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Endocrinology Vol. 145, No. 12 5580-5588
Copyright © 2004 by The Endocrine Society

Monomerization as a Prerequisite for Intramolecular Cleavage and Shedding of the Thyrotropin Receptor

R. Latif, T. Ando and T. F. Davies

Division of Endocrinology, Diabetes and Bone Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029-6574

Address all correspondence and requests for reprints to: Dr. R. Latif, Department of Medicine, Box 1055, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, New York 10029-6574. E-mail: rauf.latif{at}mssm.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The TSH receptor (TSHR) undergoes intramolecular cleavage of the ectodomain yielding a two-subunit structure on the cell surface. Subsequently, the TSHR ectodomains (the {alpha}- or A-subunits) are shed from the cell surface. In this study we first confirmed TSHR {alpha}-subunit shedding from tagged-TSHR transfected Chinese hamster ovary cells. We found that TSH exacerbated this phenomenon of TSHR subunit shedding. The 125I-TSH cross-linking technique has been suggested as useful in the assessment of dynamic changes in TSHR processing. In our hands this technique did not detect any enhancement of cleavage by TSH. However, we found that the cross-linking method had an inherent insensitivity for studying receptor dynamics as exhibited by its inability to detect even major degrees of TSHR down-regulation. We, therefore, used a cell-based, double-antibody, flow cytometric immunoassay to quantitate TSHR cleavage in real time. We then found that different lines of Chinese hamster ovary TSHR cells, when treated with TSH, showed a time- and dose-dependent increase in TSHR cleavage in addition to ectodomain shedding. We previously reported that monoclonal TSHR stimulating antibody (MS-1) did not always act like TSH. In particular, MS-1 did not enhance TSHR cleavage. However, when we used the Fab fragment of MS-1, we were able to induce cleavage in a similar time frame to TSH. These results suggested that the intact bivalent antibody immobilized the TSHRs in their multimeric state and inhibited intramolecular cleavage. In support of these observations, fluorescence recovery after photo bleaching measurements demonstrated a greater increase in TSHR mobility with MS-1 Fab fragments than with the intact MS-1 IgG. In conclusion, these data indicated that monomer formation from multimeric TSHRs might be an important requirement for TSHR cleavage and TSHR ectodomain shedding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TSH RECEPTOR (TSHR) expressed on the plasma membrane of thyroid follicular epithelial cells is the key molecule that regulates thyroid growth and function (1, 2, 3). Unlike other glycoprotein hormone receptors such as the LH/choriogonadotropin or FSH receptors, the TSHR undergoes intramolecular cleavage into covalently linked {alpha}/A and ß/B subunits (2, 4, 5). This intramolecular cleavage is followed by reduction and shedding of the extracellular {alpha}-subunit from the membrane anchored ß-subunit resulting in a ß/{alpha} ratio of 3:1 in the thyroid cell (4).

It has been previously suggested that the TSHR ectodomain shedding process involves two steps: 1) cleavage of the receptor into two subunits and 2) reduction of the disulfide bonds bridging the receptor subunits. The first step may involve a matrix metalloprotease-like enzyme acting at the cell surface (6), and the second step must involve the enzymatic reduction of the disulfide bonds that hold these cleaved receptor subunits together, most probably by protein disulfide isomerase (7). It has also been suggested that the process of shedding is up-regulated by TSH (6).

TSHR cleavage is focused on a 50-amino acid insert, which is unique to the ectodomain of the TSHR (residues 316–366) (2, 5, 8). Studies with mutant and wild-type TSHRs have revealed the initiation sites of cleavage (9, 10) and removal of this 50-amino acid insert by a sequential process (11), but the pathophysiological relevance of cleavage has remained enigmatic. The extent of TSHR holoreceptor cleavage may involve most of the TSHRs expressed in thyroid tissues, whereas heterologous transfected cells may show variable percentages of receptors retaining the holomeric form (12). In addition, different TSHR holomeric forms exist consisting of a mature approximately 120-kDa receptor and an approximately 95-kDa mannose-rich precursor (2, 12). Whereas the greater accumulation of uncleaved receptors in transfected mammalian cells is assumed to be due to inefficient processing (12), it may also be secondary to the lack of a TSH ligand in such cell cultures because it has previously been shown that addition of TSH in such TSHR transfected cells enhances cleavage (13).

Whereas there is evidence that TSHR cleavage and reduction are not necessary for signal transduction because TSH binds and activates the TSHR, even after removal of the cleaved region (14), there is evidence implying abnormal receptor trafficking of truncated receptors (15). Recently TSHR antibodies from patients with Graves’ disease have been shown to have preferential binding for the shed {alpha}-subunit when compared with the intact membrane bound receptor (16). Therefore, it has been hypothesized that the shed {alpha}-subunit may act as a systemic antigenic reservoir initiating Graves’ disease and contributing to the extra thyroidal manifestations of Graves’ disease in the retroorbital tissues and skin (17). If these processes of TSHR cleavage and shedding are dynamic changes regulated by TSH, then the above hypothesis may have important pathophysiologic consequences.

Another important posttranslational processing feature of the TSHR is the formation of multimeric species both in transfected cells and native membranes (18, 19). The regulation of these multimeric species by TSH (20) on the cell surface may have relevance in regulating the processing of the receptor for cleavage and shedding. In an earlier paper, we speculated that it was the cleaved receptors that multimerized on the cell surface (18). Subsequently we observed that the cleaved form of the TSHR in transfected Chinese hamster ovary (CHO) cells was able to bind Gs{alpha}, and, therefore, cleavage of the TSH receptor may be associated with receptor activation in these cells (21).

Recently we described a cell-based flow cytometric assay for TSHR cleavage developed using two TSHR-specific monoclonal antibodies (mAbs) to the cleaved region and the ß/B subunit (13). However, this method of measuring cleavage could not be reproduced using 125I-TSH cross-linking as a measure of receptor dynamics (22). The 125I-TSH cross-linking technique involves TSH covalently cross-linked biochemically to the receptors and has been useful in TSHR structural studies (9, 23). In this report we performed a detailed analysis of the fluorescence-activated cell sorter (FACS) cleavage assay and compared it with the cross-linking method. Using the FACS approach, we again found that TSH enhanced TSHR cleavage and that the methodology allowed us to quantitate this response. In contrast, 125I-TSH cross-linking was nonquantitative and insensitive to receptor dynamics and failed to reveal TSH enhanced cleavage. We obtained further support for TSH-induced cleavage by also confirming TSH-induced TSHR {alpha}/A subunit shedding from cells expressing tagged TSHRs.

In addition to the further evaluation of TSH-enhanced cleavage and shedding, we also compared the effects of TSH with a TSHR-stimulating mAb (MS-1). MS-1, which necessarily had dimeric binding sites like all IgG molecules, failed to act like TSH and did not accentuate TSHR cleavage and may even have decreased such constitutive activity. However, monovalent Fab fragments of MS-1 were able to accelerate TSHR cleavage suggesting that monomeric TSHRs, not multimerized TSHRs, were the subject of TSHR cleavage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The three CHO human TSHR cells used in the current study were: 1) CHO-TSHR (JPO9) cells kindly provided by Dr. G. Vassart (University of Brussels, Belgium) express approximately 90,000 receptors per cell (24). JPO2 were CHO cells transfected with an empty vector and thus used as the control. These were cultured in F12 medium supplemented with penicillin/streptomycin (100 U/ml), 10% fetal bovine serum, and 400 µg/ml of G418 (neomycin sulfate). 2) TSHR-0 cells and TSHR 10,000 cells kindly provided by Dr. B. Rapoport (Cedars Sinai Medical Center, Los Angeles, CA). These cells were maintained in F12 medium with 10% fetal bovine serum with no selection markers. 3) TSHR-0 cell line was obtained without transgenome amplification in methotrexate and expresses approximately 1.5 x 105 receptors per cell (25).

Antibodies used in this study
The antibodies used in the study included the following: 1) MS-1, a hamster-derived stimulating mAb that recognized a conformationally dependent epitope on the TSHR {alpha}-subunit (13); 2) 9F4, a hamster TSHR mAb directed to the ectodomain of the receptor, also recognizes a conformationally dependent epitope; 3) M1 and M4 are murine mAbs to the TSHR; M4 recognized an epitope in the cleaved region 322–342 (M4 or RSR4, RSR Ltd., Cardiff, UK) (26), and M1 (M1 or RSR1, RSR Ltd.) recognized residues 381–385 in the ß-subunit of the receptor; 4) 2C11, a murine mAb, also recognizes an epitope in cleaved region (residues 354–359) (Serotec, Raleigh, NC) (22, 27); 5) Antihemagglutinin (HA) is a rabbit polyclonal antibody recognizing the HA epitope (Sigma, St. Louis, MO); and 6) MS-1 Fab was made in-house as described below.

Generation of MS-1 Fab
MS-1 was dissolved with 100 mM sodium acetate (pH 5.5) supplemented with 50 mM cysteine and 1 mM EDTA. Ten micrograms of Papain (Sigma) per milligram protein were added to MS-1 and incubated overnight at 37 C. Iodoacetamide (final concentration 75 mM, Sigma) was added to stop the reaction. Fab fragment was purified using fast protein liquid chromatography from this dissociated mixture (help kindly provided by Dr. S. Wilk, Division of Pharmacology, Mount Sinai School of Medicine).

Detection of shed TSHR {alpha}-subunits
A stable line of amino terminus HA epitope tagged TSHR (HA-TSHR) was developed for this purpose using the construct HA-TSHR (wild type) (28) (kindly provided by Dr. M. W. Szkudlinski, Trophogen, Rockville, MD). The shed {alpha}-subunit of receptor in the culture supernatant from HA-TSHR cells was detected using a double-determinant immunoassay. In brief, culture supernatant was collected from cells treated with 103 µU/ml of bovine TSH-treated (Sigma) and untreated cells after 24 h and concentrated 10-fold using microcentrators (Centricon, Millipore Inc., Bedford, MA). ELISA plates coated with a polyclonal anti-HA rabbit antibody (Sigma) at a concentration of 1µg/well in carbonate-bicarbonate buffer (pH 9.6) overnight at 4 C. The plates were blocked using 2% BSA in PBS containing 0.05% Tween 20 (PBST) for 1 h at 37 C. After washing the wells twice with PBST, the concentrated culture supernatant was added to the respective wells and incubated further for 2 h at 37 C. After washing the plate three times with PBST, the bound receptor fragment was detected using 5 µg/ml of biotinylated TSHR-Ab MS-1 in PBST for 1 h at 37 C or biotinylated 9F4. The biotinylation of these purified antibodies was done by the standard protocol as described (29). Furthermore, the bound biotinylated MS-1/9F4 was detected using streptavidin HRP (Zymed Inc., South San Francisco, CA) at a dilution of 1:5000 for 30 min at 37 C. The signal was developed using 3', 5, 5'-tetramethyl benzidine (BD Bioscience, San Diego, CA) (50 µl/well) as the substrate for 30 min at room temperature. The reaction was stopped using 50 µl/well of 0.1 M Na phosphates, and absorbance was measured using an ELISA reader at 450 nm.

Flow cytometric cleavage assay
This FACS-based assay was performed as described previously (13). (see Fig. 2Go for a schematic representation). The murine mAbs to the TSHR that were used included one to the cleaved region 322–342 (M4) (26) and the other to the cleaved region 354–359 (2C11, Serotec) (22, 27). We also used a mAb recognizing residues 381–385 (M1) to detect both uncleaved holoreceptor and cleaved ß-receptors. CHO-TSHR cells were seeded at 0.4–0.6 x 106 cells/well in 6-well plates in F12 medium. After overnight, cells were further incubated in fresh medium for another 12–18 h before being treated with TSH, monoclonal thyroid stimulating TSHR antibody (MS-1), or the Fab fragment of MS-1 at increasing concentrations for 24 h at 37 C. After incubation, the cells were washed twice with PBS and detached using 1 mM EDTA/EGTA for 5 min at room temperature and washed once more in FACS buffer (PBS with 0.02% sodium azide). Equal numbers of cells were then stained by the above-mentioned antibodies for 1 h at 4 C, and bound primary antibody was detected using secondary antimouse IgG conjugated to phycoerythrin (Jackson ImmunoResearch Inc., West Grove, PA) or antimouse IgG1{gamma} (1) conjugated to fluorescein isothiocyanate (Roche Applied Sciences, Indianapolis, IN) that reacts with the heavy chain of IgG1 subclass and has no cross-reactivity for hamster IgG. The result of the enhanced cleavage, as evidenced by decreased M4 binding, was then represented as a ratio of mean fluorescent intensity (MFI) of M1 (antibody recognizing the cleaved region as well as the holoreceptor) over the mean fluorescent intensity obtained by the antibody against the cleaved region (M4).



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  		FIG. 2. Schematic diagram of the flow cytometric assay used for quantitation of TSHR cleavage. A and B, Indicated are M1 and M4 antibody binding sites before and after TSHR cleavage. The cleavage region is marked by amino acids 316–366; TM, Transmembrane region of the receptor. A (top right panel, M4 and M1 reactivity at 0 µU/ml TSH. B (bottom right panel), M4 and M1 FACS profile after treatment with 102 µU/ml TSH. Note the marked decrease in M4 reactivity. The letter B in the profiles refers to the background staining.

 
TSH blockade by M1 and M4
To study whether TSH caused any physical blockade of the antibodies used in the flow cytometric assay, we carried out a binding study using M1 and M4 after TSH treatment. JP09 cells cultured in complete medium in 100-mm dishes were treated with increasing concentrations of bovine TSH (102 to 103 µU/ml) for 2 h at 37 C. The cells, after washing with PBS (2x), were detached using 1 mM EDTA/EGTA and distributed into 0.5 x 106cells/tube and stained with increasing concentrations of antibody. Percent binding (% binding) was calculated from the MFI under different TSH treatments by taking untreated cells as 100%, and binding curves were plotted using Prism 4 (GraphPad Software Inc., San Diego, CA).

TSHR down-regulation
To study receptor loss from the cell surface in response to TSH stimulation, CHO-TSHR cells were seeded in 100-mm dishes 1 d before stimulation with increasing concentrations of TSH (10 to 103 µU/ml) for up to 24 h after washing out the unbound TSH with PBS (pH 7.4). The cells were then stained separately with mAbs M1 and M4 as described earlier. Percent binding was calculated as described previously.

125I-TSH covalent cross-linking of the TSHR
We followed the method as described by Chazenbalk et al. (22). Briefly, confluent 100-mm dishes of cells were treated with 103 µU/ml unlabeled TSH for 24 h in complete medium. The unbound TSH was washed out with cold Hanks’ buffer (x3), and dishes were then incubated with 125I-labeled TSH with specific activity of 80 µCi/µg (Kronus Inc., Boise, ID) followed by cross-linking with 1 mM disuccinimidyl suberate (Sigma) and processed further as described (22). The processed samples were electrophoresed on 10% SDS-PAGE, and prestained molecular weight markers were included to determine fragment sizes. Radiolabeled protein bands were visualized and quantitated in a PhosphorImager (Molecular Dynamics Inc., Sunnyvale, CA).

TSHR signal transduction
cAMP assays were performed as previously described (13). In brief, CHO-TSHR cells were seeded at 4 x 104 cells/well on 96-well plates 1 d before the assay. Cells were stimulated with purified mAb or Fab, TSH, and forskolin diluted with cell culture medium containing 2 mM isobutylmethylxanthine (Sigma). The intracellular cAMP was measured by enzyme immunoassay according to the manufacturers’ protocol (Amersham Bioscience, Piscataway, NJ).

Fluorescence recovery after photo bleaching (FRAP)
FRAP was performed to study the lateral diffusion of tagged TSHRs before and after treatment as previously described (20) with modifications. Subsequent movement of surrounding nonbleached fluorescent molecules into the photobleached area was recorded at 50% laser power to avoid bleaching of cells. Briefly, cells were grown on {delta} TC3 dishes (Bioptechs, Butler, PA) and maintained at 37 C on a heated thermal stage. One hour before imaging, the cells were treated with 5 µg/ml cycloheximide (Sigma) to stop the influx of new receptors to the surface from inside the cell. A preincubation of 30 min for TSH-, MS-1-, and MS-1Fab-treated dishes was carried out before performing the FRAP. The spot photo bleaching was done using x60 oil objective (1.32 N.A.) on a confocal microscope [LSM TCP-SP (UV), Leica, Québec, Canada]. Images (8 bit 512 x 512 zoom 2x) were acquired for pre- and postbleached time points. The objective was also maintained at the same temperature during the entire course of the experiment to minimize any thermal aberrations during image collection. A 2-sec exposure of an annotated spot (approximate radius of 2 µm) on the cell surface with the 488-nm Argon laser at 25 mW power produced a bleached spot. Pre- and postbleached images were collected every 3sec for a period of 1 min, and fluorescence intensity in the photobleached area and a reference point of similar area of each image were measured using the software IPLabs (version 3.5, Scanalytics, Inc., Fairfax, VA). The pre- and postbleach intensities were normalized to100% (unbleached reference point) and 0% (bleached spot), respectively. The percent recovery was calculated from the relative intensity computed for each time point by following the protocol of Tanimura et al. (30).

Statistical analyses
Results were expressed as the mean ± SD of least three experiments, and values were statistically compared using Student’s’ t test or one-way ANOVA. Results were determined to be significant with P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Constitutive and TSH-regulated shedding of the TSHR {alpha}-subunit
To study TSHR ectodomain shedding, a stable clone of transfected CHO cells expressing N terminus HA-tagged TSHRs (HA-TSHR) was developed. The expression of HA-tagged receptors in this stable clone was verified using anti-HA and TSHR-specific antibodies (Fig. 1AGo), and the cells were highly responsive to TSH as judged by cAMP generation (Fig. 1BGo). HA-TSHR cells were treated with 103 µU/ml of TSH for 24 h. We were then able to detect constitutive and TSH-induced shedding of TSHR fragments from HA-TSHR cells (Fig. 1CGo). There were no shed receptors detected in supernatants from untagged CHO-TSHR (JP09) controls. The specificity of the assay was also confirmed using biotinylated antibody to the TSHR ß-subunit (M1) (data not shown).



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  		FIG. 1. Characterization and detection of shed {alpha}-subunit using N terminus HA TSHR. A, N terminus HA-tagged TSHR clone (HA-TSHR) tested for surface expression of HA and TSHR by mAbs. CHO TSHR (JPO9) and TSHR10,000 cells were used as controls. B, cAMP dose response of the cells. C, Detection of HA-TSHR shed {alpha}/A subunit captured by anti-HA and probed with biotin-labeled MS-1 and 9F4 as detailed in Materials and Methods (n = 2).

 
TSH regulation of TSHR cleavage
The flow cytometric cleavage assay was based on the loss of binding of a TSHR mAb (M4) that selectively bound epitopes within the cleaved region of the receptor (Fig. 2Go). A second mAb (M1) that recognized both the uncleaved holoreceptor and the cleaved TSHR on the cell surface was directed at the ß-subunit of the receptor (residues 381–385) distal to cleavage site II (9) as indicated schematically in Fig. 2Go. This M1 mAb, therefore, measured all receptor forms resident on the cell membrane and served as a reference point for the assay. The binding of M1 was then compared with the binding of antibody to the cleaved region (M4). Cleavage was measured as the loss of M4 binding, which exceeded any change in M1 binding. In fact, a dose-dependent inhibition of binding by both mAbs was observed, most likely due to TSH blockade, or down-regulation of, the TSHR (Fig. 3Go) (see below), but a clear enhancement of cleavage after TSH treatment was also measurable as enhanced loss of M4 binding, compared with M1 (Fig. 3Go). Hence, the FACS-based assay suggested enhanced loss of the cleaved region after 24 h of TSH treatment (Fig. 3Go). However, this type of assay cannot easily reveal the level of constitutive TSHR cleavage without calculating the binding concentration of both mAbs in the absence of TSH.



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  		FIG. 3. TSHR cleavage and down-regulation measured by FACS. M1 and M4 binding was studied 24 h after stimulation with the indicated doses of TSH. Disproportionate loss of M4 to M1 is a measurement of TSHR cleavage. The reduction in M1 binding is a measure of down-regulation. Data are expressed as % binding of each antibody determined by MFI, compared with no treatment.

 
Factors influencing the legitimacy of the FACS cleavage assay
We consistently observed a decrease in M1 and M4 binding with increasing doses of TSH treatment (Fig. 3Go). One of the major factors that clearly may have caused this response was the physical blockade of the mAb epitopes by TSH occupancy. To examine this, CHO-TSHR (JP09) cells were treated first with increasing doses of TSH (102 to 103 µU/m) for 2 h. After washing out the unbound TSH, the cells were probed with increasing doses of antibodies and analyzed. The results (Fig. 4Go, A and B) indicated that M1 and M4 binding, after increasing doses of TSH treatment, did not change significantly. Hence, the M4 mAb, which binds to the cleaved region of the TSHR, is a neutral antibody and does not affect receptor occupancy. The residues that mAb M1 binds to (amino acids 381–385) encompass one of the domains in the TSH binding pocket (31). M1 is a potent inhibitor of TSH binding, and, therefore, it was possible that TSH treatment may itself have affected M1 binding. However, we did not observe this (Fig. 4BGo). Thus, the decrease in M1 or M4 binding seen as a result of TSH treatment was not due to TSH occupancy of residual TSHRs (22).



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  		FIG. 4. Binding profile of M1 and M4 after TSH treatment. JP09 cells were incubated with increasing doses of TSH (102 to 103 µU/ml) for 2 h and stained with increasing concentrations of M1 (A) or M4 (B) antibody. The antibody binding was determined by MFI in FACS, and binding at each concentration is expressed as % binding with MFI at saturating dose as 100%.

 
Enhanced down-regulation of receptors after ligand treatment is a common phenomenon (32, 33, 34) and could have accounted for a TSH-induced reduction in the binding of TSHR antibodies. However, a reduction in M1 vs. M4 binding was not seen after 2 h of TSH exposure. Therefore, because TSH was unable to block epitopes for both these mAbs, any reduction in antibody binding must have been accounted for by receptor down-regulation. Evaluation of the binding of TSHR antibodies M1 and M4 after 24 h of TSH treatment (Fig. 3Go) showed reduced binding to TSHR expressing cells. However, antibody M4 showed a significant further reduction in binding relative to antibody M1, i.e. an approximately 60% reduction in binding of M4, compared with an approximately 20% reduction in binding seen with M1 (P < 0.01), even at 102 µU/ml TSH. Therefore, down-regulation of the receptors due to TSH treatment reduced the binding of both M1 and M4, but a significant preferential reduction in binding of M4 over M1 was still observed. This reduction in the binding of M4 relative to M1 can be explained only by a selective and excessive loss of the M4 epitope from the cell surface. This preferential loss of the M4 epitope represents TSH-induced cleavage of the TSHR.

TSH regulation of TSHR cleavage using 125I-TSH covalent cross-linking
Previously, TSH-induced cleavage was not observed in a high-expressing CHO-TSHR cell line (TSHR-0) when examined using chemical cross-linking (22). We also examined this cross-linking technique by studying constitutive and TSH-induced cleavage in CHO-TSHR cells (JP09 and TSHR-0). For controls we used CHO cells (JPO2) transfected with vector only. By definition, the cross-linking technique can reveal only those TSHRs able to bind labeled TSH. Hence, these studies demonstrated a broad band of approximately 100 kDa holoreceptors and an approximately 50- to 55-kDa {alpha}-subunit band in both cell types (Fig. 5AGo). The presence of the {alpha}-subunits seen in this assay must be secondary to either holoreceptor degradation or cleavage. Because the bands were highly reproducible, it is unlikely that nonspecific degradation was the cause, and most investigators have concluded that constitutive nonthyroid cell-specific cleavage occurs in these TSHR-expressing cells. Densitometric quantitation of the two major bands (holoreceptor and {alpha}/A subunit forms) of the TSHR is shown in Fig. 5BGo. These data also demonstrated the extent of cleaved TSHR expression in comparison with the uncleaved holoreceptor. There was no change in the amount of cleaved TSHR detected using 125I-TSH cross-linking in JPO9 or TSHR-0 cells after 24 h of TSH treatment (103 µU/ml). In contrast, some 40–60% of TSHRs were down-regulated under the same experimental conditions by 24 h (Fig. 5CGo). However, TSHR {alpha}-subunits are known to be shed from the cells after cleavage (6, 7), so even this comparison of forms underestimates the percent of receptors constitutively cleaved. These results demonstrated that the FACS technique took into account both TSHR occupation and/or down-regulation that could not be detected with the cross-linking system. In addition, the interassay deviations obtained with the cross-linking technique should cause further hesitation in using this approach for measuring dynamic changes in receptor forms.



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  		FIG. 5. Measurement of cleavage using 125I-TSH cross-linking. Confluent layers of indicated cells grown on 100-mm dishes were preincubated with and without 103 µU/ml of TSH for 24 h. After stringent washing with buffer, the 125I-TSH was covalently cross-linked to intact cells and processed further as described in Materials and Methods. A, Autoradiograph of experiments with and without TSH (n = 4). B, Mean densitometric values for the holoreceptor and the {alpha}/A subunit of the cleaved TSHR (n = 4) C, Down-regulation of TSHR measured by M1 binding by FACS after 24 h treatment with 103 µU/ml TSH (n = 3).

 
Assessment of TSH regulated cleavage in different cell types
We tested whether our observations of TSH enhancement of TSHR cleavage was due to the type of TSHR expressing cells and/or the detecting antibodies used. When we compared JP09 and TSHR-0 cells in four experiments, we found enhancement of TSHR cleavage in both JPO9 and TSHR-0 cells when measured using antibodies M1 and M4 (Fig. 6Go). However, no significant enhancement of cleavage was observed in either cell when mAb 2C11 was used. We considered it possible that the failure of 2C11 antibody to detect cleavage in JP09 or TSHR-0 cells was due to incomplete cleavage of the expressed TSHR linear epitope (11). Because the 2C11 epitope was distal to the M4 epitope, we extended the TSH treatment until 48 h. This extended time course of TSH treatment gave similar results with the M1/2C11 ratio at 24 h being similar to that at 48 h (data not shown). Such data did not support the logic that cleavage would extend only to the 2C11 epitope with an extended incubation time. Hence, these results indicated that cleavage of the TSHR was often incomplete in these transfected cells. Thus, the choice of cleavage region antibodies for the FACS-based assay was critical.



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  		FIG. 6. Assessment of TSHR cleavage in JPO9 and TSHR-0 cells. JP09 (A) and TSHR-0 (B) cells were incubated for 24 h with 103 µU/ml TSH. The cells were stained with saturating concentrations of M1, M4, or 2C11 antibody, respectively, and antibody binding was determined by FACS assay. The results are represented as the MFI ratio of M1 to M4 or M1 to 2C11, an increase in these ratios indicates increased cleavage.

 
TSH-induced cleavage is TSHR mediated
To study whether enhancement of TSHR cleavage was a receptor-specific phenomenon or a postreceptor phenomenon, TSHR-expressing CHO cells were activated with 40 µM forskolin or 103 µU/ml TSH for 2 and 24 h separately. The cells were assayed for enhanced cleavage using the FACS assay. As indicated in Fig. 7AGo, forskolin did not enhance cleavage in these cells after 2 and 24 h of treatment, in marked contrast to 24-h TSH. Furthermore, the cAMP response generated by forskolin was robust in these studies (Fig. 7BGo).



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  		FIG. 7. Effect of forskolin on cleavage. A, JP09 cells were treated with TSH (103 µU/ml) or forskolin (40 µM) and cells assayed for TSHR cleavage. The data are indicated as the MFI ratio of M1 to M4, and increase in these ratios indicates increased cleavage. B, cAMP response with different doses of TSH and forskolin after 2 h.

 
Effect of TSHR-stimulating antibody MS-1 on TSHR cleavage
We have previously shown that pretreatment of TSHR-expressing CHO cells with increasing concentrations of TSHR-stimulating antibody MS-1 did not enhance cleavage (13). This was in marked contrast to TSH. We hypothesized that this lack of cleavage enhancement may have been secondary to the stimulating antibody MS-1 holding the receptors in a dimeric or oligomeric state and thus preventing monomer formation (20). Therefore, we incubated JPO9 cells with MS-1-Fab (5 µg/ml), which retained its bioactivity (Fig. 8BGo), and assayed for cleavage (Fig. 8AGo). Intact MS-1 (10 µg/ml) and TSH 103 µU/ml were used as controls. The results confirmed that MS-1 did not enhance cleavage, whereas cells incubated with MS-1-Fab showed significant cleavage enhancement.



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  		FIG. 8. A, Effect of MS-1 and MS-1 Fab fragment on cleavage. JOP9 cells were incubated for 24 h with MS-1 (5 µ/ml), MS-1-Fab (5 µg/ml), or TSH (102 µU/ml) and then assayed for cleavage results are expressed by the ratio of M1 to M4. Increase in the ratio indicates increased cleavage. B, Dose-dependent cAMP response of MS-1 and MS-1 Fab.

 
TSHR movement detected using FRAP
To further test the hypothesis that the TSHR antibody prevented TSHR monomer formation, we assessed TSHR movement in TSHRGFP cells treated with purified MS-1 using FRAP as described previously (20). It has been shown that higher molecular forms, such as multimers of TSHR, move more slowly than monomeric forms, and FRAP is sensitive in measuring dynamic changes in lateral movements (35, 36, 37). As indicated in Fig. 9BGo, the recovery of the receptors into the bleached area after TSH was faster, compared with that of the untreated control (P < 0.001), consistent with monomer formation from constitutive oligomers as previously demonstrated (20). However, cells treated with 20 µg/ml MS-1 showed a reduced recovery in mobility that is lower than untreated cells (P < 0.001). These data further supported our hypothesis that MS-1 may hold the receptors in their oligomeric state, thus preventing them from attaining a faster mobility. Recovery rates with MS-1 Fab were intermediate between intact MS-1 and TSH, which was consistent with our hypothesis.



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  		FIG. 9. Lateral movement of TSHRGFP treatment was studied by FRAP. TSHRGFP cells were subjected to spot photobleaching using a 488-nm Argon laser. The fluorescence intensity of the spot before and after photobleaching was calculated for a reference point and the bleached spot as indicated in the images in A. The percent fluorescence recovery of each time was thus calculated from this analysis as described in Materials and Methods. A, Images of pre- and postbleached stages showing recovery of cells. A dotted circle marks the pre- and postbleached spots in the sequence of images. B, Quantitative data showing percent recovery in the various treatments. *, P < 0.001 and P < 0.05, compared with 0 TSH.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There have been many studies trying to understand the mechanism of the posttranslational molecular dissection of the TSHR into {alpha}- and ß-subunits. This process involves the removal of a 50-amino acid insert in the ectodomain, which is unique among the family of G protein-coupled receptors (2, 4, 9, 10, 11, 38, 39). However, it remains unclear whether this region of the receptor or the process of receptor cleavage has any pathophysiologic role (2, 17, 40). Toward this end we recently reported that TSH ligand was able to enhance TSHR cleavage when measured using a flow cytometric method based on CHO cells expressing the human TSHR (13). We have now examined this FACS methodology in more detail and provided further data that suggested that monomerization of the receptors may be an important preliminary process to the cleavage event.

Earlier investigations suggested that TSH must have a role in the twin processes of cleavage and shedding (6, 7, 8). Although it has been established that the cell membrane-associated protein disulfide isomerase is involved in the reduction of disulfide bridge(s) leading to the shedding of the {alpha}-subunit (7), this would most likely have occurred if the first step of cleavage was accentuated by TSH because both processes occur at the cell membrane (12, 39). As in other known receptors such as the colony-stimulating factor 1 receptor, TNF receptor, and IL receptors, the mechanism of cleavage and shedding may be due to activation of cell membrane-associated proteases (or convertases) via second-messenger activation such as the protein kinase A pathway, etc. (41, 42, 43). The speculative association of a matrix metalloprotease (6, 23) involved in TSHR cleavage would suggest that the above explanation is a likely possibility. Our shedding assay, free from any biochemical manipulation of the TSHR-expressing cells, confirmed this influence of TSH on subunit shedding. As has also previously shown (6), this TSH-induced enhancement of subunit shedding suggested either enhanced TSHR cleavage and/ or a direct influence by TSH on receptor shedding of previously cleaved receptors. Hence, these data encouraged us to explore the influence of TSH on TSHR cleavage.

We developed a cell-based flow cytometric assay to examine, in a quantitative way, the effect of TSH and TSHR antibodies on subunit formation. Although this assay is subtractive, based on the loss of antibody epitopes in the 50-amino acid cleaved region, it has been highly reproducible in our hands. However, important factors in assay reproducibility included the choice of an appropriate cleavage region antibody. Because the assay rests on the loss of binding of epitopes within the cleaved region of the receptor, any factors changing this binding would obviously affect the assay. This could include the occupancy of the epitope by residual TSH, and, therefore, only non-TSH competing antibodies could be used. In this regard the choice of 2C11, which is a weak blocker of TSH action (27), was not an appropriate choice for this assay. The TSH binding pocket has been defined by three major epitopes (amino acids 246–260, 277–296, and 381–385) situated on either side of the 50-amino acid insert (31). Earlier findings also emphasized that this 50-amino acid insert (amino acids 317–366) was unlikely to be involved in TSH binding (44). Another possible cause of interference in the FACS-based cleavage assay would be alteration of the binding affinities of the mAbs employed. However, this was not the case, as shown by almost identical binding of the mAbs after TSH treatment, suggesting that the conformational changes that had occurred were not significant enough to alter the antibody binding sites. The choice of a polyclonal reference antibody as used by Chazenbalk et al. (22), with a mixture of affinities, may also interfere with the sensitivity of this assay.

A major factor that may affect cells exposed to TSH is marked down-regulation of TSHRs on the cell surface. We found that CHO-TSHR cells had markedly reduced TSHR expression after 24 h of TSH treatment. It has also been shown previously, in primary thyrocytes and transfected cells, that TSH treatment leads to internalization of the TSH receptors (45). However, using the appropriate antibodies, there was a significant preferential decrease in antibody binding to the cleaved region (M4) of the TSHR after TSH treatment, compared with the binding of antibody to the intact holoreceptor and ß-subunit (M1). This preferential decrease in binding observed as a result of TSH treatment could be accounted for only by TSHR cleavage. However, the faster internalization of truncated receptors, compared with holoreceptors, has also been observed (15), and this may have contributed to the decrease in M1 and not M4 binding. Hence, down-regulation may cause an underestimate of the extent of cleavage.

Using a 125I-labeled derivative of TSH binding to porcine thyroid membranes, Kajita et al. (46) showed for the first time that the TSH receptor consisted of two subunits. This technique was first introduced to delineate the different structural forms of the TSHR. Subsequently, this technique was adopted by others (9) to delineate the sites of TSHR cleavage (9, 23, 38, 47, 48) using mutant and chimeric TSHRs. However, this system has never before been used for examining dynamic changes in TSHR forms as occurs after intramolecular cleavage. A recent report (22) claimed that the extent of TSHR cleavage and changes in the constitutive level of cleaved receptors can be best detected by 125I-TSH cross-linking. We, therefore, incorporated into our studies the simultaneous use of this cross-linking technique. The technique was unable to detect the major loss of TSHRs (40–60%) after TSH-induced down-regulation. It is also important to recognize that 125I-TSH can only bind to unoccupied TSHRs, i.e. receptors without labeled antibody bound as result of pretreatment, and this further restricted the population of receptors that could be detected in the TSHR-expressing cell lines. Furthermore, the phenomenon of shedding was also not detectable by cross-linking, most likely because the constitutive level of TSHR cleavage estimated by this technique, when using cell cultures, was the result of a combination of an increase in {alpha}/A subunit production after cleavage and a decrease in {alpha}-subunits after their loss from the cell secondary to shedding. The large standard interassay errors observed in our study and that of Chazenbalk et al. (22) indicated that this method was also subjective and only semiquantitative. Hence, it appears that 125I-TSH covalent cross-linking to the TSHR may be a satisfactory method to study TSHR structure but cannot detect changes in constitutive and regulated cleavage.

Having established that the flow cytometric assay predictably detected cleavage, we also analyzed its efficacy in light of a previous report questioning the methodology (22). We, therefore, used a second line of TSHR overexpressing CHO cells (TSHR-0) and a second antibody to the cleaved region referred to earlier (TSHR mAb 2C11), which had failed to demonstrate TSH-induced TSHR cleavage when used together (22). We also failed to observe a TSH-induced change in the binding of 2C11, compared with M4, in both JP09 and TSHR0 cells, indicating the importance of antibody choice in this assay, rather than the level of TSHR expression, as also pointed out by others (22, 25). Studies have shown cleavage to be incomplete in transfected cells (12), suggesting that the choice of antibody to the cleaved region would be critical in the FACS assay. Because the epitope of 2C11 is amino acids 354–359 (27), compared with the upstream epitope of M4 (amino acids 322–341), these data suggested that M4 loss may be more pronounced if cleavage was sequential, as reported by others (11), beginning around amino acid 301 (2, 9, 10) in the proximity of site 1 (23). Furthermore, our data suggested that the concept of a C-peptide, i.e. an intact 50-amino acid, being released as a result of intramolecular cleavage was incorrect. Thus, the choice of cleavage region antibodies for the FACS-based assay was critical.

We have shown previously (13) that increasing doses of stimulating antibody MS-1 were unable to enhance cleavage of the receptor as seen with TSH. This led us to hypothesize that the antibody may be holding the receptors in dimeric or higher order states, which prevented cleavage from occurring. To test this hypothesis, we used Fab fragments of MS-1 and measured cleavage. In contrast to the intact antibody, the Fab fragment was able to induce cleavage. We then performed FRAP to study lateral movement of receptors as previously used to demonstrate the dissociation of TSHR multimers after TSH treatment (20). FRAP measurements showed receptor movement enhanced by TSH and MS-1 Fab but not by the intact MS-1, which was consistent with our hypothesis. In contrast, it has been shown that GH binding to the GH receptor induced GHR dimerization. However, the dimerized GHR was more resistant to proteolysis than the monomeric GHR (49), consistent with our own observations of the TSHR.

In summary, these studies have shown that the flow cytometric method has proven to be a simple cell-based quantitative method for studying dynamic changes in surface TSHR cleavage and down-regulation, whereas 125I-TSH cross-linking should be used only to study TSHR structure. The differential effects of TSH and stimulating TSHR antibody on cleavage and receptor movement of TSHRs are consistent with the need for monomer formation before TSHR cleavage.


   Acknowledgments
 
We thank Drs. Yaron Tomer, Reigh-Yi Lin, and Samira Daniel from our division for critical review of this manuscript and comments. We thank Dr. Scott Henderson [shared facility at Mount Sinai School of Medicine (MSSM)] for the confocal studies performed at the MSSM-Microscopy Shared Research Facility, supported, in part, with funding from a National Institutes of Health-National Cancer Institute shared resources grant (1 R24 CA095823-01). We also thank Dr. Sherwin Wilk (Department of Pharmacology, MSSM) for helping us with the fast protein liquid chromatography.


   Footnotes
 
This work was supported by National Institutes of Health Grants DK52464, DK45011, and AI 24671 (to T.F.D.), the David Owen Segal Endowment, and the Marvin Sinkoff Endowment.

Abbreviations: CHO, Chinese hamster ovary; FACS, fluorescence-activated cell sorter; FRAP, fluorescence recovery after photo bleaching; HA, hemagglutinin; mAb, monoclonal antibody; MFI, mean fluorescent intensity; MS-1, monoclonal TSHR stimulating antibody; PBST, PBS containing 0.05% Tween 20; TSHR, TSH receptor.

Received June 25, 2004.

Accepted for publication August 12, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Milgrom E, de Roux N, Ghinea N, Beau I, Loosfelt H, Vannier B, Savouret JF, Misrahi M 1997 Gonadotrophin and thyrotrophin receptors. Horm Res 48(Suppl 4):33–37
  2. Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM 1998 The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr Rev 19:673–716[Abstract/Free Full Text]
  3. Vassart G, Dumont JE 1992 The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr Rev 13:596–611[CrossRef][Medline]
  4. 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]
  5. Rees Smith B, McLachlan SM, Furmaniak J 1988 Autoantibodies to the thyrotropin receptor. Endocr Rev 9:106–121[Abstract]
  6. Couet J, Sar S, Jolivet A, Hai M-TV, Milgrom E, Misrahi M 1996 Shedding of human TSH 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. Biochem 35:14800–14805[CrossRef][Medline]
  8. Misrahi M, Milgrom E 1997 Cleavage and shedding of the TSH receptor. Eur J Endocrinol 137:599–602[CrossRef][Medline]
  9. Chazenbalk GD, Tanaka K, Nagayama Y, Kakinuma A, Jaume JC, McLachlan S, Rapoport B 1997 Evidence that the TSH receptor ectodomain contains not one, but two, cleavage sites. Endocrinology 138:2893–2899[Abstract/Free Full Text]
  10. Graves P, Pritsker A, Davies TF 1999 Post-translational processing of the natural human TSH receptor: demonstration of more than two cleavage sites. Endocrinology 84:2177–2181
  11. de Bernard S, Misrahi M, Huet JC, Beau I, Desroches A, Loosfelt H, Pichon C, Pernollet JC, 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]
  12. 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]
  13. 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]
  14. 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]
  15. Quellari M, Desroches A, Beau I, Beaudeux E, Misrahi M 2003 Role of cleavage and shedding in human thyrotropin receptor function and trafficking. Eur J Biochem 270:3486–3497[Medline]
  16. Chazenbalk GD, Pichurin P, Chen CR, Latrofa F, Johnstone AP, McLachlan SM, Rapoport B 2002 Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor. J Clin Invest 110:209–217[CrossRef][Medline]
  17. Vassart G, Costagliola S 2004 A physiological role for the posttranslational cleavage of the thyrotropin receptor? Endocrinology 145:1–3[Free Full Text]
  18. Latif R, Graves P, Davies TF 2001 Oligomerization of the human thyrotropin receptor: fluorescent protein-tagged hTSHR reveals post-translational complexes. J Biol Chem 276:45217–45224[Abstract/Free Full Text]
  19. Graves PN, Vlase H, Bobovnikova Y, Davies TF 1996 Multimeric complex formation by the natural TSH receptor. Endocrinology 137:3915–3920[Abstract]
  20. Latif R, Graves P, Davies TF 2002 Ligand-dependent inhibition of oligomerization at the human thyrotropin receptor. J Biol Chem 277:45059–45067[Abstract/Free Full Text]
  21. Ciullo I, Latif R, Graves P, Davies TF 2003 Functional assessment of the thyrotropin receptor-ß subunit. Endocrinology 144:3176–3181[Abstract/Free Full Text]
  22. Chazenbalk GD, Chen CR, McLachlan SM, Rapoport B 2004 Does thyrotropin cleave its cognate receptor? Endocrinology 145:4–10[Abstract/Free Full Text]
  23. Chazenbalk GD, Rapoport B 1994 Cleavage of the thyrotropin receptor does not occur at a classical subtilisin-related proprotein convertase endoproteolytic site. J Biol Chem 269:32209–32213[Abstract/Free Full Text]
  24. Costagliola S, Swillens S, Niccoli P, Dumont JE, Vassart G, Ludgate M 1992 Binding assay for thyrotropin receptor autoantibodies using the recombinant receptor protein. J Clin Endocrinol Metab 75:1540–1544[Abstract]
  25. 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]
  26. Oda Y, Sanders J, Roberts S, Maruyama M, Kato R, Perez M, Petersen VB, Wedlock N, Furmaniak J, Rees SB 1998 Binding characteristics of antibodies to the TSH receptor. J Mol Endocrinol 20:233–244[Abstract]
  27. 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
  28. Zhang M, Tong KP, Fremont V, Chen J, Narayan P, Puett D, Weintraub BD, Szkudlinski MW 2000 The extracellular domain suppresses constitutive activity of the transmembrane domain of the human TSH receptor: implications for hormone-receptor interaction and antagonist design. Endocrinology 141:3514–3517[Abstract/Free Full Text]
  29. Harlow E, Lane D 1998 Antibodies: a laboratory manual. Long Island, NY: Cold Spring Harbor Laboratory
  30. Tanimura N, Nagafuku M, Liddicoat DR, Hamaoka T, Kosugi A 2003 Analysis of the mobility of signaling molecules in lymphocytes using fluorescence photobleaching techniques. Sci STKE 2003:p110
  31. Jeffreys J, Depraetere H, Sanders J, Oda Y, Evans M, Kiddie A, Richards T, Furmaniak J, Rees SB 2002 Characterization of the thyrotropin binding pocket. Thyroid 12:1051–1061[CrossRef][Medline]
  32. Kawakami N, Miyoshi K, Horio S, Fukui H 2004 ß(2)-Adrenergic receptor-mediated histamine h(1) receptor down-regulation: another possible advantage of ß(2) agonists in asthmatic therapy. J Pharmacol Sci 94:449–458[CrossRef]
  33. Singh SP, McDonald D, Hope TJ, Prabhakar BS 2004 Upon thyrotropin binding the thyrotropin receptor is internalized and localized to endosome. Endocrinology 145:1003–1010[Abstract/Free Full Text]
  34. Von Zastrow M 2001 Role of endocytosis in signalling and regulation of G-protein-coupled receptors. Biochem Soc Trans 29:500–504[CrossRef][Medline]
  35. Azpiazu I, Gautam N 2004 A FRET based sensor indicates that receptor access to a G protein is unrestricted in a living mammalian cell. J Biol Chem 279:27709–27718[Abstract/Free Full Text]
  36. Lippincott-Schwartz J, Snapp E, Kenworthy A 2001 Studying protein dynamics in living cells. Nat Rev Mol Cell Biol 2:444–456[CrossRef][Medline]
  37. Schaaf MJ, Cidlowski JA 2003 Molecular determinants of glucocorticoid receptor mobility in living cells: the importance of ligand affinity. Mol Cell Biol 23:1922–1934[Abstract/Free Full Text]
  38. 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]
  39. 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]
  40. Graves PN, Davies TF2000 New insights into the thyroid-stimulating hormone receptor. The major antigen of Graves’ disease. Endocrinol Metab Clin North Am 29:267–286
  41. Mui AL, Kay RJ, Humphries RK, Krystal G 1992 Ligand-induced phosphorylation of the murine interleukin 3 receptor signals its cleavage. Proc Natl Acad Sci USA 89:10812–10816[Abstract/Free Full Text]
  42. Pandiella A, Massague J 1991 Multiple signals activate cleavage of the membrane transforming growth factor-{alpha} precursor. J Biol Chem 266:5769–5773[Abstract/Free Full Text]
  43. Tedder TF 1991 Cell-surface receptor shedding: a means of regulating function. Am J Respir Cell Mol Biol 5:305–306
  44. Tanaka K, Nagayama Y, Yamasaki H, Hayashi H, Namba H, Yamashita S, Niwa M 1996 Epitope-tagging of a functional thyrotropin receptor: detection of the native receptor on intact cells. Biochem Biophys Res Commun 228:21–28[CrossRef][Medline]
  45. Baratti-Elbaz C, Ghinea N, Lahuna O, Loosfelt H, Pichon C, Milgrom E 1999 Internalization and recycling pathways of the thyrotropin receptor. Mol Endocrinol 13:1751–1765[Abstract/Free Full Text]
  46. Kajita Y, Rickards CR, Buckland PR, Howells RD, Rees SB 1985 Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane. Biochem J 227:413–420[Medline]
  47. Chazenbalk GD, Nagayama Y, Russo D, Wadsworth HL, Rapoport B 1990 Functional analysis of the cytoplasmic domains of the human thyrotropin receptor by site-directed mutagenesis. J Biol Chem 265:20970–20975[Abstract/Free Full Text]
  48. Tanaka K, Chazenbalk GD, McLachlan SM, Rapoport B 1999 The shed thyrotropin 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]
  49. Baumann G 2002 When should we measure growth hormone binding protein? J Pediatr Endocrinol Metab 15:959–961[Medline]



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