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Endocrinology Vol. 147, No. 6 3107-3113
Copyright © 2006 by The Endocrine Society

Evidence that Human Thyroid Cells Express Uncleaved, Single-Chain Thyrotropin Receptors on Their Surface

Chun-Rong Chen, Gregorio D. Chazenbalk, Kolja A. Wawrowsky, Sandra M. McLachlan and Basil Rapoport

Autoimmune Disease Unit, Cedars-Sinai Research Institute and University of California, Los Angeles School of Medicine, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Basil Rapoport, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: rapoportb{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The prevailing concept is that, in human thyroid tissue in vivo, all cell-surface TSH receptors (TSHR) cleave into disulfide linked A and B subunits. Because this viewpoint is based on studies using homogenized thyroid tissue and because of TSHR fragility, we studied TSHR subunit structure in intact thyroid cells, primary human thyrocyte cultures, FRTL-5 rat thyroid cells, and WRO (follicular) and NPA (papillary) thyroid cancer cell lines. To overcome the handicap of very low TSHR expression in thyroid cells, we generated a TSHR-expressing adenovirus (TSHR-Ad-RGD) with an integrin-binding RGD motif enabling efficient entry into cells lacking the coxsackie-adenovirus receptor. Two days after TSHR-Ad-RGD infection, [125I]TSH cross-linking to intact cells revealed uncleaved, single-chain TSHR as well as cleaved TSHR A subunits on the surface of all four thyroid cell types. The extent of TSHR cleavage, which is independent of the level of TSHR expression, was consistently lower in the human thyroid cancer cell lines than in the other cell lines. In flow cytometry studies after TSHR-Ad-RGD infection, strong signals were detected in all four thyroid cell types using a monoclonal antibody that primarily recognizes the uncleaved TSHR. Finally, using the same monoclonal antibody, confocal microscopy confirmed the presence of single-chain TSHR on TSHR-Ad-RGD-infected thyroid cells. In summary, TSH covalent cross-linking, flow cytometry, and confocal microscopy demonstrate the presence of uncleaved TSHR on the human thyrocyte surface. These data provide stronger evidence for this alternative than the contrary view based on the finding of only cleaved TSHR in homogenized thyroid cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS AT present a vigorous debate, with potential implications for understanding the pathogenesis of Graves’ disease, regarding the in vivo subunit structure of the TSH receptor (TSHR). That the TSHR in thyrocyte homogenates contains A and B subunits linked by disulfide bonds has been known for more than 20 yr (1). Translation from a single mRNA transcript (2, 3) indicates that these subunits are formed by intramolecular cleavage. The textbook view based on an evaluation of the literature is that "all wild-type TSHR receptors on the surface of thyroid follicular cells seem to be in (the) cleaved form" (4). The present study reexamines this conclusion.

Initial TSH cross-linking studies to membrane preparations from homogenized thyrocytes (1) or nonthyroidal cells expressing the recombinant TSHR (5) indicated that TSHR cleavage into A and B subunits was complete. Also in cell extracts, immunodetection with TSHR-specific monoclonal antibodies (mAb) confirmed complete cleavage into subunits (renamed {alpha} and ß) (6). In contrast, TSH cross-linking to intact cells in monolayer culture, but not to membrane preparations from the same cells, revealed a proportion (typically 30–50%) of single-chain, uncleaved TSHR on the cell surface (5). This finding, consistent with previous studies on intact FRTL-5 rat thyroid cells (7), raised the possibility that, in vivo, without cell disruption, most TSHR on the cell surface were in the uncleaved form (5). Subsequently, however, elimination of TSHR cleavage by mutagenesis (8), as well as precursor labeling studies (9), revealed that TSHR cleavage into A and B subunits is not an artifact consequent to cell homogenization. However, because cell damage or sickness increases TSHR intramolecular cleavage (5, 10), the extent of this cleavage on intact thyrocytes in vivo remains an unanswered question. Because of the absence of uncleaved TSHR in human thyrocyte preparations, it has been argued that detection of single-chain recombinant receptors in transfected nonthyroidal cells reflects the "mistaken" detection of monomeric immature receptors (11). Alternatively, it is considered probable that the high level of recombinant TSHR expression overwhelms capacity of the cleavage enzyme (12).

It is obviously not possible to determine directly whether human thyrocytes in vivo express uncleaved TSHR on their surface. Moreover, ex vivo studies require removing the thyroid and enzymatically dispersing cells or homogenizing tissue. Study of intact thyroid cells in culture offers the closest alternative to the in vivo situation. However, TSHR expression on cultured thyrocytes is very low, and such cells are difficult to transfect with TSHR plasmid DNA. To overcome this handicap, we expressed the human TSHR in human thyroid cells by means of an adenovirus vector with an RGD motif in the HI loop of its fiber knob (13). Binding of this motif to integrin facilitates infection of cells lacking the coxsackie-adenovirus receptor (CAR). Our finding of uncleaved TSHR on the surface of cultured thyroid cells provides stronger evidence for this phenomenon than the prevailing contrary view that is based on the finding of only cleaved TSHR in homogenized thyroid cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
TSHR adenovirus construction
The TSHR cDNA in the vector pECE-neo (3) was digested with EcoRI, blunted with DNA polymerase Klenow fragment (USB, Cleveland, OH), digested with XbaI, and ligated into the transfer vector pHMCMV6 (kindly provided by Dr. H. Mizuguchi, Tokyo, Japan). Except when indicated, restriction enzymes were from New England Biolabs (Beverly, MA). Insert-positive plasmids were digested with I-CeuI and PI-SceI (BD Biosciences Clontech, Palo Alto, CA) and ligated into the same sites of an adenovirus containing an RGD motif on the HI loop of its fiber knob (13) (also provided by Dr. H. Mizuguchi). The adenovirus vector with the TSHR cDNA (TSHR-Ad-RGD) was linearized with PacI and transfected into HEK293 cells (American Type Culture Collection, Manassas, VA) with SuperFect (Qiagen, Valencia, CA). TSHR-Ad-RGD was propagated in HEK293 cells and purified by CsCl density gradient centrifugation, and viral particle concentration was determined by measuring the absorbance at 260 nm.

Cell cultures
CHO cells were purchased from American Type Culture Collection. FRTL-5 is a line of well-differentiated rat thyroid cells (14). Two human thyroid carcinoma lines were kindly provided by Dr. Jerome Hershman (Los Angeles, CA). NPA and WRO are differentiated papillary and follicular carcinoma cell lines, respectively (15). Aliquots of a primary culture of human thyroid cells had been prepared previously and cryopreserved (16, 17). Sterile human thyroid tissue suitable for primary cultures is no longer available. CHO, untransfected or stably expressing the TSHR (3, 18), and FRTL-5 cells were cultured in Ham’s F-12 medium. WRO, NPA, and primary human thyroid cells were cultured with DMEM. Media were supplemented with 10% fetal calf serum (5% for FRTL-5 cells), penicillin (100 U/ml), gentamycin (50 µg/ml), and fungizone (2.5 µg/ml). FRTL-5 cell medium also contained 2.5 mU/ml bovine TSH, 5 µg/ml transferrin, and 10 mU/ml insulin (all from Sigma (St. Louis, MO). When indicated, cell monolayers were infected with TSHR-Ad-RGD (1010 particles/ml). After approximately 48 h, the medium was removed and the cells were rinsed before performing the assays described below.

Covalent cross-linking of radiolabeled TSH
Highly purified bovine TSH (purchased from the National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, Dr. A. F. Parlow) was radiolabeled with 125I to a specific activity of approximately 60 µCi/µg using Bolton-Hunter reagent (4400 Ci/mmol; PerkinElmer Life Sciences, Boston, MA). Confluent 100-mm-diameter dishes of cells infected with or without TSHR-Ad-RGD for 48 h were rinsed, and 5 µCi [125I]TSH in 5 ml modified Hanks’ buffer (without NaCl) supplemented with 220 mM sucrose and 0.25% BSA (binding buffer) was added under conditions described in the text. Unbound [125I]TSH was removed by rinsing the cells three times with ice-cold binding buffer. Disuccinimidyl suberate (1 mM; Sigma) in 10 mM Na phosphate buffer (pH 7.4) was then added for 20 min at room temperature. The cross-linking reaction was terminated by the addition of 50 mM ammonium acetate (final concentration), and the cells were rinsed twice with PBS and scraped into 10 mM Tris, (pH 7.5) containing phenylmethylsulfonyl fluoride (100 µg/ml) and leupeptin (1 µg/ml) (both from Sigma). Cells were homogenized using a Polytron homogenizer and centrifuged for 5 min at 4 C (500 x g). The supernatant was centrifuged (15 min, 10,000 x g, 4 C), and the pellet was resuspended in 40 µl 10 mM Tris (pH 7.5). After the addition of Laemmli buffer containing 0.7 M ß-mercaptoethanol (30 min at 50 C), the samples were electrophoresed on 7.5% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). Radiolabeled proteins were visualized by autoradiography on Biomax MS x-ray film (Eastman Kodak, Rochester, NY).

Flow cytometry
After infection for 48 h with TSHR-Ad-RGD (1010 particles/ml), cells in monolayer were resuspended with 1 mM EDTA and 1 mM EGTA in calcium and magnesium-free PBS and 10 mM HEPES (pH 7.4). After washing with PBS containing 10 mM HEPES (pH 7.4) 2% heat-inactivated fetal calf serum, and 0.05% NaN3, the suspended cells (~106) were incubated for 30 min at room temperature in 100 µl of the same buffer containing the antibodies mentioned below, all at 10 µg/ml final concentration. After rinsing, the cells were incubated for another 30–45 min at 4 C with fluorescein isothiocyanate-conjugated, affinity-purified goat antimouse IgG (1:100 dilution; Caltag Laboratories, Burlingame, CA). Cells were then washed and analyzed using a Beckman FACScan flow cytometer (BD Biosciences, San Jose, CA). Cells stained with propidium iodide (1 µg/ml) were excluded from analysis. Specific geometric mean fluorescence values were calculated after subtraction of background fluorescence obtained using the second antibody alone.

Murine mAb
The following mAb were used for flow cytometry and confocal microscopy. The first was mAb 2C11 to the TSHR (19) (Serotec, Raleigh, NC). The 2C11 linear epitope includes TSHR amino acid residues 354–359 (20) within the C-peptide region that is deleted from most receptors undergoing intramolecular cleavage (21). In our laboratory, 2C11 recognizes a synthetic peptide encompassing TSHR amino acid residues 352–371 (our unpublished observation). The second was mouse mAb 17 (our unpublished observation) generated by immunizing BALB/c mice with the TSHR A subunit in an adenovirus vector (22). This mAb (nonstimulatory and with minimal TSH binding inhibitory activity) recognizes the native TSHR on the surface of stably transfected cells. The third was polyclonal normal mouse IgG.

Confocal immunofluorescence microscopy
The cells indicated in the text were cultured in Lab-Tek II chamber slides (Nalge Nunc International, Naperville, IL). After culture for 48 h in medium containing TSHR-Ad-RGD (1010 particles/ml), cells were fixed for 30 min at 4 C with prechilled, sterile 4% paraformaldehyde in PBS and then permeabilized for 15 min with 0.01% Triton X-100 at room temperature. Slides were incubated for 1 h at room temperature in PBS containing 1% BSA followed by the indicated antibody (10 µg/ml final concentration) in 0.2 ml of 0.5% BSA in PBS. Slides were rinsed, and bound antibody was detected with Alexa Fluor 488 chicken antimouse IgG (H+L) (1:200 dilution; Invitrogen, Carlsbad, CA). Nuclei were counterstained with 7-amino actinomycin D (5 µg/ml final concentration; Invitrogen). Images were acquired with a Leica (Mannheim, Germany) TCSSP confocal microscope and a PlanApo x100, numerical aperture 1.4 lens.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Covalent cross-linking of radiolabeled TSH to thyroid cells
Radiolabeled TSH covalent cross-linking to intact CHO cells in monolayer culture reveals the subunit structure of the mature TSHR on the cell surface (5, 23). Because of evidence that the degree of TSHR cleavage into A and B subunits is greatly influenced by cell manipulation (5, 10), we evaluated the influence of TSH binding conditions before TSH cross-linking. As observed in numerous previous studies with CHO cells stably expressing the TSHR, autoradiography of [125I]TSH-TSHR complexes electrophoresed under reducing conditions revealed two forms of receptor (Fig. 1Go). An approximately 80-kDa complex represents the TSHR A subunit of the cleaved receptor dissociated from the B subunit (the latter is not visible because it cross-links minimally, if at all, with [125I]TSH). A second complex of greater mass ({approx}130 kDa) indicates TSH cross-linked to the uncleaved TSHR that remains as a single chain despite disulfide bond reduction. Binding of [125I]TSH to TSHR-CHO cells at either 37 or 4 C before covalent cross-linking influenced the ratio of cleaved vs. uncleaved TSHR on the cell surface. Thus, a greater proportion of TSHR A subunits were evident after TSH binding for 2 h at 37 C (Fig. 1AGo) than at 4 C (Fig. 1BGo). Densitometric quantitation of these data are depicted in Fig. 1CGo. TSH binding for 1 h at 4 C before cross-linking yielded a relatively high proportion of uncleaved vs. cleaved TSHR, and this condition was used in subsequent experiments.


Figure 1
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  		FIG. 1. Radiolabeled TSH covalent cross-linking to the TSHR on the surface of CHO cells. [125I]TSH was bound to monolayer cultures of cells stably expressing the TSHR at either 37 C (A) or 4 C (B) for the indicated periods before covalent cross-linking (see Materials and Methods). Detergent-extracted proteins were electrophoresed on 7.5% polyacrylamide gels under reducing conditions followed by autoradiography for ~16 h. C, Densitometric quantitation of the extent of TSHR cleavage. For each lane, net densitometric units for the uncleaved and cleaved TSHR A subunit are expressed as a percentage of the sum of these two values. Data shown are the mean ± range of duplicate determinations.

 
To enhance TSHR expression in a variety of cell types that may lack the CAR, we generated a TSHR-expressing adenovirus vector that also expresses an RGD motif (TSHR-Ad-RGD). This motif enables adenovirus entry into cells by binding to integrin, a ubiquitous matrix protein. In preliminary flow cytometry studies, we confirmed expression of TSHR-Ad-RGD in CHO cells, which was indeed considerably greater than TSHR expression using standard adenovirus (pAdHMCMV) vector (24) (data not shown).

Turning to the primary goal of the present study, as determined by [125I]TSH cross-linking, TSHR-Ad-RGD infection induced TSHR expression in each of four different thyroid cell types, namely human thyrocyte primary cultures, FRTL-5 rat thyroid cells, and WRO (follicular) and NPA (papillary) thyroid cancer cell lines (15). All four thyroid cell types expressed uncleaved, single-chain TSHR as well as cleaved TSHR A subunits on their surface (Fig. 2AGo). Of interest, the extent of TSHR cleavage was consistently lower in the human thyroid cancer cell lines (WRO and NPA) than in the other cell lines (Fig. 2BGo). Without TSHR-Ad-RGD infection, TSHR expression levels in these human thyroid cell types was too low to be detected except for FRTL-5 rat thyroid cells after prolonged autoradiographic exposure (2 wk, with consequent high background) (Fig. 2CGo). To avoid overexposure with the TSHR-Ad-RGD-infected FRTL-5 cells, the sample applied to the gel was diluted 10-fold. Most important, the proportion of cleaved TSHR was not reduced in the TSHR-Ad-RGD infected vs. uninfected FRTL-5 cells despite the greater level of TSHR expression in the former. This observation is consistent with previously published evidence (25) that the proportion of cleaved to uncleaved TSHR remains unchanged in transfected CHO cells despite a 10-fold variation in TSHR cell-surface expression (Fig. 2DGo).


Figure 2
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  		FIG. 2. Radiolabeled TSH cross-linking to thyroid cells. A, TSH cross-linking performed 2 d after infection with TSHR-Ad-RGD of four different thyroid cell types: human thyrocyte primary cultures (HT primary), FRTL-5 rat thyroid cells, WRO follicular thyroid carcinoma cells, and NPA papillary thyroid cancer cells. Also included are similarly infected CHO cells. Detergent-extracted proteins were electrophoresed on 7.5% polyacrylamide gels under reducing conditions followed by autoradiography for ~16 h. B, Densitometry on the data shown in A. Data represent the mean ± SD of triplicate values. C, [125I]TSH cross-linking to uninfected FRTL-5 cells (left lane) and FRTL-5 cells 2 d after TSHR-Ad-RGD infection (right lane). Because of the very weak signal with uninfected cells, autoradiographic exposure was for 2 wk with resulting high background. To avoid overexposure with the infected FRTL-5 cells, the amount of radioactivity applied to the gel was reduced. D, Previously published data indicating that the proportion of cleaved to uncleaved TSHR remains unchanged in transfected CHO cells despite a 10-fold variation in TSHR cell-surface expression. [Reproduced with permission from Chazenbalk et al.: Endocrinology 137:4586–4591 (25 ). © The Endocrine Society.]

 
Flow cytometric detection of uncleaved TSHR on the surface of thyroid cells
If TSHR intramolecular cleavage on the surface of thyroid cells is complete, then receptors lacking the C-peptide region should not be detected by flow cytometry. The presence of the C-peptide region can be determined using mAb 2C11 whose epitope (amino acid residues 354–359) lies within this region (Fig. 3AGo). We previously confirmed the validity of this determination using a TSHR in which the C-peptide region has been deleted by mutagenesis (26). After TSHR-Ad-RGD infection of FRTL-5 rat thyroid cells, human thyrocyte primary cultures, and differentiated human thyroid cancer cells (NPA and WRO), strong signals were detected on flow cytometry using mAb 2C11 (Fig. 3BGo). These data clearly indicate that a significant proportion of TSHR remain uncleaved on the surface of thyroid cells. As controls, a mAb to the A-subunit region (mAb 17) also provided strong fluorescence signals in these thyroid cells, as well as in a stably transfected CHO cell line. We want to emphasize that we regard these data as qualitative rather than quantitative. Potential differences in mAb affinities, concentrations, and recognition by second antibody make it impossible to assign a quantitative value for the extent of TSHR cleavage by flow cytometry.


Figure 3
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  		FIG. 3. Flow cytometric detection of uncleaved TSHR on the surface of thyroid cells. A, The epitope of mAb 2C11 (19 ) (amino acid residues 354–359) lies within the C-peptide region that is mostly deleted during intramolecular cleavage of the TSHR into A and B subunits (11 21 ). For this reason, mAb 2C11 recognizes primarily the single-chain, uncleaved TSHR. Specificity of mAb 2C11 for this epitope has been established by ELISA (20 ) as well as by TSHR mutagenesis (26 ). In contrast, mAb 17, generated by immunization with the A-subunit alone and lacking other portions of the TSHR, cannot distinguish between cleaved and uncleaved TSHR. Incidentally, unlike mAb 2C11, the mAb 17 epitope is conformational and cannot be mapped using synthetic TSHR peptides (our unpublished data). B, After TSHR-Ad-RGD infection of FRTL-5 rat thyroid cells, human thyrocyte primary cultures, and differentiated human thyroid cancer cells (NPA and WRO), strong signals were detected on flow cytometry using both mAb 2C11 and mAb 17. Flow cytometry was performed 2 d after TSHR-Ad-RGD infection of FRTL-5 rat thyroid cells, human thyrocyte primary cultures, and differentiated human thyroid cancer cells (NPA and WRO), as well with uninfected CHO cells stably expressing the TSHR. As a negative control, cells were probed with normal mouse IgG.

 
Confocal microscopy
Antibody binding to intact cells in monolayer culture detected by confocal microscopy involves even less cell manipulation than TSH cross-linking and flow cytometry. Cellular proteins are fixed with paraformaldehyde immediately after removing culture medium. To confirm cell-surface expression of single-chain, uncleaved TSHR, we studied TSHR-Ad-RGD-infected WRO human thyroid cells and, as a control, CHO cells. Normal mouse IgG yielded extremely low background staining (Fig. 4Go, left column). The mAb to the C-peptide region (2C11) revealed clear cell-surface signals on both WRO thyroid and CHO cell lines, indicating the presence of uncleaved TSHR (Fig. 4Go, middle column). As expected, the mAb to the A subunit also detected TSHR on the cell surface and both mAb revealed TSHR within the cells, presumably in the synthetic pathway (Fig. 4Go, right column).


Figure 4
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  		FIG. 4. Confocal microscopic detection of uncleaved TSHR on the thyroid cell surface. Two days after TSHR-Ad-RGD infection of WRO human thyroid cells and CHO cells in chamber slides, medium was removed and the cells were fixed immediately with paraformaldehyde and permeabilized with 0.01% Triton X-100 (see Materials and Methods). Cells were probed with mAb to the C-peptide region (2C11; recognizes primarily the single-chain, uncleaved TSHR) and to the A subunit (mAb 17 binds to both cleaved and uncleaved TSHR). The same concentration of normal mouse IgG (NmIgG; 10 µg/ml) was used as a control. Primary antibody binding was assessed using Alexa Fluor 488 chicken antimouse IgG, and nuclei were counterstained with 7-amino actinomycin D. Images were acquired with a Leica TCSSP confocal microscope and a PlanApo x100, numerical aperture 1.4 lens.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Intramolecular cleavage of the TSHR is an enigmatic phenomenon. TSH binds with similar high affinity to both cleaved and uncleaved receptors on the cell surface (5). Cleavage is not necessary for either TSHR constitutive activity (27) or receptor activation by TSH (28). However, TSHR cleavage may, perhaps fortuitously, be related to development of Graves’ disease, a disease that occurs only in humans. In transfected, nonthyroidal cells, TSHR cleavage into A and B subunits is followed by shedding of some of the A subunits linked to the largely transmembrane B subunit (29, 30). In an animal model of Graves’ disease, A subunits are more potent than the wild-type TSHR in inducing thyroid-stimulating antibodies (22). For these reasons, it is of interest to understand the extent of TSHR cleavage on the surface of thyrocytes.

The fragility of the TSHR requires appreciation to interpret data on TSHR cleavage and A-subunit shedding. As mentioned above, uncleaved recombinant TSHR are only detected on the surface of intact nonthyroidal cells but not in the same cells after homogenization (5). Culture of transfected nonthyroidal cells under suboptimal conditions (serum deprivation) increases TSHR cleavage, degradation, and A-subunit shedding (10). Although A-subunit shedding (studied in serum-poor medium) has been attributed to disulfide bond disruption by protein disulfide isomerase (29), there is also evidence for A-subunit release by spontaneous N-terminal degradation of the B subunit (23). Finally, TSH binding activity is greatly reduced (~70-fold) in TSHR extracted by detergent from cell monolayers compared with cells detached from the culture dish by scraping (31). For these reasons, we suggest caution in accepting the current consensus that, in thyroid tissue in vivo, essentially all TSHR are cleaved into A and B subunits (4, 12, 32, 33) when based on studies involving thyroid tissue freezing, thawing, and homogenization (11) or "sick" cultured cells.

Although it is not possible to obtain direct information on the extent of TSHR cleavage in the thyroid in vivo, in the present study, we addressed this question using the closest alternative, namely a variety of thyroid cell types including our last remaining vials of primary human thyroid cells (administrative requirements preclude additional preparations). Moreover, we overcame the obstacle of very low TSHR expression on thyrocytes by using a TSHR-expressing adenovirus whose binding to cells does not require the CAR (13). We recognize that this approach, although necessary for TSHR detection, is not "physiological" and represents a limitation to interpretation of our findings. Thus, it is possible that TSHR overexpression contributed to our detection of uncleaved TSHR on the cell surface because of "saturation of the cellular machinery" for TSHR intramolecular cleavage (12). We cannot exclude the possibility that TSHR in untransfected human thyroid cells (were they detectable) would be entirely cleaved. However, we point out that there is no evidence that TSHR cleavage enzyme activity is limiting in human thyroid cells. Conversely, our present data indicate that TSHR overexpression is not associated with cleavage enzyme saturation in well-differentiated FRTL-5 thyroid cells. We confirm the presence of uncleaved TSHR on the surface of these cells (7) and demonstrate further that the proportion of cleaved to uncleaved receptors is similar in wild-type cells and cells overexpressing the TSHR. In addition, because of the importance of this point, we present previously published data (25) that clearly refute the concept of cleavage enzyme saturation in nonthyroidal cells (Fig. 2DGo). It is also of interest that the extent of receptor cleavage appears to vary among different thyroid cell types. The relative amount of cleaved TSHR is proportionately greater in FRTL-5 cells than in two thyroid cancer cell lines (WRO and NPA), whereas human thyrocyte primary cultures display an intermediate degree of cleavage. Speculation on the significance of this observation is beyond the scope of the present study.

Our study demonstrates uncleaved TSHR on the surface of intact thyroid cells using three different methodological approaches. First, unlike immunoprecipitations and immunoblots on cell homogenates, TSH cross-linking to intact cells only visualizes mature cell-surface TSHR and not intracellular precursors or degradation products. Moreover, we optimized our TSH cross-linking protocol to maximize cell "health" after binding in NaCl-free medium (necessary to enhance TSH binding to attain reasonable autoradiographic exposures). A shorter time period in this medium, as well as incubation at 4 C rather than 37 C, is less damaging to cells. TSH binding at 4 C will also avoid receptor internalization and will minimize activity of the presently unknown TSHR cleavage enzyme, thereby providing a more accurate "snapshot" of the extent of TSHR cleavage at one particular time. TSH cross-linking under these conditions revealed a greater degree of uncleaved vs. cleaved TSHR on the cell surface. These data again emphasize the difficulty in concluding that TSHR cleavage is complete in homogenized thyroid cell preparations.

The second approach that we used, perhaps more gentle than TSH cross-linking, was flow cytometric detection of uncleaved TSHR on the surface of intact thyroid cells by means of an mAb whose epitope is in the C-peptide region. This region is not present in TSHR that have undergone intramolecular cleavage into A and B subunits (11, 21). TSHR recognition by the C-peptide region-specific mAb was very similar to that of an mAb generated to the A subunit alone, a component of both cleaved and uncleaved receptors. Previous flow cytometry studies on TSHR-expressing nonthyroidal cells (not thyroid cells as in the present study) interpreted differential mAb recognition as a quantitative assay for the extent of TSHR cleavage (34). On the basis of the latter assay, approximately half of TSHR on the surface of thyroid cells are cleaved into subunits (present study). However, two different mAb can vary in concentration, affinity, and second antibody recognition. Therefore, we believe that our flow cytometry data should be viewed as qualitative, not quantitative. We can only conclude that a readily detectable proportion of TSHR on the surface of cultured thyroid cells transfected with the TSHR remains uncleaved. Confocal microscopy, the third approach that we used, involves even less thyrocyte manipulation than TSH cross-linking or flow cytometry. Proteins in healthy cells in monolayer culture are instantaneously fixed before TSHR immunodetection. The presence of uncleaved TSHR on the surface of WRO thyrocytes (strong signal with a C-peptide region-specific mAb) is unequivocal.

In summary, our study using four different thyroid cell types raises an alternative to the prevailing concept that TSHR on the thyrocyte surface in human thyroid tissue are fully cleaved into A and B subunits in vivo. No ethical procedure can definitely determine TSHR subunit structure in human thyroids in vivo. Ex vivo studies are necessary. The present concept is based on TSHR extracted after freezing, thawing, and homogenizing thyroid tissue. Although not ideal, studying transfected, intact cultured cells approaches more closely the in vivo situation. TSH covalent cross-linking, flow cytometry, and confocal microscopy clearly demonstrate the presence of uncleaved TSHR on the surface of cultured thyrocytes. Finally, the observation that the extent of TSHR cleavage may differ in normal and neoplastic thyrocytes raises interesting questions for additional investigation.


   Acknowledgments
 
We thank Drs. H. Mizuguchi (Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan) and Y. Nagayama (Nagasaki University School of Biomedical Sciences, Nagasaki, Japan) for making the RGD-adenovirus vector available to us. We are also grateful for contributions by Dr. Boris Catz (Los Angeles, CA).


   Footnotes
 
This work was supported by National Institutes of Health Grants DK 19289 (to B.R.) and DK 54684 (to S.M.M.).

C.-R.C., G.D.C., K.A.W., S.M.M., and B.R. have nothing to declare.

First Published Online February 23, 2006

Abbreviations: CAR, Coxsackie-adenovirus receptor; mAb, monoclonal antibody; TSHR, TSH receptor; TSHR-Ad-RGD, TSHR-expressing adenovirus vector expressing an RGD motif.

Received November 29, 2005.

Accepted for publication February 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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G. Kleinau and G. Krause
Thyrotropin and Homologous Glycoprotein Hormone Receptors: Structural and Functional Aspects of Extracellular Signaling Mechanisms
Endocr. Rev., April 1, 2009; 30(2): 133 - 151.
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