Association of the thyrotropin receptor with calnexin,
calreticulin and BiP
Effects on the maturation of the receptor
Sandrine Siffroi-Fernandez*, Annie Giraud, Jeanne Lanet and Jean-Louis Franc
U555 INSERM, Faculte
´
de Me
´
decine, Universite
´
de la Me
´
diterrane
´
e, Marseille, France
The thyrotropin receptor (TSHR) is a member of the G
protein-coupled receptor superfamily. It has by now been
clearly established that the maturation of the glycopro-
teins synthesized in the endoplasmic reticulum involves
interactions with molecular chaperones, which promote
the folding and assembly of the glycoproteins. In this
study, we investigated whether calnexin (CNX), calreti-
culin (CRT) and BiP, three of the main molecular chap-
erones present in the endoplasmic reticulum, interact with
the TSHR and what effects these interactions might have
on the folding of the receptor. In the first set of experi-
ments, we observed that in a K562 cell line expressing
TSHR, about 50% of the receptor synthesized was
degraded by the proteasome after ubiquitination. In order
to determine whether TSHR interact with CNX, CRT
and BiP, coimmunoprecipitation experiments were per-

maturation of the oligosaccharides in the Golgi apparatus,
the TSHR is cleaved at the cell surface by a metallopro-
tease [4]. This cleavage leads to the formation of an
extracellular A subunit (53 kDa) and a membrane span-
ning domain (B subunit) (38 kDa); the two subunits are
held together by disulfide bridges [5]. A subunit can be
shed into the extracellular space after reduction by the
protein disulfide isomerase. In transfected L-cells, only one-
third of the receptor was found to be able to reach a
mature form [6].
It is well known that the lumen of the endoplasmic
reticulum is a critical site in the process of protein
maturation. The endoplasmic reticulum contains proteins
called molecular chaperones that facilitate the folding and
prevent the aggregation of the newly synthesized protein.
Molecular chaperones interact longer with protein which is
unable to attain a normal conformation. These interactions
lead to the retention of the protein in the endoplasmic
reticulum and then to its retranslocation into the cytoplasm
and its degradation by the proteasome after ubiquitination
[7,8]. Little is known so far, however, about the interac-
tions occurring between molecular chaperones and G
protein receptors, apart from the association of the V2
vasopressin receptor with calnexin (CNX) and calreticulin
(CRT), and that of the gonadotropin receptor with these
two molecular chaperones and with GRP94 and BiP,
which have been previously described [9,10]. In the present
study, the interactions between TSHR and three of the
main endoplasmic reticulum molecular chaperones, BiP,
CNX, and CRT, were analyzed and it was attempted to

obtained from Roche Molecular Biochemicals (Le Meylan,
France); protein A-Sepharose was obtained from Zymed
Laboratories (San Francisco, CA, USA); Expre35S35S
protein labeling mix [referred to as [
35
S](Met + Cys)] was
obtained from NEN Life Science Products (Paris, France).
Calnexin rabbit polyclonal antibody (SPA-860), calreticulin
rabbit polyclonal antibody (SPA-600), BiP rabbit polyclo-
nal antibody (SPA 826), and KDEL mouse monoclonal
antibody (SPA-827) were obtained from Stressgen (Victoria,
Canada). Monoclonal anti-mouse immunoglobulin peroxi-
dase conjugate was obtained from Amersham Pharmacia
Biotech (Les Ullis, France). TSHR-pcDNA3 and TSHR-
K562 cell lines were given by S. Costagliola (Bruxelles,
Belgium) [11,12], BiP-CHO cell lines and parental cells
(DUKX) were kindly supplied by A. J. Dorner and were as
previously described [13], TSHR monoclonal antibody
(A10) was kindly supplied by P. Banga (London, UK), as
previously described [14].
Cloning of CNX and CRT and complementary DNAs
A full-length 1.8-kb cDNA coding for rabbit CRT (provi-
ded by Dr Michalak, Alberta, Canada) was cloned into the
KpnIandXbaI sites of the pcDNA3.1/Hygro expression
vector. A full-length 2.5-kb cDNA coding for dog CNX (a
gift from Dr D. Thomas, Montreal, Canada) was cloned
into the KpnIandNotI sites of the expression vector
pcDNA3.1/Hygro.
Cell culture and transfection procedure
BiP-CHO and DUKX cells were transfected with TSHR-

M
castanospermine (CST). They were then pulsed for 1 h in the
same medium supplemented with [
35
S](Met + Cys)
(66 lCiÆmL
)1
). After the pulse, the radiolabeling medium
was removed, the cells were washed twice with suitable
medium and then chased for various times in HAM’s F12 or
RPMI medium supplemented with 10% fetal bovine serum,
penicillin (100 IUÆmL
)1
), streptomycin (100 lgÆmL
)1
)with
or without the corresponding drug (MG132 or CST). When
the chase was completed, TSHR-BiP-CHO and TSHR-
DUKX cells were washed twice with 2 mL ice-cold NaCl/
P
i
, then scraped into 1 mL ice-cold NaCl/P
i
and centrifuged
for 5 min at 200 g and CNX-TSHR- K562, CRT-TSHR-
K562 and TSHR-K562 cells were centrifuged for 5 min at
200 g. All cells were resuspended in 200 lLextraction
buffer containing 1% Triton X-100, 10 m
M
Tris/HCl

buffer containing 62 m
M
Tris/HCl (pH 6.8), 2% SDS, 5%
glycerol and 5% 2-mercaptoethanol. The samples were then
subjected to SDS/PAGE (7.5%). The radioactivity was
detected and quantified using a Phosphorimager (Fudjix
BAS 1000, Japan).
Immunoblotting of CNX, CRT and BiP
TSHR-K562, TSHR-CNX-K562 and TSHR-CRT-K562
cells (2 · 10
6
cells) were centrifuged for 5 min at 200 g.
TSHR-BiP-CHO or TSHR-DUKX cells obtained from
9.6 cm
2
dishes were washed twice with 2 mL of ice-cold
NaCl/P
i
, then scraped and resuspended in 1 mL ice-cold
NaCl/P
i
and centrifuged (200 g, 5 min). The pellets were
resuspended in 100 lL buffer containing 50 m
M
Tris/HCl
(pH 7.4), 0.15
M
NaCl, 1% Triton-X100, 0.3% deoxycholic
acid, and protease inhibitor cocktail. The cells were then
tumbledfor20minat4°C (vortexing every 2 min) and

buffer containing 1% CHAPS, 10 m
M
Tris/HCl (pH 7.4),
0.15
M
NaCl, protease inhibitor cocktail, and in the case
of the BiP immunoprecipitation procedure, 25 UÆmL
)1
apyrase. After being left to stand for 1 h at 4 °C, this
preparation was centrifuged for 15 min at 10 000 g. Four
microliters of anti-CNX, anti-CRT, or anti-BiP antibodies
were added to the supernatant and after a 2-h incubation
period at 4 °C, 20 lL of protein A Sepharose were added
and the preparation was left for 1 h at 4 °C. The immune
complexes were retrieved by performing a brief centrifu-
gation at 10 000 g andwashedfourtimeswith1mLof
extraction buffer. The antigens were then eluted using
100 lL of Laemmli sample buffer and heated at 45 °C
for 30 min. After performing Western blotting on a
poly(vinylidiene difluoride) membrane, the nonspecific
sites were blocked as described above and the TSHR
coimmunoprecipitated with molecular chaperones was
revealed using mAb A10 labeled with horseradish peroxi-
dase along with the Zenon antibody labeling kit
(Molecular Probes).
RESULTS
Synthesis, maturation and degradation of TSHR
at the endoplasmic reticulum level
To study the maturation of TSHR after its synthesis, K562
cells stably transfected with human TSHR [11] were used.

After the pulse, proteasome inhibition significantly
increased the amount of TSHR bearing high mannose
type structures (Fig. 2A–C). As can be seen in Fig. 2A
and B, in the presence of the proteasome inhibitor, there
was an increase in apparition of high molecular weight
bands forming a regularly spaced ladder. This was
0h 1h 5h 22h 48h
TSHR bearing complex type
structures
TSHR bearing high mannose
type structures
Subunit A
50403020
Time (h)
10 20 30 40 50 0
0
50
100
150
% of 35S TSHR immunoprecipitated
A
B
Fig. 1. Synthesis and maturation of the TSHR in K562 cells. Two
million cells were preincubated for 2 h in 1 mL of cysteine- and
methionine-free DMEM supplemented with 10% dialyzed fetal bovine
serum, then pulse labeled for 1 h with 66 lCi of [
35
S](Met + Cys).
Cells were then chased for the times indicated in RPMI medium with
10% fetal bovine serum. After the chase, cells were centrifuged for

chaperone/substrate complexes: CHAPS was used as the
detergent and a reduction of ATP level was obtained by
adding apyrase during the BiP immunoprecipitation
procedure. A negative control was also carried out using
nonimmune rabbit serum. After the immunoprecipitation
step, the complex was dissociated and the TSHR detected
after performing Western blotting using mAb A10. A
band corresponding to the molecular mass of the imma-
ture form of the receptor was observed in the lanes
corresponding to the immunoprecipitation with antimo-
lecular chaperone antibody but practically not in the
negative control material (Fig. 3). It should be noted that
a greater amount of receptor was coimmunoprecipitated
with CNX and CRT than with BiP.
Effects of interactions between TSHR and CNX
and CRT
We then attempted to determine the effects of these
interactions on the maturation of the receptor. CNX is an
integral membrane protein, and CRT, a soluble luminal
protein. Both are present in the endoplasmic reticulum and
bind to monoglucosylated glycoproteins. CNX and CRT
have been described as being necessary to the folding and
oligomeric assembly of various glycoproteins [17].
To study the role of CNX and CRT in the folding of
newly synthesized TSHR molecules, we performed pulse-
chase analyses on TSHR-K562 cells treated with and
without 1 m
M
of CST, which is known to inhibit the
trimming of the three glucoses from the core oligosaccha-

0
Time (h)
35S-TSHR immunoprecipitated (arbitrary units)
D
Fig. 2. Synthesis, maturation and degradation of TSHR in the presence
or absence of MG132. Pulse-chase analysis was carried out as described
in Fig. 1. Cells were preincubated in 1 mL cysteine- and methionine-
free DMEM with (B, d)orwithout(A,s)100l
M
MG132, then
pulsed with [
35
S](Met + Cys) and chased in medium either supple-
mented or not with MG132. Samples were analyzed by SDS/PAGE
after a reduction step (A, B) and quantified by performing phos-
phorimaging (C, D). (C) TSHR bearing high mannose and complex-
type structures; (D) TSHR bearing multiple ubiquitin molecules. The
experiment was repeated three times and similar results were obtained
in each case.
1 2 3 4
97 kDa
Fig. 3. Detection of TSHR after the coimmunoprecipitation procedure
using anti-CNX, anti-CRT and anti-BiP antibodies. Extracts of TSHR-
K562 cells were subjected to immunoprecipitation using anti-CNX
(lane 2), anti-CRT (lane 3), and anti-BiP (lane 4) antibodies. Non-
immune precipitation was performed using nonimmune rabbit serum
(lane 1). After SDS/PAGE and Western blotting, the TSHR coim-
munoprecipitated was revealed using mAb A10 coupled to horseradish
peroxidase.
Ó FEBS 2002 TSH receptor and molecular chaperones (Eur. J. Biochem. 269) 4933

and intermolecular aggregation [17]. To investigate the
role of BiP in the TSHR folding process, we used a CHO
cell line overexpressing this molecular chaperone [13]. It
was observed after performing Western blotting using
antibodies directed against BiP that these cells expressed
five times more Bip than the parental cells (data not
shown). These two cell lines were transiently transfected
with the TSHR-pcDNA3. Forty-eight hours later, these
two cell lines were pulse-chased using [
35
S] (Met + Cys).
The TSHR was immunoprecipitated with the mAb A10
and analyzed by SDS/PAGE. At the end of the pulse,
approximately the same quantity of high mannose-type
structure was recovered in the two cell lines. During the
chase, the TSHR bearing high mannose-type structure
disappeared more rapidly in the cell overexpressing BiP.
This decrease ranged between 20 and 50%, depending on
the chase time and the experiments. The formation of
TSHR bearing complex-type structures also decreased in
Time (h)
35S-TSHR immunoprecipitated (arbitrary units)
5 10 15 20 0
50
100
150
0
Fig. 4. Effects of castanospermine on the degradation rate of TSHR.
Pulse-chase analysis was performed as described in Fig. 1. Cells were
preincubated, incubated with [

and d, m) or with pcDNA3.1/Hygro alone (A, B and, s, n). After
48 h, pulse-chase analysis was performed as described in Fig. 1. s and
d, TSHR bearing high mannose type structures; n and m,TSHR
bearing complex-type structures plus A subunit. The maximum
intensity of TSHR band recorded in the control assay was taken to be
100%. The experiment was repeated four times and similar results were
obtainedineachcase.
4934 S. Siffroi-Fernandez et al. (Eur. J. Biochem. 269) Ó FEBS 2002
these cells (Fig. 6) by approximately 20%. This indicates
that BiP increased the degradation of TSHR at the
endoplasmic reticulum level. These data suggest that BiP
has a negative effect on the folding of TSHR. The
interaction of this receptor with BiP leads to an increase in
the degradation of TSHR and also to a decrease in the
amount of TSHR able to reach the Golgi apparatus,
where the complex-type structures are acquired.
DISCUSSION
The aim of this study was to analyze the mechanism
involved in the folding and degradation of newly synthe-
sized TSHR at the endoplasmic reticulum level. In the first
set of experiments, we observed that in the newly synthes-
ized TSHR-K562 cell line, about 50% of the TSHR were
able to acquire the complex mature structure. The remain-
ing 50% of the receptors, which were not able to enter the
maturation pathway were certainly degraded by the
proteasome after being retanslocated into the cytosol
(Fig. 2). Similar results were obtained using a CHO cell
line (DUKX) transfected with the TSHR (Fig. 6) or with a
recombinant GPI-anchored TSHR extracellular domain
[19] (data not shown). This finding is in agreement with the

and/or CRT stabilize the receptor and slow down its
degradation. But these interactions do not seem to be
essential to the final folding of the receptor, because in
cells overexpressing CNX or CRT, the same quantity of
TSHR was able to reach the Golgi apparatus as in the
control cells.
The molecular chaperone BiP interacts with a wide
variety of unrelated nascent polypeptides. These peptides
usually show a high degree of hydrophobicity, which is
consistent with the likelihood that BiP interacts with
sequences normally located within the completely folded
protein. It has also been established that BiP binds to
misfolded proteins and may mediate their retrograde
translocation prior to proteasome degradation [28,29]. In
order to study the potential role of BiP in the folding of
TSHR, we used a CHO cell line overexpressing higher levels
of BiP than those obtained by stress induction [13]. In these
cells, larger amounts of the newly synthesized TSHR are
degraded than in the parent cells. In addition, the fact that a
smaller proportion of the TSHR is able to reach the Golgi
apparatus indicates that interactions between TSHR and
BiP do not contribute positively to the proper folding of the
receptor.
Molinari and Helenius [30], using Semliki forest virus
and influenza hemagglutinin expressed in CHO cells,
observed that during the protein synthesis, direct interac-
tions with CNX and CRT occur if glycans are present
within about 50 residues from the protein NH
2
-terminus.

mannose type structures; n, m, TSHR bearing high-mannose type
structures plus A subunit. The experiment was repeated three times
and similar results were obtained in each case.
Ó FEBS 2002 TSH receptor and molecular chaperones (Eur. J. Biochem. 269) 4935
Other molecular chaperones and folding catalysts are
certainly required for the receptor to be able to fold
properly. For example, GRP94 associates with advanced
folding intermediates [31,32], and cytoplasmic chaperones
such as Hsp70 or Hsp90 can interact with the polypeptide
chains during their synthesis and can bind to the cytoplas-
mic parts of transmembrane proteins [33]. Further research
is now required to determine which other molecular
chaperones participate in the folding of TSHR and how
exactly these various molecules contribute to the folding or
degradation of the receptor.
ACKNOWLEDGEMENTS
S. Siffroi-Fernandez was supported by Association pour le Develop-
pement des Recherches Biologiques et Medicales and by Association
pour la Recherche contre le Cancer. We thank G. Vassart and
S. Costagliola for kindly providing the TSHR-pcDNA3 and TSHR-
K562cellline,P.BangaforthemAbA10,M.MichalakfortheCRT
cDNA, D.Y. Thomas for CNX cDNA and A.J. Dorner for Bip-CHO
and DUKX cells.
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