help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-1002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/1/4    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chazenbalk, G. D.
Right arrow Articles by Rapoport, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chazenbalk, G. D.
Right arrow Articles by Rapoport, B.
Endocrinology Vol. 145, No. 1 4-10
Copyright © 2004 by The Endocrine Society

Does Thyrotropin Cleave Its Cognate Receptor?

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

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

Address all correspondence and requests for reprints to: Basil Rapoport, M.B., 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
 
A recent report of major pathophysiological significance, and opposed to present concepts, is that TSH (but not MS-1, a hamster monoclonal thyroid-stimulating antibody), cleaves the single-chain TSH receptor (TSHR) on the cell surface into its two-subunit form. We reassessed the issue using two approaches. First we wished to confirm the flow-cytometric assay previously used to quantitate TSHR cleavage. We used CHO cell lines expressing large (TSHR-10,000 cells) or conventional (TSHR-0 cells) numbers of TSHR. Cells were preincubated (16 h) in either control medium or medium supplemented with TSH (5 x 10-8 M) or MS-1 (10 µg/ml). After stringent washing to maximize removal of residual ligand, we performed flow cytometry with two antibodies, one recognizing only the single-chain TSHR, the other recognizing all (cleaved and uncleaved) TSHRs. TSH pretreatment did not appear to increase TSHR cleavage. Instead we observed ligand occupancy of the TSHR (with MS-1) or fewer receptors on the cell surface (down-regulation), particularly with the TSHR-0 cells. Second, we covalently cross-linked [125I]TSH to monolayers of these cells, an unequivocal method to determine directly the proportion of single-chain and two-subunit TSHR forms. Pretreatment of TSHR-10,000 and TSHR-0 cells with TSH had no effect on the degree of TSHR cleavage. MS-1 slightly reduced spontaneous cleavage. In conclusion, in contrast to a recent report, we show that TSH does not alter the subunit structure of its cognate receptor, and we provide insight into the difficulties associated with the flow-cytometric assay for TSHR cleavage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TSH RECEPTOR (TSHR), unlike the gonadotropin receptors, undergoes intramolecular cleavage on the cell surface into A and B subunits that remain linked by disulfide bonds (1, 2). The extent of this posttranslational modification is variable, with different proportions of single-chain and two-subunit forms of the TSHR detected in different experiments and in different tissues (for example, Refs.3, 4, 5). TSHR cleavage is followed by shedding of some of the ligand-binding A subunits (6), for which different mechanisms have been proposed (7, 8, 9). The pathophysiological significance of TSHR cleavage into subunits has drawn considerable attention and speculation. TSH binds with similar affinity to both the single-chain and two-subunit forms of the TSHR (4). Moreover, cleavage into subunits does not appear to be required for receptor activation by ligand (10). On the other hand, only the two-subunit form of the TSHR is reported to form multimers (11). Cleavage is also necessary (but by itself insufficient) for high ligand-independent activity (12, 13), an unusual functional characteristic of the TSHR (14, 15). Of relevance to the pathogenesis of Graves’ disease, the shed A subunit (rather than the holoreceptor) appears to be the critical autoantigen in the generation of thyroid-stimulating autoantibodies (TSAbs) (16).

Recent data have led to provocative new proposals regarding the ligand-binding properties and functional activities of the single-chain and cleaved forms of the TSHR. In contrast to previous observations, Davies et al. (17) suggest that only the cleaved, two-subunit TSHR binds TSH and can, therefore, be activated. Even more controversial is the recent report that TSH, unlike TSHR autoantibodies, cleaves the single-chain TSHR into subunits (18). Previously, based on the discovery that thrombin cleaves its cognate receptor (19), as well as on the observation of homology between a segment in the TSHR cleavage region and the thrombin receptor tethered ligand, we considered the possibility that TSH (like thrombin) possessed enzymatic activity and also cleaved its cognate receptor (12). However, we discarded this notion because TSHR cleavage occurs on the surface of cells cultured in the absence of TSH. Furthermore, we were unable to see an effect of added TSH on TSHR structure (Chazenbalk, G. D., and B. Rapoport, unpublished observations).

In the present study, to reconcile these discrepant data, we reinvestigated the issue of TSH-induced cleavage of the TSHR. The need to resolve this issue is increased by the suggestion of Ando et al. (18) that the inability of TSAb (unlike TSH) to cleave the TSHR could be a novel explanation for prolonged thyroid stimulation in Graves’ disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Generation of two different Chinese hamster ovary (CHO) cells lines stably expressing the human TSHR has been described previously. One cell line (TSHR-10,000) has an amplified transgenome and expresses on the cell surface approximately 1.9 x 106 receptors per cell (20). The name of this cell line derives from transgenome amplification with selection at a final concentration of 10,000 nM methotrexate. The other standard cell line without transgenome amplification in methotrexate (hence TSHR-0) expresses approximately 1.5 x 105 receptors per cell (20, 21). Cells were cultured in Ham’s F-12 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), gentamicin (50 µg/ml), fungizone (2.5 µg/ml), and amphotericin B (2.5 mg/ml).

Flow cytometry
Before flow cytometry, cell monolayers in 60-mm-diameter dishes were incubated for 16 h at 37 C in cell culture medium (see above) with or without added bovine TSH (bTSH; Sigma-Aldrich, St. Louis, MO) or a hamster thyroid-stimulating monoclonal antibody, MS-1 (18), both at 5 x 10-8 M final concentration. The cells were then rinsed three times with Dulbecco’s PBS, pH 7.4 (37 C for 5 min) and detached using 1 mM EDTA/1 mM EGTA in PBS, Ca2+ and Mg2+ free. The suspended cells (~3–5 x 105) were rinsed once with PBS containing 10 mM HEPES, pH 7.4, 2% heat-inactivated fetal bovine serum, and 0.05% NaN3 and were then incubated for 30 min at room temperature in 100 µl of the same buffer containing the following TSHR antibodies: 1) murine monoclonal antibody (mAb) 2C11 (Serotec Ltd., Oxford, UK) and 2) polyclonal antibody (termed anti-D1-NET in this report) generated by immunizing a mouse with an adenovirus expressing a noncleaving TSHR lacking the C-peptide region (Fig. 1Go) (16). After rinsing, the cells were incubated for another 30 min at 4 C with fluorescein isothiocyanate-conjugated, affinity-purified goat antimouse IgG (1:100; Caltag Laboratories Inc., Burlingame, CA), washed, and analyzed using a Beckman FACScan flow cytometer (Beckton Dickinson Immunocytometry Systems, San Jose, CA). Cells stained with propidium iodide (1 µg/ml) were excluded from analysis. Net mean fluorescence values for mAb 2C11 and polyclonal Ab D1-NET were calculated after subtraction of background fluorescence obtained using the second antibody alone.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 1. Schematic representation of the two antibodies used in the flow cytometric assay for TSHR cleavage. Single-chain TSHRs reaching the cell surface undergo intramolecular cleavage into disulfide-linked A and B subunits with the loss of an intervening C-peptide region of the TSHR (not recovered intact) (9 22 24 ). Polyclonal antibody anti-D1-NET recognizes the ectodomain of the TSHR but its epitopes cannot include the C-peptide region because the antibody was generated by immunization with a TSHR lacking the latter segment. Anti-D1-NET will, therefore, recognize both single-chain and two-subunit forms of the TSHR on the cell surface. In contrast, mAb 2C11 (25 ) can recognize only the uncleaved, single-chain TSHR because its epitope lies within the C-peptide region.

 
Radiolabeled TSH binding to cell monolayers
Confluent cells in 24-well plates were preincubated for 16 h at 37 C in culture medium alone or supplemented with bTSH or with mAb MS-1 (both at 5 x 10-8 M) and rinsed as described above. Cells were then incubated for 1 h at 4 C in 250 µl binding buffer (Hanks’ buffer with 250 mM sucrose substituting for NaCl to maintain isotonicity and 0.25% BSA) containing approximately 10,000 cpm [125I]TSH (Kronus, Boise, ID). After rapid rinsing three times with binding buffer (4 C), cells were solubilized with 0.5 ml 1 N NaOH, and radioactivity was measured in a {gamma}-counter. Nonspecific binding, determined in the presence of excess TSH (10-7 M), was subtracted to provide specific counts bound.

Covalent cross-linking of radiolabeled TSH
The procedure used in the present study has been described previously in detail (22), with minor modifications. In brief, highly purified bTSH (National Hormone and Peptide Program, Torrance, CA) was radiolabeled with 125I to a specific activity of approximately 60 µCi/µg using Bolton-Hunter reagent (Perkin-Elmer Life Sciences, Boston, MA). Confluent 100-mm-diameter dishes of cells were preincubated for 16 h at 37 C in culture medium alone or supplemented with 5 x 10-8 M TSH or mAb MS-1. After three rinses in Dulbecco’s PBS, pH 7.4 (37 C for 5 min), the cells were incubated for 1 h at 4 C with 2–5 µCi [125I]TSH, followed by cross-linking with 1 mM succinimidyl suberate (Sigma-Aldrich) and processed as described previously. The resuspended crude cell membranes were solubilized in Laemmli sample buffer (23) containing 0.7 M ß-mercaptoethanol (30 min at 50 C), the samples were electrophoresed on 7.5% SDS-polyacrylamide gels (Bio-Rad Laboratories Inc., Hercules, CA). Prestained molecular-weight markers (Bio-Rad) were included in parallel lines. Radiolabeled proteins were visualized by autoradiography on Biomax MS x-ray film (Eastman Kodak Co., Rochester, NY) Background density, measured at a site within the lanes but distant from any bands, was subtracted to provide net density values.

Statistical analysis
Where indicated in the text, ANOVA with Dunnett’s method for pairwise multiple comparisons was used for nonparametric values. Significance between groups of normally distributed values was determined by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Assessment of TSHR cleavage by flow cytometry
We applied the previously reported flow cytometric assay of Ando et al. (18) in which quantitation of receptor cleavage is determined indirectly by disappearance of the single-chain receptor. TSHR intramolecular cleavage into A and B subunits is associated with loss of a C-peptide region (22, 24). The mAb 2C11 (25) can recognize only the uncleaved, single-chain TSHR on the cell surface because its epitope lies within the C-peptide region (Fig. 1Go). The second antibody, polyclonal anti-D1-NET, was raised in mice to a TSHR lacking the C-peptide region (26). This antibody will recognize both cleaved and uncleaved receptors, that is all TSHRs on the cell surface. In principle, the signal with the latter antibody is the reference value against which is measured the decline in the signal of the C-peptide region antibody following cleavage. Incidentally, it should be pointed out that the need for this subtractive determination is the lack of an antibody (in the present and previous studies) that can recognize only the cleaved, two-subunit receptor.

After overnight preincubation in a high concentration of TSH (5 x 10-8 M) or with MS-1, a hamster thyroid-stimulating mAb (10 µg/ml), CHO cells expressing the human TSHR (TSHR-10,000) were resuspended and subjected to flow cytometry using mAb 2C11 (single-chain TSHR only) and anti-D1-NET (all TSHRs). To optimize quantitation of TSHR expression we wished, if possible, to use a saturating concentration of each antibody. Saturation was clearly attained with 10 µg/ml 2C11 (Fig. 2AGo) but could not be attained with anti-D1-NET even at 1:20 serum dilution, the highest practical concentration (Fig. 2BGo). In three separate experiments, preincubation of cells in either TSH or in MS-1 had no significant effect on the total number of TSHRs (anti-D1-NET) (Fig. 2CGo) or on the number of uncleaved TSHRs (2C11) (Fig. 2DGo). Therefore, under these conditions, we were unable to observe TSH-induced cleavage of the single-chain TSHR.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 2. Flow cytometric detection of single-chain and cleaved forms of the TSHR on CHO cell line (TSHR-10,000). This cell line, with an amplified transgenome, expresses approximately 1.9 x 106 TSHRs per cell. Cell monolayers were preincubated for approximately16 h with control medium without additive (Con) or with the same medium supplemented with bTSH (5 x 10-8 M) or with mAb MS-1 (10 µg/ml). Cells were extensively rinsed, resuspended, and subjected to flow cytometry using the indicated antibody concentration or dilution. Representative experiments are shown for mAb 2C11 (A) and for anti-D1-NET (B). Immunofluorescence values (expressed as a percentage of control) for three experiments performed at or near TSHR saturating concentrations are shown for mAb 2C11 (C) and for anti-D1-NET (D). Bars, mean + SE.

 
One possible explanation for the difference between our data and those of the Davies group was that we used a cell line (TSHR-10,000) expressing large numbers of TSHR (~1.9 x 106 per cell) (20). We, therefore, repeated these experiments with a standard TSHR-expressing CHO cell line without an amplified transgenome (hence TSHR-0). In this case, we could attain saturation, or near saturation, with anti-D1-NET (Fig. 3BGo), as with 2C11 (Fig. 3AGo). Overnight preincubation of these cells in TSH reproducibly decreased the number of TSHRs subsequently detected with a C-peptide region-specific mAb (2C11) by approximately 30% relative to control cells not preincubated in TSH (Fig. 3CGo). At face value, this finding would support the previous conclusion that TSH cleaves the TSHR into subunits. However, three differences between the two studies negated this conclusion. Most important, preincubation with TSH reduced the fluorescence signal with both antibodies (2C11 and anti-D1-NET) (Fig. 3Go, C and D), consistent with a reduction in total number of TSHRs on the cell surface rather than the single-chain receptor alone. Second, in contrast to the previous report (18), preincubation with monoclonal TSAb MS-1 mimicked the effect of TSH on TSHR cell surface expression (Fig. 3Go, A–D). Finally, the TSH-induced reduction in the fluorescent signal with C-peptide region-specific mAb 2C11 was considerably less, approximately 30% vs. approximately 75% reported by Ando et al. (18).



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 3. Flow cytometric detection of single-chain and cleaved forms of the TSHR on CHO cell line (TSHR-0). This standard cell line does not have an amplified transgenome and expresses approximately 1.5 x 105 TSHRs per cell. Cell monolayers were preincubated for approximately 16 h with control medium without additive (Con) or with the same medium supplemented with bTSH (5 x 10-8 M) or with mAb MS-1 (10 µg/ml). Cells were extensively rinsed, resuspended, and subjected to flow cytometry using the indicated antibody concentration or dilution. Representative experiments are shown for mAb 2C11 (A) and for anti-D1-NET (B). Immunofluorescence values (expressed as a percentage of control) for three experiments performed at or near TSHR saturating concentrations are shown for mAb 2C11 (C) and for anti-D1-NET (D). Bars, mean + SE; *, P < 0.05 compared with control (ANOVA with Dunnett’s method for pairwise comparisons).

 
TSHR occupancy or down-regulation by ligand
Two possible reasons for our inability to confirm the conclusions of Ando et al. were persistent ligand occupancy of receptors or a ligand-induced decrease in receptor number (down-regulation) after prolonged exposure to ligand. To eliminate or minimize the former in the flow cytometry studies described above, we extensively rinsed cells in medium warmed to 37 C to facilitate ligand dissociation. Nevertheless, we evaluated the efficacy of this procedure. After preincubation in TSH, [125I]TSH binding to TSHR-10,000 cells was similar to that of control cells not exposed to TSH (Fig. 4AGo). However, with TSHR-0 cells expressing far fewer receptors, radiolabeled TSH binding was reduced by approximately 30%, suggesting TSHR down-regulation or (less likely) residual ligand occupancy (Fig. 4BGo). Preincubation of TSHR-0 cells with the thyroid-stimulating mAb MS-1 produced a similar reduction in radiolabeled TSH binding (Fig. 4BGo). However, with TSHR-10,000 cells, MS-1 (unlike TSH) significantly reduced [125I]TSH binding (Fig. 4AGo).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 4. TSHR occupancy or down-regulation by ligand. TSHR-10,000 cells and TSHR-0 cells were preincubated in control medium without additive (Con) or in medium with TSH or with MS-1 added, followed by extensive rinsing (5 min x 3 at 37 C), as described in Figs. 2Go and 3Go. Subsequently, instead of resuspending the cells for flow cytometry, [125I]TSH binding was assessed on the adherent cells (see Materials and Methods). As noted in the text, such stringent washing following the preincubation removes most residual TSH, but not MS-1. Therefore, in the case of TSH, reduced binding is likely to reflect a decrease in cell surface TSHR number (down-regulation), although some TSH occupancy cannot be excluded. *, P < 0.05 compared with preincubation in medium without additive (Con). Pairwise comparisons were performed by ANOVA with Dunnett’s method.

 
Also of relevance, we observed that even our stringent washing procedure was insufficient to fully remove TSHR-occupied MS-1, a hamster mAb with powerful TSH-binding inhibitory activity (18). This was evident on flow cytometry using mouse antibodies to quantitate TSHR expression (see Figs. 2Go and 3Go). We observed a high background of 192 ± 18 fluorescence units (mean ± SE; n = 6) in control samples with second antibody alone after preincubation of cells with MS-1. After preincubation of cells in TSH, background with second antibody alone was only 12.7 ± 0.9 fluorescence units (mean ± SE, n = 6). The only explanation for the finding with MS-1 is that antimouse IgG cross-reacts with residual hamster IgG on the cell surface. This background value, subtracted to calculate specific antibody signals, is relatively substantial with TSHR-0 cells expressing the conventional number of TSHRs with a maximal signal of only 600 fluorescence units (see Fig. 3Go).

TSHR cleavage assessed by radiolabeled TSH covalent cross-linking
Unlike deducing the extent of TSHR cleavage by flow cytometry, covalent cross-linking of [125I]TSH to intact cells is an unequivocal method to determine directly the proportion of single-chain and two-subunit forms of the TSHR expressed on the cell surface. TSH binds equally well to both forms of the TSHR (4). TSH cross-linking to control CHO cells preincubated in medium without added ligand revealed 70–80% of the TSHRs in the single-chain form with the balance (20–30%) cleaved into subunits, as detected by the ligand-binding A subunit. This degree of cleavage was similar in the TSHR-10,000 and TSH-0 cells; that is, the extent of cleavage was independent of the level of TSHR expression (representative autoradiograms shown in Fig. 5Go, A and B; densitometric quantitation of autoradiograms indicated in Fig. 5Go, C and D; n = 4 and n = 3, respectively). Overnight incubation of both TSHR-expressing cell types in 5 x 10-8 M TSH did not increase the amount of A subunits relative to that in control cells. Similar results were observed in cells pretreated with a lower TSH concentration (10-9 M) as used by Ando et al. (18) (data not shown). Neither did thyroid-stimulating mAb MS-1 increase TSHR cleavage. Instead, this antibody tended to decrease the quantity of cleaved vs. single-chain TSHRs, although this difference did not achieve statistical significance. However, on pooling the densitometric data for the two cell lines and expressing the data as a ratio (single-chain/two-subunit forms of the TSHR), mAb MS-1 significantly reduced TSHR cleavage relative to cells incubated in medium without additive (means of 4.6 ± 1.0 and 11.2 ± 2.0, respectively; P = 0.008, Student’s t test). Preincubation in TSH did not significantly alter this ratio (3.1 ± 0.69 for TSH and 4.6 ± 1.0 for medium; P = 0.238).



View larger version (68K):
[in this window]
[in a new window]
  		FIG. 5. TSHR cleavage assessed by radiolabeled TSH covalent cross-linking. TSHR-10,000 cells and TSHR-0 cells were preincubated in control medium without additive (Con) or supplemented with TSH or MS-1 under conditions identical to those described in Figs. 2–4GoGoGo with the exception that, after stringent washing, [125I]TSH was covalently cross-linked to the intact cells in monolayer (see Materials and Methods). Representative autoradiograms are shown for the TSHR-10,000 cells (A) and TSH-0 cells (B). Densitometric quantitation of [125I]TSH bound to the ectodomain of the single-chain (SC) receptor, and to the A subunit of the cleaved TSHR, is shown for three separate experiments with the TSHR-10,000 cells (C) and the TSHR-0 cells (D) (mean + SE).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The recent report of Ando et al. (18) that TSH, unlike a monoclonal TSAb, cleaves the single-chain TSHR on the cell surface into its two-subunit form (18) conflicts with previous concepts on the mechanism of TSHR intramolecular cleavage (reviewed in Ref.27) and, if substantiated, represents an important advance. It is suggested that TSAb, by not cleaving the TSHR, prolongs the half-life of the latter on the cell surface and represents a novel mechanism to explain overstimulation of the gland in Graves’ disease (18). We point out that, by the same logic, an elevated TSH would decrease thyroid function in primary hypothyroidism. Moreover, there is presently no evidence for a difference in the turnover rates of the single-chain and two-subunit forms of the TSHR.

The principle of the flow cytometry assay used by Ando et al. (18) to quantitate the extent of TSHR cleavage is complex. No antibody can specifically detect the cleaved receptor. Instead, the assay is subtractive, comparing the signal of a reference antibody that detects all TSHRs on the cell surface with the signal of another antibody whose epitope (in the C-peptide region) is lost upon TSHR cleavage (Fig. 1Go). In addition, the C-peptide region-specific antibody does not detect receptors already cleaved before the addition of TSH. Without this information, it cannot be concluded that TSH increases cleavage by, for example, 75% (18). Finally, the B subunit-specific reference antibody used by Ando et al. (18) will overestimate the number of receptors with cleaved or uncleaved ectodomains because it will detect receptors that have shed their A subunits, reportedly the majority of TSHR species on the cell surface (2).

These principles aside, we attempted to reproduce the data of Ando et al. (18), introducing three modifications. First, we sought to use saturating concentrations of each antibody to make the number of receptors on the cell surface, rather than antibody concentration, a limiting factor. Second, we used a polyclonal reference antibody raised against the entire ectodomain but with the C-peptide region deleted. TSHR recognition by this antibody cannot, therefore, be influenced by the presence of absence of the C-peptide region, a function of receptor intramolecular cleavage (Fig. 1Go). Finally, after preincubation with TSH or with the monoclonal TSAb (MS-1) before flow cytometry, we washed the cells more thoroughly than Ando et al. (18) to minimize the possibility of residual ligand occupancy of receptors.

Using these stringent conditions, we detected no significant effect of TSH or of MS-1 on the differential recognition of TSHR antibodies to the entire ectodomain and to the C-peptide region. In the case of cells overexpressing the TSHR, there was no change in the flow cytometric signal with either antibody. With a conventional cell line expressing approximately 105 TSHRs per cell, we did observe a 30–40% decrease in signal on flow cytometry, as well as by radiolabeled TSH binding. However, unlike the findings of Ando et al. (18), this decreased signal was not limited to TSH and was quantitatively similar for both the reference antibody and the cleavage-specific antibody. These data do not indicate TSH induced TSHR cleavage but, rather, suggest TSHR down-regulation and/or residual TSHR occupancy. In the case of TSH, extensive rinsing of cell monolayers (particularly at 37 C to enhance dissociation) effectively reverses TSH activation of the receptor (for example, Ref.28). Moreover, the previous observation that TSHR cleavage required prolonged exposure to TSH (24 h for a full effect) (18) is consistent with the time course of TSHR down-regulation (29). For mAb MS-1, the residual high background that we observed with second antibody alone clearly indicates substantial receptor occupancy, which could influence interpretation of the TSHR cleavage assay (18). Indeed, the preliminary report (30) that an MS-1 Fab cleaves the TSHR (in contrast to MS-1 IgG, and like TSH) may reflect lack of cross-reactivity of the second antibody with the Fab.

In contrast to the flow cytometric cleavage assay, covalent cross-linking of [125I]TSH to intact cells in monolayer directly detects single-chain and two-subunit forms of the TSHR, quantifiable by autoradiography. By this means, we observed no effect of TSH on TSHR intramolecular cleavage. This was the case regardless of whether we used a cell line expressing very large numbers of TSHRs or a cell line with a conventional number of TSHRs [as used by Ando et al. (18)]. Indeed, the TSH cross-linking data suggested the reverse of the previous report. Rather than TSH cleaving the TSHR, we observed that hamster TSAb (MS-1) (18) reduced TSHR cleavage. It is possible that MS-1 may protect the single-chain TSHR from cleavage by a matrix metalloprotease-related enzyme (5, 24), likely membrane associated (31).

It is a common view that TSHR cleavage into subunits is incomplete in transfected cells because overexpression in the latter overwhelms the cleavage mechanism (for example, Refs.5 and 11). The present TSH cross-linking data confirm previous evidence that do not support this hypothesis (20). Thus, the extent of TSHR cleavage was similar (20–30%) in two cell lines despite an approximately 10-fold difference in their levels of TSHR expression (Fig. 5Go). As noted above (Introduction), for reasons not fully understood there is considerable inter-study variability in the extent of TSHR cleavage even when using the same cell line (reviewed in Ref.27). We have suggested that cleavage is greater after cell homogenization (4) or in sick cells (8). In our experience, the 20–30% degree of cleavage consistently observed in the present series of experiments is lower than in previous studies (~50%), perhaps reflecting the good health of the cells. Of importance, this low degree of cleavage optimizes the ability to answer the question of whether an agent such as TSH enhances cleavage. Obviously, if initial cleavage is substantial, it will be more difficult to observe enhancement of cleavage by ligand.

In conclusion, using the same approach as Ando et al. (18) as well as covalent cross-linking of TSH to the TSHR, we find that TSH does not increase the extent of cleavage of the single-chain TSHR on the cell surface into A and B subunits. In addition, rather than having no effect on the TSHR cleavage process, a hamster monoclonal TSAb may diminish TSHR intramolecular cleavage.


   Acknowledgments
 
We are grateful to Dr. Terry Davies and Dr. Takao Ando for providing us with an aliquot of MS-1, as well as to B.R.A.H.M.S. (Berlin, Germany), for providing some of the radiolabeled TSH used in this study. We thank the National Hormone and Distribution Program of the National Institute of Diabetes and Digestive and Kidney Diseases and Dr. A. F. Parlow for access to highly purified bovine TSH for radioiodination.


   Footnotes
 
This work was supported by NIH Grant DK19289.

Abbreviations: bTSH, Bovine TSH; CHO, Chinese hamster ovary; mAb, monoclonal antibody; TSAb, thyroid-stimulating autoantibodies; TSHR, TSH receptor.

Received August 5, 2003.

Accepted for publication August 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Buckland PR, Rickards CR, Howells RD, Jones ED, Rees Smith B 1982 Photo-affinity labelling of the thyrotropin receptor. FEBS Lett 145:245–249[CrossRef]
  2. Loosfelt H, Pichon C, Jolivet A, Misrahi M, Caillou B, Jamous M, Vannier B, Milgrom E 1992 Two-subunit structure of the human thyrotropin receptor. Proc Natl Acad Sci USA 89:3765–3769[Abstract/Free Full Text]
  3. Furmaniak J, Hashim FA, Buckland PR, Petersen VB, Beever K, Howells RD, Rees Smith B 1987 Photoaffinity labelling of the TSH receptor on FRTL5 cells. FEBS Lett 215:316–322[CrossRef][Medline]
  4. Russo D, Chazenbalk GD, Nagayama Y, Wadsworth HL, Seto P, Rapoport B 1991 A new structural model for the thyrotropin (TSH) receptor as determined by covalent crosslinking of TSH to the recombinant receptor in intact cells: evidence for a single polypeptide chain. Mol Endocrinol 5:1607–1612[Abstract]
  5. Misrahi M, Ghinea N, Sar S, Saunier B, Jolivet A, Loosfelt H, Cerutti M, Devauchelle G, Milgrom E 1994 Processing of the precursors of the human thyroid-stimulating hormone receptor in various eukaryotic cells (human thyrocytes, transfected L cells and baculovirus-infected insect cells). Eur J Biochem 222:711–719[Medline]
  6. Couet J, Sokhavut S, Jolivet A, Vu Hai M-T, Milgrom E, Misrahi M 1996 Shedding of human thyrotropin receptor ectodomain: Involvement of a matrix metalloprotease. J Biol Chem 271:4545–4552[Abstract/Free Full Text]
  7. Couet J, de Bernard S, Loosfelt H, Saunier B, Milgrom E, Misrahi M 1996 Cell surface protein disulfide-isomerase is involved in the shedding of human thyrotropin receptor ectodomain. Biochemistry 35:14800–14805[CrossRef][Medline]
  8. Tanaka K, Chazenbalk GD, McLachlan SM, Rapoport B 1999 The shed component of the TSH receptor is primarily a carboxyl terminal truncated form of the A subunit, not the entire A subunit. Mol Cell Endocrinol 150:113–119[CrossRef][Medline]
  9. Tanaka K, Chazenbalk GD, McLachlan SM, Rapoport B 1999 Subunit structure of thyrotropin receptors expressed on the cell surface. J Biol Chem 274:33979–33984[Abstract/Free Full Text]
  10. Chazenbalk GD, McLachlan SM, Nagayama Y, Rapoport B 1996 Is receptor cleavage into two subunits necessary for thyrotropin action? Biochem Biophys Res Commun 225:479–484[CrossRef][Medline]
  11. Latif R, Graves P, Davies TF 2001 Oligomerization of the human thyrotropin receptor. Fluorescent protein-tagged hRSHR reveals post-translational complexes. J Biol Chem 276:45217–45224[Abstract/Free Full Text]
  12. Chazenbalk GD, Tanaka K, McLachlan SM, Rapoport B 1999 On the functional importance of thyrotropin receptor intramolecular cleavage. Endocrinology 140:4516–4520[Abstract/Free Full Text]
  13. Chen C-R, Chazenbalk GD, McLachlan SM, Rapoport B 2003 Targeted restoration of cleavage in a noncleaving thyrotropin receptor demonstrates that cleavage is insufficient to enhance ligand-independent activity. Endocrinology 144:1324–1330[Abstract/Free Full Text]
  14. Van Sande J, Swillens S, Gerard C, Allgeier A, Massart C, Vassart G, Dumont J 1995 In Chinese hamster ovary K1 cells dog and human thyrotropin receptors activate both the cyclic AMP and the phosphatidylinositol 4,5-bisphosphate cascades in the presence of thyrotropin and the cyclic AMP cascade in its absence. Eur J Biochem 229:338–343[Medline]
  15. Van Sande J, Parma J, Tonacchera M, Swillens S, Dumont J, Vassart G 1995 Genetic basis of endocrine disease: somatic and germline mutations of the TSH receptor gene in thyroid diseases. J Clin Endocrinol Metab 80:2577–2585[CrossRef][Medline]
  16. Chen C-R, Pichurin P, Nagayama Y, Latrofa F, Rapoport B, McLachlan SM 2003 The thyrotropin receptor autoantigen in Graves’ disease is the culprit as well as the victim. J Clin Invest 111:1897–1904[CrossRef][Medline]
  17. Davies T, Marians R, Latif R 2002 The TSH receptor reveals itself. J Clin Invest 110:161–164[CrossRef][Medline]
  18. Ando T, Latif R, Pritsker A, Moran T, Nagayama Y, Davies TF 2002 A monoclonal thyroid-stimulating antibody. J Clin Invest 110:1667–1674[CrossRef][Medline]
  19. Vu TK, Hung DT, Wheaton VI, Coughlin SR 1991 Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057–1068[CrossRef][Medline]
  20. Chazenbalk GD, Kakinuma A, Jaume JC, McLachlan SM, Rapoport B 1996 Evidence for negative cooperativity among human thyrotropin receptors overexpressed in mammalian cells. Endocrinology 137:4586–4591[Abstract]
  21. Kakinuma A, Chazenbalk G, Filetti S, McLachlan SM, Rapoport B 1996 Both the 5' and 3' noncoding regions of the thyrotropin receptor messenger RNA influence the level of receptor protein expression in transfected mammalian cells. Endocrinology 137:2664–2669[Abstract]
  22. Chazenbalk GD, Tanaka K, Nagayama Y, Kakinuma A, Jaume JC, McLachlan SM, Rapoport B 1997 Evidence that the thyrotropin receptor ectodomain contains not one, but two, cleavage sites. Endocrinology 138:2893–2899[Abstract/Free Full Text]
  23. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  24. de Bernard S, Misrahi M, Huet J-C, Beau I, Desroches A, Loosfelt H, Pichon C, Pernollet J-C, Milgrom E 1999 Sequential cleavage and excision of a segment of the thyrotropin receptor ectodomain. J Biol Chem 274:101–107[Abstract/Free Full Text]
  25. Johnstone AP, Cridland JC, DaCosta CR, Harfst E, Shepherd PS 1994 Monoclonal antibodies that recognize the native human thyrotropin receptor. Mol Cell Endocrinol 105:R1–R9
  26. Tanaka K, Chazenbalk GD, McLachlan SM, Rapoport B 1998 Thyrotropin receptor cleavage at site 1 does not involve a specific amino acid motif but instead depends on the presence of the unique, 50 amino acid insertion. J Biol Chem 273:1959–1963[Abstract/Free Full Text]
  27. Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM 1998 The thyrotropin receptor: interaction with thyrotropin and autoantibodies. Endocr Rev 19:673–716[Abstract/Free Full Text]
  28. Rapoport B 1976 Dog thyroid cells in monolayer tissue culture: adenosine 3',5'-cyclic monophosphate response to thyrotropic hormone. Endocrinology 98:1189–1197[Abstract]
  29. Foti D, Catalfamo R, Russo D, Costante G, Filetti S 1991 Lack of relationship between cAMP desensitization and TSH receptor down-regulation in the rat thyroid cell line FRTL-5. J Endocrinol Invest 14:213–218[Medline]
  30. Latif R, Ando T, Marians R, Davies TF Intramolecular cleavage of the TSH receptor: impact of TSHR antibodies. Program of the 85th Annual Meeting of The Endocrine Society, Philadelphia, PA, 2003, p 172 (Abstract P1-163)
  31. Tanaka K, Chazenbalk GD, McLachlan SM, Rapoport B 2000 Evidence that cleavage of the thyrotropin receptor involves a molecular ruler’ mechanism: deletion of amino acid residues 305–320 causes a spatial shift in cleavage site 1 independent of amino acid motif. Endocrinology 141:3573–3577[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
R. Latif, T. Ando, and T. F. Davies
Lipid Rafts Are Triage Centers for Multimeric and Monomeric Thyrotropin Receptor Regulation
Endocrinology, July 1, 2007; 148(7): 3164 - 3175.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
R. Latif, T. Ando, and T. F. Davies
Monomerization as a Prerequisite for Intramolecular Cleavage and Shedding of the Thyrotropin Receptor
Endocrinology, December 1, 2004; 145(12): 5580 - 5588.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
G. Vassart and S. Costagliola
A Physiological Role for the Posttranslational Cleavage of the Thyrotropin Receptor?
Endocrinology, January 1, 2004; 145(1): 1 - 3.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/1/4    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chazenbalk, G. D.
Right arrow Articles by Rapoport, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chazenbalk, G. D.
Right arrow Articles by Rapoport, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals