Endocrinology, doi:10.1210/en.2002-220884
Endocrinology Vol. 144, No. 7 3176-3181
Copyright © 2003 by The Endocrine Society
Functional Assessment of the Thyrotropin Receptor-ß Subunit
I. Ciullo,
R. Latif,
P. Graves and
T. F. Davies
Division of Endocrinology, Diabetes and Bone Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10128
Address all correspondence and requests for reprints to: Dr. T. F. Davies, Department of Medicine, Box 1055, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029-6574. E-mail: Terry.Davies{at}mssm.edu.
 |
Abstract
|
|---|
Posttranslational processing of the TSH receptor (TSHR) involves proteolysis of a single chain holoreceptor into TSHR-
(or A) and TSHR-ß (or B) subunits, which remain associated via disulfide bonds and which may then form oligomers. As both uncleaved and cleavage-derived forms of this receptor have been reported to bind TSH and transduce signals, reasons for this cleavage into - and ß-subunits have remained enigmatic. Recently we suggested that TSHR cleavage was related to receptor oligomerization and now we have asked if cleavage influenced the binding of G proteins to this receptor. Furthermore, as TSHR- subunits are subject to shedding from the cell surface membrane, we have examined whether the remaining TSHR-ß subunits could mediate signaling themselves, either constitutively and /or ligand-induced. We found that only the cleaved form of the TSHR in transfected Chinese hamster ovary cells was able to bind Gs protein, suggesting that cleavage of the native TSH receptor was associated with receptor activation. We also found that independently expressed TSHR-ß subunits on stable cell lines were unable to mediate either constitutive or TSH-induced signaling, as monitored by their inability to induce cAMP accumulation.
These data suggested that receptor cleavage was intimately associated with receptor activation in the wild-type TSH receptor and that the residual TSHR-ß subunits left on the thyroid cell membrane, after TSHR cleavage and subsequent TSHR- shedding, were essentially silent and did not participate in signal transduction.
|
Introduction
|
|---|
THE TSH RECEPTOR (TSHR) is a G protein-coupled receptor residing in the plasma membrane of thyrocytes and a variety of other cells (1, 2). It confers responsiveness to the pituitary hormone TSH and is a common antigen target of T cells and autoantibodies (TSHR-Ab) in autoimmune thyroid diseases. Depending on the binding epitope, TSHR-Ab may stimulate or block growth and activity of thyroid cells, consequently stimulating or blocking thyroid hormone production by the gland. We, and others (1, 2, 3, 4, 5), have previously shown that TSH receptors are present as both uncleaved holoreceptors and as cleavage-derived, disulfide-linked TSHR- and TSHR-ß subunits. The former subunit contains the amino terminus and comprises most of the large, glycosylated extracellular domain (ectodomain) of this receptor. TSHR-ß is nonglycosylated and comprises a short extracellular segment followed by seven transmembrane helices (and connecting loops), plus an intracellular cytoplasmic tail. In thyroid tissue preparations, TSHR-ß subunits were detected both as monomers and as disulfide-linked homodimers and higher order complexes (6, 7). Recently, we used fluorescence resonance energy transfer and coimmunoprecipitation to show that TSHRs existed as constitutive oligomers and suggested that cleavage may be associated with oligomerization (8, 9).
The presence of multiple forms of the TSHR on thyrocytes and TSHR-transfected cells raised new questions about their roles in signaling and autoimmunity. Here we addressed two of them. First, we asked whether TSHR cleavage had an effect on the ability of this receptor to bind G proteins as a requirement for signaling. This was examined by determining which form(s) of the TSHR could be coimmunoprecipitated using G protein antibodies. Second, we asked whether TSHR-ß subunits, stably expressed in the absence of TSHR-, had any signaling capacity (either constitutive and/or ligand-induced). Earlier mutational analysis had suggested that TSHR-, in addition to its crucial role in binding TSH, functioned to dampen constitutive activity of this receptor via interactions with TSHR-ß (10, 11). Furthermore, many TSHR-ß subunits exist autonomously, as a result of TSHR- shedding from the plasma membrane (12, 13). To determine whether TSHR-ß subunits were functional and capable of mediating signal transduction, TSHR-ß cDNA was ligated behind the TSHR signal peptide cDNA, to direct trafficking, and was expressed in Chinese hamster ovary cells (CHO) to assess surface expression and function. As several size variants of TSHR-ß (presumably processing variants) have been reported (14), three variants encompassing the range of reported sizes (residues 316–764, 366–764, and 409–764) were chosen for this study. These cDNAs were expressed as such or as fusion proteins linked to green fluorescent protein (GFP) at the carboxyl termini. Surface fluorescence and immunoblotting were used to assess membrane TSHR-ß expression in stable CHO clones, whereas binding to G proteins and cAMP production (after stimulation with TSH) provided the functional assays. Our results demonstrated that, although each TSHR-ß was stably expressed on cell membranes, none of the cell lines was able to mediate signal transduction in response to TSH. Nor were there any increases in the basal rates of cAMP synthesis compared with nontransfected controls. This suggested that TSHR-ß subunits present on thyrocytes, as the result of TSHR shedding (12, 13), were unlikely to play a major role in either constitutive or TSH-induced signaling and that both of these processes were, therefore, TSHR- subunit dependent.
|
Materials and Methods
|
|---|
Truncated TSHR-ß construction and tagging
Because the TSHR undergoes several sequential cleavages around, and within, a 50-amino-acid residue (317–366) insert, absent from other members of this receptor family, three different size variants of TSHR-ß were selected for the study (Fig. 1
). These were obtained by PCR amplification from full-length TSHR cDNA in pBluescript SK+ as the template. A single reverse primer, corresponding to the TSHR carboxyl terminus was used in each case (5'-CATAGGCGCCGCCCAGGTCCCTGGGCACGTCGAG-3'). Three forward primers 5'-CATAGGCGCCTTGAATAGCCCCCTCCACCAGGAA-3; 5'-CATACCCGGGCAGGAGCTCAAAAACCCCCAGGAA-3'; 5'-GAAGACATAATGGGCTACAAGTTCCTGAGA-3' with added restriction enzyme sites were used to generate TSHR-ß subunits beginning with residue 316, 366, and 409. The restricted products were ligated in-frame to the 3' end of the TSHR signal peptide cDNA generated by annealing forward and reverse signal peptide oligonucleotides and their sequences verified by direct sequencing. Each truncated TSHR-ß was then transferred independently into pcDNA 3.1 (Invitrogen, Carlsbad, CA) and pEGFP-N1 (CLONTECH Laboratories, Inc., Palo Alto, CA). For cloning into pEGFP-N1, stop codons at the 3' end of the insert were omitted and the modified inserts ligated in-frame to the amino terminus GFP.
View larger version (11K):
[in this window]
[in a new window]
|
Figure 1. TSHR-ß subunit constructs. A, Full length TSHR containing amino acids 1–764. B, TSHR-ß316 containing amino acids 316–764. C, TSHR-ß366 containing amino acids 366–764. D, TSHR-ß409 containing amino acids 409–764.
|
|
Cell culture and transfection
Chinese hamster ovary (CHO-K1) cells maintained in Ham’s F12 medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin and streptomysin were seeded at a density of 3 x 106 cells/100-mm dish for transfection. cDNA constructs were then transfected into CHO cells using a Ca2+ phosphate protocol, and the cells were selected with 500 µg/ml neomycin sulfate (G418) to yield stable clones. CHO cells stably expressing full-length TSHR (JPO9, kind gift from Dr. G. Vassart, Brusselles, Belgium) were used as controls.
Preparation of membranes
Adherent cells were detached by gently scraping with a rubber policeman and suspended in 5 ml of ice cold HB buffer (250 mM sucrose; 50 mM Tris HCl, pH 7.6; 1.25 mM EGTA) containing a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN). After briefly homogenizing in a polytron, the samples were centrifuged at 760 x g for 15 min. The supernatants were ultracentrifuged at 140,000 x g for 2 h. The resulting pellets were then solubilized in immunoprecipitation buffer (100 mM KCl; 50 mM Tris-HCl, pH 7.5; 0.05% ß-mercaptoethanol; and 0.5% Nonidet P-40).
Immunoprecipitations and Western blot analyses
Solubilized membranes were normalized for protein content in each experiment and incubated with 1µg/ml of Gs antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 C overnight, followed by incubation with protein A agarose (Roche Biochemicals, Indianapolis, IN) for 2 h. The beads were washed with lysis buffer and the bound immune complexes eluted by boiling in SDS-PAGE sample containing 2% of 2ß-mercaptoethanol. The reduced samples were fractioned by 10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes. Blocked (5% dry milk, PBS 0.05% Tween) membranes were then incubated with primary antibody (MAb-A10 to the TSHR N-terminal end, kind gift from Dr. Paul Banga, London, UK, at 1 µg/ml) for 1 h at room temperature and followed by HRP-conjugated secondary antibodies diluted (1:3000) in blocking buffer. The blots were developed by ECL (ECL-PLUS, Amersham Pharmacia Biotech, Piscataway, NJ).
cAMP measurements
CHO cells stably expressing hTSHR-ß constructs (105 cells/well) were tested for cAMP generation using the Biotrak cAMP enzyme immunoassay system (Amersham Pharmacia Biotech). JPO9 and JP02 cells served as positive and negative controls. All cells were lysed after 1 h of stimulation with 10 or 100 µU/ml of bovine TSH (Sigma, St. Louis, MO) and intracellular cAMP levels assessed.
FACS analysis
Cells expressing GFP-tagged TSHR-ß subunits were detached by treating with 1 mM EDTA/EGTA for 5 min at room temperature, washed twice in PBS, and resuspended in PBS containing 0.2% BSA and 0.02% sodium azide. The cells were analyzed for fluorescence under FL1 (fluorescein isothiocyanate channel). For detecting untagged TSHR-ß surface expression, and the cells were stained with a TSHR-ß specific monoclonal antibody (RSR1, amino acids 381–385, kind gift from Dr. B. Rees Smith, Cardiff, UK) and detected using antimouse phycoerythrin-labeled secondary antibody under FL2 (PB channel).
Lipophilic tracer staining for membrane expression of TSHR constructs
A stock solution (1 mg/ml) of the lipophilic tracer carbocyanine (CM-DiI, Molecular Probes, Inc., Orlando, FL) was prepared in di-methylformamide. Fresh working dilutions in Dulbecco’s balanced salt solution of (1 µg/ml) were prepared for each experiment. This was then added to cells grown on chamber slides, incubated for 2 min at 37 C, and immediately shifted to 4 C for an additional 10 min. To stop further staining, the cells were washed with PBS and fixed in 2% paraformaldehyde.
|
Results
|
|---|
Analysis of G protein binding to TSHR
Because both uncleaved TSH holoreceptors and cleavage-derived heterodimers (-ß) have been reported to bind TSH and transduce signals (15), it was of interest to assess G protein binding to these two species. This was examined by coimmunoprecipitation of TSHR species present in solubilized TSHR membranes using Gs antibody, followed by immunoblot detection (under reducing conditions) of TSHR species in the complex using a TSHR- antibody recognizing the amino terminus (residues 21–35) of the TSHR (16, 17). Although no uncleaved, full-length TSH holoreceptors were detected in the immunoprecipitates, the TSHR- antibody showed specific reactivity to an approximately 55-kDa species (Fig. 2), the size expected for the long form of the TSHR- subunit. Because TSHR- is an ectodomain fragment, incapable of binding Gs directly, the binding must have occurred via disulfide-linked TSHR-ß subunits before samples were reduced for immunoblot analysis. Further, the amount of Gs associated with cell membranes and detected by TSHR- antibody was increased by addition of TSH to the culture (Fig. 3A); this was not the case with Gq (Fig. 3B). Taken together, these results suggested that TSHR cleavage was required for binding to G protein and that signaling was mainly through Gs in this system.
Expression of truncated TSHRs
We next investigated whether TSHR-ß subunit cDNAs of different lengths (Fig. 1
), ligated to TSHR signal peptide cDNA and transfected into CHO cells, were expressed as protein on the cell surface. Where possible, this was assessed using FACS analysis of cells containing the GFP-tagged constructs. The clones showed up to 50% GFP expression for ß366 (Fig. 4A), but ß316 was not easily detected. The surface expression of the TSHR-ß constructs, using the monoclonal antibody to amino acids 381–385 (RSR1) was, therefore, also ascertained in cells transfected with the ß316 and ß366 constructs (Fig. 4B) and both were detectable. Finally, the surface localization of each of the TSHR-ß GFP variants was ascertained by plasma membrane staining using a red lipophilic dye (CMDiI). The localized spots of yellow staining seen on the surface of these cells (Fig. 5) was due to colocalization of the red dye and GFP, confirming the expression of all three TSHR-ß subunits on the plasma membrane, although ß316 expression was also weak in this system. Immunoblots of cell membrane preparations from these clones, developed with a polyclonal GFP peptide antibody, detected expression of each predicted TSHR-ßGFP fusion protein. Figure 6 (upper panel, lane 1) shows a TSHR-ßGFP (366–764aa) signal of 87 kDa size from lysates of CHO cells transfected with the corresponding cDNA, and its absence from CHO cells transfected with empty vector (lane 2). A band of similar size was detected in cells transfected with full-length TSHR-GFP (lane 3), as expected in reduced samples. All three samples contained free GFP of 27 kDa size (Fig. 6, lower panel), apparently due to some degree of cleavage between the fusion partners.
View larger version (32K):
[in this window]
[in a new window]
|
Figure 4. Expression analysis of TSHR-ß subunits by FACS. A, GFP expression in cells transfected with truncated TSHR-ß GFP fusion constructs. The level of GFP expression in these cells is shown by log fluorescent intensity as depicted by FL1. The percentage positive cells are marked within each quadrant. B, The same cells probed with MAb RSRS1 (381–385 amino acids) and showing surface expression of TSHR-ß subunits. The secondary antibody was labeled with phycoerythrin and detected by FL2.
|
|
View larger version (37K):
[in this window]
[in a new window]
|
Figure 5. Cell surface expression of TSHR-ß variants. Colocalization of TSHR-ßGFP fluorescence and plasma membrane staining with red lipophilic dye (CM-DiI). Upper panel shows GFP fluorescence in cells transfected with TSHR-ßGFP variants. The middle panel shows the same cells that have taken the CM-DiI. Patches of yellow peripheral fluorescence seen in some transfected cells is from the colocalization of GFP and CM-DiI as shown by the merged images in the bottom panel.
|
|
View larger version (40K):
[in this window]
[in a new window]
|
Figure 6. Immunoblot analysis of TSHR-ßGFP protein expression. Membranes prepared from cells transfected with GFP vector alone (lane 2), full-length TSHRGFP (lane 3), and TSHR-ß GFP (lane 1) were probed with polyclonal GFP antibody. The antibody detected a band corresponding in size to the TSHR-ß subunit in TSHR-ßGFP (lane 1) and TSHRGFP (lane 3) transfected cells as shown in lane 2 of the top panel. The control 27-kDa GFP band in vector transfected and other cells are indicated in the bottom panel.
|
|
Absence of signal transduction by truncated TSHRs
To determine if expression of the truncated TSHR variants mediated signaling, cAMP synthesis was monitored, both in the basal state and after stimulation with different concentrations of TSH. Compared with untransfected cells, there was no difference in the basal levels of cAMP generated by clones expressing full-length or truncated TSHR (Table 1). Upon stimulation with TSH, cells expressing the full-length receptor had a large increase in cAMP production, but there was no increase in cells expressing any of the three truncated TSHR-ß subunits. Similar data were obtained after stimulation with serum from patients with Graves’ disease and known to have stimulating TSHR antibodies (Fig. 7). Taken together, these experiments indicated that only cells expressing the full-length TSHR were able to increase cAMP accumulation after incubation with TSH or serum containing TSHR-stimulating autoantibodies.
View this table:
[in this window]
[in a new window]
|
Table 1. The levels of cAMP generated by cells expressing full-length or truncated TSHR-ß were measured under basal conditions (medium without serum) and with 1 mU/ml of TSH
|
|
View larger version (9K):
[in this window]
[in a new window]
|
Figure 7. Absence of cAMP responses in TSHR-ß transfected after treatment with Graves’ serum. TSHR-ß and TSHR full-length transfected cells were incubated in the presence and absence of Graves’ serum (2 mg/ml for 1 h) before the measurement of intracellular cAMP. There was an 8-fold increase in cAMP in full-length TSHR transfected subunits (bars 5 and 6), whereas neither the empty vector transfected (bars 1 and 2) nor the TSHR-ß transfected cells (bars 3 and 4) gave any response to stimulation with the serum. The values shown are the mean ± SE of cAMP measurement in three experiments.
|
|
|
Discussion
|
|---|
This study was prompted by recent observations in our laboratory demonstrating that the TSHR is present in multiple forms on thyrocytes and transfected cells, and that these can undergo rapid association/dissociation within the plasma membrane (6, 7, 8, 9). To better understand how these possible interactions may impact function, CHO cells expressing different TSHR constructs were used as a model system. Our studies were designed to determine which forms of the TSHR were coupled to G proteins, and whether one of the posttranslational cleavage products (TSHR-ß) had any autonomous signaling capacity. Our results suggested that only cleaved receptors were coupled to G proteins, as assessed by coimmunoprecipitation with G protein antibodies. Furthermore, CHO cells stably expressing TSHR-ß chains in the absence of TSHR- showed no evidence of either constitutive or ligand-induced activity, as assessed by cAMP generation.
Previously, we showed that cleaved TSHRs had the capacity to dimerize and oligomerize in this model system (8). Taken together, the results of the present study suggest that cleavage is likely to be associated with both function and self-association of this receptor, although the precise relationship remains unclear. As the vast majority of TSHRs are present as cleavage-derived subunits in thyroid tissue-derived membrane preparations (17), this is not unexpected. However, the situation is reversed in the CHO-TSHR model, in which uncleaved TSHRs may predominate, due to reduced trafficking and processing efficiencies of this receptor in nonthyroidal cells (14). In this model, it has also been reported that artificially uncleaved TSHRs were competent to bind TSH and to signal (15). The latter implied coupling to Gs, but this may have been aided by an abnormal ectodomain secondary to the sequence changes applied to prevent cleavage. Coimmunoprecipitation of cleaved but not uncleaved TSHRs with Gs antibodies in the present study suggested distinct coupling differences. For example, it could be that coupling to TSH holoreceptors was also less efficient and/or less stable, or that they were less amenable to immunoprecipitation due to conformational differences from cleaved receptor-G protein complexes. While intrinsically interesting, it is important not to lose sight of the fact that it is the cleaved receptor that predominates in the native thyroid cell.
The next issue addressed in this report involved a single subunit of this two-subunit receptor, the TSHR-ß component. Several observations on the status of this component have suggested that it might have innate activity, independent of TSHR- (18, 19). First are the simple facts of cleavage and shedding, making this receptor unlike its otherwise closely related glycoprotein hormone family members, the LH and FSH receptors, which do not cleave (1, 2, 20). The end result of both events is the creation of two physically independent entities, TSHR- and TSHR-ß subunits (5). In theory, either or both could retain autonomous function, either in the circulation (TSHR-) and/or on the plasma membrane (TSHR-ß). A second observation was related to the constitutive activity of this receptor. One model, supported by mutational analysis has suggested that nonliganded TSHR- functions to dampen the constitutive activity of the receptor via direct interaction with TSHR-ß (21, 22) and as previously demonstrated with truncated TSHR (19). This predicted that independent expression of TSHR-ß, without TSHR-, as in the present study, should have enhanced constitutive signaling. This was not the case using three size variants of TSHR-ß selected for our experiments as stable cells. One explanation may be that the levels of expression were less than in cells expressing full-length TSHR (JP09), which may exhibit readily detectable constitutive signaling (compared with JP02 cells lacking the TSHR) perhaps secondary to an unphysiological degree of overexpression. However, our data did not support the observations that TSHR-ß has the capacity for normal autonomous signaling, either constitutively or TSH-induced. Whether constitutive activation is an artificial result of overexpressing the TSHR, an effect of only transient expression systems, or a normal physiologically relevant phenomenon remains to be further determined.
|
Acknowledgments
|
|---|
We thank Alla Pritsker for technical assistance and the help of Dr. Scott Henderson of the Mount Sinai confocal laser scanning unit. R.L. is supported by the David Owen Segal Endowment.
|
Footnotes
|
|---|
This work was supported by NIH Grants DK-52464, DK-35764, and DK-45011 (to T.F.D.).
Abbreviations: Ab, Autoantibody; CHO, Chinese hamster ovary; CM-DiI, lipophilic tracer carbocyanine; GFP, green fluorescent protein; TSHR, TSH receptor.
Received August 23, 2002.
Accepted for publication April 1, 2003.
|
References
|
|---|
- Graves PN, Davies TF 2000 New insights into the thyroid-stimulating hormone receptor. The major antigen of Graves’ disease. Endocrinol Metab Clin North Am 29:267–86[CrossRef][Medline]
- Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM 1998 The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies [Erratum (1999) 20:100]. Endocr Rev 19:673–716[Abstract/Free Full Text]
- Sanders J, Oda Y, Roberts SA, Maruyama M, Furmaniak J, Smith BR 1997 Understanding the thyrotropin receptor function-structure relationship. Baillieres Clin Endocrinol Metab 11:451–479[CrossRef][Medline]
- Davies T, Marians R, Latif R 2002 The TSH receptor reveals itself. J Clin Invest 110:1–4
- 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]
- Graves PN, Vlase H, Davies TF 1995 Folding of the recombinant hTSH receptor extracellular domain: identification of folded monomeric and tetrameric complexes which bind TSH receptor autoantibodies. Endocrinology 136:521–527[Abstract]
- Graves PN, Vlase H, Bobovnikova Y, Davies TF 1996 Multimeric complex formation by the natural TSH receptor. Endocrinology 137:3915–3920[Abstract]
- 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]
- 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]
- Duprez L, Parma J, Costagliola S, Hermans J, Van Sande J, Dumont JE, Vassart G 1997 Constitutive activation of the TSH receptor by spontaneous mutations affecting the N-terminal extracellular domain. FEBS Lett 409:469–474[CrossRef][Medline]
- Kopp P, Muirhead S, Jourdain N, Gu WX, Jameson JL, Rodd C 1997 Congenital hyperthyroidism caused by a solitary toxic adenoma harboring a novel somatic mutation (serine281->isoleucine) in the extracellular domain of the thyrotropin receptor. J Clin Invest 100:1634–1639[Medline]
- Couet J, Misrahi M, Milgrom E, Shedding of the extracellular domain of the human TSH receptor from thyrocytes and transfected L cells. Program of the 77th Annual Meeting of The Endocrine Society, Washington, DC, 1995, p 78 (Abstract 24-5)
- 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]
- Misrahi M, Ghinea N, Sar S, Saunier B, Jolivet A, Loosfelt H, Cerutti M, Devauchelle G, Milgrom E 1994 Processing of precursors of the human TSH receptor in various eukaryotic cells. Eur J Biochem 222:711–719[Medline]
- 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]
- Vlase H, Nakashima M, Graves PN, Tomer Y, Morris JC, Davies TF 1995 Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice: evidence for intramolecular epitope spreading. Endocrinology 136:4415–4423[Abstract]
- Graves P, Pritsker A, Davies TF 1999 Post-translational processing of the natural human thyrotropin receptor: demonstration of more than two cleavage sites. J Clin Endocrinol Metab 84:2177–2181[Abstract/Free Full Text]
- Szkudlinski MW, Fremont V, Ronin C, Weintraub BD 2002 Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships. Physiol Rev 82:473–502[Abstract/Free Full Text]
- 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]
- Rees Smith B, McLachlan SM, Furmaniak J 1988 Autoantibodies to the thyrotropin receptor. Endocr Rev 9:106–121[Abstract/Free Full Text]
- Vassart G, Dumont JE 1992 The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr Rev 13:596–611[Abstract/Free Full Text]
- Vassart G, Van Sande J, Parma J, Tonacchera M, Duprez L, Swillens S, Dumont J 1996 Activating mutations of the TSH receptor gene cause thyroid diseases. Ann Endocrinol (Paris) 57:50–54[Medline]
This article has been cited by other articles:
|
|
|
|
 
G. Kleinau, H. Jaschke, S. Neumann, J. Lattig, R. Paschke, and G. Krause
Identification of a Novel Epitope in the Thyroid-stimulating Hormone Receptor Ectodomain Acting as Intramolecular Signaling Interface
J. Biol. Chem.,
December 3, 2004;
279(49):
51590 - 51600.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
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]
|
|
|
Top
Abstract
Introduction
