Processing of the Transforming Growth Factor beta  Type I and II Receptors
BIOSYNTHESIS AND LIGAND-INDUCED REGULATION*

(Received for publication, November 22, 1996, and in revised form, December 19, 1996)

Katri M. Koli Dagger § and Carlos L. Arteaga Dagger §par **

From the Departments of Dagger  Medicine and  Cell Biology, Vanderbilt University School of Medicine, § Vanderbilt Cancer Center and the par  Department of Veteran Affairs Medical Center, Nashville, Tennessee 37232-5536

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Three cell surface transforming growth factor beta  (TGFbeta ) receptor (R) proteins regulate the effects of TGFbeta isoforms on growth and differentiation. TGFbeta -IR and -IIR are transmembrane serine/threonine kinases directly mediating the signaling across the plasma membrane. Both TGFbeta and its receptors are ubiquitously expressed, hence the fine regulation of the multiplicity of responses most likely involves several levels of control including the regulation of expression, complex formation, and down-regulation of the receptor proteins. In mink lung epithelial cells, TGFbeta -IIR was first synthesized as a ~60-kDa endoglycosidase H-sensitive precursor protein, which was converted to a mature ~70-kDa protein. The half-life of metabolically labeled mature TGFbeta -IIR was estimated to be 60 min and was further reduced to ~45 min in the presence of exogenous TGFbeta 1. Minimal internalization of 125I-TGFbeta 1 at 37 °C was detected suggesting that the rapid turnover was not due to endocytosis and degradation of the ligand-receptor complexes. TGFbeta -IR was synthesized as a ~53-kDa precursor protein, which was processed to a mature ~55-kDa receptor protein. The half-life of TGFbeta -IR was >12 h. A fraction of tunicamycin-treated type I and II receptors that reach the cell surface was able to associate in the presence of ligand suggesting that heteromeric complexes can form in a post-endoplasmic reticulum compartment before full glycosylation is achieved. These results show differential processing and turnover of TGFbeta -IR and TGFbeta -IIR providing a potential additional mechanism for modulation of cellular responses to TGFbeta s.


INTRODUCTION

The TGFbeta 1 family of proteins participates in the regulation of a variety of biological activities including regulation of cellular growth and phenotype (1-3). Most cells can produce latent forms of TGFbeta , and their activation plays an important regulatory role in TGFbeta actions (4, 5). In epithelial cells, TGFbeta treatment leads to inhibition of growth, regulation of the production of extracellular matrix proteins, and modulation of proteolysis (4). The cell surface signaling receptor complex is composed of two transmembrane serine/threonine kinases named type I (~55 kDa) (6) and type II (~70 kDa) (7) TGFbeta receptors. TGFbeta -IIR binds the ligand first, after which TGFbeta -IR is recruited to a heteromeric complex most likely containing several receptor molecules (8, 9). Ligand-dependent phosphorylation of the GS-domain of TGFbeta -IR leads to the propagation of the signal downstream (10, 11). Type III TGFbeta receptor, a proteoglycan also known as betaglycan, functions mostly as a storage protein as well as in presenting the ligand for the signaling receptors (12). Both TGFbeta -IR and TGFbeta -IIR are needed to mediate the biological effects of TGFbeta ligands. Recent reports, however, suggest separate signaling pathways for the antiproliferative and the matrix modulatory effects of TGFbeta with the latter only requiring TGFbeta -IR signaling in some cell systems (13, 14).

Since most cell types can produce both TGFbeta receptors and ligand(s), the regulation of cellular responsiveness relies on the production of active TGFbeta and its presentation to signaling receptors. Therefore, the role of receptor protein associations, turnover, and down-regulation is likely critical for the control of TGFbeta signals and the modulation of overall cellular responsiveness. TGFbeta -IIR levels have been shown to correlate with TGFbeta responsiveness (15, 16). Cancer cells refractory to TGFbeta 's antiproliferative action have often lost TGFbeta -IIR expression (17-19). Although TGFbeta -IR can bind ligand only in association with TGFbeta -IIR, it is indispensable for TGFbeta responses since phosphorylation of its GS domain provides possible binding sites for intracellular substrates (10, 20). Interestingly, cellular transformation by Ha-ras oncogene as well as tropic hormones can down-regulate cell surface binding sites for TGFbeta , thus altering TGFbeta responsiveness (21-23). We have examined the biosynthesis and ligand-induced modulation of naturally expressed TGFbeta -IR and TGFbeta -IIR in CCL-64 mink lung epithelial cells, which are known to express abundant amounts of all three TGFbeta receptors and are potently growth inhibited by exogenous TGFbeta .


EXPERIMENTAL PROCEDURES

Cell Culture and Metabolic Labeling

CCL-64 mink lung epithelial cells (American Type Culture Collection, Rockville, MD) were cultured in McCoy's medium supplemented with 5% fetal bovine serum (JRH Biosciences, Lenexa, KS). Subconfluent (~80%) cell monolayers were labeled with 500 µCi/ml Tran35S-label (>1000 Ci/mmol, ICN Biomedicals, Inc., Irvine, CA) for 20 min in improved minimal essential medium lacking cysteine and methionine. After a quick wash with chase medium (McCoy's medium: 5% fetal calf serum, 300 µg/ml cysteine, 500 µg/ml methionine), the cells were incubated in chase medium for the indicated times. For steady-state labeling, cells were incubated with 50 µCi/ml Tran35S-label for 2.5 h in improved minimal essential medium lacking cysteine and methionine. Following labeling the cell monolayers were prepared as described below.

Cell Lysis and Immunoprecipitation

Cell monolayers in 100-mm tissue culture plates were solubilized with 1 ml of lysis buffer (20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 25 mM NaF, 1% Triton X-100, 1 mM dithiothreitol, 2 mM NaMo4, 2 mM NaVO4, 1 µg/ml aprotinin, 1 µg/ml leupeptin) for 30 min at 4 °C. After a 12,000 × g centrifugation for 15 min, the lysates were precleared with protein A-Sepharose (Sigma) for 30 min at 4 °C and precipitated overnight with a polyclonal TGFbeta -IR (V-22, Santa Cruz Biotechnology, Santa Cruz, CA), TGFbeta -IIR (C-16, Santa Cruz Biotechnology), or TGFbeta -IIR 2732 antibodies. The latter was raised against the extracellular domain of the human type II receptor overexpressed in Sf9 cells as a HIS-tagged protein and provided by Dr. Xiao-Fan Wang (Duke University, Durham, NC). This incubation was followed by a 1.5-h incubation with protein A-Sepharose. The Sepharose particles were washed three times with radioimmune precipitation buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) and the immune complexes dissociated with lysis buffer containing 1% SDS for 5 min at 95 °C. The concentration of SDS was diluted to 0.1% with lysis buffer and a second immunoprecipitation with the same antibody was performed for 2.5 h at room temperature, the last 1.5 h in the presence of protein A-Sepharose. The immune complexes were eluted with Laemmli sample buffer, boiled, resolved by 8% SDS-PAGE, and visualized by autoradiography.

Deglycosylation Procedures

Cell lysates were subjected to overnight immunoprecipitation with TGFbeta -IR or TGFbeta -IIR antibodies as described above. After washes the immune complexes bound to protein A-Sepharose beads were treated with 50 milliunits of endoglycosidase H (Sigma) in 17 mM phosphate-buffered saline, pH 5.5 (170 mM NaCl, 10 mM sodium phosphate buffer) for 24 h at 37 °C or deglycosylated using an enzymatic deglycosylation kit containing NANase II, O-glycosidase DS, and PNGase F according to the manufacturer's protocol (Bio-Rad). Control samples were treated with the same buffers lacking enzymes. After deglycosylation, a second immunoprecipitation was performed as described above, and the samples were resolved by 8% SDS-PAGE and visualized by autoradiography.

Internalization of 125I-TGFbeta

Cell monolayers in 6-well plates were preincubated in a binding buffer (128 mM NaCl, 5 mM KCl, 5 mM MgSO4, 1.2 mM CaCl2, 50 mM Hepes, pH 7.5, 0.2% BSA) for 15 min at 37 °C. Fresh binding buffer containing 1 ng/ml 125I-TGFbeta 1 (specific activity, 173 µCi/µg; DuPont NEN) with or without 100-fold excess unlabeled TGFbeta 1 (Genentech, South San Francisco, CA) was added and the plates incubated in a 37 °C water bath for different times ranging from 1 to 30 min. After incubation at 37 °C the plates were quickly put on ice and washed twice with cold phosphate-buffered saline/0.2% BSA. 125I-TGFbeta 1 bound to cell surface receptors was acid washed with 50 mM glycine, pH 2.4, 100 mM NaCl, 2 mg/ml polyvinylpyrrolidone, M urea twice for 3 min at 4 °C. After two washes with phosphate-buffered saline, 0.2% BSA, cells were lysed with 1 M NaOH for 30 min at 37 °C. 125I measurements from acid-washed and NaOH-lysed cells, representing both the surface bound not internalized and internalized ligand, respectively, were determined in a Gamma 7000 counter (Beckman Instruments).

125I-TGFbeta Binding and Affinity Cross-linking

Binding was performed on adherent cells in 100-mm tissue culture dishes as described previously (24). Cells were incubated in binding buffer (see above) containing 1 ng/ml 125I-TGFbeta 1 with or without 100-fold excess unlabeled TGFbeta 1 for 4 h at 4 °C with gentle rocking. After two washes with ice-cold binding buffer without BSA on ice, the bound 125I-TGFbeta 1 was cross-linked to cell surface receptors with 50 µM disuccinimidyl suberate (Pierce) for 15 min at 4 °C in 10 ml of binding buffer without BSA. Cells were solubilized in lysis buffer as described above (cell lysis and immunoprecipitation) and the lysates subjected to overnight immunoprecipitation with TGFbeta -IR or TGFbeta -RII antibodies followed by incubation with protein A-Sepharose for 1.5 h. Immune complexes were then resolved by 5-15% gradient SDS-PAGE and visualized by autoradiography. Exposures on PhosphorImager screens and image analysis with ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA) were used to visualize labeled proteins in some cases.

Western Blotting

For Western blotting, 100-µg aliquots of cellular protein were separated by 8% SDS-PAGE and transferred to nitrocellulose membranes by semi-dry electrophoretic transfer (Bio-Rad). Nonspecific binding was blocked with 5% nonfat milk in TTBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. The membranes were incubated with antibodies in the same buffer for 1 h at room temperature and washed three times with TTBS for 10 min each. Bound antibodies were detected using peroxidase-conjugated anti-immunoglobulins (Amersham Corp.) and an enhanced chemiluminescence detection system (Kirkegaard & Perry Laboratories, Gaithersburg, MD).


RESULTS AND DISCUSSION

The Biosynthesis and Turnover of TGFbeta -IR and -IIR

Recent studies using cycloheximide to block cellular protein synthesis have suggested that the turnover rate of TGFbeta binding sites in bone cells is relatively fast (25). Since TGFbeta -IR cannot bind TGFbeta 1 alone, these affinity binding studies provide clues only on the turnover of TGFbeta -IIR protein. We have followed the fate of metabolically labeled receptors in CCL-64 mink lung epithelial cells. Subconfluent cultures were labeled with Tran35S-label for 20 min followed by chase with medium lacking label for variable times. The newly synthesized TGFbeta -IIR first appeared as a ~60-kDa form with a half-life of <30 min (Fig. 1A). This form was sensitive to treatment with endoglycosidase H (Fig. 1B). Since endoglycosidase H cleaves only high mannose oligosaccharides but not more complex structures, this result suggests that this ~60-kDa form represents an ER, pre-Golgi precursor form. Within 15 min of the chase, a ~70-kDa endoglycosidase H-resistant smear appeared with a longer half-life of approximately 60 min. This form was sensitive to deglycosylation by enzymes that remove all N- and O-linked oligosaccharides (Fig. 1B), indicating it represents the mature type II receptor.


Fig. 1. Biosynthesis of TGFbeta -IIR. Cells were labeled with 500 µCi/ml Tran35S-label for 20 min and chased for the indicated times with growth medium containing unlabeled methionine (300 µg/ml) and cysteine (500 µg/ml). Lysis of cells was followed by double immunoprecipitation with TGFbeta -IIR antibodies as described under "Experimental Procedures." A, chase time ranging from 0 to 4 h. B, after a 30-min chase and immunoprecipitation, the immune complexes bound to protein A-Sepharose were treated with 50 milliunits of endoglycosidase H (endo H) or a combination of NANase II, O-glycosidase, and PNGase F (deglyc) overnight followed by a second immunoprecipitation with the same antibodies. The immune complexes were analyzed by 8% SDS-PAGE and autoradiography. The arrows indicate the migration of receptor proteins. Molecular mass markers in kDa are shown at the left of each panel.
[View Larger Version of this Image (41K GIF file)]


TGFbeta -IR was also first synthesized as a precursor form of molecular mass ~53 kDa that was chased to a mature ~55-kDa protein (Fig. 2A). The observed half-life of both precursor and mature forms was considerably longer than that of TGFbeta -IIR. The longer persistence of the type I receptor precursor form indicates a less efficient ER processing compared with that of TGFbeta -IIR precursor. The steady-state 35S-labeled mature TGFbeta -IR was sensitive to N- and O-linked deglycosylation (Fig. 2B), thus indicating that the 55-kDa form represents the fully processed cell surface receptor. The higher molecular mass band was not blocked by competing immunizing peptide, indicating it is nonspecific (Fig. 2B).


Fig. 2. Biosynthesis of TGFbeta -IR. A, cells were labeled with 500 µCi/ml Tran35S-label for 20 min and chased for the indicated times with growth medium containing unlabeled methionine (300 µg/ml) and cysteine (500 µg/ml). Lysis of cells was followed by double immunoprecipitation with TGFbeta -IR antibodies. B, cells were labeled with 50 µCi/ml Tran35S-label for 2.5 h, lysed, and precipitated with TGFbeta -IR antibodies in the presence (+) or absence (-) of the immunizing peptide. Immune complexes bound to protein A-Sepharose beads were deglycosylated (deglyc) as in Fig. 1B followed by a second immunoprecipitation with the same antibody. For panels A and B, the immune complexes were analyzed by 8% SDS-PAGE and autoradiography. The arrows indicate the migration of receptor proteins. Molecular mass markers in kDa are shown at the left of each panel.
[View Larger Version of this Image (33K GIF file)]


Effect of TGFbeta on TGFbeta -IIR and -IR Turnover

We analyzed next whether TGFbeta could further influence the rapid turnover of TGFbeta -IIR. For this purpose, subconfluent cells were labeled with Tran35S-label for 2.5 h in methionine/cysteine-free medium followed by chase with full medium. At the end of the labeling (time 0) both the precursor and the mature TGFbeta -IIR could be immunoprecipitated from the cell lysates (Fig. 3A). The amount of the mature ~70-kDa TGFbeta -IIR was higher compared with the precursor form indicating that it represents the predominant and/or more stable receptor species. In cells that were treated with 10 ng/ml TGFbeta 1 for the last 20 min of labeling as well as throughout the chase, two other proteins coprecipitated with TGFbeta -IIR: a ~50-kDa and a ~37-kDa protein, the latter corresponding to the size of TRIP-1 (TGFbeta receptor interacting protein-1 (26)). The identity of these proteins was not further confirmed, but their ability to coprecipitate with TGFbeta -IIR antibodies in a ligand-dependent manner under stringent double immunoprecipitation conditions suggests a strong association induced by exogenous ligand. In the presence of TGFbeta 1 the half-life of the mature TGFbeta -IIR was shortened to approximately 45 min (Fig. 3B).


Fig. 3. Effect of TGFbeta on TGFbeta -IIR turnover. Cells were labeled with 50 µCi/ml Tran35S-label for 2.5 h and chased for the indicated times with growth medium containing an excess of unlabeled methionine and cysteine. TGFbeta 1 (10 ng/ml) was present in indicated samples (+) during the last 20 min of 35S labeling and throughout the chase period. Immunoprecipitation was performed with TGFbeta -IIR antibodies. The immune complexes were analyzed by 8% SDS-PAGE and autoradiography. The arrows indicate the migration of TGFbeta -IIR proteins as well as two coprecipitating proteins of molecular mass ~50 and ~37 kDa. Molecular mass markers in kDa are shown at left. PhosphorImager analysis of the ~70-kDa band in control (-) and TGFbeta 1-treated (+) cells using ImageQuant software is shown in the graph. The results are expressed as a percent of control of respective zero time points.
[View Larger Version of this Image (26K GIF file)]


Labeling with Tran35S-label for 2.5 h in methionine/cysteine-free medium followed by chase with full medium indicated that the ~55-kDa mature TGFbeta -IR form was the predominant one (Fig. 4). Some TGFbeta -IR protein could still be immunoprecipitated 18 h after the 2.5-h steady-state labeling indicating that TGFbeta -IR half-life was considerably longer than that of TGFbeta -IIR. Contrary to the results with TGFbeta -IIR, the half-life of steady-state 35S-labeled TGFbeta -IR was not considerably affected by TGFbeta 1 treatment (Fig. 4).


Fig. 4. Effect of TGFbeta on TGFbeta -IR turnover. Cells were labeled with Tran35S-label and chased for indicated times with (+) or without (-) TGFbeta 1 as in Fig. 3. Double immunoprecipitation was performed with TGFbeta -IR antibodies. The immune complexes were analyzed by 8% SDS-PAGE and autoradiography. The arrow indicates the migration of TGFbeta -IR protein. Molecular mass markers in kDa are shown at left.
[View Larger Version of this Image (38K GIF file)]


Internalization of 125I-TGFbeta 1

We addressed whether the rapid turnover of TGFbeta receptors in the presence of ligand was due to endocytosis and degradation. To measure the rate of 125I-TGFbeta 1 internalization, cells were exposed to the radiolabeled ligand for different times at 37 °C. Surface-bound ligand was released from the cell surface by an acid wash procedure. This buffer (pH 2.4) was shown to remove ~95% of specifically bound 125I-TGFbeta 1 after 2 h of binding at 4 °C, conditions under which no ligand internalization should occur (Fig. 5A, bars 2). To confirm the efficacy of our receptor stripping procedure, binding was performed in intact cells after acid wash. Cell membranes were not affected by the transient exposure to low pH, since 125I-TGFbeta 1 binding was comparable with that in non-pretreated cells (Fig. 5A, bars 3 versus bar 1).


Fig. 5. Internalization of 125I-TGFbeta 1. A, 125I-TGFbeta 1 binding at 4 °C and acid wash procedure. Cell monolayers grown in 6-well plates were incubated in triplicate with 1 ng/ml of 125I-TGFbeta 1 ± 100 ng/ml unlabeled TGFbeta 1 at 4 °C. After 2 h incubation, cells were either lysed in 1 M NaOH (bar 1) or acid washed to remove surface bound ligand and then lysed with NaOH (bars 2). In bars 3, an acid wash step preceded binding, which was followed by acid wash and NaOH lysis. B, kinetics of 125I-TGFbeta 1 binding and internalization. Cells were incubated with 1 ng/ml of 125I-TGFbeta 1 ± 100 ng/ml unlabeled TGFbeta 1 for the indicated times at 37 °C after which surface-bound non-internalized ligand was removed by an acid wash procedure. The remaining internalized ligand was measured by solubilizing cells with 1 M NaOH. C, Western blot of TGFbeta -IIR. Cells grown on 100-mm dishes were treated with or without 10 ng/ml TGFbeta 1 overnight in improved minimal essential medium/10% fetal calf serum. Cell were lysed and 100 µg aliquots were subjected to 8% SDS-PAGE followed by a TGFbeta -IIR immunoblot (2732 antibody). Molecular mass markers in kDa are shown at left.
[View Larger Version of this Image (16K GIF file)]


A time-dependent increase in acid-washable specific binding of 125I-TGFbeta 1 was observed (Fig. 5B).Very little ligand was internalized during the first 10 min of the experiment, and even after a 30-min incubation at 37 °C the ratio of surface/internalized ligand was 0.25 (Fig. 5B). These results suggest that the rapid turnover of TGFbeta receptors in the presence of ligand may not be explained by endocytosis and subsequent degradation. This was further supported by Western blot analysis of TGFbeta 1-treated cells. An overnight incubation with exogenous ligand did not alter TGFbeta -IIR content in CCL-64 cells (Fig. 5C).

The possibility that the short half-life results from the release of the extracellular domain of TGFbeta -IIR into the medium was investigated by immunoprecipitation of the chase medium after labeling with Tran35S-label (see above) with the polyclonal TGFbeta -IIR antibody (#2732) raised against the receptor's extracellular domain. Even with a long exposure time no proteins were detected in the precipitated chase medium 1-3 h after cell labeling (data not shown), and small proteolytic fragments also were not detected in the precipitated cell lysates (Fig. 1A).

Association of TGFbeta -IR with TGFbeta -IIR

Chen et al. (27) reported recently that the cytoplasmic domains of type I and II receptors have an inherent affinity for each other even in the absence of the ligand. The interaction was shown to require kinase activity and thus depended on phosphorylation. Part of the receptors at cell surface exist as hetero-oligomers although TGFbeta -IIR homo-oligomers predominate (28, 29). We studied the stage at which TGFbeta -IR can associate with TGFbeta -IIR by stripping oligosaccharide chains from receptor proteins with tunicamycin followed by 125I-TGFbeta 1 binding at 4 °C, covalent cross-linking, and precipitation with TGFbeta -IIR or -IR antibodies. Tunicamycin inhibits the formation of N-glycosidic linkages during protein synthesis with the newly synthesized proteins mimicking the precursor/ER form of the receptors. The trafficking of receptors to the cell surface was not eliminated by tunicamycin. Treatment with 5 µg/ml tunicamycin for 5 h was enough to deglycosylate TGFbeta -IIR, whereas 24 h of treatment was needed for TGFbeta -IR (Fig. 6), consistent with the longer half-life and slower processing in the ER of the type I receptor (Fig. 2). Deglycosylated TGFbeta -IIR was able to bind exogenous ligand and associate with both fully processed TGFbeta -IR (5 h, lane 2) and deglycosylated TGFbeta -IR as judged by coprecipitation by TGFbeta -IIR antibodies (24 h, lane 2) and TGFbeta -IR antibodies (24 h, lane 4). However, deglycosylated TGFbeta -IIR did not coprecipitate with TGFbeta -IR antibodies (5 h, lane 4). This could well reflect a lower precipitation efficiency of the latter since TGFbeta -IIR antibodies coprecipitated both deglycosylated TGFbeta -IIR and mature TGFbeta -IR (5 h, lane 2). The lesser amounts of deglycosylated TGFbeta -IR that could be detected in the presence of tunicamycin (24 h panel) may reflect diminished trafficking to the cell surface, an alternation in the half-life of the ER form of TGFbeta -IR, and/or a critical need of N-linked glycosylation in TGFbeta -IR for ligand binding. These not mutually exclusive possibilities will require further study.


Fig. 6. Effect of tunicamycin on 125I-TGFbeta 1 binding. Cells were treated with 5 µg/ml tunicamycin for 5 or 24 h followed by 125I-TGFbeta 1 binding and cross-linking as indicated under "Experimental Procedures." Cell lysates were subjected to immunoprecipitation with TGFbeta -IIR or TGFbeta -IR antibodies and immune complexes analyzed by 5-15% gradient SDS-PAGE. The labeled proteins were visualized by autoradiography either on x-ray film (left panel) or PhosphorImager screens (right panel). The molecular mass markers are shown on the left of each panel. Ip, immunoprecipitate.
[View Larger Version of this Image (62K GIF file)]


In summary, native TGFbeta type I and II receptors are processed differently and separately in mink lung epithelial cells, with the TGFbeta -IIR protein exhibiting a more efficient ER processing and a much shorter half-life (approximately 60 min versus >12 h) than type I receptor. Studies with exogenous labeled ligand suggested that the fast turnover of TGFbeta -IIR protein is not due to receptor endocytosis and subsequent degradation. This short metabolic half-life of native TGFbeta -IIR, as measured directly by metabolic labeling, agrees with a recent study in osteoblasts. In this study, suppression of protein synthesis with cycloheximide reduced 125I-TGFbeta 1 binding to types I and II receptor with a half-life of 2 h (25). The rapid reduction in binding to TGFbeta -IR in this study may not have reflected the stability of newly synthesized type I receptor but could be explained by the reduction in type II receptor, critical for TGFbeta -IR binding. Our direct biosynthetic studies suggest a more prolonged half-life for TGFbeta -IR in epithelial cells. It is possible perhaps that the turnover of TGFbeta receptors may be different in cells of different lineage and/or altered by endogenous secretion of receptor ligands. This speculation requires further study. The short metabolic half-life of TGFbeta -IIR may have important implications for the reversible and rapid modulation of the many TGFbeta -mediated cellular responses. In addition its different processing with that of TGFbeta -IR allows for a possible additional mechanism of regulation of TGFbeta s actions.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant R0I CA62212, MERIT REVIEW and CLINICAL INVESTIGATOR grants from the Department of Veteran Affairs, and the T. J. Martell Foundation.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.
**   To whom correspondence should be addressed: Div. of Medical Oncology, Vanderbilt University, 1161 22nd Ave. South, 1956 TVC, Nashville, TN 37232-5536. Tel.: 615-936-1919; Fax: 615-343-7602; E-mail: carlos.arteaga{at}mcmail.vanderbilt.edu.
1   The abbreviations used are: TGFbeta , transforming growth factor beta ; TGFbeta -IR or IIR, TGFbeta -I or -II receptor; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; NANase II, alpha II-3,6-N-acetylneuraminidase; PNGase F, peptide-N-[N-acetyl-beta -glucosaminyl]-asparagine amidase; O-glycosidase, endo-alpha -N-acetylgalactosaminidase.

Acknowledgments

We thank Teresa C. Dugger for expert technical assistance and Dr. Peter Nørgaard for helpful discussions.


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