The Functional Expression of Recombinant Human Thyrotropin Receptors in Nonthyroidal Eukaryotic Cells Provides Evidence that Homologous Desensitization to Thyrotropin Stimulation Requires a Cell-Specific Factor*

Chazenbalk, Gregorio D.; Nagayama, Yuji; Kaufman, Keith D.; Rapoport, Basil

doi:10.1210/endo-127-3-1240

Cited by 59 publications

(20 citation statements)

References 22 publications

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“…In contrast, chimeras TSH-LHR-2 (residues 83-170), TSH-LHR-4 (residues 261-360), and TSH-LHR-5 (residues 361-418) bound TSH with high affinity. As with the wild-type TSH receptor (22), hCG did not bind to chimeras TSH-LHR-2, TSH-LHR-4, and TSH-LHR-5, indicating that these chimeras retained their specificity for TSH binding. Functionally, as determined by the intracellular cAMP response to TSH stimulation (Table 1) chimeras TSH-LHR-1 and TSH-LHR-3 were totally inactive, as expected in view of their inability to bind the hormone with high affinity.…”

Section: Resultsmentioning

confidence: 81%

Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function.

Nagayama

Wadsworth

Chazenbalk

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

179

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To define the sites in the extracellular domain of the human thyrotropin (TSH) receptor that are involved in TSH binding and signal transduction we constructed chimeric thyrotropin-luteinizing hormone/chorionic gonadotropin (TSH-LH/CG) receptors. The extracellular domain of the human TSH receptor was divided into five regions that were replaced, either singly or in various combinations, with homologous regions of the rat LH/CG receptor. The chimeric receptors were stably expressed in Chinese hamster ovary cells.The data obtained suggest that the carboxyl region of the extracellular domain (amino acid residues 261418) and particularly the middle region (residues 171-260) play a role in signal transduction. The possibility is also raised of an interaction between the amino and carboxyl regions of the extracellular domain in the process of signal transduction. With respect to hormone binding, substitution of the entire extracellular domain of the LH/CG receptor for the corresponding region of the TSH receptor resulted in high-affinity human CG binding with complete loss of TSH binding. Surprisingly, however, there was at least one chimera with a substitution at each of the five domains that still retained high-affinity TSH binding. Substitution of residues 1-170 of the TSH receptor with the corresponding region of the LH/CG receptor was associated with the retention of high-affinity TSH binding but ligand specificity was lost in that TSH and human CG could interact functionally with the receptor. In summary, these studies suggest that the middle region and carboxyl half of the extracellular domain of the TSH receptor are involved in signal transduction and that the TSH-binding region is likely to span the entire extracellular domain, with multiple discontinuous contact sites. 902The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Section: Resultsmentioning

confidence: 81%

Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function.

Nagayama

Wadsworth

Chazenbalk

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

179

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“…These data were compatible with previous reports showing TSHR desensitization in vitro by TSHR-Ab's (from Graves disease patients) (24-26) using thyroid cell cultures. However, with TSHR-transfected CHO cells there have been conflicting reports regarding desensitization induced by TSH (39)(40)(41)(42), and this most likely depended upon the characteristics of the CHO cells used (43). As far as we know, there are no reports showing TSHR downregulation induced by TSHR-Ab's in such models.…”

Section: Discussionmentioning

confidence: 99%

Concentration-dependent regulation of thyrotropin receptor function by thyroid-stimulating antibody

Ando¹,

Latif²,

Davies³

2004

J. Clin. Invest.

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Thyrotropin receptor (TSHR) Ab's of the stimulating variety are the cause of hyperthyroid Graves disease. MS-1 is a hamster mAb with TSHR-stimulating activity. To examine the in vivo biological activity of MS-1, mice were treated with purified MS-1 intraperitoneally and the thyroid response evaluated. MS-1 induced a dose-dependent increase in serum thyroxine (T4), with a maximum effect after 10 μg of MS-1 was administered. MS-1-secreting hybridoma cells were then transferred into the peritoneum of nude mice to study chronic thyroid stimulation. Serum MS-1 levels detected after 2 weeks were approximately 10-50 μg/ml, and the serum TSH was suppressed in all animals. Serum triiodothyronine levels were elevated, but only in animals with low serum MS-1 concentrations. In addition, there was a negative correlation between serum T4 and the serum MS-1 concentrations. These in vivo studies suggested a partial TSHR inactivation induced by excessive stimulation by MS-1. We confirmed this inactivation by demonstrating MS-1 modulation of TSHR function in vitro as evidenced by downregulation and desensitization of the TSHR at concentrations of MS-1 achieved in the in vivo studies. Thus, inactivation of the TSHR by stimulating TSHR autoantibodies (TSHRAb's) in Graves disease patients may provide a functional explanation for the poor correlation between thyroid function and serum TSHR-Ab concentrations.

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“…Thyrotropin-induced stimulation of cAMP synthesis was then initiated with the indicated concentrations of bovine thyrotropin. After 60 min at 37"C, at which point maximal thyrotropin-induced CAMP accumulation is attained [26], the reaction was stopped by the addition of 1 ml 5% trichloroacetic acid. CAMP was isolated by ion-exchange chromatography and measured by a protein-binding assay as described previously [27].…”

Section: Methodsmentioning

confidence: 99%

Enhanced cAMP Accumulation by the Human Thyrotropin Receptor Variant with the Pro52Thr Substitution in the Extracellular Domain

Loos¹,

Hagner²,

Bohr³

et al. 1995

European Journal of Biochemistry

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Recently, a naturally occurring variant of the human thyrotropin receptor with a Pro52Thr substitution in the N-terminal extracellular domain of the receptor has been identified. To determine the functional significance of this substitution, cDNAs of wild-type and variant thyrotropin receptors were stably expressed in Chinese hamster ovary cells. The Pro52Thr substitution did not affect synthesis and membrane localization of the receptor, as evidenced by '251-thyrotropin binding analysis to intact cells. The variant receptor and the wild-type receptor were expressed in equivalent numbers and displayed identical binding affinity for thyrotropin. Strikingly, thyrotropin increased cAMP accumulation to a much greater extent in cells expressing the variant receptor as compared to the wild-type receptor-expressing cells. Basal and cholera toxin-stimulated or forskolin-stimulated cAMP levels were not different. It is concluded that the Pro52Thr substitution in the N-terminal region of the human thyrotropin receptor produces a receptor protein with enhanced coupling to cAMP production. This naturally occurring hyperactive thyrotropin receptor may participate in hyperthyroidism of patients with Graves' disease.Keywords: thyrotropin receptor variant; CAMP; Chinese hamster ovary cells ; Graves' disease.Thyrotropin exerts its effects on the thyroid cell by binding to specific receptors on the cell surface. The thyrotropin receptor belongs to the superfamily of seven-transmembrane receptors that activate signal-transduction pathways through their interaction with guanine-nucleotide-binding regulatory proteins (G proteins) [I -41. The receptor represents one member of a subfamily of glycoprotein hormone receptors that also includes the follitropin receptor and the lutropin receptor [5-lo], and members of this subfamily are characterized by a large N-terminal extracellular domain. As demonstrated for the thyrotropin receptor, this extracellular domain is essential for ligand binding and has also been implicated in signal transduction [I1 -1 31. In contrast to the glycoprotein hormone receptors, the majority of G-protein-coupled receptors, such as rhodopsin, adrenergic, and muscarinic receptors, have a short N-terminal region that does not play a major role in ligand binding and signal transduction [14, 151. In both subgroups, however, the cytoplasmic regions play a pivotal role in G protein coupling and subsequent effector activation [14-181. The mechanism by which binding of thyrotropin to the extracellular region of the receptor is transduced via the membrane-spanning regions to the cytoplasmic segments of the receptor that interact with the G proteins is unknown. In human thyroid cells, signal transduction by the thyrotropin receptor is predominantly mediated by the stimulatory G, protein resulting in enhanced cAMP synthesis by adenylyl cyclase, hormone secretion, and proliferation [ 191.The thyrotropin receptor plays a pivotal role in function and growth of thyroid cells [19]. The receptor is also thought to be the central...

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The Functional Expression of Recombinant Human Thyrotropin Receptors in Nonthyroidal Eukaryotic Cells Provides Evidence that Homologous Desensitization to Thyrotropin Stimulation Requires a Cell-Specific Factor*

Cited by 59 publications

References 22 publications

Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function.

Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function.

Concentration-dependent regulation of thyrotropin receptor function by thyroid-stimulating antibody

Enhanced cAMP Accumulation by the Human Thyrotropin Receptor Variant with the Pro52Thr Substitution in the Extracellular Domain

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