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J Biol Chem, Vol. 273, Issue 50, 33423-33428, December 11, 1998
From the Departments of The amino-terminal ectodomain of thyrotropin
(TSH) receptor (TSHR) is heavily glycosylated with asparagine-linked
(N-linked) oligosaccharides. The present studies were
designed to evaluate how acquisition and processing of N-linked
oligosaccharides play a role in the functional maturation of human
TSHR. A glycosylation inhibitor tunicamycin, which inhibits the first
step of N-linked glycosylation (acquisition of
N-linked oligosaccharides), and a series of mutant Chinese
hamster ovary (CHO)-Lec cells defective in the different steps of
glycosylation processing were used. Inhibition of acquisition of
N-linked oligosaccharides by tunicamycin treatment in CHO
cells stably expressing TSHR produced nonglycosylated TSHR, which was
totally nonfunctional. In contrast, all of the TSHRs synthesized in
mutant CHO-Lec1, 2, and 8 cells (mannose-rich, sialic acid-deficient,
and galactose-deficient oligosaccharides, respectively) bound TSH and
produced cAMP in response to TSH with an affinity and an
EC50 similar to those in TSHR expressed in parental CHO
cells (CHO-TSHR; sialylated oligosaccharides). However, Lec1-TSHR and
Lec2-TSHR were not efficiently expressed on the cell surface, whereas
the expression levels of Lec8-TSHR and CHO-TSHR were essentially
identical. All of the TSHRs expressed in CHO-Lec cells cleaved into two
subunits. Finally, anti-TSHR autoantibodies from Graves' patients
interacted with all of the TSHRs harboring different oligosaccharides
to a similar extent. These data demonstrate that acquisition and
processing of N-linked oligosaccharides of TSHR appear to
be essential for correct folding in the endoplasmic reticulum and for
cell surface targeting in the Golgi apparatus. We also show that
complex type carbohydrates are not crucially involved in the
interaction of TSHR with TSH and anti-TSHR autoantibodies.
The receptors for glycoprotein hormones (thyrotropin receptor
(TSHR),1 lutropin receptor
(LHR), and follitropin receptor (FSH)) are members of a subfamily of G
protein-coupled receptors, characterized by an extremely large
amino-terminal extracellular domain, which is the high affinity binding
site for the respective ligands (1, 2) and is, in the case of TSHR, the
primary autoantigen in autoimmune thyroid disease, including Graves'
disease (1). Like many other secreted and cell surface proteins, these
receptors undergo a series of posttranslational modifications such as
disulfide bonding (1), glycosylation (3-9), palmitoylation (10, 11), and proteolytic cleavage (subunit formation in TSHR) (12-14), many of
which appear to play roles in protein maturation and/or intracellular trafficking.
In human TSHR, the receptor ectodomain has asparagine-linked
(N-linked) oligosaccharides, which represent 30~40% of
its molecular weight (15). Earlier studies with in vitro
site-directed mutagenesis suggest that, among six potential
N-linked glycosylation sites, the first (amino acid 77) and
third (amino acid 113) glycosylation sites are essential for the cell
surface expression and function of the full-length TSHR (16). The
possibility, however, cannot be excluded in this paper that the amino
acid substitutions introduced per se affected receptor
function (9). Subsequent studies on the functional role of
N-linked oligosaccharides of TSHR have been performed with
the truncated form of TSHR ectodomain expressed in mammalian cells and
insect cells, yielding controversial data (15, 17, 18). However, it is
difficult to interpret these data, because conformational integrity of
TSHR protein has not been verified in these studies. Conformationally
intact TSHR protein showing high affinity TSH binding has so far been
expressed only in mammalian cells as the full-length receptor or the
ectodomain fused to a membrane-anchoring peptide (19-21).
The process of N-linked glycosylation of proteins has been
well documented (see Fig. 1) (22). Briefly, a dolichol pyrophosphate precursor (Glc3Man9GlcNAc2) is at
first transferred to Asn side chain of Asn-X-Ser/Thr
consensus sequence for N-linked oligosaccharides in a
nascent polypeptide in the endoplasmic reticulum. Processing is
initiated by the removal of the three terminal glucose residues and at
least one mannose residue in the endoplasmic reticulum, followed by
transportation to Golgi apparatus, where mannose residues are further
trimmed, and N-acetylglucosamine, galactose, and sialic acid
residues are sequentially added. The newly synthesized glycoproteins then exit the Golgi and are transported to their final destination.
In the present studies, to further clarify the functional roles of
N-linked oligosaccharides in human TSHR, we evaluated the functional properties of nonglycosylated TSHR and TSHRs with different types of N-linked oligosaccharides. Nonglycosylated TSHR was
produced by treatment of TSHR expressed in Chinese hamster ovary (CHO) cells with tunicamycin, and TSHRs with different oligosaccharides were
expressed in a series of mutant CHO-Lec cell lines harboring mutations
in the distinct steps in carbohydrate processing (see Fig. 1) (23).
CHO Cell Lines--
Mutant CHO cell lines, CHO-Lec1, 2, and 8, originally established by Dr. Pamela Stanley (23), were obtained from
the American Type Culture Collection (Rockville, MD). CHO-Lec1 cells
(ATCC CRL-1735) have no detectable
N-acetylglucosaminyl-transferase I activity, and proteins
expressed carry oligosaccharides bearing mannose-rich intermediates
(Man5GlcNAc2) at sites normally occupied by
complex carbohydrates
(Sia2Gal2GlcNAc2Man3GlcNAc2)
in parental cell line CHO cells (Fig. 1).
CHO-Lec2 (ATCC CRL-1736) and -Lec8 (ATCC CRL-1737) cells lack
CMP-sialic acid and UDP-galactose translocases and are incapable of
transporting CMP-sialic acid and UDP-galactose from the cytosol to the
Golgi, thereby producing sialic acid-deficient (Gal2GlcNAc2Man3GlcNAc2)
and sialic acid- and galactose-deficient (GlcNAc2Man3GlcNAc2)
oligosaccharides, respectively. These cells were maintained at 5%
CO2 in minimal essential medium Transfection and Cell Culture--
The human full-length TSHR
cDNA (24) was ligated into the expression vector pCAGGS (25) to
yield pCAG-TSHR. The plasmid was transfected into the cells described
above with Lipofectin reagent (Life Technologies, Inc.). The cells were
selected with 500 µg/ml G418 (Geneticin; WAKO, Osaka, Japan) and
pooled. Surviving clones (12 clones for CHO cells and 24 for CHO-Lec
cells) were also isolated with cloning cylinders, and the clonal cell
lines expressing the highest levels of the receptor, determined by
[125I]TSH binding (see below), were selected.
Tunicamycin Treatment--
The cells were treated with 5 µg/ml
tunicamycin (WAKO) for 3 days. The medium was replaced daily to purge
the cells of wild-type TSHR. Tunicamycin inhibits transfer of dolichol
pyrophosphate precursor to Asn in the consensus sequence for
N-linked glycosylation (Asn-X-Ser/Thr).
Immunoprecipitation of TSHR--
Metabolic labeling with
[35S]methionine/cysteine (Trans 35S Label;
ICN, Irvine, CA), immunoprecipitation of TSHR with mouse anti-TSHR monoclonal antibodies A10 and A11 (26), deglycosylation of TSHR with
endoglycosydase H (endo H) and N-glycosidase F, and SDS-polyacrylamide gel electrophoresis were performed as described previously (4, 11, 27).
Signals were obtained with a Fujix Bioimaging Analyzer Bas-5000 (Fuji,
Tokyo, Japan).
Immunoblot analysis of TSHR--
Extraction of the crude cell
membrane and immunoblotting were performed as described previously (28)
with mouse anti-TSHR monoclonal antibodies A10 and A11 (26).
[125I]TSH Binding and cAMP
Measurements--
125I-labeled bovine TSH was from the
TRAb kit (Cosmic, Tokyo, Japan). [125I]TSH binding to
intact cells and intracellular cAMP measurement with a cAMP
radioimmunoassay kit (Yamasa, Tokyo, Japan) were performed as described
previously (11). Unlabeled TSH used in TSH binding studies was of
bovine origin (Sigma). [125I]TSH binding to the
detergent-solubilized extracts was performed with polyethylene glycol
(PEG) precipitation previously described (29). Briefly, the
detergent-solubilized extract was prepared by solubilization of the
crude membrane (11) with 1% Triton X-100 at 4 °C for 1 h.
One-hundred µl of detergent-solubilized extract from 100 µg of
crude membrane was incubated with 100 µl of [125I]TSH
(~15,000 cpm) for 1 h at 37 C, and solubilized extract and [125I]TSH complexes were precipitated with PEG.
Thryoid Stimulatory Autoantobody (TSAb) and TSH Binding
Inhibitory Autoantibody (TBIAb) Assays--
Two Graves' sera positive
for TSAb activity, another two Graves' sera positive for TBIAb, and a
mixture of three normal sera were used. Preparation of crude IgG
fraction with PEG and measurements of TSAb activity were performed as
described previously (30). For the TBIAb assay, detergent-solubilized
extract (100 µl) from 100 µg of crude membrane was incubated with
100 µl of serum and 100 µl of [125I]TSH (~15,000
cpm) for 1 h at 37 C, and solubilized extract and [125I]TSH complexes were precipitated with PEG as
described above. TBIAb activity was calculated as follows: [1 Immunoprecipitation and Immunoblot of TSHR in the Clonal Cell Lines
Stably Expressing TSHR--
Using clonal cell lines stably expressing
the highest levels of receptors, we determined the molecular masses and
glycosylation patterns of TSHR expressed in CHO cells treated with and
without tunicamycin and in a series of CHO-Lec cells. In
immunoprecipitation experiments (2-h pulse followed by 2-h chase), TSHR
protein expressed in parental CHO cells (CHO-TSHR) was visualized as an
~95-kDa precursor and an ~120-kDa mature protein (Fig.
2A, lane 1) as previously
reported (27). Both decreased in size to ~85 kDa on
N-glycosidase F treatment (Fig. 2A, lane 3), a
size compatible with that of the core polypeptide chain of TSHR (24)
and also identical to that of nonglycosylated TSHR (Fig. 2A,
compare with lane 4; see below). The ~95-kDa protein was
sensitive to endo H, indicating that this has high mannose type
oligosaccharides, whereas the ~120-kDa species possesses complex type
oligosaccharides because of its resistance to endo H (Fig. 2A,
lane 2).
Tunicamycin treatment completely inhibited addition of oligosaccharides
to TSHR (Fig. 2A, lanes 4-6). Thus, TSHR was detected as an
~85-kDa single species in CHO cells treated with 5 µg/ml tunicamycin.
With respect to mutant cell lines, CHO-Lec1 cells produced a TSHR that
migrated as an ~90-kDa band, slightly smaller than a TSHR with high
mannose oligosaccharides (Fig. 2A, compare lanes 1 and 7) and was sensitive to endo H (Fig. 2A,
lane 8). This finding is consistent with the fact that proteins
synthesized in CHO-Lec1 cells have oligosaccharides of mannose-rich
intermediates (Man5GlcNAc2), and high mannose
type oligosaccharides in the ~95-kDa TSHR in parental CHO cells are
presumably Man8-9GlcNAc2. CHO-Lec8 cells
produced a TSHR migrating as a doublet with molecular masses of ~90
and ~95 kDa (Fig. 2A, lane 10); the ~90-kDa species is likely a TSHR with galactose- and sialic acid-deficient
oligosaccharides (GlcNAc2Man3GlcNAc2) because of its
resistance to endo H (Fig. 2A, lane 11). In CHO-Lec2 cells,
TSHR was visualized as a doublet of ~105 and ~95 kDa (Fig.
2A, lane 13). The ~105-kDa species is likely a TSHR with
sialic acid-deficient oligosaccharides
(Gal2GlcNAc2Man3GlcNAc2).
Although it is well known that TSHR cleaves into two subunits (12-14),
TSHR subunits were detectable only after longer chase and
N-glycosidase F digestion in our immunoprecipitation (27). Therefore, the subunit structures of TSHRs with different
oligosaccharides were analyzed on immunoblotting.
On immunoblotting, the single polypeptide of TSHR, for example, the
~95- and ~120-kDa species in immunoprecipitation of CHO-TSHR, were
not visualized. Instead, several nonspecific reactions were observed
between 70 and 120 kDa (Fig. 2B). However, the diffuse "A
subunit" with the mean molecular mass of ~55 kDa could be clearly detected in CHO-TSHR. The majority of TSHRs expressed on the cell surface probably cleave into two subunits at the steady state levels. A
similar diffuse band, slightly smaller than that in CHO-TSHR, was also
present in Lec2-TSHR (Fig. 2B, lane 5). The diffuse banding
pattern of TSHR A subunit in CHO and CHO-Lec2 cells may be attributable
to different degrees of glycosylation of the core polypeptides. These
oligosaccharides may contain various outer chain sequences including
different sizes of polylactosamine (22). In contrast, TSHR A subunit
was observed as a doublet in Lec1-TSHR and particularly in Lec8-TSHR
(Fig. 2B, lanes 3 and 4), in which
oligosaccharides must be homogeneous. This finding agrees with our
recent hypothesis of "two cleavage sites in TSHR" (27). Thus, these
data suggest that all of the TSHRs expressed in CHO-Lec cells can
cleave into two subunits. Cleavage seems to occur at the cell surface
and to be mediated by matrix metalloprotease(s) (31). No cleaved
product was observed in tunicamycin-treated, nonglycosylated TSHR (Fig.
2B, lane 2), suggesting that nonglycosylated TSHR may not be
expressed on the cell surface.
These data are in agreement with the expected consequences of the
mutations represented by the cell lines used.
Effect of Tunicamycin on Expression and Function of TSHR Stably
Expressed in CHO Cells--
Pooled clones of CHO cells stably
expressing TSHR were treated with 5 µg/ml tunicamycin for 3 days and
subjected to [125I]TSH binding and TSH-induced cAMP
production experiments. As shown in Fig. 2A, 5 µg/ml
tunicamycin completely inhibited transfer of dolichol precursor to Asn
in the nascent TSHR. Although effect of 3-day treatment with
tunicamycin should be evaluated with immunoblot, not
immunoprecipitation, immunoblot showed no specific signal as mentioned
above. However, this period of treatment has been previously
demonstrated to be enough to purge the cells of wild-type recombinant
LHR (9). As shown in Fig. 3A,
[125I]TSH binding determined with intact cells was of
high affinity in CHO-TSHR. Untransfected CHO cells also showed
nonspecific TSH binding of low affinity. This specific, high affinity
TSH binding was largely abolished in CHO-TSHR treated with tunicamycin.
Consistent with TSH binding data, little or no TSH-induced cAMP
production was observed in tunicamycin-treated CHO-TSHR (Fig.
3C). In addition, as shown in Fig. 3B, the
solubilized extract from tunicamycin-treated CHO-TSHR also showed no
TSH binding, although the solubilized extract from CHO-TSHR cultured in
the absence of tunicamycin demonstrated significantly higher TSH
binding than untransfected CHO cells. Thus, together with the
immunoblot data, nonglycosylated TSHR seems to accumulate
intracellularly in an unfolded form, thereby suggesting an essential
role for acquisition of oligosaccharides in correct folding and
membrane expression of TSHR.
[125I]TSH Binding and cAMP Production in CHO and
CHO-Lec Cells Stably Expressing TSHR--
As shown in Fig.
4A, [125I]TSH
binding experiments with pooled clones of cells revealed that Lec8-TSHR
bound to TSH with an affinity and a Bmax similar
to those of CHO-TSHR. In contrast, specific TSH binding was
significantly decreased in Lec1-TSHR and Lec2-TSHR. However, not only
the detergent-solubilized extracts from CHO and CHO-Lec8 cells but also
those from CHO-Lec1 and CHO-Lec2 cells demonstrated significantly
higher [125I]TSH binding than that from untransfected CHO
cells (Fig. 4B). Consistent with the TSH binding data in
intact cells, CHO-TSHR and Lec8-TSHR produced intracellular cAMP in
response to TSH stimulation to a similar extent, and Lec1-TSHR and
Lec2-TSHR showed blunted TSH-induced intracellular cAMP synthesis
(~10 and ~30% of that in CHO-TSHR, respectively) (Fig.
4C).
To further evaluate the function of TSHR expressed in CHO-Lec cells,
particularly in Lec1 and Lec2, clonal cell lines expressing the highest
levels of receptors were used. The clonal cells lines of Lec1-TSHR and
Lec2-TSHR, as well as those of CHO-TSHR and Lec8-TSHR, demonstrated
specific TSH binding, as shown in Fig.
5A. In these cells, the
binding affinity for TSH was inversely correlated with the receptor
number expressed. Thus, TSH binding affinity was highest in Lec1- and
Lec2-TSHR and lowest in CHO-TSHR (Table
I). In functional studies, however, the
EC50 for TSH-induced cAMP production was very similar in
all of the cells (Fig. 5B and Table I), thereby suggesting
that the apparently lower binding affinity for TSH in CHO-TSHR appears
to be related to its higher expression levels as previously reported
(32). Therefore, we interpret these data as that TSH binding is likely
of high affinity in TSHR expressed in all of the cells.
These data indicate that Lec8-TSHR is correctly folded and fully
expressed on the cell surface, and that Lec1-TSHR and Lec2-TSHR accomplish conformational maturation but are trapped intracellularly. However, some of these receptors can reach the cell surface and normally transduce a signal.
TSAb and TBIAb Activities in the Clonal Cell Lines Stably
Expressing TSHR--
The effect of different oligosaccharides on the
ability of TSHR to interact with anti-TSHR autoantibodies was examined.
In the TBIAb assay shown in Fig.
6A, two Graves' IgG, one with
potent TBIAb activity (Graves 1) and the other with moderate
activity (Graves 2) in CHO-TSHR, comparably inhibited
[125I]TSH binding in all of the CHO-Lec cells. TSAb
activity in two Graves' sera (Fig. 6B, Graves 3 and
Graves 4) was also similar in all of the cells. Therefore,
receptor recognition by autoantibodies does not involve complex type
oligosaccharides in TSHR.
In the present studies, using a glycosylation inhibitor,
tunicamycin, and a series of mutant CHO-Lec cells defective in the different steps of glycosylation process, we evaluated how acquisition and modification of oligosaccharides affect the functional maturation of TSHR.
Our results indicate that nonglycosylated TSHR produced by inhibition
of the first step of glycosylation with tunicamycin treatment is not
folded correctly. In contrast, high affinity TSH binding to Lec1-TSHR
clearly indicates that TSHR with mannose-rich intermediates
(Man5GlcNAc2) is conformationally intact.
Therefore, the acquisition of N-linked oligosaccharides
appears to allow the nascent, highly unfolded TSHR protein to
accomplish conformational maturation in the endoplasmic reticulum.
Several roles of addition of N-linked oligosaccharides to
nascent proteins have been demonstrated; for example,
N-linked oligosaccharides attach to lectin-like molecular chaperons such as calnexin and calreticulin, facilitating correct protein folding, and also play a role in the "quality control" system of the endoplasmic reticulum that ensures selective
transportation of the properly folded proteins for the Golgi complex
(33). Therefore, unfolded proteins such as nonglycosylated TSHR
mentioned above may be trapped in the endoplasmic reticulum.
Impaired cell surface expression in Lec1-TSHR suggests that processing
of N-linked oligosaccharides from high mannose type to
complex type in the Golgi apparatus appears to participate in cell
surface targeting of TSHR. However, efficient cell surface expression
of TSHR is not necessarily dependent on completion of complex
oligosaccharides, because TSHR with sialic acid- and galactose-deficient oligosaccharides (Lec8-TSHR) is efficiently expressed on the cell surface. Contrarily, TSHR with sialic
acid-deficient oligosaccharides (Lec2-TSHR) is not efficiently
expressed on the cell surface. We cannot explain these data at present,
although oligosaccharides terminating in galactose residues may somehow interfere with cell surface targeting of TSHR in the Golgi apparatus. Alternatively, lack of sialylation in the trans-Golgi network may
affect a role of the trans-Golgi network as an important sorting site
for different proteins destined for the apical or basolateral surfaces
of cell membranes (34). For any reason, these data are in agreement
with the facts that N-linked oligosaccharides generally play
critical roles in protein folding and intracellular trafficking in the
endoplasmic reticulum and the Golgi apparatus, respectively (33, 35).
However, our data on TSHR may not be generalized to other membrane
proteins, because (i) the membrane receptor for Newcastle disease virus
is reported not to be properly expressed in mouse mammary carcinoma
Had-1 cells deficient in UDP-galactose transporter like Lec8 cells
(36), and (ii) the expression levels of rat LHR were decreased by
30-40% of that in CHO cells in all of the CHO-Lec
cells.2
Our data also indicate that complex type oligosaccharides do not appear
to constitute TSH or autoantibody binding sites or to participate in
signal transduction, although glycosylation is reported to be related
to function in some proteins (37-39). It has recently been reported
that the truncated type of TSHR ectodomain, which is not secreted,
possesses high mannose type carbohydrates and is incapable of binding
to TSH or anti-TSHR autoantibodies in CHO cells (15). However, whether
this lack of binding was related to polypeptide misfolding or to the
type of oligosaccharides on the protein was unknown in this study. From
our results, it is now clear that misfolding is the primary event.
Thus, TSHR ectodomain by itself may not be able to accomplish correct
folding, thereby being trapped in the endoplasmic reticulum and unable
to acquire complex oligosaccharides. Recent studies also suggest that
only the TSHR ectodomain fused to a membrane-anchored peptide seems to
be correctly folded (19-21).
A nonessential role of galactose and sialic acid in function of TSHR
shown in Lec8 cells is reminiscent of our previous finding (4, 40) of
high affinity TSH binding to TSHR expressed in 293 human embryonal
kidney cells. These cells express
N-acetylgalactosaminyl-transferase and
N-acetylgalactosaminyl-N-acetylglucosaminyl-mannosyl-sulfotransferase and produce N-linked oligosaccharides terminating in sulfate
in >70% of the chains (41). Of interest is that some glycoproteins synthesized in the thyroid gland have been reported to be sulfated (42,
43). TSHR expressed in the thyroid gland may also be, at least in part,
sulfated. Furthermore, N-linked oligosaccharides in bovine
pituitary TSH that we used in the present studies are exclusively
sulfated, whereas human pituitary TSH terminates both in sulfation and
sialylation (44). Biological activity of sulfated TSH has been reported
to be more potent than sialylated TSH (45). The carbohydrates,
especially the terminal sialic acids, of TSH, have also been
demonstrated to attenuate its biological activity (45, 46). In this
regard, it will be interesting to compare the relative potency of TSH
with different types of oligosaccharides to bind and stimulate TSHR
with various oligosaccharides.
In FSH receptor, although removal of N-linked
oligosaccharide from the wild-type receptor does not affect FSH binding
(5, 7), the deglycosylated receptor produced by in vitro
mutagenesis or tunicamycin treatment shows no FSH binding activity (7). Thus, the N-linked oligosaccharides seem to be necessary for
the proper folding rather than hormone binding in FSH receptor.
However, the role of N-linked oligosaccharides in LHR is
controversial; although high affinity human chorionic gonadotropin
binding to LHR does not appear to involve the carbohydrates on LHR, it
remains unclear whether the oligosaccharides are necessary for the
correct folding of the nascent LHR (6, 8, 9).
In conclusion, our data show that acquisition and processing of
N-linked oligosaccharides in TSHR are essential for correct folding in the endoplasmic reticulum and for intracellular trafficking in the Golgi apparatus. However, binding of TSH and anti-TSHR autoantibodies to TSHR does not appear to involve the oligosaccharides of the receptor.
We thank Dr. J. P. Banga (Kings College
School of Medicine, London, UK) for providing mouse anti-TSHR
monoclonal antibodies and Dr. J. Miyazaki for the expression vector
pCAGGS. We are also grateful to Dr. B. Rapoport (Cedars-Sinai Medical
Center, Los Angeles, CA) for critical review of the manuscript.
*
This work was supported in part by Grant-in-Aid 09671064 for
General Scientific Research from the Ministry of Education, Culture, and Science of Japan (to Y. N.).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: Dept. of Pharmacology
1, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki,
852-8523, Japan. Tel.: 81-95-849-7043; Fax: 81-95-849-7044; E-mail:
nagayama{at}net.nagasaki-u.ac.jp.
The abbreviations used are:
TSHR, thyrotropin
receptor; LHR, lutropin receptor; FSH, follitropin; CHO, Chinese
hamster ovary; endo H, endoglycosidase H; PEG, polyethylene glycol; TSAb, thyroid stimulatory autoantibody; TBIAb, TSH binding inhibitory
autoantibody; TSH, thyrotropin.
2
Y. Nagayama et al., unpublished data.
Role of Asparagine-linked Oligosaccharides in Protein Folding,
Membrane Targeting, and Thyrotropin and Autoantibody Binding of the
Human Thyrotropin Receptor*
§,
,
Pharmacology 1, ¶ Nature
Medicine, and Internal Medicine 1, Nagasaki University
School of Medicine, Nagasaki, 852-8523, Japan
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
supplemented with 10%
fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). Parental CHO cells were grown in Ham's F-12 medium with 5%
fetal calf serum and antibiotics described above.
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Fig. 1.
Scheme of N-linked
oligosaccharide biosynthetic pathway in the endoplasmic reticulum and
the Golgi apparatus. The inhibition site of tunicamycin and
defects in CHO-Lec cells are shown.
(radioactivity with test IgG/radioactivity with normal IgG)] × 100 (%).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 2.
Immunoprecipitation and immunoblot of TSHR
expressed in CHO and CHO-Lec cells. A, the cells were
metabolically labeled with [35S]methionine/cysteine (2-h
pulse and 2-h chase) followed by immunoprecipitation of TSHR with
anti-TSHR monoclonal antibodies A10 and A11 (see "Materials and
Methods"). Precipitated samples were left untreated (lanes 1, 4, 7, 10, and 13) or treated with endo H (lanes
2, 5, 8, 11, and 14) or N-glycosidase F
(lanes 3, 6, 9, 12, and 15) and subjected to
7.5% SDS-polyacrylamide gel electrophoresis under reducing conditions
and autoradiography. B, crude membrane preparations
(50 µg) of the cells were subjected to 7.5% SDS-polyacrylamide gel
electrophoresis under reducing conditions. After transfer to a
membrane, proteins were probed with A10 and A11. Signals were
developed with streptoavidin-horseradish peroxidase and
3,3'-diaminobenzidine/H2O2.
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Fig. 3.
[125I]TSH binding and cAMP
response to TSH stimulation in tunicamycin-treated CHO-TSHR.
A and C, the cells treated with or without 5 µg
of tunicamycin for 3 days were subjected to [125I]TSH
binding and cAMP response assays as described under "Materials and
Methods." B, the detergent-solubilized extracts from the
cells treated with or without 5 µg of tunicamycin for 3 days were
used for [125I]TSH binding as described under
"Materials and Methods." [125I]TSH used in each
experiment was ~15,000 cpm. The data are mean ± S.E.
(n = 4) of two separate experiments determined in
duplicates. Open circles, control CHO-TSHR; open
squares, CHO-TSHR treated with tunicamycin; closed
circles, untransfected CHO. *, significantly higher than
untransfected CHO cells (Student's t test).
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Fig. 4.
[125I]TSH binding and cAMP
response to TSH stimulation in pooled clones of CHO and CHO-Lec cells
stably expressing TSHR. [125I]TSH binding
(A and B) and cAMP response (C) assays
were performed in pooled clones of the cells as described in the legend
to Fig. 3. Open circles, CHO-TSHR; closed
circles, untransfected CHO; open squares, Lec1-TSHR;
closed squares, Lec2-TSHR; open triangles,
Lec8-TSHR. *, significantly higher than untransfected CHO cells
(Student's t test).
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Fig. 5.
[125I]TSH binding and cAMP
response to TSH stimulation in clonal cell lines of CHO and CHO-Lec
cells stably expressing TSHR. [125I]TSH binding and
cAMP response assays were performed in clonal cell lines of the cells
as described in legends to Figs. 3 and 4. Open circles,
CHO-TSHR; closed circles, untransfected CHO; open
squares, Lec1-TSHR; closed squares, Lec2-TSHR;
open triangles, Lec8-TSHR.
Summary of functional properties of TSHR expressed in clonal cell lines
of CHO and CHO-Lec cells
View larger version (23K):
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Fig. 6.
TSAb and TBIAb activities in clonal cell
lines of CHO and CHO-Lec cells stably expressing TSHR. A,
the solubilized extracts from 100 µg of crude membranes were
incubated with [125I]TSH (~15,000 cpm) and a mixture of
normal sera or two Graves' sera (Graves 1 and Graves
2) for 1 h at 37 °C, precipitated with PEG, and counted as
described under "Materials and Methods." B, the cells
were stimulated with 10 
8 M bovine TSH, crude
IgG fractions from a mixture of normal sera or two Graves' sera
(Graves 3 and Graves 4) for 2 h at 37 °C.
cAMP released into the medium was determined. The data are mean ± S.E. (n = 4) of two separate experiments determined in
duplicate.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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