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- 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 degradation of the receptor at the endoplasmic reticulum level. Correspondence to J. L. Franc, U555 INSERM, Faculte ´ de Me ´ decine, 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’Hoˆ pital, 67091 Strasbourg cedex, France. (Received 12 April 2002, revised 22 July 2002, accepted 20 August 2002) Eur. J. Biochem. 269, 4930–4937 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03192.x MATERIALS AND METHODS 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 [ 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- 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 37 °Cfor16 hwith10 m M sodium butyrate, the cells (2 · 10 6 ) were preincubated for 2 h in Met- and Cys-free DMEM supplemented with 10% dialyzed fetal bovine serum, 10 m M sodium butyrate, with or without 100 l M MG132 and with or without 1 m 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 (pH 7.4), 0.15 M NaCl and protease inhibitor cocktail for 1h at 4°C (vortexing every 2 min), and 600 lLof immunoprecipitation buffer (1% NP40, 20 m M Hepes, 0.3 M NaCl, 2 m M EDTA 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 4 °C with protein A-Sepharose and centrifuged for 2 min at 10 000 g. The supernatant was incubated for 2 h at 4 °C with mAb A10, and after 25 lLof protein A-Sepharose had been added, it then was incubated for 1 h at 4 °C. 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 m M Tris/HCl, 2 m M EDTA and 0.1% SDS buffer. The precipitated proteins were separated from the antibody-protein A-Sepharose complex by heating the preparation at 45 °C for 30 min in the Laemmli sample 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 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 4 °C with calnexin rabbit polyclonal antibody (SPA-860), calreticulin rabbit polyclonal antibody (SPA-600) or KDEL Ó FEBS 2002 TSH receptor and molecular chaperones (Eur. J. Biochem. 269) 4931 mouse 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 37 °Cin RPMI 1640 medium supplemented with 10% fetal bovine serum, and 10 m M butyrate. Cells were centrifuged for 5 min at 200 g and resuspended in 200 lLextraction 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. Pulse-chase experiments were performed using [ 35 S]Met and [ 35 S]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 [ 35 S](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 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 5 min at 10 000 g. The supernatant was incubated for 1 h at 4 °Cwith 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 resultswereobtainedineachcase. 4932 S. Siffroi-Fernandez et al. (Eur. J. Biochem. 269) Ó FEBS 2002 confirmed 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 m M 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 A B 0h 3h 5h 22h 0h 3h 5h 22h Time (h) 5 10 15 20 20 40 60 80 0 0 35S-TSHR im m unoprecipitated (arbitrary units) C 5 10 15 20 0 50 100 150 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 the 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 [ 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 [ 35 S](Met + Cys), and chased with (d) or without (s)1 m M CST. Samples were analyzed by SDS/PAGE and 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) 35S-TSHR immunoprecipitated 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 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 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 NH 2 -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) % of 35S-TSHR immunoprecipitated 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)andthe 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-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. 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(1994) Association of Hsp47, Grp78, and Grp94 with procollagen supports the successive or coupled action of molecular chaperones. J. Cell. Biochem. 56, 518–526. 32. Melnik, J., Dul, J.L. & Argon, Y. (1994) Sequential interaction of the chaperones BiP and GRP94 with immunoglobulin chains in the endoplasmic reticulum. Nature 370, 373–375. 33.Loo,M.A.,Jensen,T.J.,Cui,L.,Hou,Y.,Chang,X.B.& Riordan, J.R. (1998) Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome. EMBO J. 17, 6879–6887. Ó FEBS 2002 TSH receptor and molecular chaperones (Eur. J. Biochem. 269) 4937 . 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. 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. . 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