help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 Nguyen, L. Q.
Right arrow Articles by Kopp, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nguyen, L. Q.
Right arrow Articles by Kopp, P.
Endocrinology Vol. 143, No. 2 395-402
Copyright © 2002 by The Endocrine Society

TRH-TSH-THYROID

Cloning of the Cat TSH Receptor and Evidence Against an Autoimmune Etiology of Feline Hyperthyroidism

Lynda Q. Nguyen, Onur Karamanoglu Arseven, Hans Gerber, Barbara S. Stein, J. Larry Jameson and Peter Kopp

Division of Endocrinology, Metabolism & Molecular Medicine (L.Q.N., O.K.A., J.L.J., P.K.), Northwestern University Medical School, Chicago, Illinois 60611; Department of Clinical Chemistry Inselspital (H.G.), University of Berne, 3010 Berne, Switzerland; and Chicago Cat Clinic (B.S.S.), Chicago, Illinois 60646

Address all correspondence and requests for reprints to: Peter Kopp, M.D., Division of Endocrinology, Metabolism & Molecular Medicine, Northwestern University, Tarry 15, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: p-kopp{at}northwestern.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cats are the only nonhuman mammalian species with a high incidence of hyperthyroidism, and a better understanding of the pathogenesis of feline hyperthyroidism is of clinical relevance for veterinary medicine. The etiology of this disease in cats remains controversial. Both an intrinsic autonomy of growth and function of follicular cells as well as an autoimmune-related mechanism have been proposed. To explore the role of the autologous TSH receptor (TSHR) in the pathogenesis of hyperthyroidism in cats, we cloned the coding sequence of the feline TSHR by RT-PCR. The open reading frame consists of 2292 nucleotides and encodes a 763-amino acid protein, one amino acid less than the human TSHR. Species comparison reveals that the cat TSHR is most closely related to the canine TSHR, with 96% identity and 97% similarity in amino acid sequence. cAMP accumulation, inositol phosphate production, and TSH binding were similar in the feline TSHR, compared with the human receptor. Analogous to the human TSHR, the cat TSHR also displays basal constitutive activity. To test the possibility that hyperthyroid cats develop antibodies that stimulate the autologous receptor, transfected cells expressing the feline TSHR were treated with sera or purified IgG obtained from 16 hyperthyroid cats. There was no increase in cAMP-dependent luciferase activity in the hyperthyroid cats, suggesting the absence of stimulatory autoantibodies. These sera were also negative for TSH-binding inhibitory Igs in an RRA. At least in the animals included in this study, there is no evidence for the presence of circulating thyroid stimulating factors as a mechanism underlying the pathogenesis of feline hyperthyroidism, and the findings support a model involving intrinsic autonomy of thyroid follicular cell growth and function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ASIDE FROM HUMANS, cats are the only other mammalian species in which spontaneous thyrotoxicosis occurs relatively frequently, and feline hyperthyroidism is particularly common among aging cats (1, 2, 3, 4, 5, 6). The pathogenic mechanisms underlying feline hyperthyroidism remain controversial, as both an autonomous mechanism of growth and function and an autoimmune etiology have been proposed (7, 8).

In Graves’ disease, the most prevalent form of hyperthyroidism in humans, excessive hormone production of the thyroid gland is caused by thyroid-stimulating antibodies that bind to the TSH receptor (TSHR) (9). Thyroid autoantibodies against the TSHR were thought to be present in sera of hyperthyroid cats, in studies using indirect immunofluorescence (10). Furthermore, purified IgG preparations from hyperthyroid cats caused a significant increase in [3H]- thymidine incorporation into DNA, as well as a strong stimulation of cell proliferation in the Fischer rat thyroid cell line FRTL-5 (11). However, none of these studies provided evidence that the tested sera or IgG directly stimulated hormone production.

In contrast to these observations suggesting a resemblance to human autoimmune hyperthyroidism, histologic and autoradiographic analyses of surgically removed feline thyroids demonstrated that the vast majority of thyrotoxic cats have multinodular goiters (3, 7). The nodules, ranging in size from less than 1 mm to 3 cm, have an increased propensity for iodine organification and [3H]-thymidine incorporation and, in contrast to the surrounding normal paranodular tissue, seem to be TSH-independent (3, 7, 12). In addition, xenotransplantation studies of goitrous tissue from hyperthyroid cats onto athymic (nu/nu) mice revealed that the cells are hyperfunctioning and proliferate autonomously, even when the endogenous TSH of the host is suppressed by concomitant administration of T4 (7). Goitrous tissue from hyperthyroid cats may thus provide a useful experimental model to elucidate the pathogenesis leading to hyperplasia and hyperfunction (1, 5, 7).

In addition to its important role in thyroid follicular cell function and as a target of stimulatory autoantibodies, the TSHR can also be activated by somatic and germline mutations in the TSHR gene (13, 14). In humans, somatic TSHR mutations have been found in patients with autonomously functioning toxic adenomas (14, 15), whereas germline TSHR mutations are associated with an autosomal dominant syndrome of hereditary toxic thyroid hyperplasia or sporadic congenital hyperthyroidism (14, 16, 17). Mutational analysis of the transmembrane domain of the feline TSHR gene in cases of sporadic and familial feline thyrotoxicosis suggest that TSHR gene mutations are not involved in the pathogenesis of feline hyperthyroidism (18).

In this study, we cloned the coding sequence of the feline TSHR and investigated whether sera or purified IgG from hyperthyroid cats contains stimulatory autoantibodies against the autologous receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of the coding region of the feline TSHR
Total RNA was isolated from cat thyroid tissue using the thiocyanate method. Reverse transcription was performed with 4 µg RNA. Two overlapping fragments, A and B, were then amplified by PCR. Fragment A was generated using primers specific for the human TSHR at the 5' untranslated region (-43 to -23 bp, relative to the A of the initiation codon: 5'-GCGATTTCGGAGGATGGAGAA-3'), and a reverse primer covering nucleotides 1595–1575 of the human coding sequence (5'-CCGGTCCAGGCGCATGGCG-3'). Fragment B was generated with a sense primer covering nucleotides 900–921 (5'-GTGTAATGACAGCAGTATTCGG-3') in the human coding sequence and a degenerate primer based on the alignment of the human, mouse, and rat TSHR sequences in the 3' untranslated region (5'-GGGAT/CTGGAA/GT/CGCATA/GTTCA/TAGAAAC-3'; 2342–2368 in the human sequence). The PCR products were cloned into the TA cloning vector PCRII.I (Stratagene, La Jolla, CA), and six clones of each fragment were subjected to direct DNA sequence analysis using FS AmpliTaq DNA polymerase with the ABI prism dye primer cycle sequencing kit, following the protocol of the supplier (PE Applied Biosystems, Foster City, CA). Sequencing products were analyzed using a 373A DNA sequencer (PE Applied Biosystems).

After sequence analysis of fragment A and B, the full-length coding region (-21 to +2315) of the feline TSHR was generated by PCR using Pfu polymerase and cloned into the XhoI and BamHI sites of the expression vector pSVL (Pharmacia Biotech, Piscataway, NJ). The final construct was verified by direct DNA sequencing.

Assays of TSHR function: transient expression; cAMP and luciferase assays; determination of inositol phosphates
TSA-201 cells, a clone of human embryonic kidney 293 cells (19), were maintained in DMEM supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were seeded into 12-well plates and transfected using the calcium-phosphate method. The wild-type feline or human TSHR cDNAs were transfected using 500 ng/well plasmids. The wild-type human TSHR cDNA in the expression vector pSVL was a gift from Prof. Gilbert Vassart (Brussels, Belgium). The empty pSVL vector was included as negative control.

For luciferase assays, a cAMP-dependent reporter plasmid (500 ng), containing 846 bp of the human glycoprotein hormone {alpha}-promoter fused to the luciferase gene ({alpha}-846-Luc), was cotransfected with the pSVL vector expressing wild-type feline or human TSHR, or pSVL vector only. Cells were harvested after 48 h for luciferase assays, cAMP, inositol phosphates, and TSH binding studies. Luciferase assays were performed as previously described (20, 21).

For cAMP accumulation, cells were incubated for 1 h in PBS containing 0.5 mM 3-isobutyl-1-methyl-xanthine ± 10 mIU/ml bovine TSH (Sigma, St. Louis, MO). The medium was then removed, and cAMP was extracted with ice-cold 95% ethanol (22). The cell extracts were dried under vacuum, and cAMP was determined with an enzyme immunoassay (BIOTRAK; Amersham Pharmacia Biotech, Arlington Heights, IL).

For determination of inositol phosphates, cells were incubated with 4 mCi/ml [3H]-myoinositol (American Radiolabeled Chemicals, St. Louis, MO) in DMEM supplemented with 3% FBS. The medium was removed after 24 h and replaced by PBS containing 20 mM LiCl for 30 min at 37 C. Cells were then treated with ± 100 mIU/ml bovine TSH for 30 min. After removal of the medium, ice-cold 0.4-M HClO4 was added. [3H]-Labeled inositol phosphates were isolated by AG1-X8 resin chromatography (23) and measured in a scintillation counter. All experiments were performed at least four times in triplicate; groups were compared by ANOVA (Statview; Microsoft Corp., Redmond, WA).

TSH-binding
Binding of TSH in transfected TSA cells was measured using 125I-labeled bovine TSH (20, 21). Cells were incubated for 2 h at 22 C in 400 µl assay buffer (222 mM sucrose-supplemented NaCl-free HBSS with 0.5% BSA and 20 mM HEPES, pH 7.4) containing 2–4 x 104 cpm (45 mCi) 125I-bovine TSH (a gift from BRAHMS Diagnostica, Berlin, Germany) in the presence of increasing amounts of unlabeled bovine TSH. Cells were washed with assay buffer and solubilized with 1 N NaOH. Bound radioactivity was measured, and specific binding was calculated by subtracting nonsaturable binding in the presence of 10-7 M unlabeled TSH. The binding studies were performed in duplicate and repeated more than six times. Data analysis and computation of the 50% inhibitory concentration (IC50) and total receptor number (Bmax) was performed with the Prism 2.0 software (GraphPad Software, Inc., San Diego, CA).

cAMP-Dependent luciferase assay with serum or purified IgG from hyperthyroid cats
Serum was obtained from cats with untreated hyperthyroidism and patients with Graves’ disease. IgGs were purified using a protein A column according to the protocol and with buffers provided by the supplier (Sigma). In brief, the protein column was regenerated with HEPES buffer and a regeneration buffer and then equilibrated with binding buffer at a flow rate of 1 ml/min. A total of 1–2 ml serum mixed with 2–4 ml of the binding buffer were then loaded onto the column with an approximate flow rate of 0.5 ml/min. The column was subsequently rinsed with binding buffer and attached to a desalting cartridge. Elution of the IgGs was performed by passing 5 ml elution buffer through the column and a desalting cartridge. Subsequently, the eluted proteins were dialyzed against PBS overnight, and protein concentration was determined by the Bradford assay. Cells were incubated with serum or increasing amounts of purified IgGs as indicated, for 6–24 h. Luciferase assays were performed as described above.

Determination of TSHR antibodies by RRA
Determination of TSHR binding inhibitory Igs (TBII) was performed using a commercial RRA, following the protocol of the supplier (Kronus, San Clemente, CA). Serum from patients with Graves’ disease and serum samples from the hyperthyroid cats were incubated with 125I-labeled bovine TSH and solubilized porcine TSHRs and compared with a standard curve established with samples with known amounts of TBII. After incubation, receptor-bound 125I-labeled TSH was separated from unbound 125I-labeled TSH.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sequence comparison
The amino acid sequence of the feline TSHR, aligned to all known TSHR sequences, is shown in Fig. 1Go. The coding sequence has been deposited in GenBank (accession no. AF218264). The feline TSHR is most closely related to the canine receptor, with 96% identity and 97% similarity in amino acid sequence. The amino acid sequence of the cat TSHR is 90% identical and 92% similar to the human TSHR. The feline TSHR lacks glutamate 360, a residue that is conserved in all other species, making its overall length only 763 residues.



View larger version (103K):
[in this window]
[in a new window]
  		Figure 1. TSHR amino acid sequences. Amino acid (aa) sequence alignment of the feline, canine, human, ovine, murine, and rat TSHRs. The feline (aa 360) and the bovine (aa 329) TSHRs are shorter, by 1 aa, and only have 763 residues. The amino acid sequence of the human TSHR is taken from Libert et al. (32 ) and differs at position 601 (Y601H) from the sequence reported by Nagayama et al. (34 ). Potential N-glycosylation sites are shown in bold and are underlined; the transmembrane domains are shown in bold. The coding sequence has been deposited in GenBank (accession no. AF218264).

 
The feline sequence reported here differs from the published transmembrane domain sequence of Pearce et al. (18) at codons 487 (E487D), 624 (E624K), and 635 (I635M). This may reflect the presence of polymorphisms in the cat TSHR DNA sequence. In DNA extracted from three cell lines obtained from autonomous nodules (Gerber, H., and H. J. Peter; unpublished results), we also found a sequence variation at codon 558 (V558L) in one allele of one cell line. Functional analysis of the V558L TSHR, however, revealed that this conservative amino acid change has no apparent effect on function, and probably represents a simple polymorphism (data not shown).

Functional properties of the feline TSHR
Functional assays revealed that the feline TSHR has characteristics similar to those of the human TSHR (Fig. 2–4GoGoGo). Basal and TSH-stimulated cAMP accumulation were similar in cells transfected with the wild-type feline TSHR, compared with cells transfected with the wild-type human TSHR (Fig. 2Go, A and B). Measuring cAMP accumulation after 1 h, there was detectable constitutive basal activity (Fig. 2AGo), but this was more readily apparent in the cAMP-dependent luciferase assay (Fig. 2CGo). This observation is consistent with previous studies (20, 21), and it suggests that, in addition to the differences in duration of cAMP accumulation in the two experimental systems, the luciferase assay may be more sensitive because of the integration of amplified downstream signals (24).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 2. cAMP accumulation and cAMP-dependent luciferase assays. A and B, TSA cells were cotransfected with pSVL vector and wild-type human or feline pSVL TSHRs. cAMP was measured using an enzyme-based immunoassay, 1 h after treatment with medium (A) or medium supplemented with 10 IU/ml bovine TSH (B). Cells transfected with the wild-type human and feline TSHRs show constitutive basal activity (A) and a similar response to TSH (B). Values are the mean ± SEM from triplicates. C, TSA cells were cotransfected with pSVL vector, wild-type human or feline pSVL TSHRs, and the {alpha}-846-luc reporter. Cells were treated ± TSH (10 mIU/ml for 6 h). Both the human and the feline TSHRs display constitutive basal activity and have a similar increase in response to TSH. Values are the mean ± SEM from triplicates.

 


View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Inositol phosphate accumulation. TSA cells were transfected with pSVL vector or wild-type human and feline pSVL TSHRs. Twenty-four hours after transfection, cells were treated with 100 mIU/ml bovine TSH for 30 min. 3H-labeled inositol phosphates were isolated by AG1-X8 resin chromatography and measured in a scintillation counter. Basal and TSH-stimulated inositol phosphate accumulation did not differ significantly between the human and feline wild-type TSHRs. Values are the mean ± SEM from triplicates.

 


View larger version (19K):
[in this window]
[in a new window]
  		Figure 4. TSH binding to TSHRs. Binding of TSH in transfected TSA cells was measured using 125I-labeled bovine TSH and increasing amounts of unlabeled TSH. The IC50 value for the feline TSHR was 1.91 x 10-10 vs. 3.61 x 10-10 M for the human receptor, suggesting a slightly higher affinity of the cat TSHR for bovine TSH. The Bmax values were 1.4 x 10-9 mol/well for the human and 1.2 x 10-9 mol/well for the feline receptor, suggesting the presence of similar receptor numbers. Values are the mean ± SEM from duplicates.

 
Basal inositol phosphate accumulation did not differ between the human and the feline TSHRs. Cells transfected with the feline TSHR displayed a significant increase in inositol phosphate accumulation in response to TSH, albeit to a slightly lesser degree than for the human TSHR (Fig. 3Go).

The IC50 value for the feline TSHR was 1.91 x 10-10 vs. 3.61 x 10-10 M for the human receptor, suggesting a slightly higher affinity of the cat TSHR for bovine TSH (Fig. 4Go). The Bmax values were 1.4 x 10-9 mol/well for the human and 1.2 x 10-9 mol/well for the feline receptor, suggesting the presence of similar receptor numbers.

Stimulation with sera and IgG fractions from hyperthyroid cats
To determine whether hyperthyroid cats contain stimulatory antibodies to their autologous receptor, TSA cells transiently cotransfected with the feline TSHR and a cAMP-dependent luciferase reporter ({alpha}-846-Luc) were treated with sera or purified IgG derived from 16 thyrotoxic cats or from patients with Graves’ disease (Fig. 5AGo). cAMP-dependent luciferase accumulation did not increase in response to exposure of the cells to serum of hyperthyroid cats. In contrast, luciferase activity was significantly increased when cells were treated with sera from patients with Graves’ disease or bovine TSH. Transfection results from cells treated with purified IgG derived from two hyperthyroid cats are shown in Fig. 5BGo. Increasing doses of purified IgG did not stimulate cAMP-dependent luciferase activity. However, there was a significant increase in luciferase activity when cells were treated with bovine TSH.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. Treatment with sera and purified IgG from hyperthyroid cats. A, Cells transiently transfected with the feline TSHR were treated with medium without or with FBS (16 sera from hyperthyroid cats, 3 sera from patients with Graves’ disease) or 10 mIU/ml bovine TSH. In contrast to the Graves’ sera and bovine TSH, sera from hyperthyroid cats did not result in an increase of cAMP-dependent luciferase activity. The sera from 16 hyperthyroid cats and 3 patients with Graves’ disease have been tested individually, and the results have then been pooled. B, Purified IgG from hyperthyroid cats were added in increasing amounts to cells transiently transfected with the feline TSHR. Increasing amounts of purified IgG did not stimulate cAMP-dependent luciferase activity. Values are the mean ± SEM from triplicates.

 
Determination of TSHR antibodies by RRA
Determination of TBII, using an RRA, did not reveal the presence of significant inhibition of 125I-labeled TSH binding to porcine TSHRs, and TBII values were below 13 U/liter in all cats included in the study (data not shown). In contrast, control sera values from two patients with Graves’ disease were 50 and 80 U/liter, respectively.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The TSHR has been the subject of intense scrutiny because of its role in thyroid follicular cell physiology and as a target of autoantibodies in human autoimmune thyroid disease (9, 25, 26, 27). More recently, it has been the focus of interest because of the detection of gain-of-function mutations that provide a molecular explanation for the development of toxic adenomas, nonautoimmune familial hyperthyroidism, and sporadic nonautoimmune congenital hyperthyroidism (13, 14). Loss-of-function mutations in this receptor are recognized to result in partial or complete resistance to TSH, with compensated or overt hypothyroidism (13, 14, 28).

The cloning of a functional cat TSHR provides an essential tool for further characterizing the molecular mechanisms underlying feline hyperthyroidism, an important clinical entity in veterinary medicine. The availability of the feline TSHR enabled us to test the effects of sera and purified IgG from hyperthyroid cats on the autologous receptor. All previous studies addressing autoimmune mechanisms in hyperthyroid cats used either rat or porcine tissue. Therefore, it was conceivable that the negative results observed are attributable to discrete differences in epitopes among the different species (29, 30, 31). The biochemical properties of the cat TSHR are similar to those of the human TSHR. Transiently transfected mammalian cells expressing the cat TSHR failed to respond to treatments with sera or IgG purified from the hyperthyroid cats included in this study. In contrast, sera from patients with Graves’ disease stimulated the feline receptor and resulted in marked increases of cAMP-dependent luciferase activity. These results suggest that the hyperthyroidism in the animals included in this study does not have an autoimmune etiology. Together with the typically multinodular goiter found in cats with hyperthyroidism, this observation is consistent with models proposing an autonomous mechanism of growth and function intrinsic to thyroid follicular cells (1, 3, 12). This observation, however, does not exclude the possibility that the pathogenesis of feline hyperthyroidism may be heterogeneous, as it is the case in humans.

Pearce et al. (18) isolated the cDNA of the transmembrane domain of the feline TSHR and analyzed this segment of the receptor for activating mutations that may account for sporadic and familial feline thyrotoxicosis. However, no mutations were found in this region of the TSHR, suggesting that gain-of-function mutations in the TSHR probably are not a common cause of feline hyperthyroidism. Direct sequence analysis of DNA isolated from cell lines obtained from hyperthyroid cats is congruent with the study of Pearce et al., because there were no mutations in the transmembrane domain of the TSHR. The only sequence alteration we detected in DNA obtained from a cat cell line derived from an autonomous nodule, V558L, did not alter the function of the receptor, a finding that is not surprising, given the conservative nature of the amino acid change.

TSHR cDNAs of various mammalian species have been cloned, including man, dog, cow, sheep, rat, and mouse (20, 32, 33, 34, 35, 36, 37, 38, 39). Sequence comparison of the TSHRs from various species indicates a high degree of conservation (Fig. 1Go). A residue that was the focus of particular attention in the human TSHR is Y601, because one of the reported clones was found to have a histidine at this position (34). The other reported human cDNAs (32, 35), as well as all known TSHR sequences, identified a tyrosine at this position (Fig. 1Go). More recent studies indicated that substitution of tyrosine 601 by histidine results not only in loss of basal constitutive activity but also loss of coupling to Gq/11 (40, 41). Of note, the potential N-glycosylation sites are also highly conserved among the various TSHRs, but the human receptor has an additional N-glycosylation site at amino acids 113–115 (Fig. 1Go). The rather discrete differences in the cat TSHR do not seem to be of functional significance. Analogous to the human TSHR, the cat TSHR displays basal constitutive activity; and at higher doses of TSH, it stimulates the phospholipase C inositol phosphate pathway by coupling to Gq/11 (42).

The human TSHR is unique among the glycoprotein hormone receptors, because it is submitted to cleavage into two subunits (43, 44, 45, 46). This intramolecular cleavage seems to involve the removal of a segment of the ectodomain (46, 47, 48). Sequence comparison of the various mammalian TSHRs reveals a high degree of conservation at cleavage site 1 (human aa 302–317) and cleavage site 2 (human aa 367–369; Fig. 1Go). The human receptor differs at positions 306 and 307. In the human receptor, the hydrophobic isoleucine at position 306 is replaced by methionine. At position 307, it contains the uncharged polar glutamine residue, whereas all other TSHRs contain arginine or, in the case of the ovine receptor, the nonpolar tryptophan. Whether these differences in the human receptor are of importance for the phenomenon of cleavage, and thus possibly antigenicity of the receptor, is unclear. Of note, deletion mutants encompassing site 1 shift the cleavage site toward the aminoterminus, suggesting that cleavage is not dependent on a specific amino acid sequence but rather occurs at a fixed distance from a protease attachment site (49).

In summary, this study demonstrates that the feline TSHR has biochemical properties similar to those of the human receptor. Sera and purified IgG from hyperthyroid cats do not stimulate the autologous receptor, and, and least in the cats included in this study, the data argue against an autoimmune etiology of their hyperthyroidism. Sequence comparisons of various mammalian TSHR sequences may provide additional insight into discrete differences that may be relevant for the structure of certain antigenic epitopes in the human TSHR and thus the pathogenesis of Graves’ disease.


   Acknowledgments
 
This study is dedicated to the memory of Dr. Barbara S. Stein, D.V.M.

We are grateful to Wen-Xia Gu, Northwestern University, for help with the binding experiments; G. Vassart (Brussels, Belgium) for providing the human TSHR in pSVL; and BRAHMS Diagnostica for providing 125I-labeled bovine TSH.


   Footnotes
 
This work was supported by a Northwestern University New Investigator Award from the Howard Hughes Medical Institute (to P.K.) and NIH Grant DK-42144 (to J.L.J.).

Abbreviations: Bmax, Total receptor number; IC50, 50% inhibitory concentration; TBII, TSHR-binding inhibitory Igs; TSHR, TSH receptor.

Received August 22, 2001.

Accepted for publication October 5, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gerber H, Peter HJ, Ferguson D, Peterson ME 1994 Etiopathology of feline toxic nodular goiter. Vet Clin North Am Small Anim Pract 24:541–565[Medline]
  2. Holzworth S, Theran P, Carpenter JL, Harpster NK, Todoroff RJ 1980 Hyperthyroidism in the cat: ten cases. J Am Vet Med Assoc 176:345–353[Medline]
  3. Hoenig M, Goldschmidt MH, Ferguson DC, Koch K, Eymontt MJ 1982 Toxic nodular goiter in the cat. J Small Anim Pract 23:1–12[Medline]
  4. Peterson ME, Kintzer PF, Cavanagh PG, Fox PR, Ferguson DC, Johnson GF, Becker DV 1983 Feline hyperthyroidism: pretreatment and clinical evaluation of 131 cases. J Am Vet Med Assoc 183:103–110[Medline]
  5. Peterson M 1984 Feline hyperthyroidism. Vet Clin North Am Small Anim Pract 14:809–826[Medline]
  6. Scarlett JM 1994 Epidemiology of thyroid diseases of dogs and cats. Vet Clin North Am Small Anim Pract 24:477–486[Medline]
  7. Peter P, Gerber H, Studer H, Becker D, Peterson M 1987 Autonomy of growth and of iodine metabolism in hyperthyroid feline goiters transplanted onto nude mice. J Clin Invest 80:491–498
  8. Peter HJ, Gerber H, Studer H, Peterson ME, Becker DV, Groscurth P 1991 Autonomous growth and function of cultured thyroid follicles from cats with spontaneous hyperthyroidism. Thyroid 1:331–338[Medline]
  9. Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM 1998 The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr Rev 19: 673–716
  10. Kennedy RL 1988 Autoantibodies in feline hyperthyroidism. Res Vet Sci 45:300–306[Medline]
  11. Brown RS, Keating P, Livingston PG, Bullock L 1992 Thyroid growth immunoglobulins in feline hyperthyroidism. Thyroid 2:125–130[Medline]
  12. Peter HJ, Gerber H, Studer H, Smeds S 1985 Pathogenesis of heterogeneity in human multinodular goiter. J Clin Invest 76:1992–2002
  13. Vassart G 1998 Hypo- and hyperthyroidism caused by mutations in the TSH receptor. In: Spiegel A, ed. G proteins, receptors, and disease. Totowa, NJ: Humana Press; 119–138
  14. Kopp P 2001 The TSH receptor and its role in thyroid disease. Cell Mol Life Sci 58:1301–1322[CrossRef][Medline]
  15. Parma J, Duprez L, van Sande J, Cochaux P, Gervy C, Mockel J, Dumont J, Vassart G 1993 Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature 365:649–651[CrossRef][Medline]
  16. Duprez L, Parma J, van Sande J, Allgeier A, Leclere J, Schvartz C, Delisle M, Decoulx M, Orgiazzi J, Dumont J, Vassart G 1994 Germline mutations in the thyrotropin receptor gene cause non-autoimmune autosomal dominant hyperthyroidism. Nat Genet 7:396–401[CrossRef][Medline]
  17. Kopp P, Van Sande J, Parma J, Duprez L, Gerber H, Joss E, Jameson JL, Dumont JE, Vassart G 1995 Congenital hyperthyroidism caused by a germline mutation in the thyrotropin-receptor gene. N Engl J Med 332:150–154[Free Full Text]
  18. Pearce SH, Foster DJ, Imrie H, Myerscough N, Beckett GJ, Thoday KL, Kendall-Taylor P 1997 Mutational analysis of the thyrotropin receptor gene in sporadic and familial feline thyrotoxicosis. Thyroid 7:923–927[Medline]
  19. Margolskee R, McHendry-Rinde B, Horn R 1993 Panning transfected cells for electrophysiological studies. Biotechniques 15:906–911[Medline]
  20. Gu WX, Du GG, Kopp P, Rentoumis A, Albanese C, Kohn LD, Madison LD, Jameson JL 1995 The thyrotropin (TSH) receptor transmembrane domain mutation (Pro556-Leu) in the hypothyroid hyt/hyt mouse results in plasma membrane targeting but defective TSH binding. Endocrinology 136:3146–3153[Abstract]
  21. 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]
  22. Hirayu H, Magnusson R, Rapoport B 1985 Studies on the mechanism of desensitization of the cyclic AMP response to TSH stimulation in a cloned rat thyroid cell line. Mol Cell Endocrinol 42:21–27[CrossRef][Medline]
  23. Berridge M, Dawson R, Doumer P, Heslop J, Irvine R 1983 Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. J Biol Chem 212:473–482
  24. Jinsi-Parimoo A, Gershengorn MC 1997 Constitutive activity of native thyrotropin-releasing hormone receptors revealed using a protein kinase C- responsive reporter gene. Endocrinology 138:1471–1475[Abstract/Free Full Text]
  25. Rees Smith B, McLachlan SM, Furmaniak J 1988 Autoantibodies to the thyrotropin receptor. Endocr Rev 9:106–121[Abstract/Free Full Text]
  26. 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]
  27. Vassart G, Parma J, van Sande J, Dumont JE 1994 The thyrotropin receptor and the regulation of thyrocyte function and growth: update 1994. In: Braverman LE, Refetoff S, eds. Clinical and molecular aspects of diseases of the thyroid. Endocr Rev Monographs 3:77–80
  28. Kopp P 1999 Resistance to TSH. In: Jameson JL, ed. Hormone resistance syndromes. Totowa, NJ: Humana Press; 111–144
  29. Gerber H, Peter HJ, König MP, Studer H 1986 Species specific action of Graves’ serum on xenotransplanted human thyroid tissue. Ann Endocrinol (Paris). 15th Annual Meeting of The European Thyroid Association, Stockholm, 47 (Abstract 16)
  30. Gerber H, Peter HJ, Zakarija M 1987 Direct comparison of the stimulatory effect of Graves’s serum on thyroid tissue of 5 different species—a xenotransplantation study in nude mice. Ann Endocrinol (Paris). 16th Annual Meeting of The European Thyroid Association, 48 (Abstract 152)
  31. Gerber H, Peter HJ, Studer H 1992 Effect of human stimulating autoantibodies on the radioiodine uptake of the mouse thyroid gland. Exp Clin Endocrinol 100:45–47[Medline]
  32. Libert F, Lefort A, Gerard C, Parmentier M, Perret J, Ludgate M, Dumont JE, Vassart G 1989 Cloning, sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding of autoantibodies. Biochem Biophys Res Commun 165:1250–1255[CrossRef][Medline]
  33. Parmentier M, Libert F, Maenhaut C, Lefort A, Gerard C, Perret J, van Sande J, Dumont JE, Vassart G 1989 Molecular cloning of the thyrotropin receptor. Science 246:1620–1622[Abstract/Free Full Text]
  34. Nagayama Y, Kaufman KD, Seto P, Rapoport B 1989 Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem Biophys Res Commun 165:1184–1190[CrossRef][Medline]
  35. Misrahi A, Loosfelt H, Atger M, Sar S, Guiochon-Mantel A, Milgrom E 1990 Cloning, sequencing and expression of human TSH receptor. Biochem Biophys Res Commun 166:394–403[CrossRef][Medline]
  36. Akamizu T, Ikuyama S, Saji M, Kosugi S, Kozak C, McBride OW 1990 Cloning, chromosomal assignment, and regulation of the rat thyrotropin receptor: expression of the gene is regulated by thyrotropin, agents that increase cAMP levels, and thyroid autoantibodies. Proc Natl Acad Sci USA 87:5677–5681[Abstract/Free Full Text]
  37. Stein SA, Oates EL, Hall CR, Grumbles RM, Fernandez LM, Taylor NA 1994 Identification of a point mutation in the thyrotropin receptor of the hyt/hyt hypothyroid mouse. Mol Endocrinol 8:129–138[Abstract/Free Full Text]
  38. Silversides DW, Houde A, Ethier JF, Lussier JG 1987 Bovine thyrotropin receptor cDNA is characterized by full-length and truncated transcripts. J Mol Endocrinol 18:101–112
  39. Bockmann J, Winter C, Wittkowski W, Kreutz MR, Bockers TM 1997 Cloning and expression of a brain-derived TSH receptor. Biochem Biophys Res Commun 238:173–178[CrossRef][Medline]
  40. Biebermann H, Schoneberg T, Schulz A, Krause G, Gruters A, Schultz G, Gudermann T 1998 A conserved tyrosine residue (Y601) in transmembrane domain 5 of the human thyrotropin receptor serves as a molecular switch to determine G-protein coupling. FASEB J 12:1461–1471[Abstract/Free Full Text]
  41. Karamanoglu Arseven O, Wilkes WP, Jameson JL, Kopp P 1999 Naturally occurring variants of the human thyrotropin (TSH) receptor reveal that tyrosine 601 (Y601) plays a pivotal role in constitutive basal activity and coupling to Gsa and Gq/11. Thyroid 10:3–10
  42. Allgeier A, Offermans S, van Sande J, Spicher K, Schultz G, Dumont JE 1994 The human thyrotropin receptor activates G-protein Gs and Gq11. J Biol Chem 269:13733–13735[Abstract/Free Full Text]
  43. Loosfelt H, Pichon C, Jolivet A, Misrahi M, Caillou B, Jamous M 1992 Two-subunit structure of the human thyrotropin receptor. Proc Natl Acad Sci USA 89:3765–3769[Abstract/Free Full Text]
  44. 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. J Endocrinol Metab 138:2893–2899
  45. Tanaka K, Chazenbalk GD, McLachlan SM, Rapoport B 1999 Thyrotropin receptor cleavage at site 1 involves two discontinuous segments at each end of the unique 50-amino acid insertion. J Biol Chem 274:2093–2096[Abstract/Free Full Text]
  46. 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]
  47. 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]
  48. Chazenbalk GD, Tanaka K, McLachlan SM, Rapoport B 1999 On the functional importance of thyrotropin receptor intramolecular cleavage. J Clin Endocrinol Metab 140:4516–4520
  49. 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
 J EndocrinolHome page
D C Ferguson, Z Caffall, and M Hoenig
Obesity increases free thyroxine proportionally to nonesterified fatty acid concentrations in adult neutered female cats
J. Endocrinol., August 1, 2007; 194(2): 267 - 273.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
S G Watson, A D Radford, A Kipar, P Ibarrola, and L Blackwood
Somatic mutations of the thyroid-stimulating hormone receptor gene in feline hyperthyroidism: parallels with human hyperthyroidism
J. Endocrinol., September 1, 2005; 186(3): 523 - 537.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 Nguyen, L. Q.
Right arrow Articles by Kopp, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nguyen, L. Q.
Right arrow Articles by Kopp, P.

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