(Received for publication, November 22, 1996, and in revised form, December 19, 1996)
From the Departments of Three cell surface transforming growth factor The TGF Since most cell types can produce both TGF 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 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
TGF Cell lysates were subjected to
overnight immunoprecipitation with TGF 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-TGF 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-TGF 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).
Recent
studies using cycloheximide to block cellular protein synthesis have
suggested that the turnover rate of TGF
TGF
We analyzed
next whether TGF
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 TGF
We addressed
whether the rapid turnover of TGF
A time-dependent increase in acid-washable specific binding
of 125I-TGF The possibility that the short half-life results from the release of
the extracellular domain of TGF 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 TGF
In summary, native TGF We thank Teresa C. Dugger for expert
technical assistance and Dr. Peter Nørgaard for helpful
discussions.
Medicine and ¶ Cell
Biology,
Department of
Veteran Affairs Medical Center, Nashville, Tennessee 37232-5536
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
(TGF
) receptor (R) proteins regulate the effects of TGF
isoforms
on growth and differentiation. TGF
-IR and -IIR are transmembrane
serine/threonine kinases directly mediating the signaling across the
plasma membrane. Both TGF
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, TGF
-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 TGF
-IIR was estimated to
be 60 min and was further reduced to ~45 min in the presence of
exogenous TGF
1. Minimal internalization of
125I-TGF
1 at 37 °C was detected suggesting that
the rapid turnover was not due to endocytosis and degradation of the
ligand-receptor complexes. TGF
-IR was synthesized as a ~53-kDa
precursor protein, which was processed to a mature ~55-kDa receptor
protein. The half-life of TGF
-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 TGF
-IR and TGF
-IIR
providing a potential additional mechanism for modulation of cellular
responses to TGF
s.
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 TGF
, and their activation plays an
important regulatory role in TGF
actions (4, 5). In epithelial
cells, TGF
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) TGF
receptors.
TGF
-IIR binds the ligand first, after which TGF
-IR is recruited
to a heteromeric complex most likely containing several receptor
molecules (8, 9). Ligand-dependent phosphorylation of the
GS-domain of TGF
-IR leads to the propagation of the signal
downstream (10, 11). Type III TGF
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
TGF
-IR and TGF
-IIR are needed to mediate the biological effects
of TGF
ligands. Recent reports, however, suggest separate signaling
pathways for the antiproliferative and the matrix modulatory effects of TGF
with the latter only requiring TGF
-IR signaling in some cell
systems (13, 14).
receptors and ligand(s),
the regulation of cellular responsiveness relies on the production of
active TGF
and its presentation to signaling receptors. Therefore,
the role of receptor protein associations, turnover, and
down-regulation is likely critical for the control of TGF
signals
and the modulation of overall cellular responsiveness. TGF
-IIR
levels have been shown to correlate with TGF
responsiveness (15,
16). Cancer cells refractory to TGF
's antiproliferative action have
often lost TGF
-IIR expression (17-19). Although TGF
-IR can bind
ligand only in association with TGF
-IIR, it is indispensable for
TGF
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 TGF
, thus altering TGF
responsiveness (21-23). We have
examined the biosynthesis and ligand-induced modulation of naturally
expressed TGF
-IR and TGF
-IIR in CCL-64 mink lung epithelial
cells, which are known to express abundant amounts of all three TGF
receptors and are potently growth inhibited by exogenous TGF
.
Cell Culture and Metabolic Labeling
-IR (V-22, Santa Cruz Biotechnology, Santa Cruz, CA), TGF
-IIR
(C-16, Santa Cruz Biotechnology), or TGF
-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.
-IR or TGF
-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.
1 (specific
activity, 173 µCi/µg; DuPont NEN) with or without 100-fold excess
unlabeled TGF
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-TGF
1 bound to cell surface
receptors was acid washed with 50 mM glycine, pH 2.4, 100 mM NaCl, 2 mg/ml polyvinylpyrrolidone, 2 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).
Binding and Affinity
Cross-linking
1 with or without 100-fold excess unlabeled
TGF
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-TGF
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
TGF
-IR or TGF
-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.
The Biosynthesis and Turnover of TGF-IR and -IIR
binding sites in bone cells
is relatively fast (25). Since TGF
-IR cannot bind TGF
1 alone,
these affinity binding studies provide clues only on the turnover of
TGF
-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 TGF
-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 TGF-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 TGF
-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)]
-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 TGF
-IIR. The
longer persistence of the type I receptor precursor form indicates a less efficient ER processing compared with that of TGF
-IIR
precursor. The steady-state 35S-labeled mature TGF
-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 TGF-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 TGF
-IR
antibodies. B, cells were labeled with 50 µCi/ml Tran35S-label for 2.5 h, lysed, and precipitated with
TGF
-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)]
on TGF
-IIR and -IR Turnover
could further influence the rapid turnover of
TGF
-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 TGF
-IIR could be
immunoprecipitated from the cell lysates (Fig.
3A). The amount of the mature ~70-kDa TGF
-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 TGF
1 for the last 20 min of
labeling as well as throughout the chase, two other proteins
coprecipitated with TGF
-IIR: a ~50-kDa and a ~37-kDa protein,
the latter corresponding to the size of TRIP-1 (TGF
receptor
interacting protein-1 (26)). The identity of these proteins was not
further confirmed, but their ability to coprecipitate with TGF
-IIR
antibodies in a ligand-dependent manner under stringent double immunoprecipitation conditions suggests a strong association induced by exogenous ligand. In the presence of TGF
1 the half-life of the mature TGF
-IIR was shortened to approximately 45 min (Fig. 3B).
Fig. 3.
Effect of TGF on TGF
-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. TGF
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 TGF
-IIR antibodies. The
immune complexes were analyzed by 8% SDS-PAGE and autoradiography. The
arrows indicate the migration of TGF
-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
TGF
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)]
-IR form was the predominant
one (Fig. 4). Some TGF
-IR protein could still be
immunoprecipitated 18 h after the 2.5-h steady-state labeling
indicating that TGF
-IR half-life was considerably longer than that
of TGF
-IIR. Contrary to the results with TGF
-IIR, the half-life
of steady-state 35S-labeled TGF
-IR was not considerably
affected by TGF
1 treatment (Fig. 4).
Fig. 4.
Effect of TGF on TGF
-IR turnover.
Cells were labeled with Tran35S-label and chased for
indicated times with (+) or without (
) TGF
1 as in Fig. 3. Double
immunoprecipitation was performed with TGF
-IR antibodies. The immune
complexes were analyzed by 8% SDS-PAGE and autoradiography. The
arrow indicates the migration of TGF
-IR protein.
Molecular mass markers in kDa are shown at left.
[View Larger Version of this Image (38K GIF file)]
1
receptors in the presence of
ligand was due to endocytosis and degradation. To measure the rate of
125I-TGF
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-TGF
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-TGF
1 binding
was comparable with that in non-pretreated cells (Fig.
5A, bars 3 versus bar 1).
Fig. 5.
Internalization of 125I-TGF1.
A, 125I-TGF
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-TGF
1 ± 100 ng/ml
unlabeled TGF
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-TGF
1 binding and internalization. Cells were
incubated with 1 ng/ml of 125I-TGF
1 ± 100 ng/ml
unlabeled TGF
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 TGF
-IIR. Cells grown on 100-mm dishes were treated with or without 10 ng/ml TGF
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 TGF
-IIR immunoblot (2732 antibody). Molecular mass markers in kDa are shown at
left.
[View Larger Version of this Image (16K GIF file)]
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 TGF
receptors in the
presence of ligand may not be explained by endocytosis and subsequent
degradation. This was further supported by Western blot analysis of
TGF
1-treated cells. An overnight incubation with exogenous ligand
did not alter TGF
-IIR content in CCL-64 cells (Fig.
5C).
-IIR into the medium was investigated
by immunoprecipitation of the chase medium after labeling with
Tran35S-label (see above) with the polyclonal TGF
-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).
-IR with TGF
-IIR
-IIR
homo-oligomers predominate (28, 29). We studied the stage at which
TGF
-IR can associate with TGF
-IIR by stripping oligosaccharide
chains from receptor proteins with tunicamycin followed by
125I-TGF
1 binding at 4 °C, covalent cross-linking,
and precipitation with TGF
-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
TGF
-IIR, whereas 24 h of treatment was needed for TGF
-IR
(Fig. 6), consistent with the longer half-life and
slower processing in the ER of the type I receptor (Fig. 2). Deglycosylated TGF
-IIR was able to bind exogenous ligand and associate with both fully processed TGF
-IR (5 h, lane 2)
and deglycosylated TGF
-IR as judged by coprecipitation by TGF
-IIR antibodies (24 h, lane 2) and TGF
-IR antibodies (24 h,
lane 4). However, deglycosylated TGF
-IIR did not
coprecipitate with TGF
-IR antibodies (5 h, lane 4). This
could well reflect a lower precipitation efficiency of the latter since
TGF
-IIR antibodies coprecipitated both deglycosylated TGF
-IIR and
mature TGF
-IR (5 h, lane 2). The lesser amounts of
deglycosylated TGF
-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
TGF
-IR, and/or a critical need of N-linked glycosylation
in TGF
-IR for ligand binding. These not mutually exclusive
possibilities will require further study.
Fig. 6.
Effect of tunicamycin on
125I-TGF1 binding. Cells were treated with 5 µg/ml tunicamycin for 5 or 24 h followed by
125I-TGF
1 binding and cross-linking as indicated under
"Experimental Procedures." Cell lysates were subjected to
immunoprecipitation with TGF
-IIR or TGF
-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)]
type I and II receptors are processed
differently and separately in mink lung epithelial cells, with the
TGF
-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 TGF
-IIR protein is not due to receptor
endocytosis and subsequent degradation. This short metabolic half-life
of native TGF
-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-TGF
1 binding to types I and II receptor with a
half-life of 2 h (25). The rapid reduction in binding to TGF
-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 TGF
-IR binding. Our direct biosynthetic studies suggest a more prolonged half-life for TGF
-IR in epithelial cells. It is possible perhaps that the turnover of TGF
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 TGF
-IIR may have important
implications for the reversible and rapid modulation of the many
TGF
-mediated cellular responses. In addition its different
processing with that of TGF
-IR allows for a possible additional
mechanism of regulation of TGF
s actions.
*
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: TGF, transforming
growth factor
; TGF
-IR or IIR, TGF
-I or -II receptor; PAGE,
polyacrylamide gel electrophoresis; BSA, bovine serum albumin; NANase
II,
II-3,6-N-acetylneuraminidase; PNGase F,
peptide-N-[N-acetyl-
-glucosaminyl]-asparagine
amidase; O-glycosidase,
endo-
-N-acetylgalactosaminidase.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.