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 t
Trang 1Association 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-formed TSHR was found to be associated with all three
molecular chaperones To study the role of the
inter-actions between CNX and CRT and the TSHR, we used
castanospermine, a glucosidase I and II inhibitor that blocks the interactions between these chaperones and glycoproteins In K562 cells expressing the TSHR, these drugs led to a faster degradation of the receptor, which indicates that these interactions contribute to stabilizing the receptor after its synthesis The overexpression of calnexin and calreticulin in these cells stabilizes the receptor during the first hour after its synthesis, whereas the degradation of TSHR increased in a cell line over-expressing BiP and the quantity of TSHR able to acquire complex type oligosaccharides decreased These results show that calnexin, calreticulin and BiP all interact with TSHR and that the choice made between these two chaperone systems is crucial because each of them has distinct effects on the folding and stability of this receptor
at the endoplasmic reticulum level
Keywords: thyrotropin receptor, molecular chaperones, BiP, calnexine, calreticuline, degradation
The thyrotropin receptor (TSHR) belongs to the G protein
coupled receptor family, which share a common structure
of seven transmembrane domains [1–3] Human TSHR is a
glycoprotein consisting of 764 amino acids residues
inclu-ding a 20 amino acid signal peptide It has a large
extracellular domain consisting of 398 residues, containing
six potential N-glycosylation sites After being synthesized,
the TSHR, like the other transmembrane N-glycoproteins,
is N-glycosylated in the endoplasmic reticulum After the
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 determine the effects of these interactions on the matur-ation and degradmatur-ation of the receptor at the endoplasmic reticulum level
27 Bd J Moulin, 13385 Marseille cedex 5, France.
E-mail: jean-louis.franc@medecine.univ-mrs.fr
Abbreviations: CNX, calnexin; CRT, calreticulin; CST,
castano-spermine; TSHR, thyrotropin receptor.
*Present address: INSERM-Universite´ Louis Pasteur E9918,
Centre Hospitalier Universitaire Re´gional, 1 Place de l’Hoˆpital,
67091 Strasbourg cedex, France.
(Received 12 April 2002, revised 22 July 2002,
accepted 20 August 2002)
Trang 2M A T E R I A L S A N D M E T H O D S
Materials
The following materials were supplied by Sigma: MG132,
monoclonal anti-rabbit immunoblobulin peroxidase
conju-gate; Life Technologies, Inc (Grand Island, NY, USA),
provided LipofectAMINE PLUS reagent, penicillin and
streptomycin; castanospermine was obtained from Alexis
(San Diego, CA, USA); protease inhibitor cocktail was
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 [35S](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 monoclopolyclo-nal
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) 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
KpnI and XbaI 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 KpnI and NotI sites of the expression vector
pcDNA3.1/Hygro
Cell culture and transfection procedure
BiP-CHO and DUKX cells were transfected with
pcDNA3 using lipofectAMINE PLUS reagent
TSHR-K562 cells were transfected with CNX-pcDNA3.1/Hygro
or CRT-pcDNA3.1/Hygro or pcDNA3.1/Hygro alone
using lipofectAMINE PLUS reagent Cells were then
cultured in Ham’s F-12 medium in the case of CHO cell
lines and RPMI medium in that of K562 cell lines
supplemented with 10% fetal bovine serum, penicillin
(100 IUÆmL)1) and streptomycin (100 lgÆmL)1)
Forty-eight hours after the transfection procedure, experiments
were carried out using TSHR-DUKX, TSHR-BiP-CHO,
CNX-TSHR-K562, CRT-TSHR-K562 or pcDNA3.1/
Hygro-K562 cell pools
Metabolic labeling and extraction of TSHR
After being incubated at 37C for 16 h with 10 mMsodium
butyrate, the cells (2· 106) were preincubated for 2 h in
Met- and Cys-free DMEM supplemented with 10%
dialyzed fetal bovine serum, 10 mMsodium butyrate, with
or without 100 lM MG132 and with or without 1 mM
castanospermine (CST) They were then pulsed for 1 h in the
same medium supplemented with [35S](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, BiP-CHO and TSHR-DUKX cells were washed twice with 2 mL ice-cold NaCl/
Pi, then scraped into 1 mL ice-cold NaCl/Piand centrifuged for 5 min at 200 g and CNX-TSHR- K562, CRT-TSHR-K562 and TSHR-CRT-TSHR-K562 cells were centrifuged for 5 min at
200 g All cells were resuspended in 200 lL extraction buffer containing 1% Triton X-100, 10 mM Tris/HCl (pH 7.4), 0.15MNaCl and protease inhibitor cocktail for
1 h at 4C (vortexing every 2 min), and 600 lL of immunoprecipitation buffer (1% NP40, 20 mM Hepes, 0.3MNaCl, 2 mMEDTA and 0.1% SDS) was then added and the preparation was centrifuged for 15 min at 10 000 g Immunoprecipitation and electrophoresis
The radiolabeled supernatant obtained was saved and incubated for 1 h at 4C with protein A-Sepharose and centrifuged for 2 min at 10 000 g The supernatant was incubated for 2 h at 4C with mAb A10, and after 25 lL of protein A-Sepharose had been added, it then was incubated for 1 h at 4C Immune complexes were retrieved by performing a brief centrifugation at 10 000 g and washed four times with 1 mL of immunoprecipitation buffer and twice with 10 mMTris/HCl, 2 mMEDTA and 0.1% SDS buffer The precipitated proteins were separated from the antibody-protein A-Sepharose complex by heating the preparation at 45C for 30 min in the Laemmli sample buffer containing 62 mMTris/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· 106 cells) were centrifuged for 5 min at 200 g TSHR-BiP-CHO or TSHR-DUKX cells obtained from 9.6 cm2 dishes were washed twice with 2 mL of ice-cold NaCl/Pi, then scraped and resuspended in 1 mL ice-cold NaCl/Pi and centrifuged (200 g, 5 min) The pellets were resuspended in 100 lL buffer containing 50 mMTris/HCl (pH 7.4), 0.15MNaCl, 1% Triton-X100, 0.3% deoxycholic acid, and protease inhibitor cocktail The cells were then tumbled for 20 min at 4C (vortexing every 2 min) and centrifuged for 3 min at 10 000 g Twenty microliters of the Laemmli sample buffer (Cx5) and 5% 2-mercaptoethanol were added to the supernatant and the samples were reduced by boiling for 5 min The samples were then run on 7.5% SDS/PAGE After performing Western blotting on a poly(vinylidiene difluoride) membrane, any nonspecific sites were blocked with 3% nonfat milk powder in Tris-buffered saline (NaCl/Tris) containing 0.1% Tween 20 Membranes were incubated for 2 h at room temperature or overnight at
4C with calnexin rabbit polyclonal antibody (SPA-860), calreticulin rabbit polyclonal antibody (SPA-600) or KDEL
Trang 3mouse monoclonal antibody (SPA-827) in NaCl/Tris
sup-plemented with 0.1% Tween 20 and 0.3% non fat milk
powder After being washed, the membranes were
incu-bated for 2 h at room temperature with monoclonal
anti-rabbit immunoglobulins peroxidase conjugate or
monoclonal anti-mouse immunoglobulins peroxidase
con-jugate in NaCl/Tris, 0.1% Tween 20, 0.3% non fat milk
powder After four washes in the same medium without IgG
and two washes with NaCl/Tris, the signal was developed
using SuperSignal developing medium (Pierce)
Coimmunoprecipitation of molecular chaperones
and TSHR
TSHR-K562 cells were incubated for 16 h at 37C in
RPMI 1640 medium supplemented with 10% fetal bovine
serum, and 10 mM butyrate Cells were centrifuged for
5 min at 200 g and resuspended in 200 lL extraction
buffer containing 1% CHAPS, 10 mMTris/HCl (pH 7.4),
0.15M 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 4C, 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 4C, 20 lL of protein A Sepharose were added
and the preparation was left for 1 h at 4C The immune
complexes were retrieved by performing a brief
centrifu-gation at 10 000 g and washed four times with 1 mL of
extraction buffer The antigens were then eluted using
100 lL of Laemmli sample buffer and heated at 45C
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)
R E S U L T S
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
Pulse-chase experiments were performed using [35S]Met and
[35S]Cys, and after the extraction step, an
immunoprecipi-tation step was carried out using a monoclonal antibody
directed against the extracellular part of the TSHR (mAb
A10) The results were similar to those obtained previously
by M Misrahi and colleagues [6] and showed that the
TSHR bearing high mannose type structures (97 kDa)
largely disappeared during the first five hours of chase
(Fig 1A and B) After 1 h of chase, the TSHR bearing
complex-type structures (115 kDa) and free A subunit
(55 kDa) began to appear At least 50% of the total TSHR
synthesized were able to acquire complex N-glycans (sum of
the TSHR bearing complex-type structures and the free A
subunit), and the remaining proportion was degraded It is
worth noting that only a small proportion (1–3%) of the
free A subunit was recovered in the cell culture medium
(data not shown)
Because the proteasome pathway has recently been found
to mediate the degradation of many endoplasmic reticulum proteins, we focused here on the possible involvement
of the proteasome in the degradation of TSHR In order
to test this hypothesis, cells were pulse labeled with [35S](Met + Cys) in the presence or absence of MG132, a proteasome inhibitor [15]
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
50 40 30 20 Time (h)
10 20 30 40 50 0
0 50 100 150
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
Cells were then chased for the times indicated in RPMI medium with 10% fetal bovine serum After the chase, cells were centrifuged for
protein A-Sepharose and centrifuged for 2 min at 10 000 g The supernatant was immunoprecipitated with mAb A10 Samples were analyzed by SDS/PAGE after reduction: (A) SDS/PAGE analysis; (B) quantification using a Phosphorimager, s, TSHR bearing high-man-nose type structures; d, TSHR bearing complex-type structures; m, A subunit These experiments were repeated three times and very similar results were obtained in each case.
Trang 4confirmed by quantifying this part of the gel (Fig 2D) It
seems likely that the ladder and the accompanying high
molecular weight smear correspond to polyubiquitinated
forms of the receptor The decrease in the amount of
TSHRs observed during the chase (Fig 2C) in the
presence of MG132 could be explained by the fact that
the TSHRs were retranslocated into the cytoplasm and
showed up in the form of polyubiquitinated molecules It
should also be noticed that as described by others [16], the
use of MG132 unexpectedly blocks the formation of
complex-type structures
Interactions between TSHR and CNX, CRT, and BiP
During and after their synthesis, glycoproteins interact
with a number of molecular chaperones The latter have
been found to mediate the folding and/or retention of the
protein in the endoplasmic reticulum In order to study
the possible interactions between TSHR and CRT, CNX,
and BiP, TSHR was coimmunoprecipitated with each of
these molecular chaperones These experiments were
performed on cell extracts using anti-CNX, anti-CRT
and anti-BiP antibodies under conditions that preserve the
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 mM of CST, which is known to inhibit the trimming of the three glucoses from the core oligosaccha-ride and the subsequent association between CNX or CRT and the glycoprotein substrate At the end of the pulse and at each chase time, cells were lysed and immunoprecipitation was carried out using mAb A10 The data obtained indicate that CST enhances the degradation of TSHR (Fig 4) The association with CNX and/or CRT therefore seems contribute to the stability and/or the maturation of the TSHR To obtain further insights into the contribution of CNX and CRT to
0h 3h 5h 22h 0h 3h 5h 22h
Time (h)
5 10 15 20
20
40
60
80
0
C
5 10 15 20 0
50 100 150
0
Time (h)
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
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 nonNon-immune rabbit serum (lane 1) After SDS/PAGE and Western blotting, the TSHR coim-munoprecipitated was revealed using mAb A10 coupled to horseradish peroxidase.
Trang 5the folding of TSHR and to determine whether these
molecular chaperones are a limiting factor in K562 cells,
we overexpressed CNX or CRT by transiently transfecting
TSHR-K562 cells In the Western blotting analyses
performed, the levels of CNX and CRT expression were
five times greater in the transfected cells than in the
control cells (data not shown) TSHR-K562 cells
over-expressing CNX or CRT and TSHR-K562 cells
trans-fected with pcDNA3.1/Hygro alone were pulse-chased as
previously described Under these conditions, we observed
a greater amount (+124% for CNX and +158% for
CRT) of TSHR immunoprecipitate at the end of the pulse
(Fig 5A and B) CNX and CRT therefore protect TSHR
from being degraded immediately after synthesis But this
protection had disappeared completely after 5 h of chase
and did not lead to an increase in the proportion of the
TSHRs able to acquire complex type N-glycans (Fig 5A
and B) These results show that interactions with CNX
and/or CRT prevent the TSHR from being rapidly
degraded just after its synthesis However it does not
seem likely that these interactions are absolutely necessary
for a proportion of the receptor to be able to fold
properly
Effects of interactions between TSHR with BiP
To further investigate the folding and maturation of
TSHR, we studied the possible involvement of BiP, one of
the main molecular chaperones of the endoplasmic
reticu-lum, in these events BiP is a member of the Hsp70 family
and promotes the folding and assembly of protein by
recognizing unfolded polypeptides and inhibiting
intra-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 [35S] (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) 0 5 10 15 20
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
quantified using a Phosphorimager The experiment was repeated
three times and similar results were obtained in each case.
0 5 10 15 20 0
50 100 150 200 250
Time (h)
A
Time (h)
35S-TSHR immunoprecipitated
0 5 10 15 20 0
50 100 150 200 250
B
Fig 5 Effects of calnexin and calreticulin overexpression on the folding
of TSHR in K562 cells K562-TSHR cells were transfected with CNX-pcDNA3.1/Hygro (A and d, m), CRT-pcDNA3.1/Hygro (B 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 obtained in each case.
Trang 6these 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
D I S C U S S I O N
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
data published by Schubert and colleagues [20] These
authors recently suggested that at least 30% of the newly
synthesized proteins were degraded by the proteasome The
degradation rate varied, depending on the proteins CFTR
[21], tyrosinase [22] and thyroperoxidase [23] have been
reported to be more unstable than TSHR after their
synthesis The fact that 50% of the TSHR was degraded
indicates that half of the newly synthesized TSHR does not
fold correctly and is unable to exit from the endoplasmic
reticulum
The folding of newly synthesized proteins in vivo is facilitated at the endoplasmic reticulum level by molecular chaperones and folding catalyst [8] Three of the most thoroughly characterized molecular chaperones present in the endoplasmic reticulum are CNX, CRT and BiP CNX and CRT interact with glycoproteins bearing monogluco-sylated high-mannose type oligosaccharides [24] and certainly also via polypeptide based interactions [25] It
is known that N-glycosylation of the TSHR is an important prerequisite for its transport and/or functional efficiency Both other authors and we have previously demonstrated that the inhibition of N-glycosylation by tunicamycine leads to a decrease in the amount of TSHR present at the cell surface [26,27] In the case of two other G-protein coupled receptors (V2 vasopressin receptor and gonadotropin receptor), interactions have been found to occur with various molecular chaperones, but the effects
of these interactions on the maturation of these receptors have not yet been determined In the present study, it was established that interactions between TSHR and CNX 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 NH2-terminus Glycoproteins, that have their glycans nearer to the COOH end of the sequence, associate first with BiP During the translocation of a glycoprotein, a choice therefore has to be made between these two chaperone systems As TSHRs have their first N-glycan on the Asn77, it can be hypothesized that competition occurs between these two pathways We then established in this study that depending on which of these two pathways is chosen, this glycoprotein will undergo either maturation degradation The interactions with CNX or CRT are bound to stabilize the receptor because these molecular chaperones, with the help of Erp57, will promote the proper folding of the receptor, while the association with BiP will have destabilizing effects on at least some of the receptors
Time (h)
0 5 10 15 20
0
20
40
60
80
100
Fig 6 Effects of BiP overexpession on the folding of TSHR in CHO
cells A cell line overexpressing BiP (BiP-CHO cells; m, d) and the
parent cell line (DUKX cells; n, s) were transfected with
TSHR-pcDNA3 After 48 h, pulse-chase analysis was performed as described
in Fig 1 Samples were analyzed by SDS/PAGE after a reduction step
and quantified by phosphorimaging s, d, TSHR bearing
high-mannose type structures; n, m, TSHR bearing high-high-mannose type
structures plus A subunit The experiment was repeated three times
and similar results were obtained in each case.
Trang 7Other 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
A C K N O W L E D G E M E N T S
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 pcDNA3 and
TSHR-K562 cell line, P Banga for the mAb A10, M Michalak for the CRT
cDNA, D.Y Thomas for CNX cDNA and A.J Dorner for Bip-CHO
and DUKX cells.
R E F E R E N C E S
1 Vassart, G & Dumont, J (1992) The thyrotropin receptor and the
regulation of thyrocyte function and growth Endocrine Rev 13,
61–80.
2 Sanders, J., Oda, Y., Roberts, S.A., Maruyama, M., Furmaniak,
J & Rees Smith, B (1998) Understanding the thyrotropin receptor
function-structure relationship Baille`re’s Clin Endocrinol Met.
11, 451–479.
3 Rapoport, B., Chazenbalk, G.D., Jaume, G.D & McLachlan,
S.M (1998) The thyrotropin (TSH) receptor: interaction with
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