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Endocrinology, doi:10.1210/en.2003-1432
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Endocrinology Vol. 145, No. 3 1464-1472
Copyright © 2004 by The Endocrine Society

Sphingolipid-Cholesterol Domains (Lipid Rafts) in Normal Human and Dog Thyroid Follicular Cells Are Not Involved in Thyrotropin Receptor Signaling

M. J. Costa, Y. Song, P. Macours, C. Massart, M. C. Many, S. Costagliola, J. E. Dumont, J. Van Sande and V. Vanvooren

Institute of Interdisciplinary Research, Free University of Brussels, School of Medicine (M.J.C., Y.S., P.M., C.M., S.C., J.E.D., J.V.S., V.V.), B-1070 Brussels B-1070, Belgium; and Faculty of Medicine, University of Porto (M.J.C.), Porto 4200-319, Portugal; and Histology Unit, Catholic University of Leuven, Medical School (M.C.M.), B-1200 Brussels, Belgium

Address all correspondence and requests for reprints to: Dr. J. Van Sande, Institute of Interdisciplinary Research, Free University of Brussels, School of Medicine, Campus Erasme, 808 Route de Lennik, Building C, B-1070 Brussels, Belgium. E-mail: jvsande{at}ulb.ac.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Partition of signaling molecules in sphingolipid-cholesterol-enriched membrane domains, among which are the caveolae, may contribute to signal transduction efficiency. In normal thyroid, nothing is known about a putative TSH/cAMP cascade compartmentation in caveolae or other sphingolipid-cholesterol-enriched membrane domains. In this study we show for the first time that caveolae are present in the apical membrane of dog and human thyrocytes: caveolin-1 mRNA presence is demonstrated by Northern blotting in primary cultures and that of the caveolin-1 protein by immunohistochemistry performed on human thyroid tissue. The TSH receptor located in the basal membrane can therefore not be located in caveolae. We demonstrate for the first time by biochemical methods the existence of sphingolipid-cholesterol-enriched domains in human and dog thyroid follicular cells that contain caveolin, flotillin-2, and the insulin receptor. We assessed a possible sphingolipid-cholesterol-enriched domains compartmentation of the TSH receptor and the {alpha}- subunit of the heterotrimeric Gs and Gq proteins using two approaches: Western blotting on detergent-resistant membranes isolated from thyrocytes in primary cultures and the influence of 10 mM methyl-ß-cyclodextrin, a cholesterol chelator, on basal and stimulated cAMP accumulation in intact thyrocytes. The results from both types of experiments strongly suggest that the TSH/cAMP cascade in thyroid cells is not associated with sphingolipid-cholesterol-enriched membrane domains.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EXISTENCE of clusters rich in cholesterol and sphingolipids in the exoplasmic leaflet, connected to phospholipids and cholesterol in the cytoplasmic leaflet of the lipid bilayer, was described more than 10 yr ago (1). The hydrocarbon chains of the lipids forming these microdomains are mostly long and saturated, which allows the formation of highly packed patches, less fluid than the bulk membrane. These membrane microdomains are characterized by their resistance to solubilization in nonionic detergents and a low buoyant density in sucrose or OptiPrep density gradients (2, 3, 4, 5) (to avoid nomenclature confusion, in this work we call detergent-resistant membranes (DRMs) the insoluble fraction recovered in Triton X-100/OptiPrep experiments). Although similar in lipid composition, two different types of membrane microdomains can be recognized on the basis of morphological and biochemical properties. The first, the caveolae, are flask-shaped invaginations of the plasma membrane, defined by the presence of the marker protein caveolin, a 22-kDa integral membrane protein that binds cholesterol and plays a role in the caveolae biogenesis (6, 7). In contrast, lipid rafts lack caveolin and cannot be morphologically distinguished from the plasma membrane (8, 9).

In recent years it has been shown that these lipid microdomains may play a role in several cellular processes (reviewed in Refs. 6 , 9 , and 10), such as cholesterol traffic, protein sorting, potocytosis, transcytosis, and signal transduction. With regard to signal transduction, certain proteins with saturated lipid tails, such as {alpha}-subunits of heterotrimeric G proteins, glycosyl phosphatidylinositol (GPI)-anchored proteins, and Src tyrosine kinases, tend to partition in the liquid-ordered environment. As a result, there is an increase in the effective concentration of the signaling partners, with the expected consequences on the speed of the signal transduction mechanisms (reviewed in Ref. 9). In contrast, most transmembrane proteins are excluded from these domains (11). Nevertheless, sphingolipid-cholesterol domains were found to be enriched in some transmembrane proteins (12), such as the insulin (13) and T cell (14, 15) receptors.

TSH controls thyroid gland physiology and development, mainly through the cAMP generated in response to TSH binding to its receptor (16). The TSH receptor belongs to the glycoprotein hormone subfamily of G protein-coupled receptors that also includes FSH and LH receptors. However, the TSH receptor differs from the latter by the presence of an additional specific segment in the C-terminal region of its ectodomain (17). A posttranslational cleavage in this insertion yields two subunits: an A-subunit of approximately 53 kDa, corresponding to the ectodomain and a membrane-spanning B-subunit, of which the reported experimental molecular size varies between about 38 kDa (18) and about 52 kDa (19). The subunits are bound by a disulfide bridge. The cleavage, performed by a metalloprotease, is unique among G protein-coupled receptors, and its role remains unclear.

In human thyroid, TSH stimulates both the adenylyl cyclase and the phospholipase C cascades (20, 21); however, this latter effect is obtained for higher concentrations of TSH (20). cAMP regulates hormone secretion and transcription of thyroglobulin, thyroid peroxidase, and sodium-iodide symporter mRNAs, whereas inositol 1,4,5-triphosphate-intracellular Ca2+ control iodide efflux, H2O2 production, and thyroglobulin iodination (16). In isolated dog and human thyroid membrane preparations, the TSH receptor activates Gs, Gq, Gi, and other G proteins (22). However, in intact dog thyroid cells, TSH does not activate the phospholipase C-Ca2+ cascade, and all of the known effects of TSH are mediated by cAMP.

Down-regulation of caveolin expression was reported in sporadic follicular thyroid cancer (23). In the present work we checked the presence of sphingolipid-cholesterol-enriched domains (including caveolae) in thyroid cell plasma membrane and the association of signaling proteins, TSH receptor, {alpha}-subunit of Gs and Gq, and insulin receptor, with these lipid microdomains. Most experiments aimed at defining proteins associated with sphingolipid-cholesterol-enriched domains have been performed in cell lines overexpressing the proteins of interest. However, cell lines membranes may have lipid and protein compositions different from those found in normal thyrocytes as a consequence of dedifferentiation and differences in protein expression. For instance, cells of the FRT thyroid cell line do not express caveolin-1 (24, 25, 26). Due to the low concentration of TSH receptor in the thyroid tissue, nearly all studies of this receptor in intact cells have been performed in transfected cells. However, several important differences have been found between the thyroid native receptor and the receptor overexpressed in cell lines (27). First, in human thyroid tissue the receptor posttranslational cleavage is virtually complete, as no uncleaved receptor was detected, whereas in L cells stably transfected with the receptor, some uncleaved mature receptor is still present. Second, in transfected cells, but not in human thyroid tissue, there is an abnormal accumulation inside the cells of an unprocessed mannose-rich monomeric precursor. Third, in L cells, the A-subunit is more glycosylated, and two B-subunits are detected instead of one in thyroid cells. Last, in thyroid cells, most of the receptors are located at the plasma membrane, whereas in L cells, the TSH receptor is highly concentrated in the endoplasmic reticulum and the Golgi apparatus. Therefore, in this study we used primary cultures of thyrocytes because they are closer to physiology than the rat immortalized cell lines or transfected cells that are generally used. For reasons of availability, dog thyroid cells were also used, because they present the closest similitude to their human counterpart (28), and the results were then extended to human cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Streptomycin/penicillin, fungizone, sodium pyruvate, fetal bovine serum, and all culture media were obtained from Life Technologies, Inc. (Paisley, UK). BSA, deoxyribonuclease, collagenase, bovine TSH, bovine insulin, forskolin, methyl-ß-cyclodextrin (MßCD), and Triton X-100 were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Brij 96 was obtained from Fluka (Bornem, Belgium), Nonidet P-40 and dodecyl-ß-D-maltoside were obtained from Calbiochem (Darmstadt, Germany). Protease inhibitor cocktail was purchased from Roche (Mannheim, Germany). Optiprep 60% (wt/vol) in water was obtained from Axis-Shield (Oslo, Norway). Mouse monoclonal antibodies against adaptin-ß (clone 74), flotillin-2 (clone 29), and caveolin-1 (clone 2297) and rabbit polyclonal antibodies against caveolin and the insulin receptor ß-subunit were purchased from Transduction Laboratories (Erembodegem, Belgium). Rabbit polyclonal antibodies against G{alpha}s (sc-823) and G{alpha}q (sc-393) proteins were purchased from Santa Cruz Laboratory, Inc. (Santa Cruz, CA). Noncommercial mouse monoclonal antibodies against the TSH receptor ectodomain (28.1) (29) or the membrane-spanning domain (15.2) (30) were developed by S. Costagliola, in our institute, Brussels, Belgium.

Cell lines stably expressing the TSH receptor
Chinese hamster ovary (CHO) cells stably expressing the whole wild-type TSH receptor (JP19) or the TSH receptor ectodomain anchored to the membrane by GPI (GT14) were described previously (31, 32).

Primary culture
Cells were obtained from human and dog thyroid tissue. Normal human thyroid tissue was obtained from patients undergoing surgery for partial thyroidectomy, following the rules of the University Hospital ethical committee. Follicles were isolated by mild digestion of fresh tissue with collagenase (375 µg/ml) and dispase (100 µg/ml). The follicles were cultured in the following medium: DMEM/Ham’s F-12 medium/MCDB 104 medium (2:1:1, vol/vol/vol) supplemented with 2 mM sodium pyruvate, 40 µg/ml ascorbic acid, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml fungizone, and 5 µg/ml insulin. Human thyrocytes were cultured for the first 24 h with 1% serum to ensure optimal spreading of the follicles. Dog thyroid follicles were obtained by collagenase digestion as previously described (33). After washing in basal medium (Eagle’s), the follicles were isolated by centrifugation at 100 x g for 10 sec and seeded at a density of 2 µl pellet/ml culture medium in petri dishes. The human cells were cultured for 5 d, and the dog cells were cultured for 4 d in basal conditions or in the presence of 3 µM forskolin.

Total RNA extraction
The cells were washed with PBS (140 mM NaCl, 4.5 mM Na2HPO4, 2.7 mM KCl, and 1.5 mM KH2PO4, pH 7.4), and total RNA was extracted by TRIzol (Invitrogen, Carlsbad, CA) and purified using RNeasy columns (Qiagen, Leusden, The Netherlands).

Northern blot analysis of caveolin-1 expression
After denaturation of total RNA (34), the RNAs (5 µg) were separated by electrophoresis on a 1% agarose gel in 10 mM phosphate buffer, pH 7.0. After migration for 3 h at 120 V, the RNAs were blotted overnight at room temperature to a nylon membrane using 20x standard saline citrate (3 M NaCl and 0.3 M sodium citrate) as blotting solution. After baking at 80 C, the blots were prehybridized and hybridized (35). The probe used was the cDNA coding for human caveolin-1. After the hybridization procedure, the membranes were first washed in 2x standard saline citrate/0.1% sodium dodecyl sulfate three times for 15 min each time at room temperature and then in 0.1x standard saline citrate/0.1% sodium dodecyl sulfate, at 55 C for the same periods. The membranes were then autoradiographed at -80 C using MP films (Amersham Pharmacia Biotech, Little Chalfont, UK) in the presence of intensifying screens.

RT-PCR analysis of caveolin-1 expression
Total RNA was used as a template for cDNA synthesis using the Superscript II ribonuclease H RT kit (Invitrogen, Merelbeke, Belgium). Two microliters of the cDNA were subjected to amplification in a final volume of 50 µl containing 1.5 mM MgCl2, 10 pmol of each primer, 2.5 U Taq DNA polymerase (Invitrogen), 1x polymerase buffer [10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, and 0.2 mM deoxy-NTPs]. The sequence of the primers was chosen based on the sequence of the human caveolin-1 (accession number NM_001753), from which a fragment of 305 bp was amplified (forward primer, 5'-GGGGCAAATACGTAGACTCG-3'; reverse primer, 5'-AGACAGCAAGCGGTAAAACC-3'). The polymerization reaction was started by a denaturation step at 93 C for 2 min 30 sec, followed by 35 cycles at 93 C for 1 min, 55 C for 1 min, and 72 C for 1 min and a final elongation step of 6 min at 72 C.

Solubilization of membrane proteins and TSH receptor immunoblot
All steps were carried out at 4 C. Briefly, confluent cells from 10 dishes of 10-cm diameter were washed twice with PBS, scrapped in 5 ml PBS-EDTA-EGTA (PBS, 5 mM EDTA, and 5 mM EGTA), pelleted by centrifugation at 1,000 x g, resuspended in 5 ml lysis buffer [0.02 M Tris-HCl (pH 7.5), 0.1 M (NH4)2SO4, 0.1% (v/v) glycerol, and protease inhibitors], and lysed by 10 strokes in a Dounce homogenizer (Kontes Co., Vineland, NJ) equipped with a Teflon pestle. The whole cell homogenate was centrifuged at 16,000 x g for 30 min, and the resulting membrane pellet was resuspended in 500 µl lysis buffer. Dodecyl-D-maltoside (DDM; 1%) was added to the suspension, and the membranes were solubilized with end over end rotation for 1 h, followed by centrifugation at 100,000 x g for 1 h. The supernatant containing the solubilized proteins was frozen in liquid nitrogen and stored at -80 C in aliquots. For Western blots, 20 µl of this supernatant were mixed with 6 µl 5x denaturation buffer [0.3 M Tris-HCl (pH 6.8), 50% (wt/vol) glycerol, 8% (wt/vol) dithiothreitol, and 10% (wt/vol) sodium dodecyl sulfate] and denatured at 50 C for 1 h.

Total protein extraction
All the steps were carried out at 4 C. Primary cultures of dog or human thyrocytes (10-cm-diameter petri dishes) were kept in regular medium until confluence. For total protein extraction, the cell monolayer was rinsed twice with PBS and scrapped with 1 ml 2x denaturation buffer, followed by boiling and immediate liquid N2 freezing. Then the lysate was thawed, homogenized by five passages through a 21-gauge needle, frozen again in liquid N2, and stored at -80 C.

Preparation of detergent-resistant membranes by OptiPrep gradient centrifugation
DRMs were prepared from thyrocytes in primary culture using a modification of the protocol reported by Tansey et al. (36). All procedures were carried out at 4 C. Briefly, confluent cells of two 6-cm diameter dishes per condition were rinsed twice with PBS, lysed, and scraped in 400 µl TNE buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, protease inhibitors, and Triton X-100 or Brij 96 at various concentrations]. The lysate was homogenized by passage through a 21-gauge needle 20 times. After 15-min solubilization, it underwent centrifugation at 800 x g for 10 min. The nuclear pellet was discarded, and the supernatant was adjusted to 35% (vol/vol) OptiPrep by adding 585 µl of a 60% stock solution of OptiPrep, transferred to the bottom of a centrifuge tube, and overlayed successively with 8 ml 30% OptiPrep (diluted with 0.5x TNE buffer) and 1 ml TNE buffer. After centrifugation at 197,000 x g for 4 h, 1-ml fractions were collected from the top of the gradient [designated fractions 1 (top) to 10 (bottom)]. Protein samples of equal volume taken from each fraction were concentrated with StrataClean (Stratagene, Amsterdam, The Netherlands), resuspended in 1x denaturation buffer, and boiled for 5 min, except for the samples used for TSH receptor detection, which were denatured in 5x denaturation buffer for 1 h at 50 C.

Western blotting experiments
Western blots of protein samples from OptiPrep gradient fractions or solubilized membrane proteins were performed as previously described (37). Briefly, protein samples were resolved by SDS-PAGE and blotted onto polyvinylidene fluoride membranes (NEN Life Science Products, Zaventem, Belgium), which were incubated in 5% nonfat dry milk dissolved in TBS/Nonidet P-40 buffer [20 mM Tris-HCl (pH 7.6), 140 mM NaCl, and 0.05% (vol/vol) Nonidet P-40] for 1 h at room temperature to block the nonspecific sites. Incubations with primary antibodies, diluted in the referred buffer, were carried out for 2 h at room temperature. After several washings, the membranes were incubated for 1 h with the respective peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech) diluted in TBS/Nonidet P-40. The membranes then underwent another extensive washing in TBS/Nonidet P-40, and the bound antibodies were detected using a chemiluminescence method (Western Lighting, NEN Life Science Products, Boston, MA).

MßCD treatment and cAMP measurement
Thyrocytes cultured in 3.5-cm diameter dishes were rinsed in Krebs-Ringer-HEPES (KRH) medium [25 mM HEPES (pH 7.4), 1.25 mM KH2PO4, 124 mM NaCl, 1.25 mM MgSO4, 8 mM glucose, 1.45 mM CaCl2, and 5 mM KCl] and preincubated with the same medium containing, or not, 10 mM MßCD at 37 C for 30 min. Then, the media were removed, and cells were incubated with fresh KRH medium containing 25 µM rolipram (a phosphodiesterase inhibitor) and TSH (0.1, 1, and 10 mU/ml) for 30 min. The incubation was stopped by withdrawal of the medium and addition of 0.1 M HCl to the cells. The samples were dried in a Speed-Vac concentrator (Jouan RC10.10, St. Nazaire, France), and cAMP was measured by RIA (38).

Cholesterol quantification
Thyrocytes (four 10-cm diameter dishes/condition) were incubated in KRH medium with or without 10 mM MßCD as described above. The cells were then washed with PBS, scraped in PBS-EDTA-EGTA, and pelleted by centrifugation, and total lipids were extracted with hexane/isopropanol (1:1) for 1 h at room temperature. After solvent evaporation, the extracts were derivatized with Hydroxy-sil in tetrahydrofuran for 1 h, resuspended in hexane, and analyzed by gas chromatography (Hewlett-Packard 6890, Chrompack, Sint-Katelijne-Waver, Belgium) using cholesterol acetate as an internal standard and standard cholesterol solutions for calibration.

Caveolin immunohistochemistry
Paraffin-embedded human normal thyroid sections were mounted on gelatin-covered glass slides, deparaffinized, and hydrated. Antigen retrieval was performed by boiling the slides immersed in citrate buffer containing 1% Triton X-100. The tissues were washed in PBS containing 1% BSA (PBS-BSA). Blocking of nonspecific binding sites was performed by incubation in goat preimmune serum diluted 1:50 in PBS for 20 min at room temperature. The incubation with primary antibody was carried out at 4 C for 3 h, followed by washing in PBS-BSA. Bound antibodies were visualized using the En-Vision-AEC kit, following the manufacturer’s instructions. Nuclei were counterstained with hematoxylin. The staining of the preparations was evaluated by three independent observers, and pictures were taken from representative results.

Reproducibility
All experiments were carried out at least three times with triplicate determinations for each condition in quantitative assays. A representative experiment is shown for each protocol.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of expression of the mRNA of caveolin-1 in thyroid cells
The possible expression of caveolin-1 mRNA in dog and human thyrocytes was checked by two independent methods after total RNA extraction. Caveolin-1 messenger (2.7 kb) was identified by Northern blot using caveolin-1 human cDNA sequence as probe (Fig. 1Go, upper panel). Rat and human caveolin-1 sequences present minimal differences. To confirm this by another technique, total RNA was reverse transcribed, and a fragment of 305 bp from caveolin-1 cDNA was amplified by PCR using specific primers (Fig. 1Go, lower panel). In both experiments, total RNA from CHO cells was used as a positive control, and total RNA from FRTL-5 cells was used as a negative control. The results show the expression of caveolin-1 mRNA in dog and human thyroid cells and in CHO cells, but not in FRTL-5 cells, by both techniques.



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  		FIG. 1. Caveolin-1 expression in human and dog thyroid cells. Total RNA from CHO and FRTL-5 cell lines and from dog and human thyrocytes in primary culture was extracted and purified. Caveolin-1 expression was analyzed by Northern blot (upper panel) and RT-PCR (lower panel). In the Northern blot, 5 µg total RNA were loaded in a 1% agarose gel, separated by electrophoresis, and blotted onto a nylon membrane, which was probed with cDNA coding for the human caveolin-1 sequence. In the RT-PCR, PCR amplification of caveolin-1 cDNA from human and dog thyrocytes in primary culture was performed; total RNA was reverse transcribed, and a fragment of 305 bp from caveolin-1 cDNA was amplified using the described specific primers.

 
Protein analysis in DRMs prepared with different detergents
Classically, the DRMs are concentrated on the top of the density gradients (fractions 2–4), and the solubilized proteins are found in the bottom of the tube (fractions 9 and 10). The amount and nature of proteins and lipids recovered in the DRM fraction can differ dramatically depending on the nonionic detergent used as a consequence of their different selectivities (39). Furthermore, the detergent:protein ratio may influence the protein solubilization and DRM composition (40, 41). Therefore, to optimize the solubilization conditions to obtain the maximum yield and purity of DRMs, we compared the use of Triton X-100 and Brij 96 at several concentrations (0.1%, 0.3%, 0.5%, and 1%, vol/vol) to prepare DRMs from dog and human thyrocytes in primary culture. Detergent extractions were performed at 4 C for 15 min, followed by OptiPrep centrifugation. As in the previous experiments we found that thyrocytes express caveolin, samples from the gradient fractions were immunoblotted with antibodies against this protein, as a DRM marker, and adaptin-ß, as a clathrin-associated soluble-membrane marker, as positive and negative controls, respectively. No differences were observed between the detergents, but DRMs were contaminated with adaptin-ß when the detergent concentration was 0.3% (vol/vol) or less. Caveolin was maximally enriched in lower density fractions at the lowest detergent concentration tested. The results (data not shown) suggest that 0.5% (vol/vol) Triton X-100 provided us a satisfactory compromise for detergent extraction of DRMs from primary thyrocytes, i.e. a maximal concentration of caveolin in DRMs, without detection of adaptin-ß in the same fraction. For each OptiPrep centrifugation experiment (representative results in Fig. 2Go), caveolin and adaptin-ß immunoblots were performed on all fractions as controls. In our experiments, DRMs were found in fraction 2, and soluble material was found in fractions 9 and 10. As expected, DRMs isolated from thyrocytes contain caveolae, as they are enriched in caveolin (Fig. 2Go).



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  		FIG. 2. Western blot analysis of caveolin (DRMs marker; lower panel) and adaptin-ß (clathrin-associated membrane marker; upper panel) distribution in 0.5% Triton X-100/OptiPrep density gradient fractions prepared from human thyrocytes cultured with 3 µM forskolin for 3 d. Equal volumes from each OptiPrep density gradient fraction were concentrated, subjected to 13% SDS-PAGE, and blotted. Blots were first incubated with the mouse monoclonal adaptin-ß antibody (dilution, 1:1000) and then reprobed with the rabbit polyclonal caveolin antibody (dilution, 1:5000).

 
TSH receptor, G{alpha}s, and G{alpha}q detection in DRMs prepared from thyrocytes
To keep thyrocytes under conditions as close to their differentiation status as possible, 3 µM forskolin was added to the dishes 24 h after seeding the follicles and until the end of the culture (42). In these conditions, the thyrocytes show a higher expression (assessed by Western blot; results not shown) of thyroid differentiation markers, such as sodium/iodide symporter, thyroid peroxidase, and thyroglobulin (42). For the TSH receptor detection, we solubilized membrane proteins from human (or dog) thyrocytes in 1% DDM, and we detected the receptor using two different specific mouse antibodies, recognizing either the ectodomain/A-subunit (28.1) or the membrane-spanning domain/B-subunit (15.2). The observed pattern (Fig. 3Go), which was the same in the two species, was similar to that described in the literature for thyroid tissue (43, 44, 45) and for the receptor immunopurified from human thyrocytes in primary cultures (27), with a band corresponding to the cleaved A-subunit (~50 kDa) recognized by antibody 28.1 and two bands recognized by the antibody 15.2, corresponding to the B-subunit (~44–45 kDa and slightly over ~50 kDa) result of different cleavage sites. With regard to the size of the B-subunit, our results thus confirm the data reported by Tanaka et al. (19), in which primary cleavage of the TSH receptor at site 1 generates an initial B-subunit with a molecular mass of about 51 kDa and, after the removal of the C peptide, of 44–45 kDa. Interestingly, no smear was detected between the two B-subunit bands, which suggests that after cleavage at upstream site 1, the C peptide disintegrates rapidly, as previously discussed by Tanaka et al. (19). A band of about 120–130 kDa detected by antibody 28.1 should be taken as nonspecific binding (and not the uncleaved, mature receptor), as this band is not recognized by the antibody against the B-subunit. Therefore, in human or dog thyrocytes in primary culture, there is a preponderance of the cleaved receptor. Results obtained with human thyrocytes are presented in Fig. 3AGo.



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  		FIG. 3. A, Western blot analysis of the TSH receptor prepared from human thyrocytes in primary culture. Thyrocytes were homogenized, and membrane proteins were solubilized in 1% DDM, denatured, and separated in a 9% SDS-PAGE. After blotting on polyvinylidene difluoride membranes, the receptor was revealed with monoclonal mouse antibodies against the A-subunit (28.1) or the B-subunit (15.2) of the TSH receptor. B, Western blot analysis of TSH receptor distribution between DRMs (fraction 2) and soluble material (fraction 10) of a 0.5% Triton X-100/OptiPrep density gradient prepared from thyrocytes cultured with 3 µM forskolin for 3 d. C+ refers to the DDM-solubilized receptor, as an immunodetection reference. From each OptiPrep density gradient fraction, equal volumes were concentrated, subjected to 9% SDS-PAGE, and blotted as described. Blots were then incubated with a mouse monoclonal antibody (28.1), diluted 1:1000, and directed to an epitope localized in the ectodomain of the receptor. Numbers at the left indicate the molecular mass (kilodaltons) markers.

 
For detection of the TSH receptor by Western blotting in fractions 2 and 10 of OptiPrep density gradients prepared from primary culture of dog and human thyrocytes, we used the monoclonal antibody directed against the ectodomain (28.1) because it proved to be more sensitive. Using specific antibodies, the presence of G{alpha}s and G{alpha}q was also checked in the same fractions. In these experiments we included as positive controls a total protein extract of thyrocytes (~25 µg) for G{alpha} proteins and 20 µl of the 1% DDM-solubilized TSH receptor. All of the results shown are representative of both human and dog thyrocytes, as we observed no differences between these species. The preponderant (cleaved) form of the receptor is mainly concentrated in the heavier fractions of the gradient (Fig. 3BGo). A faint band could only be detected in the DRM fraction after 20 times longer film exposure (data not shown). In a similar way, G{alpha}q proteins are entirely localized in fraction 10, as shown in Fig. 4Go. These results indicate that the TSH receptor and G{alpha}q proteins have the same localization as adaptin-ß, i.e. outside of the sphingolipid-cholesterol-enriched domains. On the other hand, G{alpha}s proteins are not restricted to either of the two fractions. Instead, these proteins show partition in both DRMs and heavy fractions of the gradient, preferably in the latter (Fig. 4Go). Addition of 1 mU/ml TSH for 1 or 24 h did not change the distribution of any of these proteins in the gradient fractions (data not shown).



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  		FIG. 4. Western blot analysis of the distribution of G{alpha}s and G{alpha}q between DRMs (fraction 2) and soluble material (fraction 10) of a 0.5% Triton X-100/OptiPrep density gradient prepared from dog thyrocytes cultured in control conditions. As immunoblot positive controls, total protein extracts from thyrocytes in primary culture (~25 µg) were included. From each OptiPrep density gradient fraction, equal volume of samples were concentrated, separated by 12% SDS-PAGE, and blotted as described. Blots were then incubated with the indicated antibodies, both diluted 1:1000. Numbers at the left indicate molecular mass (kilodaltons).

 
Insulin receptor and flotillin-2 detection in DRMs prepared from thyrocytes
To validate our negative results with TSH receptor and G{alpha}q, we attempted to detect the insulin receptor in thyrocytes. The cells were cultured in the regular medium, but supplemented with 1 mU/ml TSH and depleted of insulin to enhance the expression of this receptor (46, 47). A polyclonal antibody directed against the ß-subunit of the insulin receptor was used. As shown in other tissues (13, 48, 49), we found that DRMs of thyrocytes are substantially enriched in insulin receptor (Fig. 5Go, upper panel). As in other cell types (48), stimulation of the cells with insulin did not significantly affect the distribution of the receptor between DRMs and Triton-soluble fractions (data not shown). The ubiquitously expressed flotillin-2 (50, 51) was used as another positive control. This protein is expressed in thyroid, and at least half of it copurified with DRMs, as assessed by immunoblot (Fig. 5Go, lower panel), using a monoclonal antibody. In these immunoblot experiments, total protein extracts (~25 µg) from thyrocytes were included as a positive control.



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  		FIG. 5. Western blot analysis of the insulin receptor and flotillin-2 distribution between DRMs and soluble fractions of a 0.5% Triton X-100/OptiPrep density gradient prepared from human and dog thyrocytes, respectively. To enhance insulin receptor expression, thyrocytes were grown in an insulin-depleted medium supplemented with 3 µM forskolin and 1 mU/ml TSH, and cells were then processed as described. Equal volumes of samples from each OptiPrep density gradient fraction were concentrated, submitted to a 7.5% (for insulin receptor) and a 12% (for flotillin-2) SDS-PAGE, and blotted as described, and the blots were probed by the indicated antibodies diluted 1:1000. As immunoblot positive controls, total protein extracts from thyrocytes in primary culture (~25 µg) were included.

 
GPI-anchored TSH receptor detection in DRMs prepared from CHO cells
We applied the same Triton X-100/OptiPrep protocol to isolate DRMs from CHO cells stably expressing the wild-type human TSH receptor (JP19 cells) or the receptor ectodomain attached to the plasma membrane by a GPI anchor (GT14 cells), and we used the same antibody (28.1) to detect the TSH receptor in both cell lines. As expected for a GPI-anchored protein (2), this recombinant protein was strongly enriched in fraction 2 of the gradient (Fig. 6Go, upper panel; ~100 kDa). In the heavier, soluble fractions, a TSH receptor of smaller molecular mass (~75 kDa) was detected, presumably a less mature form, still soluble in Triton X-100. Indeed, GPI-anchored proteins become detergent insoluble only after they have been transported to the Golgi apparatus (2, 12, 52). In JP19 cells, the TSH receptor was Triton-solubilized, followed the more dense OptiPrep fractions, and was completely excluded from the DRMs (Fig. 6Go, lower panel), as previously found in thyrocytes.



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  		FIG. 6. Western blot analysis of the TSH receptor distribution between DRMs and the soluble fractions of a 0.5% Triton X-100/OptiPrep density gradient prepared from CHO cells stably expressing recombinant TSH receptor. Upper panel, CHO cells expressing the TSH receptor ectodomain anchored to the membrane by GPI (GT14). TSH receptor ectodomain (~100 kDa) is localized in DRMs (fraction 2), whereas a less mature, lower molecular mass form (~75 kDa) of the receptor is found in the heavy, Triton-soluble fractions. Lower panel, CHO cells expressing the recombinant human TSH receptor whole protein (JP19). Noncleaved mature (~120 kDa) as well as the mannose-rich precursor (~100 kDa) and the cleaved TSH receptor (~50 kDa) are present in the heavy, soluble fractions. C+ refers to the DDM-solubilized receptor, as an immunodetection reference. Cells were homogenized, and membrane proteins were solubilized in 1% DDM, denatured, and loaded, as described. From each OptiPrep density gradient fraction, equal volumes of samples were concentrated, denatured, subjected to 9% SDS-PAGE, and blotted onto polyvinylidene difluoride membranes. As in Fig. 3Go, blots were incubated with a monoclonal antibody (28.1), diluted 1:1000, and directed to an epitope localized in the ectodomain of the receptor. Numbers at the left indicate molecular mass (kilodaltons).

 
Effect of MßCD on cAMP accumulation in thyrocytes
To evaluate whether the destruction of sphingolipid-cholesterol-enriched domains has any consequence on TSH-induced cAMP accumulation, we measured cAMP in human and dog thyrocytes pretreated with 10 mM MßCD for 30 min (37 C). MßCD is a cholesterol chelator and is widely used to reduce the cellular cholesterol. Pretreatment of the cells with MßCD did not impair the cellular cAMP accumulation induced by various TSH concentrations. Again, no significant differences were found between the species. Results for dog thyrocytes are presented in Fig. 7Go. In our conditions, the treatment of thyrocytes with MßCD depleted the cholesterol levels by 90% or more compared with the control conditions (not shown).



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  		FIG. 7. Effect of MßCD on cAMP accumulation in dog thyrocytes grown in primary culture and stimulated by TSH. The cells were rinsed and preincubated for 30 min in KRH medium at 37 C without ({square}) or with ({blacksquare}) 10 mM MßCD. The medium was removed, and cells were incubated with fresh medium containing 25 µM rolipram (a phosphodiesterase inhibitor) and various TSH concentrations for 30 min. The cAMP levels in the cells were measured by RIA.

 
Caveolin immunohistochemistry on human thyroid sections
Thyroid cells are polarized epithelial cells, and the expression of TSH receptor at the cell surface is limited to the basolateral membrane (27, 53). We analyzed the localization of caveolae in thyroid by revealing caveolin by immunohistochemistry on human thyroid sections and using the staining of endothelium as an internal positive control. The expression of caveolin was observed at the apical membrane of thyrocytes (Fig. 8Go).



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  		FIG. 8. Immunohistochemical detection of caveolin in normal human thyroid. Whenever caveolin expression is detected (noted by black arrows), it appears to be localized at the apical pole of thyroid cells. Paraffin-embedded human thyroid sections were treated for immunohistochemistry analysis as described. Caveolin was detected by a mouse monoclonal antibody (A; diluted 1:100) and a rabbit polyclonal antibody (B; diluted 1:50). Caveolin staining in endothelium (noted by a red arrow) was used as an internal positive staining control. Photos were taken from representative results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In our study we show evidence by three different approaches that sphingolipid-cholesterol-enriched domains (including those containing caveolin) are present in the membranes of normal thyroid cells in primary culture. First, mRNA of the caveolin-1 gene was detected by Northern blotting and RT-PCR. Second, caveolin, insulin receptor, and flotillin-2 were detected in the low density fractions of the Triton X-100/OptiPrep density gradients, whereas the negative control adaptin-ß was not. Last, caveolin immunoreactivity was also detected in human thyroid slices. This is the first biochemical demonstration of DRMs and caveolae in thyroid cells uncontaminated by fibroblasts and endothelial cells of the tissue (54).

Our immunohistochemical data suggest an apical localization of caveolae and caveolin, in accordance with the early electron microscopy observations of cholesterol enrichment (55) and lower fluidity (56) in the apical membrane of thyrocytes compared with the basolateral membrane. Thyroid cell caveolae therefore may not contain the TSH receptor that is expressed at the basolateral membrane (27, 53). The roles of caveolae and caveolin in thyrocytes apical domain are now under investigation in our laboratory. On the other hand, as the insulin receptor is presumed to be located in the basolateral membrane, its presence in DRMs suggests that noncaveolar, sphingolipid-cholesterol-enriched domains should also be present in the basolateral membrane.

This study shows for the first time direct solubilization of the TSH receptor from thyrocytes in primary cultures. Neither the TSH receptor nor its effectors Gs and Gq are concentrated in DRMs in human or dog thyrocytes, and TSH treatment of thyrocytes did not change the distribution of TSH receptor in OptiPrep gradients. The presence of a very small amount of TSH receptor in the DRM fraction of the OptiPrep gradient could be due to contamination of DRMs with some fluid, liquid-disordered domains during the cooling of membranes (57), i.e. could result from a technical artifact. Against this hypothesis, a protein marker of liquid-disordered domains, adaptin-ß, i.e. a control for incomplete solubilization of bulk membrane (39), is excluded from DRMs. Moreover, Triton X-100 is considered one of the most reliable detergents for analyzing a possible association of proteins with sphingolipid-cholesterol-enriched domains (39). A more likely explanation for the minor presence of TSH receptor in DRMs fractions is the existence of semiordered domains, partially detergent-resistant and lying at the margins between liquid-disordered and liquid-ordered domains (40). One might hypothesize that a small part of TSH receptor would be localized in these marginal domains. Our observation that the bulk quantity of TSH receptor is found in the soluble, heavier fractions is in accordance with the predictions of London and Brown (57) that transmembrane polypeptides are generally not expected to pack well into a liquid-ordered environment and that, as a consequence, DRMs are relatively poor in transmembrane proteins (11), although they may be enriched in some (12, 58, 59). Very little is known about how the latter may occur. Palmitoylation might contribute to sphingolipid-cholesterol domain targeting (8). Again in the case of TSH receptor, its putative palmitoylation site is not crucial for cAMP or phosphoinositide signaling (60).

Two hypothesis have been proposed concerning the relation of TSH receptor with lipid rafts 1) TSH receptor monomers, rising from receptor oligomers activation by TSH, may move into lipid rafts harboring G proteins (61). 2) More recently, the same group (62) reported that "TSH receptorGFP complexes concentrate to GM1 enriched lipid rafts microdomains." However, the latter hypothesis was based on an immunofluorescence approach, the resolution of which is considered to be lower than the lipid microdomain diameter (63, 64, 65). Moreover, BODIPY-FL dye, a fluorescent analog of the ganglioside GM1, added to the cells to visualize the rafts, will probably not preferentially stain rafts because BODIPY-FL is in the hydrocarbon chains of the ganglioside. The partition of a given protein into lipid microdomains should be assessed by several techniques. Our work has critically examined the partition of TSH receptor between DRMs and Triton-soluble membranes by standard biochemical and functional methods and demonstrated no localization of the receptor in the DRMs.

With respect to G protein partitions, the absence of G{alpha}q protein from thyrocyte DRMs shows that sequestration of G{alpha}q in such sphingolipid-cholesterol-enriched domains away from the TSH receptors does not explain the low coupling efficiency of the TSH receptor to Gq in human thyroid and the absence of coupling in dog thyroid (22). This contrasts with the concentration of Gq in lung caveolae (66). On the other hand, our results showing both short (45 kDa) and long (52 kDa) splice variants of G{alpha}s partitioning between DRMs and soluble, heavier fractions are in agreement with the observations in other cell types (67, 68, 69, 70, 71). According to Schwencke et al. (70), the large stoichiometric excess of Gs proteins over their effectors allows an indiscriminate partition of Gs between sphingolipid-cholesterol-enriched domains and the bulk of the phospholipidic membrane. Interestingly, Li and co-workers (67) reported that the G{alpha}s associated with lipid microdomains is in the inactive GDP-bound form, whereas the active G{alpha}s is Triton soluble.

We cannot exclude the possibility that our detergent solubilization conditions might have disrupted the localization of TSH receptor and G{alpha}q proteins from DRMs, leading to an underestimation of protein partition in DRMs (72). This seems unlikely, as the insulin receptor, a transmembrane protein known to be located in sphingolipid-cholesterol-enriched domains in other cell types (13, 48, 49) and very susceptible to being lost from DRMs whenever detergent extraction is used (48), is found in DRMs prepared from thyrocytes, following our protocol. Moreover, other sphingolipid-cholesterol-enriched domain-associated proteins are found in DRMs of thyrocytes, caveolin and flotillin-2 (50, 73); caveolin is exclusively located in DRMs, whereas flotillin-2 presents the same distribution as the one found in phagosomes (73). It is established that GPI-anchored proteins preferentially locate in DRMs. Indeed, whereas the mature GPI-anchored ectodomain form of the TSH receptor is only present in DRMs of CHO cells, the whole native TSH receptor is not. This result demonstrates that, independently of the cell type, CHO cells or thyrocytes, the TSH receptor is not constitutively located in DRMs, except when covalently coupled to a GPI anchor. There is therefore no argument in favor of a relation of the TSH receptor signaling complexes, either with Gq or Gs, with thyrocyte DRMs.

This conclusion is strengthened by the results of MßCD treatment. MßCD is strictly surface-acting and selectively extracts membrane cholesterol by including it in a central nonpolar cyclic cavity of a seven-glucose residue ring (74). This compound is considered the most effective agent for removing cholesterol (75), and our cholesterol quantification experiments showed that this was the case. A 30-min preincubation with 10 mM MßCD did not affect the TSH-induced generation of cAMP in thyrocytes. In other studies, MßCD impaired signaling mediated by IgE (76), T cell receptor (15), and glial cell line-derived neurotrophic factor receptor/RET (36), but it did not influence Gs signaling in rat salivary epithelial A5 cells (69). Therefore, functional experiments do not support a role for DRMs in the TSH receptor-Gs-cAMP cascade either.


   Footnotes
 
This work was supported by the Ministère de la Politique Scientifique, the Fonds National de la Recherche Scientifique, the Fonds de la Recherche Scientifique Médicale, the Fonds Cancérologique de la CGER, the Association contre le Cancer, the Association Sportive contre le Cancer, Télévie, and fellowships from Fundação para a Ciência e a Tecnologia (Portugal; to M.J.C.), the University of Brussels (to Y.S.), and Télévie (to V.V.).

M.J.C. and Y.S. contributed equally to this work.

Abbreviations: CHO, Chinese hamster ovary; DDM, dodecyl-ß-D-maltoside; DRM, detergent-resistant membrane; GPI, glycosyl phosphatidylinositol; KRH, Krebs-Ringer-HEPES; MßCD, methyl-ß-cyclodextrin.

Received October 23, 2003.

Accepted for publication December 3, 2003.


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