) (23, 24, 25) and BMPs (DAF-4 from Caenorhabditis elegans) (26) were also shown to be transmembrane serine/threonine kinases. A series of novel serine/threonine kinase receptors have also been identified and denoted activin receptor-like kinases (ALK) 1-6(10, 27, 28) , and have been shown to be type I receptors for the members in the TGF-beta superfamily. ALK-5 is a signaling TGF-beta type I receptor (TbetaR-I) (10, 11, 28, 29) , ALK-2 and ALK-4 are activin type I receptors (ActR-I and ActR-IB, respectively)(28, 30, 31) , whereas ALK-3 and ALK-6 are type I receptors for osteogenic protein-1 and BMP-4 (BMPR-IA and BMPR-IB, respectively)(32) .

The TGF-beta type IV receptor has been reported to be expressed only on GH(3) rat pituitary tumor cells(33) . Binding competition studies revealed that this receptor binds not only TGF-betas, but also activin AB and inhibin B. The molecular mass of the type IV receptor complex has been reported to be 70-74 kDa including ligand, which is similar to the sizes of type I receptor complexes. In the present study, we have studied whether the type IV receptor complex on GH(3) pituitary tumor cells contains any of the known type I receptors.


MATERIALS AND METHODS

Cell Culture

NRK cells, a fibroblast cell line derived from normal rat kidney, and GH(3) cells, a rat pituitary tumor cell line, were obtained from the American Type Culture Collection. NRK cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 50 µg/ml streptomycin. GH(3) cells were grown in DMEM supplemented with 10% horse serum and 5% fetal calf serum, 100 units/ml penicillin, and 50 µg/ml streptomycin.

Preparation of Polyclonal Antibodies

Antisera against TbetaR-II and type I receptors (ALK-1, TbetaR-I, ActR-I, ActR-IB, BMPR-IA, and BMPR-IB) were made against synthetic peptides corresponding to the C-terminal tail of TbetaR-II (amino acids 545-566; (9) ) and the intracellular juxtamembrane parts of type I receptors, respectively, as described previously(10, 28) .

Radiolabeling, Binding, and Affinity Cross-linking

Recombinant human TGF-beta1 and activin A were obtained from H. Ohashi (Kirin Brewery Co., Ltd., Japan) and Y. Eto (Ajinomoto Co., Ltd., Japan), respectively. TGF-beta1 was iodinated using the chloramine T method(34) . Cells were incubated on ice for 3 h with 200 pM of I-TGF-beta1 in the presence or absence of 200-fold excess (40 nM) of unlabeled ligands in the binding buffer (phosphate-buffered saline containing 0.9 mM CaCl(2) and 0.49 mM MgCl(2) and 1 mg/ml bovine serum albumin). After incubation, the cells were washed with the binding buffer without bovine serum albumin, and cross-linking was done in the same buffer containing 0.28 mM of disuccinimidyl suberate (DSS, Pierce) for 15 min on ice. The cells were washed once with detachment buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10% glycerol, 0.3 mM phenylmethylsulfonyl fluoride) and scraped off the dish in this buffer. The cells were centrifuged and resuspended in solubilization buffer (125 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1.5% Trasylol, 1% Triton X-100) and incubated for 20 min on ice. Samples were mixed with SDS-sample buffer (80 mM Tris-HCl, pH 8.8, 0.01% bromphenol blue, 24% glycerol, 4% SDS, 10 mM dithiothreitol), and then analyzed by SDS-gel electrophoresis using a 5-12% gradient polyacrylamide gel, and subjected to autoradiography using Hyperfilm MP (Amersham). For immunoprecipitation after cross-linking, the cells were lysed on ice for 15 min in the solubilization buffer as above and incubated with 10 µl of the specific antisera/ml for 1 h at 4 °C. For blocking, 10 µg of peptides used for immunization were added together with the corresponding antisera. Thereafter, 50 µl of a protein A-Sepharose slurry (50% packed beads in solubilization buffer) was added to immune complexes, and the mixture was incubated for 1 h at 4 °C. The beads were centrifuged and washed two times with 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 1% Triton X-100, 1% deoxycholate, and 0.2% SDS, followed by one wash in distilled water. The immune complexes were eluted by boiling 3 min in the SDS-sample buffer and subjected to SDS-gel electrophoresis, followed by autoradiography.

Deglycosylation Using Endoglycosidase F

GH(3) cells were incubated with I-TGF-beta1 and cross-linked with DSS (see above). Cross-linked complexes were immunoprecipitated or not with the TbetaR-I antiserum, and then incubated with 0.5 unit of endoglycosidase F (Boehringer Mannheim Biochemica) in a buffer containing 100 mM sodium phosphate (pH 6.1), 50 mM EDTA, 1% Triton X-100, 0.1% SDS, and 1% 2-mercaptoethanol at 37 °C for 24 h. Samples were boiled in the SDS-sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis, followed by autoradiography.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Total RNA was prepared from GH(3) cells and NRK cells by guanidinium isothiocyanate method(35) . Total RNAs (5.4 µg for GH(3) cells and 7.4 µg for NRK cells) were reverse transcribed into cDNAs using antisense oligonucleotide primers denoted 5R-2 for TbetaR-I and 2R-2 for TbetaR-II, respectively (see below). Amplification was performed using Perkin Elmer Cetus DNA Thermal Cycler with Pyrococcus furiosus DNA polymerase (Stratagene). As the primers for PCR amplification, 5R-3 (sense) and 5R-2 (antisense) were used for TbetaR-I, and 2R-1 (sense) and 2R-2 (antisense) were used for TbetaR-II. The conditions for the first PCR were 94 °C for 60 s, 50 °C for 120 s, 72 °C for 180 s for the first 25 cycles. Aliquots of the PCR products were reamplified by the second PCR under the same conditions as the first. The products were subjected to 1% agarose gel electrophoresis and visualized by ethidium bromide staining.

The used oligonucleotide primers are as follows: 5R-3 (sense), nucleotides 96-113 of TbetaR-I (5`-GCTCT AGATT TCTGC CACCT CTGTAC-3`); 5R-2 (antisense), nucleotides, 441-424 of TbetaR-I (5`-GCGAA TTCGA CAGTG CGGTT ATGGC A-3`); 2R-1 (sense), nucleotides, -58 to -41 of TbetaR-II (5`-GCTCT AGACC CGAGG CTCGT TCGCG G-3`); 2R-2 (antisense), nucleotides 585-568 of TbetaR-II (5`-GCGAA TTCCT GCTGC CGGTG GACAC G-3`).

The numbering of nucleotides start from the first nucleotide (+1) of the open reading frames of rat homologue of TbetaR-I, R4 (11) or the rat TbetaR-II(36) . The sense primers have an XbaI site (TCTAGA) in the 5` end regions, and the antisense primers have an EcoRI site (GAATTC) in the 5` end regions.

[^3H]Thymidine Incorporation Assay

NRK cells and GH(3) cells were seeded in 24-well plates at a density of 10^4 cells/well in DMEM with 10% fetal calf serum, and with 10% horse serum and 5% fetal calf serum, respectively. After 24 h, the medium was changed to DMEM with 1% fetal calf serum containing various concentrations of TGF-beta1 in the presence or absence of 0.3 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA). After 16 h of incubation, 11.1 kBq of [^3H]thymidine (3.15 TBq/mmole, Amersham) was added, and the cells were incubated for an additional 2 h. Thereafter, the cells were fixed in 5% ice-cold trichloroacetic acid for more than 1 h and solubilized with 1 M NaOH for more than 20 min. The cell extract was neutralized with 1 M HCl, and ^3H radioactivity was determined in a liquid scintillation counter using Ecoscint A (National Diagnostics).

Transcriptional Response Assay

A promoter-reporter construct suitable for quantification of transcriptional activation by TGF-beta1, p3TP-Lux, was obtained from J. Massagué. The p3TP-Lux contains a region of the human plasminogen activator inhibitor-1 gene promoter and three sets of TPA-responsive elements(13, 30) . p3TP-Lux was transfected into GH(3) cells using Transfectam (Promega), following the manufacturer's protocol. In brief, GH(3) cells were seeded into 6-well plates at a density of 10^5 cells/well and transfected with 4 µg of a plasmid in DMEM without serum on the following day. After overnight incubation, the medium was changed to DMEM with 10% fetal bovine serum. After 24 h, the cells were starved in DMEM with 0.1% fetal bovine serum for 4-6 h and then exposed to various concentrations of TGF-beta1 for 24 h. Luciferase activity in the cell lysate was measured using the luciferase assay system (Promega) according to the manufacturer's protocol and a luminometer (model 1250; LKB).


RESULTS

Identification of TbetaR-I and TbetaR-II in GH(3)Cells-Cross-linking studies using I-TGF-beta1 revealed that GH(3) cells expressed a triplet of receptor complexes of molecular masses of 69-74 kDa, including the TGF-beta1 monomer (Fig. 1, lane 1); these properties are identical to those reported for the TGF-beta type IV receptor(33) . In order to examine whether the complexes of 69-74 kDa contain any of known type I receptors for members in the TGF-beta superfamily, immunoprecipitation using antisera against the type I receptors was performed. The antiserum against TbetaR-I immunoprecipitated components which appeared to correspond to the lower one or two bands of the triplet band of 69-72 kDa observed by cross-linking without immunoprecipitation (Fig. 1). A component of 86 kDa, i.e. a size similar to that of a TbetaR-II complex, which could not be seen without immunoprecipitation because of its low abundance, was coimmunoprecipitated with TbetaR-I. Antisera to the other type I receptors did not efficiently immunoprecipitate any cross-linked complexes in GH(3) cells.


Figure 1: Analysis of cross-linked TGF-beta receptor complexes in GH(3) cells by immunoprecipitation using antisera against type I receptors. GH(3) cells were incubated with I-TGF-beta1, followed by cross-linking with DSS. Aliquots of cell lysates were analyzed directly by SDS-gel electrophoresis, followed by autoradiography (lane 1), or first subjected to immunoprecipitation (IP) using antisera against type I receptors (lanes 2-7).



The immunoprecipitated TbetaR-I and TbetaR-II complexes in GH(3) cells were compared to those in another rat cell line, NRK, which is known to express TbetaR-I, TbetaR-II, and betaglycan and to respond to TGF-beta(34, 37) . Although the binding efficiencies observed by cross-linking (Fig. 2, lanes 1 and 2) were different between these cell types, the sizes of the TbetaR-I and TbetaR-II complexes immunoprecipitated from GH(3) cells corresponded to those of NRK cells (Fig. 2, lanes 3, 5, 7, and 9). Using both GH(3) cells and NRK cells, TbetaR-II complexes were coimmunoprecipitated with TbetaR-I complexes and vice versa. The fact that similar amounts of TbetaR-I were brought down from GH(3) cells with antisera against either TbetaR-I or TbetaR-II, indicates that TbetaR-II is present in GH(3) cells at an amount equal to or higher than that of TbetaR-I. The reason for the poor efficiency in cross-linking to TGF-beta receptors on GH(3) cells, compared to NRK cells, is not known. The TbetaR-I complexes immunoprecipitated by both the TbetaR-I and TbetaR-II antisera had molecular masses of 69-72 kDa, which appear to correspond to the lower components of the triplet of bands observed without immunoprecipitation.


Figure 2: Comparison of cross-linked TGF-beta receptor complexes in GH(3) cells and NRK cells by immunoprecipitation using antisera against TbetaR-I and TbetaR-II. GH(3) cells and NRK cells were incubated with I-TGF-beta1, followed by cross-linking with DSS. Aliquots of the cell lysates were analyzed directly by SDS-gel electrophoresis (lanes 1 and 2), or first subjected to immunoprecipitation (IP) using antisera against TbetaR-I or TbetaR-II (lanes 3-10). Blocking of the antisera (Block) was performed with 10 µg each of the corresponding peptides (lanes 4, 6, 8, and 10). The gel was analyzed by autoradiography.



The 69-74-kDa Cross-linked Complexes Are Composed of TbetaR-I and Other Components

In order to further characterize the identity of the cross-linked complexes in GH(3) cells, the effects of dithiothreitol treatment and deglycosylation were investigated. The binding of TGF-beta1 to TbetaR-I has been reported to be abolished by transient treatment of the cells with dithiothreitol(10, 13, 38) . When the GH(3) cells were transiently treated with dithiothreitol, and then incubated with I-TGF-beta1, followed by cross-linking, the cross-linked complexes of 72-74 kDa could still be observed (Fig. 3A, lane 2). In contrast, the binding of I-TGF-beta1 to the 69-72-kDa complexes immunoprecipitated by TbetaR-I antiserum was abolished by the dithiothreitol treatment (Fig. 3A, lane 4).


Figure 3: Biochemical characterization of the 69-74 kDa complexes affinity cross-linked with I-TGF-beta1. A, binding of I-TGF-beta1 to GH(3) cells treated with dithiothreitol. GH(3) cells were treated with or without 1 mM dithiothreitol (DTT) in the binding buffer without bovine serum albumin at 37 °C for 8 min, and then incubated with I-TGF-beta1, followed by cross-linking with DSS. The cross-linked complexes were directly analyzed by SDS-gel electrophoresis (lanes 1 and 2) or first subjected to immunoprecipitation (IP) using the TbetaR-I antiserum (lanes 3 and 4). The gel was analyzed by autoradiography. B, deglycosylation with endoglycosidase F (Endo F) of the complexes affinity cross-linked with I-TGF-beta1. GH(3) cells were incubated with I-TGF-beta1 and followed by cross-linking with DSS. The samples were treated with 0.5 unit of endglycosidase F and directly analyzed by SDS-gel electrophoresis (lanes 1 and 2) or first subjected to immunoprecipitation (IP) with the TbetaR-I antiserum (lanes 3 and 4). The gel was analyzed by autoradiography.



TbetaR-I has been reported to have an N-linked carbohydrate chain and can be deglycosylated by endoglycosidase F(10) . When the cross-linked complexes of GH(3) cells were treated with endoglycosidase F, the upper bands of the cross-linked complexes (72-74 kDa) did not shift (Fig. 3B, lanes 1 and 2). However, when the 69-72-kDa complexes immunoprecipitated by the TbetaR-I antiserum were subjected to deglycosylation, they shifted to 68 kDa (Fig. 3B, lanes 3 and 4). The TbetaR-II component that coimmunoprecipitated with TbetaR-I also shifted from 86 to 76 kDa (Fig. 3B, lanes 3 and 4), consistent with a previous report(10) .

These results indicate that the 69-72-kDa components represent TbetaR-I, which forms a heteromeric complex with TbetaR-II. In contrast, the 72-74-kDa complexes are distinct from TbetaR-I. Since the 72-74-kDa components were not immunoprecipitated by the TbetaR-II antiserum (see Fig. 2, lane 5), they do not form a complex with TbetaR-II.

Binding Properties of the 69-74-kDa Complexes to TGF-beta and Activin A

The abilities of TGF-beta1 and activin A to compete with I-TGF-beta1 for binding to GH(3) cells were compared. The cross-linked I-TGF-beta1 complexes of 69-74 kDa were found to be abolished in the presence of a 200-fold excess of unlabeled TGF-beta1 (Fig. 4, lane 2), but only slightly inhibited by 200-fold excess of unlabeled activin A (Fig. 4, lane 3). The binding of I-TGF-beta1 to the components immunoprecipitated by the TbetaR-I antiserum from GH(3) cells was inhibited by a 200-fold excess unlabeled TGF-beta1, but was not inhibited by a 200-fold excess unlabeled activin A (Fig. 4, lanes 5 and 6). Similar inefficient competitions were obtained using activin B and inhibin A (data not shown). These results indicate that the 69-72-kDa TbetaR-I components bind only TGF-beta and not activin and that the 72-74-kDa components have only low affinities for activins.


Figure 4: Competition of the binding of I-TGF-beta1 to the 69-74-kDa components in GH(3) cells by unlabeled ligands. GH(3) cells were incubated with I-TGF-beta1 in the presence or absence of 200-fold excesses of unlabeled TGF-beta1 or activin A (Cold ligand), followed by cross-linking with DSS. Aliquots of cell lysates were directly analyzed by SDS-gel electrophoresis (lanes 1-3) or first subjected to immunoprecipitation (IP) using the antiserum against TbetaR-I (lanes 4-6). The gel was analyzed by autoradiography.



RT-PCR Analysis for the Expression of TbetaR-I and TbetaR-II mRNAs

The above results suggest that TbetaR-I is expressed on GH(3) cells and forms a heteromeric complex with TbetaR-II. This does not formally exclude the possibility that GH(3) cells express proteins that are recognized by the TbetaR-I or TbetaR-II antisera, but are different from these receptors. In order to confirm that TbetaR-I and TbetaR-II are expressed in GH(3) cells, RNAs from GH(3) cells and NRK cells were subjected to RT-PCR analysis using specific primers for rat TbetaR-I and TbetaR-II. The amplified cDNA from GH(3) cells using TbetaR-I primers had the same size (0.36 kilobase) as that from NRK cells (Fig. 5). Similarly, the amplified cDNA from GH(3) cells using TbetaR-II primers had the same size (0.65 kilobase) as that from NRK cells (Fig. 5). These results support the notion that GH(3) cells express TbetaR-I and TbetaR-II.


Figure 5: RT-PCR analysis of expression of TbetaR-I and TbetaR-II mRNAs in GH(3) cells and NRK cells. Total RNAs from GH(3) cells and NRK cells were reverse transcribed into cDNAs using oligonucleotide primers specific for the rat TbetaR-I or TbetaR-II. cDNAs were amplified by PCR using specific primers for TbetaR-I or TbetaR-II and analyzed by 1% agarose gel electrophoresis, followed by ethidium bromide staining.



Signaling Activity of TGF-beta1 in GH(3) Cells

The effect of TGF-beta1 on DNA synthesis in GH(3) cells was investigated using a [^3H]thymidine incorporation assay. GH(3) cells did not respond to TGF-beta1 at concentrations of 0.1-20 ng/ml, whereas the growth of NRK cells was inhibited by 0.2-2 ng/ml of TGF-beta1 (data not shown). A prostate carcinoma cell line, PC-3, has been shown to be relatively resistant to the growth inhibitory action of TGF-beta1, but the presence of TPA facilitated the growth inhibitory activity of TGF-beta1(39) . However, the addition of TPA did not affect the [^3H]thymidine incorporation in GH(3) cells in the presence or absence of TGF-beta1 (data not shown).

The signaling activity of TGF-beta1 was also investigated by a transcriptional assay using the p3TP-Lux promoter-reporter construct (13, 30) . In GH(3) cells transfected with the p3TP-Lux plasmid, transcriptional activation of p3TP-Lux was observed by the addition of TGF-beta1 (Fig. 6), which indicates that TGF-beta1 can transduce signals in this cell line.


Figure 6: Transcriptional activation by TGF-beta1 in GH(3) cells transfected with p3TP-Lux promoter-reporter construct. GH(3) cells were transfected with p3TP-Lux and stimulated by various concentrations of TGF-beta1 for 24 h. The cellular proteins were extracted and luciferase activity was measured using the luciferase assay system (Promega). Luciferase activity is expressed relative to a control value without stimulation.




DISCUSSION

The present study shows that TbetaR-I and TbetaR-II are expressed in GH(3) cells and form a heteromeric complex in the presence of TGF-beta. Affinity labeled complexes of 69-72 kDa including I-TGF-beta1 were immunoprecipitated by an antiserum against TbetaR-I, but not by antisera against the other type I receptors. The TbetaR-II antiserum immunoprecipitated the 69-72-kDa TbetaR-I components together with TbetaR-II. Binding of TGF-beta1 to the components of 69-72 kDa was abolished by dithiothreitol treatment of GH(3) cells before incubation with I-TGF-beta1, and the complexes shifted to 68 kDa after deglycosylation. RT-PCR analysis revealed that GH(3) cells express mRNAs for TbetaR-I and TbetaR-II. These results indicate that the 69-72-kDa components on GH(3) cells represent TbetaR-I.

The 69-74 kDa components observed by cross-linking studies contained at least three bands, and the lower one or two bands corresponded to TbetaR-I. The upper components of the 72-74-kDa multiple components in GH(3) cells were not immunoprecipitated by antisera to TbetaR-I, TbetaR-II, or the other type I receptors ( Fig. 1and Fig. 2) and were resistant to treatments with dithiothreitol or endoglycosidase F. Similarly multiple components with molecular masses of 70-77 kDa were observed in other cell types, e.g. human foreskin fibroblasts (AG1518), human lung adenocarcinoma cells (A549), and human oral squamous cell carcinoma cells (HSC-2)(10) . Also in these cell lines, only the 70-kDa affinity labeled complex was immunoprecipitated by an antiserum against TbetaR-I. The 69-74-kDa-cross-linked components in GH(3) cells were reported to bind activin AB and inhibin B in addition to TGF-betas and have been suggested to be a novel type of cell surface binding protein, denoted type IV receptor(33) . In the present study, the binding of I-TGF-beta1 to the 72-74-kDa component was only slightly competed with unlabeled excess amounts of activin A, whereas the 69-72-kDa TbetaR-I complexes were not at all competed by activin A. Taken together, the present data revealed that GH(3) cells express unidentified components capable of forming a complex of 72-74 kDa with I-TGF-beta1; the protein(s) are different from TbetaR-I and the other known type I receptors, and do not form a complex with TbetaR-II. It remains to be elucidated whether the novel binding protein is a serine/threonine kinase receptor, like TbetaR-I and TbetaR-II; alternatively, it may serve as an accessory molecule, which indirectly regulates the signal transduction by TbetaR-I and TbetaR-II.

Type II receptor-like components were not detected by the cross-linking studies using I-TGF-beta1; however, immunoprecipitation of the cross-linked complexes by an antiserum against TbetaR-II revealed that TbetaR-II is present in GH(3) cells and is able to form a heteromeric complex with TbetaR-I. That GH(3) cells express the canonical TbetaR-II is also supported by the demonstration of mRNA for TbetaR-II by RT-PCR analysis. Similar observations were reported using A549 cells and HSC-2 cells(10) , where cross-linked TbetaR-II complexes occurred at very low abundance; immunoprecipitation of the cross-linked complexes using a specific antiserum against TbetaR-II revealed the expression of TbetaR-II protein. It appears that the efficiency in cross-linking to TbetaR-II can vary (see Fig. 2). Thus, in order to study the expression of TbetaR-II by affinity cross-linking, it is important to immunoprecipitate the cross-linked complexes to avoid that the binding to TbetaR-II is hidden by a nonspecific background.

Activins and inhibins regulate the secretion of follicle-stimulating hormone from rat pituitary cells. Since activin A did not efficiently compete with I-TGF-beta1 for binding to the 72-74-kDa complex, it is unlikely that this putative receptor transduces activin responses in GH(3) cells. We have recently found that rat pituitary cells in primary culture express ActR-II and ActR-IB. (^2)

The signaling activity of TGF-beta in GH(3) cells was investigated by growth inhibition and p3TP-Lux transcriptional activation assays. GH(3) cells did not respond to TGF-beta1 with regard to the growth inhibition; analogously other transformed cell lines, e.g. HSC-2 oral squamous cell carcinoma cells (40) , PC-3 prostate carcinoma cells(39) , EJ bladder carcinoma cells, and SW 480 colon carcinoma cells (41) are also unresponsive to TGF-beta even though they express TbetaR-I and TbetaR-II on their cell surfaces. On the other hand, TGF-beta1 stimulated the transcription of p3TP-Lux. Since the TbetaR-I and TbetaR-II complex has been shown to induce the p3TP-Lux transcriptional activation by TGF-beta1(11, 13, 31) , TbetaR-I and TbetaR-II expressed on GH(3) cells may be responsible for this signal. Whether the 72-74-kDa components are involved in TGF-beta signal transduction remains to be elucidated. These data indicated that the signaling pathway for the transcriptional activation of p3TP-Lux is conserved, but the growth inhibitory signal is perturbed in the GH(3) cell line. Escape from the growth inhibition by TGF-beta may be related to the loss of growth control of cancer cells. Further studies of the signal transduction pathways for TGF-beta are important to elucidate the mechanisms for carcinogenesis.


FOOTNOTES

*
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.

§
Supported by the Japanese Eye Bank Association.

Supported by a European Molecular Biology Organization postdoctoral fellowship.

**
To whom correspondence should be addressed. Tel.: +46-18-174267; Fax: +46-18-506867.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; ActR, activin receptor; ALK, activin receptor-like kinase; BMP, bone morphogenic protein; BMPR, BMP receptor; DMEM, Dulbecco's modified Eagle's medium; DSS, disuccinimidyl suberate; RT-PCR, reverse transcriptase polymerase chain reaction; TbetaR, TGF-beta receptor; TPA, 12-O-tetradecanoylphorbol 13-acetate.

(^2)
H. Yamashita, P. ten Dijke, D. Huylebroeck, T. K. Sampath, M. Andries, J. C. Smith, C.-H. Heldin, and K. Miyazono, submitted for publication.


ACKNOWLEDGEMENTS

We thank Christer Wernstedt for preparing oligonucleotides, Hideya Ohashi for recombinant TGF-beta1, Yuzuru Eto for recombinant activin, and Joan Massagué for the p3TP-Lux plasmid.


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