(Received for publication, September 12, 1996, and in revised form, January 21, 1997)
From the Whitehead Institute, Cambridge,
Massachusetts 02142, the § Division of
Gastroenterology, Department of Medicine, Brigham and Women's
Hospital, Boston, Massachusetts 02115, and the
Department
of Biology, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139
The TGF- type I and type II receptors (T
RI
and T
RII) are signaling receptors that form heteromeric cell surface
complexes with the TGF-
s as one of the earliest events in the
cellular response to these multifunctional growth factors. Using
TGF-
-responsive mink lung epithelial cells (Mv1Lu), we have
determined the half-lives of the endoplasmic reticulum (ER) and mature
forms of these receptors. In metabolically labeled cells, approximately
90% of newly synthesized type II receptor undergoes modification of
N-linked sugars in the Golgi, with a half-life of 30-35
min; the Golgi-processed form of the receptor has a relatively short
metabolic half-life of 2.5 h. In contrast, only 50% of
pulse-labeled type I receptor is converted to the Golgi-processed
and therefore endoglycosidase H-resistant form, and the endoglycosidase
H-sensitive ER form has a half-life of 2.8-3 h. Addition of 100 pM TGF-
1 causes the Golgi-processed type II receptor to
become less stable, with a half-life of 1.7 h, and also
destabilizes the Golgi-processed type I receptor. TGF-
1 binding and
cross-linking experiments on cells treated with tunicamycin for various
times confirm different ER to cell surface processing times for T
RI
and T
RII. Our results, which suggest that stable complexes between
type I and II TGF-
receptors do not form until the proteins reach a
post-ER compartment (presumably the cell surface), have important
implications for our understanding of complex formation and receptor
regulation.
The transforming growth factor-s
(TGF-
1,1 -
2, and -
3) are the
prototypical members of a growing superfamily of peptide growth factors
that includes the activins and inhibins, bone morphogenetic proteins,
Müllerian inhibiting substance, and the product of the
Drosophila decapentaplegic (dpp) gene. Members of
the TGF-
subfamily are multifunctional, with wide-ranging effects on
growth, differentiation, immune response, and extracellular matrix
organization and biogenesis (1-3). They are among the most important
and versatile growth factors yet described.
The three major cell surface receptors for TGF- are termed types I,
II, and III (T
RI, II, and III, respectively) (4-7). T
RI and
T
RII are the signaling receptors, whereas T
RIII appears to
promote ligand binding to T
RI and T
RII (8, 9). The type I and II
receptors are serine/threonine kinases with approximately 40% identity
in their kinase domains (7). Like their ligands, they are members of a
large superfamily (10-13); receptors with related serine/threonine
kinase domains include activin receptors, bone morphogenetic protein
receptors, the Drosophila saxophone, thick veins, and
punt gene products, and the Caenorhabditis
elegans Daf-1 and Daf-4 proteins (14-26). Within this
superfamily, type I- and type II-like kinases fall into distinct
subcategories. T
RI-like receptors have a highly conserved
juxtamembrane region rich in glycine and serine residues, termed the GS
domain, whereas T
RII and similar receptors have a serine- and
threonine-rich C-terminal extension.
When overexpressed in COS cells, all three types of TGF-
receptors form homo-oligomers (likely dimers) on the cell surface even
in the absence of TGF-
(27, 28).2 It is
not known, however, whether they form homo- or heterodimers in the
absence of ligand when expressed at lower, more physiologic concentrations on the surface of nontransfected TGF-
-responsive cells. Interactions between the cytoplasmic domains of T
RI and T
RII have been detected in both the yeast two-hybrid system and in
COS cells overexpressing the cytoplasmic domains of both receptors (29,
30).3 Studies using chimeric receptors with
the extracellular domain of the erythropoietin receptor and the
cytoplasmic domains of T
RI or T
RII showed that both
homodimerization of the cytoplasmic domain of the type I TGF-
receptor and heterodimerization with the cytoplasmic domain of the type
II receptor is required for intracellular signal transduction leading
to growth inhibition (31).
The type II receptor is able to bind TGF-1 and -
3 independent of
the presence of the type I receptor; T
RI requires the presence of
T
RII to bind these ligands (32-34). Both receptors are required for
high affinity binding of TGF-
2, which appears to bind to a preformed
complex of T
RI and T
RII (35). Coimmunoprecipitation studies with
ligand-bound receptors demonstrated that T
RI, T
RII, and TGF-
ligand form a ternary complex on the cell surface (9, 34). The
association between T
RI and T
RII expressed at physiologic concentrations appears to be ligand-dependent, at least
when tested with TGF-
1 (29, 36). Several lines of evidence suggest
that this ligand-induced complex may be a heterotetramer, containing at
least two copies of each signaling receptor. Weis-Garcia and Massague
(37) showed that kinase-deficient and activation-deficient type I
receptors can functionally complement each other, implying that the
activated complex contains two molecules of T
RI. Additionally, dimers of the T
RI cytoplasmic domain are required for ligand response (31). Henis et al. (27) showed that the type II
receptor is a homo-oligomer with or without TGF-
. This, in
combination with the results of two-dimensional gel analyses (38),
makes a heterotetramer the most likely minimum cell surface receptor complex.
Once a complex has formed, signaling is initiated by the
transphosphorylation of TRI by T
RII, a constitutively active
kinase, on serine and threonine residues in the GS domain (29, 30, 36).
This phosphorylation and a functional T
RI kinase are required for
downstream signaling (36, 37, 39-41). MADR2, one member of a newly
identified class of proteins required for serine/threonine kinase
receptor signaling, is a substrate of the TGF-
receptor complex; it
becomes phosphorylated and transmits signals directly to the nucleus
(42, 43). Several other cytoplasmic proteins that interact with T
RI
or T
RII have been isolated, although their role in signaling has yet
to be determined (44-49).3
As part of our ongoing studies into early events in the TGF-
response, we have studied the biosynthesis of the type I and II TGF-
receptors in nontransfected, TGF-
-responsive, mink lung epithelial
cells (Mv1Lu). We show here that the rates and efficiencies of Golgi
processing of newly made T
RI and T
RII are very different, as are
the apparent half-lives of the Golgi-processed, mature receptors. We
also show that although TGF-
1 has no effect on Golgi processing of
either receptor, it markedly reduces the stability of the mature forms
of both. This suggests that the two receptors are synthesized
independently and neither arrive on the cell surface as a preformed
complex nor, in the presence of ligand, remain on the cell surface as a
stable complex. In addition, the rapid turnover of the type II receptor
has potential implications for regulation, especially since the Endo
H-resistant (and therefore presumably cell surface) form of T
RII is
even more unstable in the presence of ligand.
Mv1Lu mink lung epithelial cells were obtained from the American Type Culture Collection and were grown in modified Eagle's medium (Life Technologies, Inc.) with 10% decomplemented fetal calf serum (Life Technologies, Inc.) supplemented with 2 mM glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and nonessential amino acids (JRH Bioscience) in a 5% humidified CO2 atmosphere. COS7 cells (American Type Culture Collection) and HYB2 cells (a gift of Dr. A. Geiser (National Institutes of Health) (50)) were grown in Dulbecco's modified Eagle's medium with the same additives except no nonessential amino acids.
Binding and Cross-linking of Iodinated TGF-TGF-1 (a gift of R & D Systems) was iodinated by the
chloramine T method as described (51), with the following
modifications. TGF-
1 (1 µg) in 10 µl of 1 M sodium
phosphate, pH 7.2, was mixed with 2 µl of Na125I (0.2 µCi). The reaction was initiated by the sequential addition of three
2-µl portions of chloramine T (0.1 mg/ml). The reaction was stopped
by the sequential addition of 10 µl of acetyl tyrosine (100 mM), 100 µl of potassium iodide (100 mM), and
100 µl of urea-saturated acetic acid. For the binding and
cross-linking experiment shown in Fig. 5, a modification of the method
of Wang et al. (4) was used. Subconfluent Mv1Lu cells in
standard medium were incubated at 37 °C in tunicamycin (Boehringer
Mannheim; stock of 3 mg/ml in DMSO) at a concentration of 2 µg/ml for
times ranging from 0 (no tunicamycin added; duplicate plates) to
11 h. Cells were rinsed once with KRH binding buffer (50 mM Hepes, pH 7.5, 128 mM NaCl, 1.3 mM CaCl2, 5 mM MgSO4, 5 mM KCl) with 0.5% fatty acid-free bovine serum albumin
(Sigma) and then incubated at 37 °C in KRH binding buffer with
either 2 µg/ml tunicamycin or an equivalent volume of DMSO (0 time
points). After 30 min, this material was aspirated and 3 ml of ice-cold
KRH binding buffer containing 50 pM
125I-TGF-
1 was added. Plates were incubated at 4 °C
with rotation for 3 h and then rinsed 3 times with ice cold KRH.
The ligand was then cross-linked to the receptor with the addition of
0.5 mg/ml disuccinimidyl suberate (Pierce) from a 100-fold concentrated stock in DMSO, followed by incubation at 4 °C for 15 min. Glycine was added to 20 mM, and plates were incubated for 10 min at
4 °C. Plates were rinsed twice with ice-cold phosphate-buffered
saline (PBS), and lysed at 4 °C for 20-30 min in 1 ml lysis buffer
(PBS with 0.5% deoxycholate, 1% Triton X-100, 10 mM EDTA,
pH 8.0, and 1 mM phenylmethylsulfonyl fluoride). Insoluble
material was pelleted by microcentrifugation at 14,000 rpm for 10 min
at 4 °C and discarded. Cleared lysates were immunoprecipitated at
4 °C overnight with
volume of a polyclonal rabbit antiserum (
-IIC (9)) raised against the C-terminal 16 amino acids of
the human type II receptor; this epitope has 15 of 16 amino acids
identical to the mink lung receptor (34). One-twentieth volume of
protein A-Sepharose CL-4B beads (Sigma) was added, and the lysates were
incubated with rotation for 30 min. Protein A-Sepharose beads were
rinsed twice with lysis buffer and once with PBS, and bound protein was
eluted by boiling in 0.5% SDS. The eluate from one of the 0 time point
plates was treated overnight with N-glycosidase F (3000 units/50 µl; New England Biolabs). Samples were analyzed by 7.5%
SDS-polyacrylamide gel electrophoresis. Autoradiographs were quantified
with a LaCie Silverscanner II and MacBAS (Fuji) software.
Metabolic Labeling
Mv1Lu cells were grown to subconfluence
in standard medium on 10-cm plates. They were rinsed once with PBS and
then starved in serum-free Dulbecco's modified Eagle's medium minus
cysteine and methionine (ICN) with glutamine and
penicillin/streptomycin for 5-5.5 h at 37 °C. The medium was then
replaced with fresh medium supplemented with 0.2 mM
oxidized glutathione (Boehringer Mannheim) and 0.5 mCi/ml of a mixture
of [35S]methionine and [35S]cysteine
(Express; New England Nuclear) and incubated at 37 °C for 10 min
(pulse-labeling). Plates were quickly rinsed twice with warmed chase
medium (serum-free Dulbecco's modified Eagle's medium with glutamine
and penicillin/streptomycin) and then incubated for the designated
chase times at 37 °C in the presence of either 100 pM
TGF-1 ("+ ligand") or 50 µg/ml of Pan-Specific TGF-
Neutralizing antibody (R & D Systems; "
ligand"), the IgG
fraction of a polyclonal rabbit antiserum that neutralizes TGF-
1,
1.2,
2,
3, and
5. Plates were then rinsed three times with
ice-cold PBS and lysed in 1 ml of lysis buffer. Cleared lysates were
precleared overnight with protein A-Sepharose and then
immunoprecipitated with
volume of antibody
-IIC or a
polyclonal rabbit antiserum raised against the juxtamembrane
cytoplasmic domain of the human type I receptor (7) (an epitope with 22 of 22 amino acids identical to the mink lung receptor (37)), at 4 °C
for 2 h. After a 30-min incubation with protein A-Sepharose, bound
beads were rinsed three times with lysis buffer plus 0.5% SDS and then
twice with PBS. Protein was eluted into 0.5% SDS. For type II receptor
immunoprecipitates, the eluate was split in two; one half was treated
with Endo H (Genzyme, 100 mIU/ml) in the presence of 100 mM
sodium citrate, pH 6.0, at 37 °C overnight. For type I receptor
immunoprecipitates, one-third of the eluate was treated with Endo H,
and another one-third with N-glycosidase F. In cases where
the type I and II receptors had identical chase times, plates were
labeled and chased in parallel; the pairs of lysates from such a time
point were pooled and then split before immunoprecipitation to ensure
that comparison between the two receptors at a given time point was
valid. Samples were analyzed by 7.5% SDS-polyacrylamide gel
electrophoresis; gels were fluorographed with 2,5-diphenyloxazole,
dried, and placed on Kodak XAR film, which was scanned as above.
Control immunoprecipitations (not shown) were performed in the presence of an excess of the immunizing peptide to confirm the identity of the type I and II receptors. Also, because the type I receptor antibody immunoprecipitates a number of unrelated but peptide-competable proteins, COS7 cells mock transfected or transfected with the ALK-5 (type I receptor) cDNA (7) (a gift of Dr. K. Miyazono, Japanese Foundation for Cancer Research, Tokyo, Japan) were pulse-labeled and analyzed on each gel to confirm the proper size of the type I receptor.
To determine the half-life
of the native type II receptor, we metabolically labeled
TGF--responsive mink lung epithelial cells, chased them from 0 to
4 h, and immunoprecipitated detergent extracts with an anti-type
II receptor antibody. The newly synthesized receptor (Fig.
1A, left panel, 0 h time point) migrates
as a doublet that shifts from 62 and 65 kDa to 58 and 61 kDa after
treatment with Endo H (Fig. 1B, left panel), indicating that
it is present in the endoplasmic reticulum. This Endo H-sensitive
material disappears with a half-life of 30-35 min (Fig.
2A, control). A smear from 67 to 77 kDa,
representing the Endo H-resistant receptor, appears at 0.5 h of
chase (Fig. 1, A and B, left panels). This
Golgi-processed receptor has a heterogeneous population of complex
N-linked sugars. More than 50% of the steady state
population of cellular receptors, including the majority of the cell
surface form, is also O-glycosylated (52).4 During the chase, approximately 90%
of pulse-labeled Endo H-sensitive receptor is converted to the Endo
H-resistant, Golgi-processed material (Fig. 2B),
demonstrating efficient conversion of the ER to the mature form, with
minimal ER degradation.
The Golgi-processed form of TRII, like the Endo H-sensitive form,
disappears rapidly, with a half-life of approximately 2.5 h (Fig.
2B). The smear of Endo H-resistant material represents both
intracellular and cell surface receptors. Over half of the total
population of Endo-H resistant T
RII, however, is sensitive to
digestion of intact cells with proteinase K (data not shown), indicating that approximately half of the total Endo-H resistant T
RII is on the cell surface at steady state. Thus, our data suggest that the half-life of cell surface T
RII is also approximately 2.5 h.
Similar experiments (data not shown) with the HYB2 line of
TGF--responsive human cells (50) also show rapid metabolism of the
type II receptor, with a half-life of the Endo H-sensitive T
RII of
25-30 min and a half-life of the Endo H-resistant form of
approximately 3 h.
To assess the effect of TGF- on the
stability of the Endo H-sensitive and -resistant forms of T
RII, the
above experiments were performed in parallel in the presence of 100 pM TGF-
1 (Fig. 1, A and B, right
panels). The Endo H-sensitive receptor disappeared with a
t1/2 of 30-35 min, identical to the rate in
control cells (Fig. 2A). This indicates that ER stability,
processing efficiency, and the rate of ER to Golgi transport of newly
made T
RII are unaffected by the presence of TGF-
. Endo
H-resistant T
RII, however, is less stable in the presence of
TGF-
, with a half-life of 1.7 h compared to 2.5 h in
control cells (Fig. 2B). Importantly, all control
experiments (
ligand) were performed in the presence of a
neutralizing antibody against the TGF-
s (see "Materials and
Methods"); thus, it is unlikely that residual or endogenously synthesized TGF-
masked an even more marked effect of
the ligand.
Performing the same experiment on HYB2 cells, we obtained a similar
lack of effect of TGF- on the fate of the Endo H-sensitive T
RII,
and a significant although less marked effect on the stability of the
Golgi-processed receptor (data not shown).
The half-life of the type I receptor was determined
by metabolically labeling Mv1Lu cells in parallel with the experiments above (Figs. 1 and 2) and immunoprecipitating the cell lysates with an
anti-type I receptor antibody. Lysates from similarly labeled, mock-
and TRI-transfected COS7 cells (not shown) were run alongside the
Mv1Lu cell material to confirm identification of the T
RI bands.
Pulse-labeled T
RI migrates on SDS-polyacrylamide gel electrophoresis
as a single species of 52 kDa (Fig. 3A, left panel). The gel mobility shifts to 49 kDa after treatment with Endo H, indicating that newly synthesized T
RI contains a high mannose, N-linked oligosaccharide characteristic of the ER
(Fig. 3B, left panel). The Endo H-sensitive ER form of
T
RI disappears with a half-life of 2.8-3 h (Fig.
4A), markedly longer than the half-life of
30-35 min of newly made, Endo-H-sensitive type II receptor. At no time
during the chase period could we detect a specific higher molecular
weight form of T
RI; we presume that the Golgi-processed, Endo
H-resistant form of T
RI migrates within the background "smear"
above 52 kDa. By treating the T
RI immunoprecipitates with
N-glycosidase F, however, removing all N-linked
sugars, we can quantify the total (Endo H-sensitive and -resistant)
amount of metabolically labeled T
RI in the cell (Fig.
3C). We calculate the amount of mature (Endo H-resistant)
T
RI by subtracting the amount of labeled Endo H-sensitive receptor
(Fig. 3B) from the total amount (N-glycosidase
F-treated) of T
RI (Fig. 3C). Fig. 4 shows that no more
than 50% of pulse-labeled T
RI is converted to an Endo H-resistant
form, compared with approximately 90% of T
RII (Fig.
2B).
Because of the slow and incomplete conversion of pulse-labeled TRI
to an Endo H-resistant form, we cannot use the data in Fig.
4B to calculate a precise half-life of Golgi-processed
T
RI. Clearly, however, it is significantly longer than the 2.5-h
half-life of the mature, Golgi-processed type II receptor (compare
control curves in Figs. 2B and 4C).
The presence of TGF- has no effect on the metabolic stability of the
Endo H-sensitive form of T
RI (Fig. 4A), nor on the rate
by which this material is converted into an Endo H-resistant form (Fig.
4C). The Golgi-processed, Endo H-resistant form of T
RI,
however, is less stable in the presence than the absence of TGF-
1
(Fig. 4C); this is similar to the decrease in stability of
the Golgi-processed type II receptor in the presence of ligand (Fig.
2B).
We performed ligand-binding and cross-linking
experiments to confirm that the ER to Golgi half-lives calculated from
pulse-chase experiments (above) correlate with ER to cell surface
half-lives. In the experiment depicted in Fig. 5, Mv1Lu
cells were incubated in tunicamycin for times ranging from 0 to 11 h. The cells were chilled, and 125I-TGF-1 was bound and
then cross-linked to cell surface TGF-
receptors. Tunicamycin blocks
the synthesis of dolichol pyrophosphoryl N-acetyl
glucosamine, an essential intermediate in the addition of
N-linked sugars to newly synthesized polypeptides. In
preliminary experiments, we showed that the concentration of
tunicamycin used, 2 µg/ml, completely inhibited N-linked
glycosylation of both receptors.
Deglycosylated TRII appears on the cell surface as early as 1 hour
after addition of tunicamycin, indicating that it is transported from
the ER to cell surface in less than this time. The half-time for
appearance of the maximum amount of deglycosylated type II receptor on
the cell surface is approximately 2 h; the actual half-time for
its formation is less than this, since the time from addition of
tunicamycin to depletion of dolichol pyrophosphoryl N-acetyl
glucosamine is unknown. In contrast, deglycosylated type I receptor
does not appear on the cell surface until 3 h after addition of
tunicamycin; the half-time for appearance of the maximum amount of
deglycosylated type I receptor is approximately 4 h. The
half-lives of the glycosylated forms of the cell surface receptors cannot be readily determined from this type of experiment, because at
the time of addition of tunicamycin there may be substantial intracellular (ER or Golgi) pools of T
RI or T
RII that can
subsequently move to and then be removed from the cell surface. Thus,
the fact that glycosylated cell surface T
RII is depleted much more
rapidly than T
RI (Fig. 5, compare II+ and I+)
provides only an indirect indication that their actual half-lives are
different. After 4-5 h of treatment with tunicamycin, however, all
cell surface T
RII is nonglycosylated, whereas all cell surface
T
RI is fully glycosylated. (We see the same results regardless of
whether lysates are immunoprecipitated with antibodies against T
RI
or T
RII or are not immunoprecipitated; data not shown.) These
results are consistent with the different rates of processing from the
ER of newly made types I and II receptors depicted in Figs. 2 and 4.
They also indicate that complexes between types I and II TGF-
receptors do not occur until the proteins have reached a post-ER
compartment (presumably the cell surface) and have important
implications for our understanding of formation of receptor complexes,
as discussed below.
The decreased amount of cell surface receptors after long incubations in tunicamycin may represent decreased protein synthesis (a known consequence of tunicamycin treatment, affecting some proteins more than others). Receptors lacking N-linked oligosaccharides bind ligand and are immunoprecipitated as efficiently as fully glycosylated receptors,4 so the lower signal is not the result of inefficient binding and cross-linking or immunoprecipitation. Decreased formation or stability of nonglycosylated type I/II receptor complexes is unlikely, given that our findings are similar for nonimmunoprecipitated lysates.
Our principal finding is that the types I and II TGF- receptors
are metabolized very differently. The ER form of the type II receptor
has a half-life of 30-35 min and is efficiently (approximately 90%)
processed by Golgi enzymes; the Golgi-processed forms of the type II
receptor have a relatively short metabolic half-life, about 2.5 h.
In contrast, the Endo H-sensitive, ER form of the type I receptor has a
half-life of 2.8-3 h, and less than 50% of pulse-labeled type I
receptor is converted to an Endo H-resistant form. The exact half-life
of Golgi-processed type I receptor is difficult to determine because of
the large ER pool of receptor subunits being slowly processed to the
Golgi at the same time that Golgi-processed receptors are being
degraded (Fig. 4, A and C). Nonetheless, the
half-life is considerably longer than that of the type II receptor.
Addition of TGF-
1 to cells causes a decrease in the stability of the
Endo H-resistant forms of both receptors. These conclusions, derived
from experiments in which TGF-
-responsive mink lung epithelial cells
were metabolically pulse-chase labeled, were confirmed by TGF-
1
binding and cross-linking experiments on cells treated with tunicamycin
for various times. In unpublished studies, similar results were
obtained using a TGF-
-responsive human cell line (HYB2). Because
these cell lines express native, rather than transfected, receptors, it
ensures that the concentrations and ratios of the types I and II
TGF-
receptors are physiologic.
Our results are consistent with recent studies on the metabolism of the
type II receptor in bone-forming cells, which show that after
cycloheximide treatment, the amount of cell surface Type II receptor
able to bind to iodinated ligand decreased with a half-life of 2 h
(53). This is an indirect measure of receptor biosynthesis and
stability, however, and provides no information about the type I
receptor, which requires cell surface type II receptor for binding to
TGF-. To the best of our knowledge, biosynthetic studies have not
been undertaken for any other members of the receptor superfamily, and
there are no published studies of metabolically labeled receptors in
nontransfected cell lines.
Our pulse-chase experiments indicate that newly made type II receptor
moves quickly and efficiently through the ER after synthesis. This
suggests rapid and efficient folding within the ER because only
correctly folded and processed proteins are transported to the Golgi
apparatus (54). Similarly, the soluble extracellular domain of the
human TGF- type II receptor is efficiently synthesized in COS-7
cells and binds 125I-TGF-
with the same specificity and
affinity as the normal cell surface T
RII
(52).5 Thus, the rapid folding and
efficient processing of the type II receptor is a property mainly of
its extracellular domain, which contains six potential disulfide bonds
and two (three in human) N-linked oligosaccharides, as well
as an undetermined number of O-linked sugars.
The Golgi-processed form of TRII is degraded with a half-life of
approximately 2.5 h (Fig. 2B). Because more than half
of the total population of Endo-H resistant T
RII is sensitive to digestion of intact cells with proteinase K and thus at steady state is
on the cell surface, our data suggest that the half-life of cell
surface T
RII also is approximately 2.5 h. This short half-life
is similar to the half-lives reported for the mature forms of the
nonrecycling interferon receptor (3 h) (55), the IL-2 receptor (1 h)
(56), and the erythropoietin receptor (45 min) (57). In contrast, other
receptors have longer half-lives: the asialoglycoprotein receptor (20 h) (58), the low density lipoprotein receptor (11 h) (59), and the
transferrin receptor (20 h) (60). After exiting the cell surface, type
II receptors do not appear to undergo proteolytic cleavage or storage
within the cell, because experiments with
125I-TGF-
-bound receptors show no release of smaller
labeled fragments into the medium (data not shown). Furthermore, by
analysis of immunoprecipitates of metabolically labeled cells, we have
not seen specific smaller proteolytic degradation products of T
RII (data not shown).
We speculate that the short metabolic half-life of the type II receptor
has implications for regulation of TGF- signaling that go beyond its
impact on complex formation. The rapid turnover of the type II receptor
potentially allows receptor numbers to change quickly in response to
various stimuli, an important characteristic for a growth factor
receptor with wide-ranging effects on differentiation and development.
The decreased stability of the mature form of T
RII in response to
ligand suggests that TGF-
itself may regulate the numbers of its
receptor and that the type II receptor may be a limiting factor in the
cellular response to TGF-
. Phosphorylation of T
RII following
ligand binding may be a signal for receptor degradation, in a manner
analogous to many receptors spanning seven membranes, which undergo
internalization and down-modulation after ligand binding (61).
The newly made ER form of the type I receptor disappears from cells
with a half-time of nearly 3 h, 5-6 times as long as for the type
II receptor. Less than one-half of the newly made receptors ever
acquire Endo-H-resistant oligosaccharides. Processing of the type I
receptor thus is similar to that of the erythropoietin receptor, which
likely folds inefficiently (57, 62, 63). A mutant erythropoietin
receptor is transported from the ER more efficiently than the wild type
receptor and is expressed in elevated numbers at the cell surface (63);
this is the first example of a mutant polypeptide that is processed in
the ER more efficiently than its wild type counterpart. We speculate
that inefficient folding and processing of the wild type erythropoietin
receptor and potentially the type I TGF- receptor in the ER is one
mechanism for controlling the number of plasma membrane receptors.
The fact that the types I and II TGF- receptors exit the ER at
different rates implies that they do so separately. Fig. 5 provides
perhaps the most vivid demonstration of the different rates of ER exit
of the two receptors: after 4 h of treatment with tunicamycin, all
cell surface T
RII was nonglycosylated, whereas all cell surface
T
RI remained fully glycosylated. Because the rates and extents of ER
to Golgi processing of the two receptors are different and because the
mature forms of the two receptors are degraded with different
half-lives, our results imply that stable complexes between the types I
and II TGF-
receptors do not occur until the proteins have reached a
post-ER compartment, presumably the cell surface. Given our results, it
would be possible to conclude the opposite
that hetero-oligomers form
in the ER
only if one receptor was synthesized in excess and
accumulated in the ER awaiting production of the other receptor. This
scenario would imply that the first receptor could not become correctly
folded and mature to the Golgi unless complexed to the second receptor. Both the type I and type II receptors, however, mature normally to the
cell surface even when expressed alone in COS cells
(36).6 Furthermore, the soluble exoplasmic
domain of the type I receptor, like that of the type II, is efficiently
secreted by transiently transfected COS cells, as well as by stably
transfected Chinese hamster ovary cells,5 again implying
that folding of the exoplasmic domain of T
RI occurs efficiently in
the absence of T
RII. Our results showing different rates of
degradation of Golgi-processed T
RI and T
RII are also consistent
with the idea that stable complexes between the two receptors do not
occur in the ER; indeed, they suggest that even cell surface complexes
are short-lived.
Studies with TGF-1 showed that formation of a T
RI/T
RII
heteromeric complex is dependent on the presence of TGF-
(29, 36).
TGF-
2, however, binds to neither cell surface signaling receptor
expressed in the absence of the other, suggesting the existence of a
preformed T
RI/T
RII complex (35). Our present experiments imply
that if T
RI/T
RII complexes do occur on the cell surface in the
absence of TGF-
, they are relatively unstable and do not form in the
ER. Although our preliminary data suggest that the type II receptor
forms dimers cotranslationally,6 it is unlikely that
heteromeric complexes with the type I receptor also form at this
point.
The cytoplasmic domains of TRI and T
RII may have an intrinsic but
weak affinity for each other, as interactions between the two have been
detected in both the yeast two-hybrid system and COS cells
overexpressing the cytoplasmic domains of both receptors (29,
30).3 Such interactions, however, have not been detected in
TGF-
-responsive cells, and we do not know whether they affect
receptor trans-activation and signal transduction. Although
weak interactions between the cytoplasmic domains of T
RI and T
RII
expressed at physiologic concentrations are not, in themselves,
sufficient for generating growth inhibitory signals, they could, if
they exist, serve to initiate formation of heterodimers of T
RI and
T
RII that become stabilized by the binding of a TGF-
ligand.
We thank Kohei Miyazono (Japanese
Institute for Cancer Research, Tokyo, Japan) for TRI cDNA,
Andrew Geiser (National Institutes of Health) for HYB2 cells, and James
Weatherbee (R & D Systems) for TGF-
1. We are grateful to Stephanie
Watowich for helpful discussions and to Ralph Lin and Riki Perlman for
critically reviewing the manuscript.
Koli and Arteaga (Koli, M., and Arteaga,
C. L. (1997) J. Biol. Chem. 272, 6423-6427) recently
published similar data. Their data agree with ours in showing a
reduction in the half-life of TRII in the presence of TGF-
. They
see no effect of ligand, however, on the half life of the type I
receptor. This difference may be due to different assumptions about the
identity of a 55-kDa form of the type I receptor. We have identified it
as an Endo H-sensitive, ER form, whereas Koli and Arteaga identify it
as the mature, Endo H-resistant receptor.