§
* Department of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel; The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142; § Department of Medicine, Brigham and
Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115; and
Department of Biology, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139
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Abstract |
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Abstract. Transforming growth factor (TGF-
) signaling involves interactions of at least two different receptors, types I (T
RI) and II (T
RII), which form
ligand-mediated heteromeric complexes. Although we
have shown in the past that T
RII in the absence of
ligand is a homodimer on the cell surface, T
RI has not
been similarly investigated, and the site of complex formation is not known for either receptor. Several studies
have indicated that homomeric interactions are involved in TGF-
signaling and regulation, emphasizing
the importance of a detailed understanding of the homooligomerization of T
RI or T
RII. Here we have
combined complementary approaches to study these
homomeric interactions in both naturally expressing
cell lines and cells cotransfected with various combinations of epitope-tagged type I or type II receptors. We
used sedimentation velocity of metabolically labeled receptors on sucrose gradients to show that both T
RI
and T
RII form homodimer-sized complexes in the
endoplasmic reticulum, and we used coimmunoprecipitation studies to demonstrate the existence of type I homooligomers. Using a technique based on antibody-mediated immunofluorescence copatching of receptors
carrying different epitope tags, we have demonstrated
ligand-independent homodimers of T
RI on the surface of live cells. Soluble forms of both receptors are secreted as monomers, indicating that the ectodomains
are not sufficient to mediate homodimerization, although TGF-
1 is able to promote dimerization of the
type II receptor ectodomain. These findings may have
important implications for the regulation of TGF-
signaling.
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Introduction |
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TRANSFORMING growth factor- (TGF-
)1 is a multipotent cytokine involved in a wide range of biological functions including cell growth, apoptosis, production of extracellular matrix, wound healing, and
differentiation (35, 37). Three high-affinity transmembrane receptors for TGF-
were identified, first through cross-linking of radiolabeled ligand and later by cDNA
cloning: the type I (T
RI, 55 kD), type II (T
RII, 75 kD),
and type III (280 kD) receptors (3, 9, 23, 25, 39). T
RI and
T
RII appear to be the signaling receptors, while the type
III receptor presents ligand to T
RII and I (21, 26, 29, 30,
44). T
RI and T
RII are serine-threonine kinases with
cysteine-rich extracellular domains and 41% identity between their kinase domains (9, 22, 24, 33). In the absence
of T
RI, the type II receptor can bind TGF-
but does not
transduce signal (27, 43). On the other hand, T
RI can be
cross-linked to radiolabeled TGF-
only in the presence of
T
RII (9, 13, 19, 43). The T
RII kinase is constitutively
active (23), and autophosphorylation on several serine residues regulates its activity and interactions with T
RI (28).
The binding of TGF-
1 to T
RII mediates the formation
of a heteromeric complex of T
RI and T
RII and the
phosphorylation of specific serine residues in T
RI by
T
RII (36, 43, 44; Wells, R., L. Gilboa, Y. Henis, and H. Lodish, manuscript in preparation). This phosphorylation
activates T
RI kinase activity and promotes its interactions with downstream effector molecules, including members of the SMAD family (1, 2, 31, 32, 45).
Both the type II (6, 12) and the type III (12) TGF- receptors form ligand-independent homooligomers (probably dimers) on the cell surface. That this is functionally important for the type II receptor is shown by studies
demonstrating that homooligomerization of T
RII is involved in both positive and negative regulation of signal
transduction via intermolecular autophosphorylation of
specific serine residues (28). So far, there is no direct physical evidence for T
RI homomeric complexes. Two lines
of evidence, however, suggest that the TGF-
receptor signaling complex contains at least two type I receptors: chimeric proteins with the extracellular domain of the erythropoietin (Epo) receptor and the cytoplasmic domain of a
constitutively active T
RI mutant are not active unless dimerized by Epo (27), and functionally complementary
T
RI mutants falling into two classes, termed kinase defective and activation defective, have been isolated (40).
We report here a detailed investigation of TRI and
T
RII homooligomer formation. We have studied cell
lines that express native TGF-
receptors as well as cells
cotransfected with various combinations of epitope-tagged
receptors using several complementary approaches: sucrose gradient velocity centrifugation to determine the
size of the receptor complexes, coimmunoprecipitation,
and immunofluorescence copatching studies to detect receptor oligomerization on the surface of live cells. We
show that both T
RI and T
RII form homodimers in the
ER and that the extracellular region alone is insufficient
for dimerization of either receptor. Our results further
demonstrate that, similar to the type II receptor (12), T
RI forms ligand- and DTT-independent homooligomers on the surface of live COS7 cells. These results have
important implications for our understanding of the events
involved in TGF-
signaling.
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Materials and Methods |
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Materials
TGF-1 was supplied by Celltrix Laboratories (Palo Alto, CA) and R & D
Systems, Inc. (Minneapolis, MN) and was radioiodinated for affinity labeling of tagged receptors as described (34, 39). For affinity labeling of soluble receptors, radioiodination was modified as described (41). 9E10
(
-myc) mouse ascites, which recognizes a specific c-myc sequence (8),
was purchased from Harvard Monoclonals (Boston, MA). 12CA5 (
-HA)
mouse ascites, which recognizes an epitope of the influenza hemagglutinin
(HA) protein (42), was from BAbCO (Richmond, CA). IgG fractions
were prepared from mouse ascites by ammonium sulfate precipitation followed by DEAE-cellulose chromatography (10). F (ab
)2 fragments were
generated by pepsin digestion, following the protocol of Kurkela et al.
(18). Fluorophore-labeled affinity-purified antibodies and Cy3-streptavidin were obtained from Jackson ImmunoResearch Laboratories (West
Grove, PA). All the F (ab
)2 preparations were reduced by mercaptoethanol and alkylated with iodoacetamide to generate monovalent Fab
fragments (11). To eliminate possible traces of IgG, the Fab
preparations
were treated with protein A-Sepharose (for 12CA5 or goat IgG) or protein G-Sepharose (for 9E10 IgG). The resulting Fab
were free of contamination by F (ab
)2 or IgG, as judged by SDS-PAGE under nonreducing conditions.
Cell Lines
COS7 and L6 cells (CRL 1651 and CRL 1458, respectively; American Type Culture Collection, Rockville, MD) were grown in DME supplemented with 10% FCS (Biological Industries, Beit Haemek, Israel or GIBCO-BRL, Gaithersburg, MD), 100 U/ml penicillin, 100 µg/ml streptomycin, and 4 mM glutamine (Biological Industries or JRH Biosciences, Lenexa, KS).
Epitope Tagging of TRI
Epitope tagging was performed according to Kolodziej and Young (16)
using site-directed mutagenesis on uracil-containing single-stranded phagemids (17). The human TGF-RI (ALK5) cDNA (9) was inserted into the
SV-40 expression vector pcDNA-I (Invitrogen Corp., Carlsbad, CA) via
HindIII and NotI sites on the polylinker. Using this vector, which also carries an M13 origin of replication, single-stranded phagemids were grown
in a dut
ung
Escherichia coli strain (CJ236/P3; Invitrogen Corp.) with
M13K07 (Bio-Rad Laboratories, Hercules, CA) as helper phage. Site-
directed mutagenesis on this template was performed with mutagenic oligonucleotides encoding the epitope-tag sequence flanked by the sequences corresponding to nucleotides 66-81 (on the 5
end) and 82-97
(3
end) of the human T
RI (counting the A of the methionine codon of
the cDNA as 1). The tag sequences used encoded either the HA epitope
YPYDVPDYA or the human c-myc epitope EQKLISEEDL. Each tag
was inserted in-frame, nine bases downstream of the putative signal peptide sequence. Double-stranded pcDNA-I preparations with inserts of
wild-type T
RI or the epitope-tagged receptors were digested by XbaI,
cutting in two places (at the 3
of the polylinker and at position 706 in the
T
RI coding sequence). The 1.6-kb piece from the wild-type, untagged receptor encoding most of the coding region of the cDNA was ligated with
the 4.9-kb piece from the myc- or HA-tagged NH 2-terminal T
RI sequences and the cloning vector pcDNA-I. The resulting constructs contained as insert all of the T
RI coding region, most of which was derived
from the wild-type, untagged receptor clone, and the NH 2-terminal region
(including the tag) derived from the tagged receptor clone. The coding regions derived from the tagged cDNAs were verified by DNA sequencing.
Construction of HA-tagged Soluble Receptors
To construct an HA-tagged secreted form of the TRII ectodomain (II-SF), a HindIII-PstI fragment of the full-length HA-tagged T
RII (T
RII-HA; 12) containing the 5
region of the ectodomain was ligated into a
PstI-HindIII fragment of the type II-secreted receptor (containing the 3
region of the ectodomain) in pcDNA-I (24). For the experiments below,
the construct was moved into the vector pcDNA-I/Amp (Invitrogen
Corp.) via the HindIII-XbaI sites of the multiple cloning region.
A secreted form of TRI was constructed by using PCR to insert a stop
codon at the 5
end of the transmembrane domain, just upstream of a
PvuII site, replacing Leu126. The mutated cDNA was then cut with EcoRI
(at the insertion site on the 5
end) and PvuII and was inserted into the
EcoRI and EcoRV sites of pcDNA-I/Amp (untagged I-SF). The HA-tagged full-length receptor was cut with HindIII (in the vector) and XhoI
(cuts in the ectodomain sequences). The fragment containing the HA-tag was inserted into a backbone of the untagged I-SF cut with HindIII and
XhoI (partial digest, with cut in the ectodomain), to yield the HA-tagged
secreted form of T
RI (I-SF). The sequence in regions derived from PCR
amplification was confirmed by sequencing.
COS7 Cell Transfections
COS7 cells were transiently transfected by the DEAE-dextran method
(38) using pcDNA-I or pcDNA-I/Amp containing the TRI and T
RII
constructs (tagged, untagged, or the tagged soluble receptors). Cells were
used for experiments 48 h after transfection.
Metabolic Labeling
Cells were grown to subconfluence in standard media on 10 cm plates. They were rinsed once with PBS, then starved in serum-free DME minus methionine and cysteine (ICN Biomedicals, Costa Mesa, CA) for 3-4 h at 37°C. The medium was replaced with fresh starvation medium supplemented with 0.2 mM oxidized glutathione (Boehringer Mannheim Corp., Indianapolis, IN) and 0.5 mCi/ml of [35S]methionine and [35S]cysteine (Express; New England Nuclear, Beverly, MA) and incubated at 37°C for 15 or 30 min. Plates were then rinsed three times with ice-cold PBS and incubated in 0.2 M iodoacetamide (1 M stock solution in 0.5 M Tris-HCl, pH 8.8, diluted to 0.2 M in PBS) for 10 min at 4°C. Cells were transferred into Eppendorf tubes (Madison, WI), lysed in 150 µl of MNT buffer (20 mM MES pH 6.0, 30 mM Tris, pH 7.4, 100 mM NaCl) supplemented with 2% n-octyl-polyoxyethylene (octyl-POE; Bachem Biosciences, Philadelphia, PA), and cleared with a 10-15-min spin (14,000 g) at 4°C. "+SDS" samples were lysed in MNT lysis buffer with 0.5% SDS. The HA-tagged secreted receptors (II-SF and I-SF) were metabolically labeled by rinsing transfected COS7 cells with PBS, followed by incubating the cells in 6 ml/ 10-cm dish of starvation medium with oxidized glutathione and 0.1 mCi/ml 35S-Express for 10.5 h. Media from two plates were then collected, filtered with a 0.22-µm filter, brought to 20 mM Hepes, pH 7.5, and 1 mM PMSF, concentrated in a Centricon 10 concentrator (Amicon, Inc., Berverly, MA) rinsed several times with MNT, brought to 150 µl with MNT (no detergent), and loaded on gradients as below.
Velocity Sedimentation on Sucrose Gradients
50 µl of size markerscarbonic anhydrase (29 kD), BSA (66 kD), alcohol
dehydrogenase (150 kD),
-amylase (200 kD), apoferritin (443 kD), and
thyroglobulin (669 kD), each 9 mg/ml except apoferritin, which was 2.2 mg/ml (Sigma Chemical Co., St. Louis, MO)
in MNT lysis buffer were
added to the 150-µl lysates described above and layered over 4.0-ml gradients of 7.5-30% sucrose in MNT/1% octyl-POE. +SDS samples included
an additional 0.5% SDS added to each gradient. Gradients were centrifuged in an SW60 rotor (Beckman Instruments, Fullerton, CA) at 60,000 rpm for 8 h at 4°C. 250-µl fractions were removed sequentially from the
top of each gradient using a hand-held pipettor. To analyze size standards, 25-µl aliquots were removed from each fraction and were separated by
8-15% gradient SDS-PAGE, followed by Coomassie blue staining. The
remainder of each fraction was brought to 0.5 ml (1 ml for +SDS samples)
with IP buffer (0.5% deoxycholate, 1% Triton X-100, 10 mM EDTA, in
PBS, pH 8.0) and immunoprecipitated with (a) 10 µl/ml of a polyclonal
rabbit antiserum raised against the COOH-terminal 16 amino acids of
T
RII (
-IIC; reference 36); (b) 15 µl/ml of a polyclonal rabbit antiserum
raised against the juxtamembrane cytoplasmic domain of T
RI (9); or (c)
1 µl/ml
-HA ascites. After overnight incubation at 4°C, 50 µl of protein
A-Sepharose CL-4B beads (1:1 in PBS; Sigma Chemical Co.) were added,
and the lysates were incubated with rotation for an additional 30 min. Beads were rinsed twice with IP buffer (with 0.5% SDS for type II receptor immunoprecipitations) and once with PBS and eluted by boiling in
SDS-PAGE sample buffer. Control immunoprecipitants (from lysates
treated identically, but not run on gradients) were eluted into 0.5% SDS
and one half of each was digested overnight at 37°C with endoglycosidase
H (Endo H; Genzyme Corp., Boston, MA; 100 mIU/ml) in the presence of
100 mM sodium citrate, pH 6. Samples were analyzed by 7.5% SDS-PAGE; gels were fluorographed with 2,5 diphenyl-oxazole, dried, and
placed on Kodak XAR film (Rochester, NY), which was quantitated with
a LaCie Silverscanner II and MacBAS (Fuji Photo Film Co., Tokyo, Japan) software. The secreted receptors in the media collected from the cells
(see former section) were treated similarly, except that the gradients were
5-15% sucrose in MNT without detergent, and markers were cytochrome
C (12.4 kD), carbonic anhydrase (29 kD), and BSA (66 kD). Fractions
from the sucrose gradients were brought to 750 µl with IP buffer, immunoprecipitated with
-HA, and analyzed on 15% polyacrylamide/1.2%
bisacrylamide SDS gels.
Receptor Cross-Linking
Binding and cross-linking of 100 pM 125I-TGF-1 to transfected COS7
cells grown on 10-cm dishes was as described (36). Cross-linked proteins
were resolved by 7.5% SDS-PAGE under reducing conditions and exposed to Kodak XAR film at -70°C. For affinity labeling of II-SF, transfected COS7 cells were rinsed with PBS and incubated at 37°C for 10.5 h
in 6 ml/10-cm dish of DME without serum. The media from two dishes
were filtered and concentrated as for the metabolically labeled material,
brought to 1.0 ml with KRH (50 mM Hepes, pH 7.5, 128 mM NaCl, 1.3 mM
CaCl2, 5 mM MgSO4, 5 mM KCl), and incubated for 3.5 h at 4°C with 250 pM 125I-TGF-
1. Disuccinimidyl suberate (0.1 mg/ml) was added (4°C, 15 min). The resulting material was quick-spun to remove insoluble cross-linker, concentrated with several washes of MNT, and brought up to 150 µl with MNT (no detergent) before being loaded on a 5-15% sucrose gradient.
Receptor Coimmunoprecipitation
48 h after transfection, cells were washed and incubated with serum-free
medium (30 min, 37°C) to eliminate residual ligand. 2 mM DTT was
added for the last 10 min of the incubation to selected dishes. Cells were
then washed twice in ice-cold HBSS/Hepes/BSA (Hanks' balanced salt solution with 20 mM Hepes, pH 7.4, supplemented with 1% fatty acid-free
BSA; Sigma Chemical Co.) and incubated in the same medium with or
without 250 pM TGF-1 (4°C, 1.5 h). The cells were washed twice more in
HBSS/Hepes and lysed in a lysis buffer (PBS containing 1% Triton X-100,
10 mM EDTA, 1 mM p-aminoethylbenzenesulfonyl fluoride). Extracts
were precleared (4°C, 1 h) by incubation with protein A-Sepharose (Pharmacia Biotech, Piscataway, NJ) for immunoprecipitation with 12CA5 antibodies or with protein G-Sepharose (for immunoprecipitation with 9E10 antibodies). Immunoprecipitation was carried out (4°C, 2 h) with 20 µg/ml
of the appropriate IgG together with protein A- or protein G-Sepharose.
Immunoprecipitates were washed three times with the lysis buffer, dissolved in Laemmli loading buffer with or without mercaptoethanol, and
run on 7% SDS-PAGE. For N-glycosidase F treatment, immunoprecipitates were dissolved in 20 µl 0.5% SDS. 20 µl of 2× enzyme buffer (100 mM sodium phosphate, pH 7.5, 2% NP-40), and 2 U of N-glycosidase F
(Boehringer Mannheim Corp.) was added to the eluate (37°C, 4 h). The
gel was blotted onto nitrocellulose and then blocked in TBST (50 mM
Tris, 100 mM NaCl, 0.1% Tween 20, pH 7.4) with 2% BSA and 5% skim
milk (4°C, 1 h). The blot was then sequentially incubated with 20 µg/ml
-myc, 1:2,000 biotinylated G
M (goat IgG anti-mouse IgG; Jackson ImmunoResearch Laboratories), and 1:1,000 streptavidin-horseradish peroxidase (GIBCO-BRL). After each incubation, the blot was washed three
times in TBST (4°C, 10 min). Enhanced chemiluminescence was used to
visualize the precipitated receptors by autoradiography on an x-ray film.
Immunofluorescence Copatching Experiments
The method used is based on Henis et al. (12), labeling the first epitope
with monovalent Fab fragments (to avoid adsorption of the mouse antibodies against the second tag to secondary antibodies prebound to the
first tag). The method was modified by the use of a biotinylated secondary
Fab
followed by Cy3-streptavidin to mediate patching of the first epitope
as well.
COS7 cells were grown on coverslips and transfected with TRI-HA
and T
RI-myc constructs. 48 h after transfection, cells were washed twice
with serum-free DME and incubated 30 min at 37°C to allow endocytosis
of ligand-bound receptors. After washing twice with cold HBSS/Hepes/
BSA, the cells were incubated in the same buffer (4°C, 2 h) with normal
goat IgG (200 µg/ml) to block nonspecific binding. This was followed by
successive incubations (4°C, 1 h each, with three washes between incubations) with: (a)
-myc Fab
(50 µg/ml); (b) Fab
fragments of biotinylated
G
M (5 µg/ml); (c) Fab
of unlabeled G
M (200 µg/ml), used to block all free binding epitopes on the
-myc antibody; and (d)
-HA IgG (20 µg/ml);
(e) FITC-labeled G
M (20 µg/ml). The cells were then washed and fixed
by two incubations (15 min, 4°C and then 15 min, 22°C) with 3.2% paraformaldehyde in PBS, pH 7.4, supplemented with 1.1% lysine and 0.24%
NaIO4. The reaction was quenched by three incubations with 50 mM glycine in PBS, pH 7.4 (5 min, 22°C). Cells were then incubated with 0.5 µg/ml
Cy3-streptavidin and mounted with mowiol (Hoechst AG, Frankfurt,
Germany) containing 29 mM n-propyl gallate (Sigma Chemical Co.). Fluorescence digital images were acquired with a Zeiss Axioskop (63× oil
objective; Thornwood, NY), coupled to a cooled CCD camera (model
CC/CE 200; Photometrics, Tuscon, AZ). The computerized microscope
system (15) and Priism software driving the focus, excitation, and emission
filter wheels were described earlier (5, 14). Fluorescein and Cy3 images
were taken using selective filter sets that essentially eliminate leakage. Superposition of the two images and contrast enhancement were performed
after correction for shift and magnification using Priism interactive multicolor visualization (5). The images were exported in TIFF format to Photoshop (Adobe, San Jose, CA) and printed.
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Results |
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TRI Forms Homodimers in the ER
Two different experiments in the literature have provided
functional evidence that TRI forms homooligomers
within the signaling complex and that this oligomerization
is functionally relevant (27, 40). The oligomeric structure
of T
RI either in the ER or the plasma membrane has not
been thoroughly characterized, however, and there is no
conclusive evidence for physical interactions between
T
RI polypeptides in the absence of ligand and the type II
receptor. We therefore used sucrose gradient velocity centrifugation of metabolically labeled receptors to determine
relative complex size. This approach was chosen since
chemical cross-linking of T
RI and T
RII has not been
possible (data not shown). We used L6 rat myoblasts,
which are TGF-
responsive and which express both of
the signaling receptors but lack the type III TGF-
receptor that may form complexes with T
RI and T
RII and
could therefore complicate size determinations. L6 cells
were pulse-labeled for 30 min, conditions under which all
labeled T
RI is Endo H sensitive (41; Fig. 1 B). Endo H
sensitivity was used as a marker for localization in the ER
since only proteins in the ER and early Golgi carry the
high mannose sugars, which are susceptible to the enzyme. Labeled L6 cells were lysed in a buffer containing octyl-POE and analyzed by velocity sedimentation on sucrose
gradients in the same detergent, with or without SDS (see
Materials and Methods). Octyl-POE was chosen as the detergent for several reasons: (a) It is nonionic; (b) it has a
density of 1; (c) it has a high CMC; and (d) it is freely miscible in aqueous solutions, enabling analysis of the migration of detergent-solubilized proteins rather than of micelles.
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Receptors from cells lysed and run in the presence of
0.5% SDS yielded a peak at fractions 4-5, corresponding
to 50 kD, as determined by size marker analysis carried
out for each individual gradient (Fig. 1, A and C). This is
compatible with the molecular mass of the monomeric receptor, predicted to be 53 kD. Receptors from cells not
treated with SDS migrated with a peak in fractions 6-8
(Fig. 1, B and C), consistent with a size of 120 kD. Although absolute size determinations from a gradient can
be inaccurate for detergent-solubilized membrane proteins, the size of this complex compared with the SDS-treated monomers is consistent with TRI dimers. T
RI
from cells labeled similarly but chased for 4 h (such that labeled T
RI carried mature N-linked oligosaccharides and
was at the trans-Golgi or beyond; reference 41) migrated with similar velocity. We conclude that T
RI forms dimer-sized complexes in the ER. Similar results were obtained
with Mv1Lu cells (not shown) and with COS7 cells transfected with tagged or untagged T
RI (Fig. 2 and data not
shown), demonstrating the generality of T
RI dimerization. The similarity between COS7 cells and the naturally
expressing cell lines regarding T
RI dimerization indicates that overexpression in COS cells does not affect
dimerization. This allowed us to use COS7 cells for immunofluorescence copatching studies, which require receptor
density at the cell surface high enough to be visualized by
immunofluorescence.
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Coimmunoprecipitation Studies Identify Ligand- and
DTT-independent TRI Homooligomers
The velocity sedimentation experiments described above
demonstrate that TRI forms complexes of a size expected for a dimer. However, it is also possible that T
RI
associates with another protein of roughly the same size.
To demonstrate that T
RI indeed forms homooligomers,
we performed coimmunoprecipitation studies on cells
cotransfected with two T
RI forms, each carrying a different epitope tag. HA or myc epitopes were inserted at the
ectodomain NH2 terminus, three amino acids downstream
of the putative signal sequence cleavage site. They were
introduced at the NH2 terminus to enable immunofluorescence copatching studies (see next section). Fig. 3 demonstrates that the tagged receptors were expressed at the surface of COS7 cells and labeled by 125I-TGF-
1 similar to
untagged T
RI. Very low levels of T
RI and T
RII were
observed in control cells transfected with vector alone (Fig. 3, lane 5) or with T
RI cDNA in the same vector
(lane 4). These low levels may be attributed to the native
receptor population in COS7 cells. Cotransfection of
T
RI (tagged or untagged) together with T
RII resulted
in a significant increase in the labeling of T
RI (lanes 1-3).
The insertion of the epitope tags did not eliminate receptor signaling, as tested by cotransfection with p3TP-Lux (a
construct that carries the luciferase gene under a TGF-
response element) into L17 cells that lack endogenous
T
RI activity (not shown). Furthermore, in the sedimentation velocity studies (Fig. 2), the tagged T
RI was able
to form dimer-sized complexes, as observed for the untagged receptor.
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We used the tagged type I receptors in coimmunoprecipitation experiments designed to confirm that the dimer-sized TRI complexes observed in the sedimentation velocity studies did in fact represent dimers and to determine
whether exposure to ligand or DTT influence T
RI homooligomers. The latter experiment was motivated by the observation that cross-linking of iodinated TGF-
1 to
T
RI in the presence of T
RII is eliminated by pretreatment of cells with DTT (4), an effect that may involve disruption of the T
RI/T
RII heterooligomeric structure
(Rodriguez, C., R. Lin, P.E. Scherer, and H.F. Lodish,
manuscript in preparation).
COS7 cells were cotransfected with TRI-HA and
T
RI-myc and lysates were immunoprecipitated with
-HA
antibodies. The immunoprecipitates were subjected to
SDS-PAGE and Western blotting, and the blots were analyzed with
-myc antibodies followed by biotinylated
G
M and streptavidin-horseradish peroxidase. The results show that T
RI-myc coprecipitates with T
RI-HA
(Fig. 4 A, lane 5, and B, lane 2). Preincubation of cells with
TGF-
1 (Fig 4 A, lane 6, and B, lane 3), DTT (Fig 4 A,
lane 7), or both (lane 8) did not change the quantity of
T
RI-myc coprecipitated with T
RI-HA. Blots from control cells, singly transfected with T
RI-HA, were not stained by
-myc, showing the specificity of the blotting
antibody (Fig. 4 A, lane 4). Transfection with T
RI-myc
alone was used as control for the immunoprecipitating antibody,
-HA (Fig. 4 A, lane 3). A positive control of cells
singly expressing T
RI-myc, where
-myc was used both
for immunoprecipitation and blotting, is depicted in lanes
1 and 2 (Fig. 4 A). The type I receptors appear in two
bands (~59 and 53 kD; Fig. 4 A, lane 1). The upper band is
N-glycosylated T
RI since treatment of the precipitated receptors with N-glycosidase F led to disappearance of the
59-kD band, leaving only the lower band (Fig. 4 B). The
53-kD band in Fig. 4 A gets somewhat distorted and swept
downward in the coprecipitation lanes because of the presence of high amounts of the
-HA antibody used for precipitation (seen in the lowest band). This antibody, unlike
-myc, runs very close to the lower T
RI band. When the gels are run under nonreducing conditions (providing better separation from the antibody band), this distortion disappears (Fig 4 B). These results demonstrate that T
RI
forms homooligomers that are unaffected by TGF-
1 or
DTT and are stable in the presence of detergents.
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Immunofluorescence Copatching Experiments Show
Ligand-independent TRI Homodimers on the Surface
of Live COS7 Cells
The experiments described in the former sections suggest
that TRI forms dimer-sized complexes and that at least
some of them are caused by homooligomer formation. To
measure the T
RI oligomeric structure at the native cell
membrane, we performed immunofluorescence copatching studies (for method description see Methods and reference 12). These studies have the advantage that they are
performed on live cells (without any possible interference
by detergents), measure the bulk of cell surface receptors
on coexpressing cells only, and supply a semiquantitative
measure for oligomer size. In this method, a tagged receptor at the surface of live, unfixed cells is forced into
patches by a double layer of bivalent IgGs. The second receptor, which carries a different tag, is labeled by antibodies coupled to another fluorophore. The samples are then
examined to explore whether the second receptor is swept into the same micropatches or whether the two receptors
congregate in separate patches. The experiment is performed in the cold to avoid internalization. If the two fluorophores used are Cy3 (red emission) and fluorescein
(green emission), then mutual patches appear yellow,
while separate patches are either red or green. It is therefore possible to count the percentage of mutual and of separate patches, thus obtaining a semiquantitative determination of the level of complex formation and of complex
size (12). It should be noted that the tags (HA and myc)
and antibodies used in the current studies do not mediate
receptor complex formation or nonspecific sweeping into
mutual patches since similar experiments using the same
tags and antibodies did not show copatching between T
RII and T
RIII (12).
COS7 cells cotransfected with TRI-HA and T
RI-myc
were subjected to successive incubations with different antibodies to mediate patching and fluorescent labeling of
the tagged receptors, as specified under Materials and
Methods. Typical results are shown in Fig. 5. The labeling
procedure is fully specific, as can be seen in the inset of
Fig. 5 A, showing control cells singly transfected with
T
RI-myc and subjected to the same labeling procedure;
the cells exhibit Cy3 labeling and almost no fluorescein labeling, which would be associated with T
RI-HA. In all
cells expressing both T
RI-myc and T
RI-HA, ~50% of
the patches were yellow. This indicates that the majority of
T
RI at the cell surface resides in dimers. Statistically, this
is the percentage expected to reside in mutual complexes
in the case of a dimer since only half of the dimers would
carry two different tags (see Discussion). Preincubation with ligand (Fig. 5, B and D) or DTT (Fig. 5, C and D) did
not alter the percentage of mutual (yellow) patches
formed by the antibodies. It should be noted that 50% copatching is not a pattern that is commonly observed in
such experiments; thus, no copatching was detected for coexpressed T
RII and III (12), and only a low percentage
of copatching is observed for T
RI/T
RII in the absence
of ligand (Wells, R.G., L. Gilboa, Y.I. Henis, and H.F. Lodish, manuscript in preparation). The results presented in
Fig. 5 suggest that all (or most of) the type I receptors appear
as ligand-independent dimers on the surface of live cells and
that this structure is not altered by preincubation with DTT.
|
TRII Forms Homodimers in the ER
Homooligomerization of TRII might have an important
role in TGF-
signal transduction. We have previously
used immunofluorescence copatching to show that T
RII
forms small homooligomers on the surface of live COS7
cells (12), similar to T
RI (Fig. 5). To determine whether
T
RI homooligomerization occurs in the ER, we performed velocity centrifugation experiments with metabolically labeled, Endo H-sensitive type II receptors. The experiments used L6 cells as described above for T
RI,
except that a shorter labeling pulse (15 min) was required
to give a T
RII population entirely sensitive to Endo H
(Fig. 6 A; reference 41). Velocity centrifugation of T
RII
from cells lysed and run in the presence of 0.5% SDS
yielded a peak at fractions 4-6 (Fig. 6, B and D), consistent
with a monomeric T
RII. Receptors from lysates lacking SDS migrated with a peak at 150 kD (Fig. 6, C and D),
consistent with the ER form being a dimer. Metabolically
labeled Endo H-resistant receptor, measured after a 2 h
chase, migrated at the same point (data not shown), consistent with our immunofluorescence copatching experiments in COS7 cells (12). Similar results were obtained with
COS7 cells transiently expressing T
RII (tagged or untagged) as well as with Mv1Lu (not shown), confirming
that dimerization of T
RII is not cell type specific. These
findings show that the type II receptors form homodimers
in the ER, which are then transported, presumably in this
form, to the cell surface.
|
The Ectodomains of TRII and T
RI Are Monomeric,
but TGF-
1 Can Induce Dimerization of II-SF
To determine whether the ectodomain of either signaling
receptor is responsible for the homodimerization we have
observed, we performed sucrose gradient velocity centrifugation studies on the secreted ectodomains of both receptors. The soluble exoplasmic domain of TRII is efficiently secreted and has binding characteristics equivalent
to the full-length receptor (24). I-SF is also efficiently secreted, suggesting that it is correctly folded; however, it cannot be cross-linked to ligand in the presence or absence
of II-SF and likely requires transmembrane and cytoplasmic domains for full interaction with the type II receptor
and for ligand binding (Wells, R.G., L. Gilboa, Y.I. Henis,
and H.F. Lodish, manuscript in preparation). COS7 cells
were transfected with a plasmid carrying an epitope-tagged form of the soluble type II receptor (II-SF), and the
media from metabolically labeled cells were separated on
detergent-free sucrose gradients. In the absence of ligand,
II-SF peaked at fractions 3-4 (Fig. 7, A and C), predicted by size markers to be 20 kD, consistent with a monomer.
The truncated receptor has complex and heterogeneous
N-linked glycosylation, accounting for the multiple labeled
bands. The same receptor, when collected from the media
of unlabeled cells and cross-linked to 125I-TGF-
1, migrated as a 60-65-kD protein (Fig. 7, B and C), consistent with a dimeric receptor bound to the ligand. This dimerization did not occur if detergent (1% Triton X-100) was
added (not shown).
|
Analogous experiments were conducted on the soluble
form of TRI (I-SF), described under Materials and Methods. The results are depicted in Fig. 8. Metabolically labeled I-SF migrated with a peak at fractions 3-6 (Fig. 8),
consistent with a monomer of molecular mass around 24 kD.
The broad peak suggests that some I-SF migrated faster,
and there could be some I-SF dimers present. These results demonstrate that the exoplasmic domains of both T
RI and T
RII are mainly monomeric and are not sufficient for dimerization by themselves. II-SF, however, can
form dimers upon TGF-
1 binding.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TGF- elicits a broad spectrum of effects, ranging from
control of cell growth and extracellular matrix composition to differentiation and cell death (37). TGF-
signal
transduction requires two cell surface receptors, T
RI and
T
RII, which are serine-threonine kinases (9, 24). A substantial amount of evidence attests to interactions between
T
RI and T
RII upon ligand binding (3, 7, 27, 43, 44).
Homomeric interactions of TGF-
receptors, however,
have not been investigated in detail, especially for T
RI.
Although there are clear indications that homomeric interactions of type I and type II TGF-
receptors are important functionally for signal transduction and regulation,
there has thus far been no physical evidence for T
RI homooligomerization, and the cellular site of T
RII homooligomerization is not known (6, 12, 27, 28, 40). In the current studies, we combined several complementary approaches
to investigate this issue: (a) sedimentation velocity studies
to determine the size of metabolically labeled receptor complexes; (b) coimmunoprecipitation experiments on coexpressed, differentially tagged forms of T
RI to explore
homooligomer formation; and (c) immunofluorescence copatching studies on cells coexpressing differentially tagged
T
RI forms to establish the existence of receptor homodimers at the surface of intact cells.
Sedimentation velocity experiments on metabolically labeled TRI demonstrated that the receptors migrate as a
complex whose size corresponds to a dimer. This complex
was observed in the ER of several cell types, from naturally expressing L6 (Fig. 1) or Mv1Lu cells to transiently
expressing COS7 cells (Fig. 2). This indicates that the
dimer-sized complex formed is not affected by the level of
expression. The fact that the complex size fits a T
RI
dimer does not necessarily point to the existence of such dimers since some other cellular protein of a similar size
can be associated with the receptor. This issue can be resolved by the use of other methods (coimmunoprecipitation and immunofluorescence copatching) to demonstrate
that T
RI does indeed form homodimers and to establish
independently the existence of T
RI dimers at the cell
surface. The latter experiments require the expression of
epitope-tagged receptors at levels high enough for good
fluorescence visualization, and they were therefore performed in COS7 cells. The use of the overexpressing COS
cells is justified in view of the similar complex size found in
centrifugation studies for T
RI expressed transiently in
COS cells or naturally in the other cell types. Furthermore, similar results were obtained in the copatching studies on COS7 cells expressing either high (~200,000 receptors/cell at the surface) or low (~20,000) receptor levels
(determined as described in reference 12).
The coimmunoprecipitation studies (Fig. 4) using tagged
TGF- type I receptors clearly demonstrate physical association between T
RI polypeptides. Thus, the complexes
formed are relatively stable and withstand, at least partially, the detergent solubilization and immunoprecipitation conditions. Together with the centrifugation velocity
studies, which point to a dimer-sized complex, these results suggest that the complex is indeed a homodimer of
T
RI.
Coimmunoprecipitation experiments do not provide a
measure for the percentage of receptors that reside in oligomers since only part of the cell population (~40%, as
determined by immunofluorescence) coexpresses the two
differently tagged receptors. Immunofluorescence copatching studies circumvent this problem by selecting only coexpressing cells for analysis; they also provide an independent
means to determine the size of the receptor oligomers and,
most importantly, do so at the surface of live cells. By coexpressing HA- and myc-tagged TRI, we have observed
that ~50% of the patches were dyed yellow (Fig. 5), implying that the majority of T
RI at the surface of COS7
cells is homodimeric. The reasoning is as follows: For
monomers, each type of tagged receptor would be swept
into separate patches by the respective antibody, yielding
no copatching. In the case of a dimer, only half of the
dimers would be composed of receptors carrying different
tags; these will be swept mostly into copatches, while the
great majority of uniformly tagged dimers will reside in either green or red patches. For trimers and tetramers, the expected percentage of complexes carrying different tags
is 75 and 87.5%, respectively. Clearly, the results of the copatching experiments are in good agreement with the receptors being dimeric. They provide an independent proof
for the homodimeric nature of the dimer-sized complex
observed in the centrifugation experiments.
The copatching experiments, as well as the coimmunoprecipitation studies, show that TRI dimers are unaffected by the presence of TGF-
1 (Figs. 4 and 5); this is
not surprising since T
RI does not bind TGF-
1 in the absence of T
RII. Interestingly, the dimeric structure of
T
RI is not disrupted by pretreatment of the cells with
DTT under conditions known to eliminate TGF-
1 binding to these receptors (Figs. 4 and 5). These findings demonstrate that the homooligomeric complex of the type I receptors can withstand whatever structural alteration is
mediated by the DTT treatment, and which interferes with
the ability of T
RI to be cross-linked to TGF-
1 in the
presence of T
RII (reference 4; Rodriguez, C., R. Lin,
P.E. Scherer, and H.F. Lodish, unpublished results).
Sedimentation velocity studies analogous to those performed on TRI were conducted on T
RII (Fig. 6). These
experiments establish that T
RII appears in dimer-sized
complexes, which are formed in the ER. Together with
previous studies using immunofluorescence copatching
(12) and coimmunoprecipitation (6, 12), which demonstrate that T
RII chains interact with each other, we can conclude that T
RII appears both in the ER and at the
cell surface as a dimer. The copatching studies (12) indicate that the dimeric nature of T
RII is not altered after
TGF-
1 binding. Moreover, the inherent tendency of
T
RII to form dimers is independent of expression level,
as indicated by the similar results obtained in sedimentation velocity studies performed on naturally expressing cell lines and on overexpressing COS cells.
To explore the role of the exoplasmic domain in promoting homodimerization, the soluble exoplasmic domains of TRI and T
RII secreted by COS7 cells were analyzed by sedimentation velocity experiments (Figs. 7 and 8).
In both cases, the receptors migrated primarily as monomers, indicating that these domains are insufficient to promote dimerization of either receptor. TGF-
1 induced
dimerization of II-SF (Fig. 7), which binds ligand (24), in
agreement with a report that TGF-
2 and TGF-
3 also
promote dimerization of the ectodomain (20). The ligand-mediated homomeric interactions of II-SF, however, are
disrupted by the presence of nonionic detergent, indicating that they are significantly weaker than in wild-type T
RII (data not shown). Accordingly, the intact T
RII is
dimeric in the absence of ligand and does not require
ligand binding for dimerization. An immediate conclusion
is that the cytoplasmic and/or transmembrane domains of
T
RII must have a major contribution to the homomeric
interactions. This conclusion is in accord with the report
(6) that the cytoplasmic domains of T
RII overexpressed in 293 cells can form homooligomers. There is, however,
evidence that the ectodomains of both receptors also contribute to the homomeric interactions since chimeras composed of the cytoplasmic and transmembrane regions of
T
RI or II and the ectodomain of the Epo receptor required dimerization by Epo to interact (27, 28). Taken together, it seems likely that several domains scattered throughout the receptors contribute to the homomeric interactions. In the case of T
RII, it appears that ligand binding can enhance the interactions between the ectodomains.
In conclusion, we have combined several methods to
characterize homooligomer formation of TRI and T
RII.
The findings demonstrate that both receptors exist as homodimers that form in the ER and persist at the cell surface. The roles of homooligomerization and its involvement in the mechanism(s) of TGF-
signaling should be
further explored. It appears, however, that for T
RI the
homodimerization has a critical role in signal transduction
(27, 40), and for T
RII it is involved in modulating signaling (28). The variety of biological responses mediated by
TGF-
ligands raises the possibility that an intricate equilibrium between various homo- and heterocomplexes, which can be affected by the binding of various ligands,
plays a role in regulating those responses.
![]() |
Footnotes |
---|
Received for publication Received for publication 2 October 1997 and in revised form 9 December 1997..
1. Abbreviations used in this paper: ![]() |
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