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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 295:404–409, 2000
Vol. 295, No. 1
2605/853846
Printed in U.S.A.
Identification of the Sites of Asparagine-Linked Glycosylation
on the Human Thyrotropin Receptor and Studies on Their Role
in Receptor Function and Expression
YUJI NAGAYAMA, EIJUN NISHIHARA, HIROYUKI NAMBA, SHUNICHI YAMASHITA, and MASAMI NIWA
Departments of Pharmacology 1 (Y.N., M.N.) and Nature Medicine (E.N., H.N., S.Y.), Nagasaki University School of Medicine, Nagasaki, Japan
Accepted for publication June 26, 2000
This paper is available online at http://www.jpet.org
The thyrotropin receptor (TSHR) is physiologically the primary regulator of differentiated function and proliferation of
thyroid follicular epithelial cells and pathogenetically a target for the immune system in autoimmune thyroid diseases
such as Graves’ disease and Hashimoto thyroiditis. This receptor as well as the receptors for lutropin (LHR) and follitropin (FSHR) comprise a unique subfamily of a G proteincoupled receptor superfamily, characterized by a large
amino-terminal ectodomain (350 – 400 amino acid residues),
which is thought to be the high-affinity hormone-binding site
(Segaloff and Ascoli, 1993; Rapoport et al., 1998).
The ectodomain of TSHR is heavily glycosylated with asparagine N-linked oligosaccharides that represent 30 to 40%
of its molecular weight (Rapoport et al., 1996). The functional
importance of oligosaccharides in TSHR function and expression has been shown by our previous studies with in vitro
site-directed mutagenesis (Russo et al., 1991) and tunicamycin treatment (Nagayama et al., 1998). Thus, inhibition of
N-linked glycosylation blocks cell surface expression of the
functional TSHR. We have also demonstrated that the addition and processing of oligosaccharides in the endoplasmic
Received for publication February 16, 2000.
indicate that our previous data appear to result from amino acid
substitution itself, not from disruption of glycosylation. The next
series of the mutants was therefore constructed to identify at
least how many glycosylation sites are necessary. Neither TSH
binding nor cAMP response was detected in TSHR mutants
with three glycosylation sites. However, the mutants with four
glycosylation sites were fully functional in terms of TSH binding
and cAMP production, although the expression levels were 30
to 40% of that in wild-type TSHR. Finally, Western blot revealed
that all six glycosylation sites are actually glycosylated. These
data indicate that 1) TSHR ectodomain contains six N-linked
carbohydrates, and 2) glycosylation of at least four sites appears necessary for expression of the functional TSHR.
reticulum (ER) and the Golgi apparatus are crucial for protein folding and intracellular trafficking, respectively (Nagayama et al., 1998).
There are six potential N-linked glycosylation sites (the
consensus sequence is Asn-Xaa-Ser/Thr for glycosylation on
an asparagine residue, where Xaa is any amino acid except
Pro) (Kornfeld and Kornfeld, 1985) at amino acid positions
Asn-77, -99, -113, -177, -198, and -302 of human TSHR
ectodomain (Fig. 1). Among them, five sites are highly conserved in human, dog, mouse, bovine, and rat TSHR (Nagayama et al., 1989; Parmentier et al., 1989; Akamizu et al.,
1990; Stein et al., 1994; Silversides et al., 1997). Asn-113 is
an exception and unique in human TSHR. In our previous
study (Russo et al., 1991), an amino acid substitution of Asn
to Gln at the first or third glycosylation sites (amino acids 77
and 113, respectively) of human TSHR disrupted TSH binding and TSH-stimulated cAMP synthesis. Although these
data suggested that these two sites appeared important for
cell surface expression of the functional TSH, the possibility
cannot be excluded that the amino acid substitution itself
rather than disruption of glycosylation might affect the receptor function in these studies. Furthermore, the precise
number and location of N-linked carbohydrates have not yet
ABBREVIATIONS: TSHR, thyrotropin receptor; LHR, lutropin receptor; FSHR, follitropin receptor; ER, endoplasmic reticulum; CHO, Chinese
hamster ovary; wt, wild-type.
404
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ABSTRACT
The amino-terminal ectodomain of human thyrotropin receptor
(TSHR) contains six potential N-linked glycosylation sites (NXaa-S/T). This study was designed to evaluate the functional
role of TSHR carbohydrates in detail. Because our previous
mutagenesis study by Asn to Gln substitutions suggested the
critical role of the first and third glycosylation sites (amino acids
77 and 113) for expression of the functional TSHR, we first
constructed TSHR mutants having these two glycosylation
sites to elucidate whether these two sites are sufficient for
TSHR function and expression; this mutant however proved to
be nonfunctional. Also the expression levels and function of
TSHR mutants with a Ser/Thr to Ala substitution at the first or
third glycosylation site were found to be intact. These data
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N-Linked Glycosylation of Thyrotropin Receptor
405
Fig. 1. Schematic representations of the mutant
TSHRs in our previous (A) and present studies (B)
and summary of their function. Data on the mutants
in A are from Russo et al. (1991). ⫻ indicates the
mutated site. Receptor number on cell surface is
expressed by ⫹/⫺ approximations.
Materials and Methods
Cell Lines. Chinese hamster ovary (CHO) cells were grown at 5%
CO2 at 37°C in Ham’s F-12 medium with 5% fetal calf serum, penicillin
(100 U/ml), and streptomycin (100 ␮g/ml). CHO-Lec8 (ATCC CRL1737) cells, which lack UDP-galactose translocases and are incapable of
transporting UDP-galactose from the cytosol to the Golgi, thereby producing sialic acid/galactose-deficient (GlcNAc2Man3GlcNAc2 compared
with Sia2Gal2GlcNAc2Man3GlcNAc2 in CHO cells) oligosaccharides
(Stanley and Siminovitch, 1977), were maintained in minimal essential
medium supplemented with 10% fetal calf serum and antibiotics as
described above.
Construction and Expression of Mutant TSHRs. TSHRS79A
and TSHRT115A cDNAs (Fig. 1) were constructed by site-directed
mutagenesis with overlapping extension method by polymerase
chain reaction using wild-type (wt) TSHR cDNA (Nagayama et al.,
1989) as a template, and TSHRS79A/N99Q/T115A cDNA was constructed using TSHRS79/T115A cDNA (see below) as a template.
The mutations and adjacent regions were confirmed by automated
DNA sequencing (Hitachi SQ-5500DNA sequencer; Hitachi Electronics Engineering Co., Tokyo, Japan). TSHRN99/177/198/302Q cDNA
was constructed by combining the EcoRI/Aat II-fragment of
TSHRN99Q cDNA (Russo et al., 1991) (Fig. 1) and the AaT II/XbaIfragment of TSHR6xQ cDNA. TSHRN99/177/198Q cDNA was from
the EcoRI/Bfr I-fragment of TSHRN99/177/198/302Q cDNA and Bfr
I/XbaI-fragment of wt TSHR cDNA. TSHRN77/99/113Q cDNA was
from the EcoRI/AaT II-fragment of wt TSHR cDNA and the AaT
II/XbaI-fragment of TSHR6xQ cDNA. TSHRN99/302Q cDNA was
from the EcoRI/Bfr I-fragment of TSHRN99Q cDNA and the Bfr
I/XbaI-fragment of TSHRN302Q cDNA. TSHRN177/198Q cDNA was
from the EcoRI/AaT II-fragment of wt TSHR cDNA and the AaT
II/XbaI-fragment ofTSHRN99/177/198Q cDNA. TSHRS79/T115A
cDNA was from the EcoRI/SnaB I-fragment of TSHRS79A cDNA and
the SnaB I/XbaI-fragment of TSHRT115A cDNA. TSHRS79/T115A/
N302Q cDNA was from the EcoRI/AaT II-fragment of TSHRS79/
T115A cDNA and the AaT II/XbaI-fragment of TSHRN302Q cDNA.
These mutant TSHR cDNAs as well as the mutant TSHR cDNAs
with a single glycosylation site disrupted (Russo et al., 1991) (Fig. 1)
were ligated into the eukaryotic expression vector pCAGGS (Nagayama et al., 1998) in which expression of the mutant TSHRs is
controlled by the constitutive CAG promoter.
These expression plasmids as well as pCAG-TSHR expressing wt
TSHR (Nagayama et al., 1998) were transfected together with
pSV2neo into the cells with Lipofectin reagent (Life Technologies
Inc., Grand Island, NY). The cells were selected with 500 ␮g/ml G418
(Geneticin; Wako, Osaka, Japan). Pooled clones of CHO cells were
used for 125I-TSH binding and cAMP measurements (see below).
Surviving clones of CHO-Lec8 cells (⬃12 clones for each transfection)
were also isolated with cloning cylinders, and the clonal cell lines
expressing the highest levels of the receptor, determined by 125I-TSH
binding, were selected.
125
I-TSH Binding and cAMP Measurement. 125I-TSH binding
to intact cells and intracellular cAMP measurement were performed
with 125I-bovine TSH (TRAb kit; Cosmic, Tokyo, Japan) and with a
cAMP radioimmunoassay kit (Yamasa, Tokyo, Japan), respectively,
as previously described (Nagayama et al., 1998). Unlabeled TSH
used in TSH-binding study was of bovine origin (Sigma, St. Louis,
MO).
Western Blot Analysis. Extraction of the crude cell membrane
and immunoblotting were performed as previously described (Nagayama et al., 1998) with mouse anti-human TSHR monoclonal
antibody A11 (Nicholson et al., 1996).
Results
As mentioned above, we have previously shown that disruption of the first or third glycosylation sites (amino acids 77
and 113) by an Asn to Gln substitution impaired cell surface
expression of the functional human TSHR (Russo et al.,
1991). From these data we expected that two oligosaccharides at amino acids 77 and 113 might be sufficient for cell
surface expression of the functional TSHR. To explore this
possibility, we constructed the mutant TSHR harboring only
these two glycosylation sites, TSHRN99/177/198/302Q.
Somewhat unexpectedly, neither TSH binding nor cAMP response to TSH stimulation was observed in pooled clones of
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been determined, except that the sixth site has recently been
shown to be actually glycosylated in human TSHR (Tanaka
et al., 1998).
The present study was therefore designed to study further
the role of N-linked carbohydrates in human TSHR; we constructed new TSHR mutants that had either a Ser/Thr to Ala
substitution at amino acids 79 and 115 instead of an Asn to
Gln substitution at amino acids 77 and 113, or a different
number of glycosylation sites. We show herein that 1) our
previous data obtained from an Asn to Gln substitution at the
first and third glycosylation sites seem to result from an
amino acid substitution itself, not from disruption of glycosylation; 2) all six glycosylation sites are actually glycosylated; and 3) glycosylation of at least four sites appears
critical for cell surface expression of the functional receptor.
406
Nagayama et al.
Fig. 2. TSH binding and cAMP response to TSH stimulation in pooled
clones of CHO cells stably expressing wt and mutant TSHRs (N99/177/
198/302Q, S79A, and T115A). 125I-TSH binding and cAMP response to
TSH stimulation were performed in pooled clones of CHO cells stably
expressing wt and mutant TSHRs (N99/177/198/302Q, S79A, and T115A)
as described under Materials and Methods. 125I-TSH used in each experiments was ⬃12,000 cpm. The data are mean ⫾ S.E. (n ⫽ 4) of two
separate experiments determined in duplicates. E, wt TSH; F, untransfected CHO cells; 䡺, TSHRS79A; f, TSHRT115A; ‚, TSHR N99/177/198/
302Q.
single glycosylation site disrupted is slightly but reproducibly increased compared with that of wt TSHR (Fig. 3, lane 3
versus lanes 4 –9), indicating that all six sites are actually
glycosylated in the context of the native receptor expressed in
CHO-Lec8 cells.
From these data together with our previous study (Russo
et al., 1991), it is now clear that TSHR ectodomain has six
N-linked oligosaccharides, and that disruption of N-linked
glycosylation at any single site has little effect on the receptor function and expression. These results suggest the quantitative rather than qualitative (location-specific) importance
of oligosaccharides in TSHR.
To identify at least how many glycosylation sites are necessary for cell surface expression of the functional TSHR, the
next series of mutant TSHRs with three or four N-linked
glycosylation sites was constructed. TSH binding or TSHinduced cAMP production was not detected in TSHR mutants
with three glycosylation sites, TSHRN99/177/198Q,
TSHRN177/198/302Q,
TSHRS79A/N99Q/T115A,
and
TSHRS79/T115A/N302Q (Figs. 1 and 4; Table 1). However, in
TSHR mutants with four glycosylation sites, TSHRN99/
302Q, TSHRN177/198Q, and TSHRS79/115A, TSH binding
and cAMP production were clearly observed; TSH-binding
affinity and the EC50 for cAMP response were indistinguishable from those in wt TSHR, whereas the expression levels of
these mutants were 30 to 40% of that in wt TSHR (Figs. 1 and
4; Table 1). These data indicate that glycosylation of at least
four sites appears necessary for cell surface expression of the
functional TSHR.
Discussion
In this article, we show several new findings with respect
to the functional role of N-linked carbohydrates on human
TSHR. First, our results obtained with Ser/Thr to Ala substitutions revealed that interpretation of our previous data
showing impairment of cell surface expression of the functional TSHR by disruption of the first or third glycosylation
site (Russo et al., 1991) are likely incorrect. An Asn to Gln
substitution at these two sites might have altered the conformation of TSHR. Similar data have been described for
FSHR (Davis et al., 1995). Therefore, our previous (Russo et
al., 1991) and present results show that disruption of a single
N-linked glycosylation site has little effect on TSHR expression and function.
We also demonstrated that all six potential N-linked glycosylation sites on TSHR ectodomain are actually glycosylated in CHO-Lec8 cells. However, it is uncertain that TSHR
endogenously expressed in thyroid cells also contains six
N-linked carbohydrates. It has been reported that the number of the actual glycosylation sites in LHR is different in the
distinct cell types used (Zhang et al., 1995; Davis et al., 1997).
We next showed that although wt TSHR and TSHR mutants with a single glycosylation site disrupted show normal
cell surface expression, elimination of two or more glycosylation sites causes progressive reduction in cell surface expression, i.e., an increasing number of mutated glycosylation
sites is associated with decreasing surface expression of the
receptor. These data together with our previous report
(Russo et al., 1991) indicate that the number of sites glycosylated rather than specific sites of glycosylation seems to
determine the efficiency of cell surface expression of TSHR.
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CHO cells stably transfected with TSHRN99/177/198/302Q
(Figs. 1 and 2; Table 1).
These data suggest either that 1) N-linked oligosaccharides at amino acids 77 and 113 are not important for TSHR
function, i.e., our previous data resulted from amino acid
substitutions introduced per se but not from disruption of
glycosylation, or 2) although interpretation of our previous
data was correct, TSHR cannot tolerate multiple mutations
at glycosylation sites. That is, these two sites are critical, but
not sufficient, for cell surface expression of TSHR.
To distinguish these two possibilities, the TSHR mutants
TSHRS79A and TSHRT115A were next constructed in which
a Ser/Thr to Ala substitution was introduced at amino acids
79 or 115 instead of an Asn to Gln substitution at residues 77
or 113 to disrupt the first and third glycosylation sites. The
expression levels and function of TSHRS79A and
TSHRT115A stably expressed in CHO cells were indistinguishable from those of wt TSHR in CHO cells (Figs. 1 and 2;
Table 1). These data strongly indicate that the former possibility is correct. Thus, an amino acid substitution at amino
acid 77 or 113 disrupted the receptor structure.
It is so far unknown how many potential N-linked glycosylation sites actually have oligosaccharides in TSHR. Western blot analysis was therefore performed to address this
issue. TSHR is well known to cleave into two subunits (Buckland et al., 1982; Loosfelt et al., 1992; Rapoport et al., 1998),
and the anti-TSHR monoclonal antibody we used (A11) recognizes the A-subunit (Nicholson et al., 1996). We first found
it difficult to detect a size difference between TSHR with six
and five oligosaccharides expressed in CHO cells (data not
shown). This is presumably because TSHR A-subunit has
highly heterogenous carbohydrates and is detected as a
“broad” band in Western blot as previously described (Fig. 3,
lane 2) (Nagayama et al., 1998). Therefore, TSHRs were
expressed in CHO-Lec8 cells in which oligosaccharides are
sialic acid/galactose deficient. In these cells TSHR A-subunit
is homogenous and can be detected as a “sharp” band by
Western blot (Nagayama et al., 1998). Western blot analysis
with the clonal cell lines stably expressing the highest levels
of each mutant as well as wt TSHR clearly showed that the
mobility of immunoreactive bands of all the mutants with a
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2000
N-Linked Glycosylation of Thyrotropin Receptor
407
TABLE 1
Summary of TSH binding and TSH-induced cAMP production in pooled clones of CHO cells stably expressing wt and mutant TSHRs
Receptors
No. of Glycosylation Sites
Receptor No. (/Cell)
Kd for TSH Binding (nM)
EC50 for cAMP (nM)
wt
N99/177/198/302Q
N99/177/198Q
N177/198/302Q
S79A/N99Q/T115A
S79/T115A/N302Q
N99/302Q
N177/198Q
S79/T115A
S79A
T115A
6
2
3
3
3
3
4
4
4
5
5
48,100 (100%)a,b
N.D.
N.D.
N.D.
N.D.
N.D.
15,300 (31.8)
20,200 (42.0)
18,300 (38.0)
45,600 (94.8)
39,200 (81.5)
0.36
N.D.
N.D.
N.D.
N.D.
N.D.
0.30
0.32
0.33
0.38
0.40
3.4
N.D.
N.D.
N.D.
N.D.
N.D.
3.0
3.5
2.8
3.3
3.3
N.D., not detectable.
a
The mean of two separate experiments, each experiment determined in duplicate. Relative expression levels compared to wt TSHR are shown in parentheses.
b
Approximately equal to 39 fmol/mg of protein.
Fig. 4. TSH binding and cAMP response to TSH stimulation in pooled
clones of CHO cells stably expressing wt and mutant TSHRs (N99/177/
198Q, N177/198/302Q, N99/302Q, N177/198Q, and S79/T115A). 125I-TSH
binding and cAMP response to TSH stimulation were performed in pooled
clones of CHO cells stably expressing wt and mutant TSHRs (N99/177/
198Q, N177/198/302Q, N99/302Q, N177/198Q, and S79/T115A) as described in legend to Fig. 2. The data are mean ⫾ S.E. (n ⫽ 4) of two
separate experiments determined in duplicates. E, wt TSH; F, untransfected CHO; 䡺, TSHRN99/177/198Q; f, TSHRN177/198/302Q; ‚,
TSHRS79A/N99Q/T115A; Œ, TSHRS79/T115A/N302Q; 〫, TSHRN177/
198Q; ⽧, TSHRN99/302Q; ⫻, TSHRS79/T115A.
However, it should be noted that the possibility cannot be
excluded that the combined effect of three or more simultaneous amino acid substitutions at the glycosylation sites may
be structurally deleterious, although a single amino acid
substitution is tolerable.
We have recently shown that TSHR is first synthesized as
a ⬃84-kDa polypeptide chain to which high mannose type
carbohydrates attach in the ER that are processed to mature,
complex type carbohydrates in the Golgi apparatus (Rapoport et al., 1996; Nagayama et al., 1998). The mature receptor with complex type carbohydrates (⬃120 kDa) is then
cleaved into two subunits (A and B) on cell surface (Misrahi
et al., 1994; Rapoport et al., 1998). (A-subunit is ⬃55 kDa in
CHO cells and ⬃43 kDa in CHO-Lec8 cells; Fig. 3.) Because
one of the functional roles of carbohydrates on membrane
proteins in the ER is to attach to lectin-like molecular chaperones such as calnexin and calreticulin, which facilitate
correct protein folding (Helenius, 1994), we speculate that
four or more carbohydrate chains on each TSHR polypeptide
may be quantitatively necessary to bind enough amounts of
molecular chaperones required for TSHR to fold correctly in
the ER. In contrast, the mutant TSHRs with three or less
carbohydrates may not fold correctly and may be trapped in
the ER by the “quality control” system, a function of the ER
that ensures selective transportation of the properly folded
proteins from the ER to the Golgi.
Decreased number of carbohydrate moieties seems to affect
cell surface expression of TSHR but not to impair TSHR
function in terms of TSH-binding affinity and the EC50 for
cAMP response in our present study. It has also recently been
demonstrated that deglycosylation of native TSHR with PNGase F treatment does not affect autoantibody binding to
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Fig. 3. Western blot analysis of wt and
the mutant TSHRs stably expressed in
CHO and CHO-Lec8 cells. Crude membrane preparations (40 ␮g) of the cells
were subjected to 7.5% SDS-polyacrylamide gel electrophoresis under reducing
condition. After transfer to a membrane,
proteins were probed with anti-TSHR
monoclonal antibody A11. Lane 1, untransfected CHO cells; lane 2, wt TSHR in
CHO cells; lanes 3 to 9, wt TSHR,
TSHRS79A, -N99Q, -T115A, -N177Q,
-N198Q, and -N302Q in CHO-Lec8 cells,
respectively. The arrows on the right indicate TSHR A-subunits with six and five
carbohydrates in CHO-Lec8 cells.
408
Nagayama et al.
Acknowledgments
We thank Dr. J. P. Banga (Kings College School of Medicine,
London) for providing mouse anti-TSHR monoclonal antibody A11,
and Prof. Basil Rapoport (Cedars-Sinai Medical Center, Los Angeles,
CA) for the cDNAs for the mutant TSHRs and also for the critical
review of the manuscript.
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Rapoport B, McLachlan SM, Kakinuma A and Chazenbalk GD (1996) Critical relationship between autoantibody recognition and thyrotropin receptor maturation as
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Ray K, Clapp P, Goldsmith PK and Spiegel AM (1998) Identification of the sites of
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Rozell TG, Davis DP, Chai Y and Segaloff DL (1998) Association of gonadotropin
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TSHR (Atger et al., 1999), although the efficacy of PNGase F
treatment has not been verified. Thus, it is plausible that the
carbohydrates may not be part of ligand-binding site in
TSHR and may not be necessary to maintain the threedimensional structure of TSHR after the completion of correct folding.
In receptors structurally and functionally related to TSHR,
the similar results have been demonstrated for FSHR. Thus,
FSHR is glycosylated on two of three potential N-linked
glycosylation sites and glycosylation of at least one site is
necessary for cell surface expression of the functional FSH,
whereas deglycosylation of native FSHR does not impair
FSH binding (Davis et al., 1995). In contrast, the number of
actual glycosylation sites and functional role of carbohydrates are controversial in LHR (Zhang et al., 1995; Davis et
al., 1997). However, for both receptors binding of calnexin to
receptor with high mannose type carbohydrates has been
demonstrated (Rozell et al., 1998).
The functional role of N-linked carbohydrates varies
among other members of the G protein-coupled receptor superfamily. Studies with in vitro site-directed mutagenesis or
with tunicamycin treatment have revealed that impaired
glycosylation does (Rands et al., 1990; Goke et al., 1994;
Kaushal et al., 1994; Davidson et al., 1995; Garcia Rodriguez
et al., 1995; Couvineau et al., 1996; Ray et al., 1998; Walsh et
al., 1998; Ho et al., 1999; Jayadev et al., 1999; Pang et al.,
1999) or does not (van Koppen and Nathanson, 1990; Fukushima et al., 1995; Unson et al., 1995; Bisello et al., 1996;
Innamorati et al., 1996; Kimura et al., 1997) affect receptor
expression and/or function. For most receptors in the former
group, impaired glycosylation is associated with reduced cell
surface expression of otherwise normal receptors (Rands et
al., 1990; Goke et al., 1994; Kaushal et al., 1994; Davidson et
al., 1995; Garcia Rodriguez et al., 1995; Ray et al., 1998;
Walsh et al., 1998; Jayadev et al., 1999), as for TSHR. Exceptions are the receptors for vasoactive intestinal peptide,
secretin and calcitonin, in all of which significance of specific
sites for N-linked glycosylation is reported (Couvineau et al.,
1996; Ho et al., 1999; Pang et al., 1999). Furthermore, deglycosylation causes a decrease in ligand binding, not cell
surface expression, of secretin and calcitonin receptors (Ho et
al., 1999; Pang et al., 1999). At present it seems difficult to
predict the role of N-linked glycosylation in receptor function
from the primary amino acid sequences.
In summary, we demonstrate herein that all six potential
N-linked glycosylation sites on TSHR ectodomain are actually glycosylated, and that glycosylation of at least four sites
appears necessary for cell surface expression of the functional TSHR.
Vol. 295
2000
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Send reprint requests to: Dr. Yuji Nagayama, M.D., Department of Pharmacology 1, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. E-mail: [email protected]
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