Function of the Type V Transforming Growth Factor beta  Receptor in Transforming Growth Factor beta -induced Growth Inhibition of Mink Lung Epithelial Cells*

(Received for publication, September 6, 1996, and in revised form, April 23, 1997)

Qianjin Liu , Shuan Shian Huang and Jung San Huang Dagger

From the Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The type V transforming growth factor beta  (TGF-beta ) is a 400-kDa nonproteoglycan membrane protein that co-expresses with the type I, type II, and type III TGF-beta receptors in most cell types. The type V TGF-beta receptor exhibits a Ser/Thr-specific protein kinase activity with distinct substrate specificity (Liu, Q., Huang, S. S., and Huang, J. (1994) J. Biol. Chem. 269, 9221-9226). In mink lung epithelial cells, the type V TGF-beta receptor was found to form heterocomplexes with the type I TGF-beta receptor by immunoprecipitation with antiserum to the type V TGF-beta receptor after 125I-TGF-beta affinity labeling or Trans35S-label metabolic labeling of the cells. The kinase activity of the type V TGF-beta receptor was stimulated after treatment of mink lung epithelial cells with TGF-beta . TGF-beta stimulation resulted in the growth inhibition of wild-type mink lung epithelial cells and to a lesser extent of the type I and type II TGF-beta receptor-defective mutants, although higher concentrations of TGF-beta were required for the growth inhibition of these mutants. TGF-beta was unable to induce growth inhibition in human colorectal carcinoma cells lacking the type V TGF-beta receptor but expressing the type I and type II TGF-beta receptors. These results suggest that the type V TGF-beta receptor can mediate the TGF-beta -induced growth inhibitory response in the absence of the type I or type II TGF-beta receptor. These results also support the hypothesis that loss of the type V TGF-beta receptor may contribute to the malignancy of certain carcinoma cells.


INTRODUCTION

Transforming growth factor beta  (TGF-beta )1 is the most potent polypeptide growth inhibitor for epithelial cells and plays an important role in the pathophysiology of epithelial cells in human and other species (1-3). The TGF-beta -induced growth inhibition of epithelial cells is mediated by specific cell surface receptors (1-4). The type V TGF-beta receptor is a 400-kDa nonproteoglycan membrane glycoprotein that co-expresses with the type I, type II, and type III TGF-beta receptors in epithelial cells and other cell types but not in certain carcinoma cells (4, 5). The type V TGF-beta receptor as well as the type I and type II TGF-beta receptors are members of a new class of Ser/Thr-specific receptor protein kinases with distinct substrate specificities (6-11). The exact roles of these receptor kinases in the growth inhibition of epithelial cells induced by TGF-beta are unknown. Recent studies have demonstrated that the heterocomplex formation of the type I and type II TGF-beta receptors and the transphosphorylation of the type I TGF-beta receptor by the type II TGF-beta receptor are important in the TGF-beta -induced growth inhibition (9-12). The role of the type V TGF-beta receptor in the growth inhibition induced by TGF-beta has not been defined. These studies reported here suggest that the type V TGF-beta receptor forms heterocomplexes with the type I TGF-beta receptor in mink lung epithelial cells and can mediate the TGF-beta -induced growth inhibition in mink lung epithelial cells in the absence of the type I or type II TGF-beta receptor. These studies reported here also support the hypothesis that loss of the type V TGF-beta receptor may contribute to the malignancy of certain carcinoma cells.


EXPERIMENTAL PROCEDURES

Materials---Na125I (17 Ci/mg), [gamma -32P]ATP (4,500 Ci/mmol), and Trans35S-label (1,000 Ci/mmol) were obtained from ICN Biochemicals, Inc. (Irvine, CA). [gamma -32P]ATP was diluted with unlabeled ATP to have a specific radioactivity of ~104 cpm/pmol. Molecular mass protein standards (myosin, 205 kDa; beta -galactosidase, 116 kDa; phosphorylase, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 29 kDa), poly-L-lysine HBr (Mr 15,000-30,000), beta -mercaptoethanol, glycerol, Triton X-100, and other chemical reagents were obtained from Sigma. TGF-beta 1 was purchased from Austral Biologicals (San Ramon, CA). Disuccinimidyl suberate (DSS) was obainted from Pierce. Peptide antigen, a hexadecapeptide containing the ATP binding site amino acid sequence (6) was synthesized using tert-butoxycarbonyl chemistry on an Applied Biosystems model 431A peptide synthesizer. Recombinant human nonglycosylated insulin-like growth factor binding protein 3 (IGFBP-3) was provided by Celtrix Pharmaceuticals, Inc. (Santa Clara, CA). TGF-beta receptor-defective mutants (DR26 and R1-B cells) and type II TGF-beta receptor cDNA and neo-vector stably transfected hereditary human colorectal carcinoma cells (RII-37 and HCT 116 Neo cells) were provided by Drs. Joan Massagué and Michael G. Brattain, respectively. Mink lung epithelial cells have been routinely maintained in the laboratory. All cultured cells were grown in 10% fetal calf serum in Dulbecco's modified Eagle's medium.

Preparation of Antiserum to the Type V TGF-beta Receptor

The antiserum to the type V TGF-beta receptor was raised in rabbits with the conjugate of bovine thyroglobulin and peptide antigen (a hexadecapeptide), whose amino acid sequence was derived from the ATP binding site amino acid sequence of the type V TGF-beta receptor (6). The peptide antigen was conjugated to bovine thyroglobulin according to the procedure of Huang and Huang (13). The antiserum to the type V TGF-beta receptor did not show reactivity to the type I and type II TGF-beta receptors based on Western blot analysis and immunoprecipitation of 125I-TGF-beta affinity labeled or Trans35S-label metabolically labeled type I and type II TGF-beta receptors from mink lung epithelial cells in the presence of 0.1% SDS.

Purification of the Type V TGF-beta Receptor from Bovine Liver Plasma Membranes

The type V TGF-beta receptor was purified by DEAE-cellulose column chromatography after Triton X-100 extraction of bovine liver plasma membranes and wheat germ lectin-Sepharose 4B affinity column chromatography as described previously (4).

Western Blot Analysis

About 0.1 µg of the type V TGF-beta receptor from bovine liver plasma membranes was subjected to 6% SDS-polyacrylamide gel electrophoresis under reducing conditions followed by electrophoretic transblotting onto nitrocellulose membranes (Protran). Western blot analysis of the type V TGF-beta receptor was performed as described previously (14), using antiserum to the type V TGF-beta receptor (1:100 dilution) with or without peptide antigen (0.5 mM).

Immunoprecipitation of the Type V TGF-beta Receptor in [32]Orthophosphate Metabolically Labeled Mink Lung Epithelial Cells

Mink lung epithelial cells (Mv1Lu cells) were grown to confluence on P-60 Petri dishes and metabolically labeled with [32P]orthophosphate according to the procedure of Huang and Huang (13). The monolayers were then treated with 0.1 nM TGF-beta 1 in Dulbecco's modified Eagle's medium, pH 7.4, at 0 °C for 30 min. The cells were then detached and lysed in 100 µl of 1% Triton X-100 in 10 mM Tris-HCl, pH 7.0, 125 mM NaCl, and 1 mM EDTA. After centrifugation, the Triton X-100 extracts were then diluted 10-fold with Triton X-100-free buffer and incubated with antiserum or nonimmune serum (1:100 dilution) at 0 °C overnight. The immunocomplexes were precipitated with 20 µl of protein A-Sepharose (50%, v/v). After washing with 20 mM Tris-HCl, pH 7.4, 0.2% Triton X-100, the immunoprecipitates were analyzed by 6% SDS-polyacrylamide gel electrophoresis under reducing conditions and autoradiography. The relative intensity of 32P-labeled type V TGF-beta receptor on the autoradiogram was quantitated by a PhosphorImager.

125I-TGF-beta Affinity Labeling of Mink Lung Epithelial Cells and Human Colorectal Carcinoma Cells

Mink lung epithelial cells (wild-type, DR26, and R1B cells) and hereditary human colorectal carcinoma cells (HCT 116 Neo and RII-37 cells) grown on P-60 Petri dishes were incubated with 125I-TGF-beta 1 (0.5, 1, and 1.5 nM) in binding buffer (50 mM HEPES, pH 7.4, 128 mM NaCl, 5 mM KCl, 5 mM MgSO4, and 1.2 mM CaCl2) containing 0.2% bovine serum albumin at 0 °C for 2.5 h. The affinity labeling of cell surface TGF-beta receptors was carried out as described previously (4, 5). The 125I-TGF-beta 1 affinity labeled receptors were then analyzed by 5.5% SDS-polyacrylamide gel electrophoresis under reducing conditions and autoradiography.

Immunocomplex Kinase Assay of the Type V TGF-beta Receptor

The immunoprecipitation of the type V TGF-beta receptor in mink lung epithelial cells treated with and without 0.1 nM TGF-beta 1 at 0 °C for 30 min was carried out in 0.1% Triton X-100 as described above, except the cells were not metabolically labeled. The immunoprecipitates were incubated with 0.2 µM recombinant nonglycosylated human IGFBP-3 in 50 µl of 20 mM HEPES, pH 7.4, containing 10% glycerol, 0.1% Triton X-100, 5 µM [gamma -32P]ATP, 0.1% mercaptoethanol, and 2.5 mM MnCl2. An aliquot of the reaction mixture was then analyzed by 7.5% SDS-polyacrylamide gel electrophoresis under reducing conditions and autoradiography. The relative intensity of 32P-IGFBP-3 on the autoradiogram was quantitated by a PhosphorImager.

Immunoprecipitation of the Type V TGF-beta Receptor in Trans35S-label Metabolically Labeled Mink Lung Epithelial Cells

Mink lung epithelial cells were grown to confluence on P-60 Petri dishes and metabolically labeled with Trans35S-label ([35S]methionine + [35S]cysteine) according to the procedure of Huang and Huang (13). The cells were then detached and lysed with Triton X-100 buffer (1% Triton X-100 in 100 mM Tris-HCl, pH 7.0, 125 mM NaCl2, and 1 mM EDTA) or RIPA buffer (1% sodium droxycholate, 1% Nonidet P-40, 0.1% SDS, 25 mM Tris-HCl, pH 7.4, and 0.15 M NaCl). After centrifugation, the Triton X-100 (final concentration, 0.1%) or RIPA buffer extracts were immunoprecipitated with antiserum to the type V TGF-beta receptor (1:100 dilution). The immunoprecipitates were then analyzed by 5.5% SDS-polyacrylamide gel electrophoresis and fluorography.

Immunoprecipitation of TGF-beta Receptors after 125I-TGF-beta Afffinity Labeling of Mink Lung Epithelial Cells

Mink lung epithelial cells were grown to confluence on P-60 Petri dishes and affinity labeled with 125I-TGF-beta in the presence of DSS as described previously (4, 5). The cells were then lysed with Triton X-100 buffer as described above. After centrifugation, the Triton X-100 buffer extracts were immunoprecipitated with antiserum to the type V TGF-beta receptor (1:100 dilution). The immunoprecipitates were analyzed by 5.5% SDS-polyacrylamide gel electrophoresis and autoradiography.

[methyl-3H]Thymidine Incorporation Assay

Wild-type mink lung epithelial cells (Mv1Lu cells), mutants (R-1B and DR26 cells), and human colorectal carcinoma cells (HCT 116 Neo and RII-37 cells) were plated on 24-well clustered dishes and incubated with various concentrations of TGF-beta 1 in Dulbecco's modified Eagle's medium containing 0.1% fetal calf serum. After incubation at 37 °C for 16 h, the cells were pulse-labeled with 1 µCi of [methyl-3H]thymidine at 37 °C for 4 h. The cells were then washed twice with 1 ml of 10% trichloroacetic acid, washed once with 0.5 ml of ethanol:ether (2:1, v/v), and dissolved in 0.2 N NaOH. The [methyl-3H]thymidine incorporation was determined by a scintillation counter.


RESULTS AND DISCUSSION

The type I, type II, and type III TGF-beta receptors have been shown to form heterocomplexes upon ligand binding (12, 15-17). The formation of heterocomplexes appears to be important for initiation of signaling (type I and type II TGF-beta receptor complex) (9-12, 15) or presentation of ligand (type III and type I/II TGF-beta receptor complexes) (16, 17). To see whether the type V TGF-beta receptor formed heterocomplexes with other TGF-beta receptors, we performed immunoprecipitations of the type V TGF-beta receptor and other TGF-beta receptors using specific antiserum to the type V TGF-beta receptor. The specificity of the antiserum to the type V TGF-beta receptor had been validated by two types of evidence. First, the antiserum reacted with the type V TGF-beta receptor in Western blot analysis. Fig. 1 shows that the antiserum specifically reacted with the type V TGF-beta receptor and its proteolytic product (~300 kDa) from bovine liver plasma membranes on the Western blot analysis (Fig. 1A, lane 1). The reaction was blocked in the presence of 0.5 mM peptide antigen (Fig. 1A, lane 2). Second, the antiserum was also able to immunoprecipitate the type V TGF-beta receptor in normal mink lung epithelial cells (Mv1Lu cells) that were metabolically labeled with [32P]orthophosphate in response to TGF-beta 1 stimulation (Fig. 1B). The immunoprecipitated type V receptor exhibited a kinase activity toward IGFBP-3 (Fig. 1C). IGFBP-3 is a nonphysiological substrate for the type V TGF-beta receptor but contains several SXE motifs that serve as the phosphorylation sites for the type V TGF-beta receptor kinase activity (7). The radioactive band at the top of the separating gel (Fig. 1C, lanes 1 and 2) was identified as the TGF-beta 1-stimulated and -unstimulated autophosphorylated type V TGF-beta receptor that could be removed by subsequent immunoprecipitation with antiserum to the type V receptor in the presence of 0.1% SDS. The TGF-beta 1-stimulated phosphorylation of the type V TGF-beta receptor and the TGF-beta 1-stimulated kinase activity of the type V TGF-beta receptor on IGFBP-3 were both estimated to be ~1.5-fold greater than those observed without TGF-beta 1 stimulation (Fig. 1, B and C, lanes 1 and 2). This 1.5-fold stimulation was comparable with that observed for the TGF-beta 1-stimulated kinase activity of the type V TGF-beta receptor purified from bovine liver plasma membranes (7).


Fig. 1. Western blot analysis of the type V TGF-beta receptor purified from bovine liver plasma membranes (A) and TGF-beta 1-stimulated phosphorylation (B) and TGF-beta 1-stimulated kinase activity (C) of the type V TGF-beta receptor in mink lung epithelial cells. A, the type V TGF-beta receptor partially purified from bovine liver plasma membranes was subjected to 6% SDS-polyacrylamide gel electrophoresis followed by electrophoretic transblotting onto nitrocellulose membranes. Western blot analysis was performed using antiserum (immune serum) to the type V TGF-beta receptor with and without 0.5 mM peptide antigen. The arrow and bar indicate the locations of the type V TGF-beta receptor and its proteolytic product, respectively. B, mink lung epithelial cells (Mv1Lu cells) were metabolically labeled with [32P]orthophosphate and treated with (+) or without (-) 0.1 nM TGF-beta 1 at 0 °C for 30 min. The cell lysates were then immunoprecipitated with antiserum (immune serum) to the type V TGF-beta receptor or nonimmune serum. The immunoprecipitates were then analyzed by 6% SDS-polyacrylamide gel electrophoresis under reducing conditions and autoradiography. The arrow indicates the location of 32P-labeled type V TGF-beta receptor. C, mink lung epithelial cells (Mv1Lu cells) were treated with (+) or without (-) 0.1 nM TGF-beta 1. After cell lysis and immunoprecipitation of the cell lysates with antiserum (immune serum) to the type V TGF-beta receptor, the kinase activity of the immunoprecipitates was assayed with recombinant nonglycosylated IGFBP-3 as substrate. After phosphorylation, 32P-labeled IGFBP-3 was analyzed by 7.5% SDS-polyacrylamide gel electrophoresis under reducing conditions and autoradiography. The arrow indicates the location of 32P-IGFBP-3.
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Normal mink lung epithelial cells (Mv1Lu cells) have been commonly used to examine the cross-interaction of the TGF-beta receptors and TGF-beta -induced signal transduction. They express all the known TGF-beta receptors (type I, type II, type III, and type V TGF-beta receptors) and exhibit both growth inhibition and transcriptional activation in response to TGF-beta stimulation (5, 18-20). In this study, the 125I-labeled TGF-beta 1 affinity labeled cell surface receptors were immunoprecipitated with the antiserum to the type V TGF-beta receptor, after mink lung epithelial cells (Mv1Lu cells) had been incubated with 0.1 nM 125I-TGF-beta 1 for 2 1/2 h at 0 °C, exposed to the cross-linking reagent DSS, and extracted with Triton X-100 buffer. The immunoprecipitates of the Triton X-100 buffer extracts were then analyzed by 5.5% SDS-polyacrylamide gel electrophoresis under reducing conditions and autoradiography. As shown in Fig. 2A, the type I TGF-beta receptor was co-immunoprecipitated with the type V TGF-beta receptor (Fig. 2A, lane 2). The immunoprecipitation efficiency for the type V TGF-beta receptor was estimated to be approximately 30% of the total labeled receptor (Fig. 2A, lane 1). Excess peptide antigen blocked the immunoprecipitation of the TGF-beta receptors (Fig. 2A, lane 3). Nonimmune serum did not immunoprecipitate the TGF-beta receptors (Fig. 2A, lane 4). These results suggest that on the cell surface of mink lung epithelial cells, the type V TGF-beta receptor forms heterocomplexes with the type I TGF-beta receptor in the presence of the ligand.


Fig. 2. Immunoprecipitation of TGF-beta receptors with antiserum to the type V TGF-beta receptor after 125I-TGF-beta 1 affinity labeling (A) and Trans35S-label metabolic labeling (B) of mink lung epithelial cells. A, cell surface TGF-beta receptors were affinity labeled with 125I-TGF-beta 1 and DSS in the presence (+) and the absence (-) of 100-fold molar excess of unlabeled TGF-beta 1. The 125I-TGF-beta 1 affinity labeled receptors were directly analyzed by 5.5% SDS-polyacrylamide gel electrophoresis under reducing conditions and autoradiography (lane 1) or extracted with Triton X-100 and immunoprecipitated with antiserum (immune serum) to the type V TGF-beta receptor in the absence (lane 2) and the presence (lane 3) of 0.5 mM peptide antigen or with nonimmune serum (lane 4). The immunoprecipitates were then analyzed by 5.5% SDS-polyacrylamide gel electrophoresis and autoradiography. The brackets indicate the locations of the type I, type II, and type III TGF-beta receptors (Tbeta Rs). The arrows indicate the locations of the type V TGF-beta receptor and dye front. B, mink lung epithelial cells were metabolically labeled with Trans35S-label. The immunoprecipitations of the Triton X-100 and RIPA buffer extracts of the labeled cells, 5.5% SDS-polyacrylamide gel electrophoresis, and fluorography were carried out as described under "Experimental Procedures." The arrows indicate the locations of the type V and type I TGF-beta receptors, and the bracket indicates the location of the type III TGF-beta receptor. The arrowhead indicates the location of a 68-kDa protein, which is possibly an isoform or differentially glycosylated form of the type I TGF-beta receptor.
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To see whether the ligand was required for the heterocomplex formation, the TGF-beta receptors were immunoprecipitated with antiserum to the type V TGF-beta receptor after mink lung epithelial cells (Mv1Lu cells) had been metabolically labeled with Trans35S-label in the absence of exogenous ligand and extracted with Triton X-100 buffer or RIPA buffer (13). The immunoprecipitates of the Triton X-100 or RIPA buffer extracts were analyzed by 5.5% SDS-polyacrylamide gel electrophoresis under reducing conditions and fluorography. As shown in Fig. 2B, the type V and type I (53 kDa) TGF-beta receptors were found in the immunoprecipitates of the Triton X-100 buffer extracts (Fig. 2B, lane 3), whereas only the type V TGF-beta receptor was detected in the immunoprecipitates of the RIPA buffer extracts (Fig. 2B, lane 1). A 68-kDa protein was also identified in the immunoprecipitates of the Triton X-100 buffer extracts (Fig. 2B, lane 3). This protein was possibly an isoform or differentially glycosylated form of the type I TGF-beta receptor because two different molecular mass isoforms of the type I TGF-beta receptor were also identified in the 125I-TGF-beta 1 affinity labeling experiment (Fig. 2A, lanes 1 and 2). RIPA buffer contained 0.1% SDS that destabilized the heterocomplexes of the type V and type I TGF-beta receptors. In the presence of RIPA buffer, only the type V TGF-beta receptor was immunoprecipitated (Fig. 2B, lane 1). Because no endogenous TGF-beta activity (growth inhibitory activity) was detected under the cultured conditions and because exogenous TGF-beta 1 did not affect the complexation of 35S-labeled type V and type I TGF-beta receptors (data not shown), these results suggest that the type V TGF-beta receptor can form heterocomplexes with the type I TGF-beta receptor in the absence of ligand.

As described previously, mink lung epithelial cells (Mv1Lu cells) have been a useful system for studying the roles of the type I and type II TGF-beta receptors in TGF-beta -induced cellular responses. Mutant cells defective for the type I or type II TGF-beta receptor have been reported to be unable to exhibit growth inhibition and transcriptional activation following TGF-beta stimulation (18-21). However, the growth inhibition and transcriptional activation could be restored by genetic complementation between cells defective in the type I and type II TGF-beta receptors (21). No study on the expression of the type V TGF-beta receptor in these mutants has been reported. To test its presence, we investigated the expression of the type V TGF-beta receptor in these mutants.

As shown in Fig. 3 (A and B), both the type II and type I TGF-beta receptor-defective mutants (DR26 and R-1B cells, respectively) did express the type V TGF-beta receptor, which was identified as the 400-kDa 125I-TGF-beta 1 affinity labeled protein. It is of importance to note that the type I TGF-beta receptor-defective mutant (R-1B cells) expressed less of the type V TGF-beta receptor when compared with the type II TGF-beta receptor-defective mutant (DR26 cells).


Fig. 3. Identification of the type V TGF-beta receptor in TGF-beta receptor-defective mutants of mink lung epithelial cells (A and B) and growth inhibitory response of these mutants to TGF-beta 1 stimulation (C). A and B, cell surface TGF-beta receptors were affinity labeled with 125I-TGF-beta 1 (0.5, 1, and 1.5 nM) and DSS in type II and type I receptor-defective mutants (DR 26 and R-1B cells, respectively) and wild-type mink lung epithelial cells (Mv1Lu cells). The 125I-TGF-beta 1 affinity labeled receptors were analyzed by 5% (panel A) or 5.5% (panel B) SDS-polyacrylamide gel electrophoresis under reducing conditions and autoradiography. The brackets indicate the locations of the type I, type II, and type III TGF-beta receptors. The arrows indicate the locations of the type V TGF-beta receptor and dye front. C, the TGF-beta 1-induced growth inhibition of mutants (DR 26 and R-1B cells) and wild-type cells (Mv1Lu cells) was assayed on 24-well clustered dishes based on the inhibition of [methyl-3H] thymidine incorporation into DNA of these cells stimulated by various concentrations of TGF-beta 1. The [methyl-3H]thymidine incorporation in wild-type cells without treatment with TGF-beta 1 was taken as 0% inhibition (51,000 ± 1, 500 cpm/well).
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Because both mutants (DR26 and R-1B cells) express the type V TGF-beta receptor, an important role of the type V TGF-beta receptor in TGF-beta -induced cellular responses cannot be excluded in the genetic complementation experiments of the mutants (21). To determine whether the type V TGF-beta receptor mediated a TGF-beta -induced growth inhibition, we investigated the effect of higher concentrations of TGF-beta 1 on [methyl-3H]thymidine incorporation into DNA of mink lung epithelial cells (Fig. 3C). The Kd values for TGF-beta 1 or TGF-beta 2 binding to the type V receptor are approximately 40-fold higher than those for the type I and type II TGF-beta receptors (6). If the type V TGF-beta receptor can mediate a TGF-beta -induced growth inhibition, TGF-beta at higher concentrations should inhibit the DNA synthesis in the type I and type II TGF-beta receptor-defective mutants. At ~50-100 pM, TGF-beta 1 induced a small but significant inhibition of DNA synthesis (Fig. 3C) in both mutants (~20-40%). In type II TGF-beta receptor-defective mutant cells (DR26 cells), the inhibition of DNA synthesis showed a small downward trend at >= 125 pM (Fig. 3C), but higher concentrations of TGF-beta 1 (~500 pM) did not cause a further decrease (30 ± 4% inhibition). A smaller degree of inhibition of DNA synthesis by TGF-beta 1 in type II TGF-beta receptor-defective mutants was previously reported but was not discussed (20). These results suggest that the type V TGF-beta receptor may mediate the growth inhibition in the absence of the type I or type II TGF-beta receptor, although a higher level of ligand is required for the type V TGF-beta receptor-mediated growth inhibition. In the wild-type cells (Mv1Lu cells) containing all TGF-beta receptors, the maximal inhibition was observed at ~1 pM of TGF-beta 1.

In a previous study, we reported that several carcinoma cells lacked the type V TGF-beta receptor and other TGF-beta receptors (5). These cells lacking the type V TGF-beta receptor (MCF-7 and PC-12 cells) have been found not to respond to TGF-beta stimulation with respect to growth inhibitory response (5).2 Recently, heriditary human colorectal carcinoma cells (HCT 116 cells) were shown to be deficient in the type II TGF-beta receptor (22). Stable transfection of these colorectal carcinoma cells with the type II TGF-beta receptor cDNA was found to rescue the transcriptional response but failed to restore the growth inhibitory response to exogenous TGF-beta (22). The inability of the transfected colorectal carcinoma cells to exert growth inhibitory response to TGF-beta stimulation was also confirmed in our laboratory. One of the possibilities for the failure to restore the growth inhibitory response could be the lack of the type V TGF-beta receptor expression in cells stably transfected with the neo-vector only (HCT 116 Neo cells) or with vector expressing the type II TGF-beta receptor cDNA (RII-37 cells). To test this possibility, we performed the 125I-TGF-beta 1 affinity labeling of the TGF-beta receptors in these cells. No detectable type V TGF-beta receptor was found in these cells (Fig. 4, lanes 1-4). The type III and type II TGF-beta receptors were detected in RII-37 cells (Fig. 4, lane 3). The type I TGF-beta receptor migrated at the dye front and could not be identified in the 5% polyacrylamide system (Fig. 4, lane 3). Absence of the type V TGF-beta receptor was confirmed by the observation that the type V TGF-beta receptor antigen was not detected by Western blot analysis in HCT 116 Neo and RII-37 cells (data not shown). These results and the results from several experiments as shown in Fig. 3 can be summarized in Table I. The type V TGF-beta appears to be critical for TGF-beta -induced growth inhibition. The type I and type II TGF-beta receptors are required for the maximal growth inhibition induced by TGF-beta . Together with the observation that only transformed epithelial cells (carcinoma cells) have been found to lack the expression of detectable type V TGF-beta receptor (5), these results also support the hypothesis that loss of the type V TGF-beta receptor may contribute to the transformed state of certain carcinoma cells (e.g. hereditary human colorectal carcinoma cells) (5).


Fig. 4. Identification of TGF-beta receptors by 125I-TGF-beta 1 affinity labeling in neo-vector and type II TGF-beta receptor cDNA stably transfected hereditary human colorectal carcinoma cells (HCT 116 cells). Neo-vector and type II TGF-beta receptor cDNA transfected HCT 116 cells (HCT 116 Neo and RII-37 cells) were grown on P-60 Petri dishes. Cell surface TGF-beta receptors were affinity labeled with 0.1 nM 125I-TGF-beta 1 and DSS as described previously. The 125I-TGF-beta 1 affinity labeled receptors were then analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. In the experiments, mink lung epithelial cells (Mv1Lu cells) were used as positive control cells. The brackets indicate the locations of the type II and type III TGF-beta receptors. The arrows indicate the locations of the type V TGF-beta receptor and dye front. In the 5% SDS-polyacrylamide gel system, the type I TGF-beta receptor comigrates with the dye front.
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Table I. TGF-beta induced growth inhibition in wild-type mink lung epithelial cells (Mv1Lu cells), type I and type II TGF-beta receptor-defective mutants (R-1B and DR26 cells), and human colorectal carcinoma cells lacking the type V TGF-beta receptor (HCT 116 Neo and RII-37 cells)


Cell TGF-beta receptor/kinase typea Growth inhibition by TGF-beta b

%
Mv1Lu cells I+, II+, V+ 100
DR26 cells I+, II-, V+ 30  ± 10
R-1B cells I-, II+, V+ 15  ± 7
HCT 116 Neo cells I+, II-, V- 0
RII-37 cells I+, II+, V- 0

a The TGF-beta receptor types were determined by 125I-TGF-beta affinity labeling. All cell lines expressed the type III TGF-beta receptor. The absence of the type V TGF-beta receptor in HCT 116 Neo and RII-37 cells was further evidenced by Western blot analysis.
b Growth inhibition was assayed with 1 or 50 pM TGF-beta 1 based on the inhibition of [methyl-3H]thymidine incorporation into cellular DNA. The [methyl-3H]thymidine incorporation in wild-type mink lung epithelial cell (Mv1Lu cells) treated with and without 1 pM TGF-beta 1 were taken as 100 and 0% growth inhibition, respectively. Three experiments were performed for each cell line. All cell lines except Mv1Lu cells were assayed with 50 pM TGF-beta 1.

Together with the previous observations of distinct substrate specificities of the type V, type I, and type II TGF-beta receptors (7, 23), the finding of heterocomplex formation of the type V TGF-beta receptor and type I TGF-beta receptor reported here raise an important question: What role does the type V TGF-beta receptor play in various cellular responses induced by three different TGF-beta isoforms (TGF-beta 1, TGF-beta 2, and TGF-beta 3)? The cellular responses induced by the TGF-beta isoforms can vary substantially (1-3, 24, 25). The molecular mechanisms of the opposite effects of TGF-beta 3 versus TGF-beta 1 or TGF-beta 2 (25) are not easy to interpret within a model in which only the type I and type II TGF-beta receptor heterocomplex mediates the signaling. The type V TGF-beta receptor could be involved in these diverse cellular responses due to its distinct kinase substrate specificity (acidotrophic kinase activity versus the basic-trophic kinase activities of the type I and type II TGF-beta receptors) (7, 23) and its different binding affinities to TGF-beta isoforms. The distinct substrate specificity of the type V TGF-beta receptor implies a different signaling pathway from those of the type I and type II TGF-beta receptors. Segregation of growth inhibitory and transcriptional responses induced by TGF-beta has been reported (22, 26, 27). The signaling pathway mediated by the type V TGF-beta receptor could be important for growth inhibitory response but not obligatory for transcriptional response (the activation of transcription of fibronectin, collagen and plasminogen activator inhibitor-1 genes) (22). The cross-modulation of the two pathways mediated by the type V/I and type II/I TGF-beta receptors should be determined by the affinities of TGF-beta isoforms to the TGF-beta receptors and extracellular concentrations of TGF-beta isoforms. The binding affinities of TGF-beta 1, -beta 2, and -beta 3 for the type I and type II TGF-beta receptors are very similar with Kd of ~0.01 nM (24). The Kd of the type V TGF-beta receptor for TGF-beta 1, -beta 2, and -beta 3 have been estimated to be ~0.4 nM, ~0.4 nM, and ~5 nM, respectively (7).2 Low concentrations of TGF-beta 3 might favor the formation of the type I and type II TGF-beta receptor heterocomplexes that do not include the type V TGF-beta receptor. Under the condition in which the amount of the type I receptor is limiting compared with those of other TGF-beta receptors, TGF-beta 3 could alter the availability of the type I TGF-beta receptor when the formation of type I and type V TGF-beta receptor heterocomplex is required for certain cellular responses induced by TGF-beta 1 or TGF-beta 2.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant CA 38808.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.
Dagger    To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8135; Fax: 314-577-8156; E-mail: huangjs{at}wpogate.slu.edu.
1   The abbreviations used are: TGF-beta , transforming growth factor beta ; DSS, disuccinimidyl suberate; IGFBP-3, insulin-like growth factor binding protein 3.
2   Q. Liu, S. S. Huang, and J. S. Huang, unpublished results.

ACKNOWLEDGEMENTS

We thank Dr. William S. Sly for critical review of the manuscript, Drs. Joan Massagué and Michael G. Brattain for kindly providing TGF-beta receptor-defective mutants (DR26 and R-1B cells) and type II TGF-beta receptor cDNA and neo-vector transfected hereditary human colorectal carcinoma cells (HCT 116 cells), and Celtrix Pharmaceuticals, Inc., for providing recombinant human nonglycosylated IGFBP-3. We also thank Tao Zhao for performing the kinase assay of the immunoprecipitated type V receptor and Maggie Klevorn for preparing the manuscript.


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