scholarly journals The Hinge Region of the TSH Receptor Stabilizes Ligand Binding and Determines Different Signaling Profiles of Human and Bovine TSH

Endocrinology ◽  
2011 ◽  
Vol 152 (10) ◽  
pp. 3986-3996 ◽  
Author(s):  
Holger Jaeschke ◽  
Jörg Schaarschmidt ◽  
Robert Günther ◽  
Sandra Mueller
Keyword(s):  
Ligand Binding ◽  
Hinge Region ◽  
Tsh Receptor ◽  
Bovine Tsh

scholarly journals The Superagonistic Activity of Bovine Thyroid-stimulating Hormone (TSH) and the Human TR1401 TSH Analog Is Determined by Specific Amino Acids in the Hinge Region of the Human TSH Receptor

2009 ◽  
Vol 284 (24) ◽  
pp. 16317-16324 ◽  
Author(s):  
Sandra Mueller ◽  
Gunnar Kleinau ◽  
Mariusz W. Szkudlinski ◽  
Holger Jaeschke ◽  
Gerd Krause ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thyroid Stimulating Hormone ◽  
Hinge Region ◽  
Camp Production ◽  
Glycoprotein Hormone ◽  
Charged Amino Acids ◽  
Bovine Thyroid ◽  
Positively Charged ◽  
Bovine Tsh ◽  
Signaling Activity

Bovine TSH (bTSH) has a higher affinity to the human TSHR (hTSHR) and a higher signaling activity than human TSH (hTSH). The molecular reasons for these phenomena are unknown. Distinct negatively charged residues (Glu297, Glu303, and Asp382) in the hinge region of the hTSHR are known to be important for bTSH binding and signaling. To investigate the potential relevance of these positions for differences between bTSH and hTSH in the interaction to the hTSHR, we determined bTSH- and hTSH-mediated cAMP production of several substitutions at these three hinge residues. To examine specific variations of hTSH, we also investigated the superagonistic hTSH analog TR1401 (TR1401), whose sequence differs from hTSH by four additional positively charged amino acids that are also present in bTSH. To characterize possible interactions between the acidic hTSHR positions Glu297, Glu303, or Asp382 and the additional basic residues of TR1401, we investigated TR1401 binding and signaling properties. Our data reveal increased cAMP signaling of the hTSHR using TR1401 and bTSH compared with hTSH. Whereas Asp382 seems to be important for bTSH- and TR1401-mediated but not for hTSH-mediated signaling, the substitution E297K exhibits a decreased signaling for all three TSH variants. Interestingly, bTSH and TR1401 showed only a slightly different binding pattern. These observations imply that specific residues of the hinge region are mediators of the superagonistic activity of bTSH and TR1401 in contrast to hTSH. Moreover, the simultaneous localization of binding components in the glycoprotein hormone molecule and the receptor hinge region permits important reevaluation of interacting hormone receptor domains.

Bovine thyrotropin receptor cDNA is characterized by full-length and truncated transcripts

1997 ◽  
Vol 18 (2) ◽  
pp. 101-112 ◽  
Author(s):  
D W Silversides ◽  
A Houde ◽  
J-F Ethier ◽  
J G Lussier
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transmembrane Domain ◽  
Full Length ◽  
Tsh Receptor ◽  
Thyrotropin Receptor ◽  
Pcr Cloning ◽  
Specific Expression ◽  
Terminal Domain ◽  
Bovine Tsh

ABSTRACT The complete coding sequence for the bovine thyrotropin (TSH) receptor was derived using a modified PCR cloning strategy. The bovine thyrotropin receptor conforms to the pattern of receptor interacting with membrane-bound G-protein already established in other species for TSH and gonadotropins receptors. The cDNA for the bovine TSH receptor consists of an open reading frame 2289 nucleotides in length, corresponding to a protein of 763 amino acids (estimated molecular mass of 86·4 kDa) which includes a 20 amino acid putative leading signal peptide. The receptor consists of a large NH2-terminal extracellular membrane domain of 417 amino acids with 5 potential N-linked glycosylation sites, a transmembrane domain (265 amino acids) consisting of 7 putative membrane α-helix spanning segments, and an intracytoplasmic COOH-terminal domain (82 amino acids). The bovine TSH receptor is one amino acid less than the corresponding sequence in dog, human, rat and mouse. Cysteine residues (n=22) were conserved when compared with other TSH receptors. Three potential phosphorylation sites were found in the transmembrane domain and the COOH-terminal domain. As with other members of this receptor family, alternative splicing was observed. A transcribed but truncated TSH receptor of 1769 nucleotides was demonstrated, lacking half of the V segment of the transmembrane domain up to the COOH-terminal domain of the full length TSH receptor. Additionally, alternative transcriptional start sites were observed. Northern blot analysis using a probe (1170 bp) spanning part of the extracellular domain up to the first loop of the transmembrane domain showed specific expression in the bovine thyroid gland with major transcripts of 9·3 and 4·3 kb, and a minor transcript of 3·8 kb being detected.

STUDIES OF THE TSH RADIORECEPTOR ASSAY

1979 ◽  
Vol 90 (2) ◽  
pp. 217-226 ◽  
Author(s):  
Gildon N. Beall ◽  
Inder J. Chopra ◽  
David H. Solomon ◽  
W. James Irvine ◽  
Sally R. Kruger
Keyword(s):  
Protease Inhibitor ◽  
Specific Binding ◽  
Thyroid Tissue ◽  
Radioreceptor Assay ◽  
Tsh Receptor ◽  
Human Thyroid ◽  
Post Mortem ◽  
Human Thyroid Tissue ◽  
Several Variables ◽  
Bovine Tsh

ABSTRACT We have examined several variables in the reagents and procedures used in the TSH radioreceptor assay, the binding of iodinated TSH to its thyroidal receptor. We found that iodinated bovine TSH (S.A. 30 U/mg) was more effectively bound to receptor than iodinated human TSH (S.A. 7.3 U/mg). Iodination of TSH with the Bolton-Hunter acylation method apparently prevented binding to TSH receptor. Surgically removed human thyroid tissue specifically bound 10.3 ±1.0 (mean ± sem) of added [125I]TSH, but post-mortem human thyroid bound only 3.9 ± 0.4% of [125I]TSH (P< 0.001). Maximal binding of [125I]TSH was found at pH 5.8. Many tissue preparations contained activity, possibly due to proteases, which inactivated TSH, and inclusion of a protease inhibitor, aprotinin, significantly increased specific binding.

scholarly journals The Human Thyrotropin (TSH) Receptor in a TSH Binding Inhibition Assay for TSH Receptor Autoantibodies1

1997 ◽  
Vol 82 (7) ◽  
pp. 2129-2134
Author(s):  
Ayumu Kakinuma ◽  
Gregorio D. Chazenbalk ◽  
Juan Carlos Jaume ◽  
Basil Rapoport ◽  
Sandra M. McLachlan
Keyword(s):  
Intact Cell ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Tsh Receptor ◽  
Intact Cells ◽  
Ovary Cells ◽  
Large Numbers ◽  
Binding Inhibition ◽  
Excellent Source ◽  
Bovine Tsh

Seven years after the molecular cloning of the human TSH receptor (TSHR), the porcine TSHR remains in general use in the TSH binding inhibition (TBI) assay for autoantibodies to the TSHR. We compared porcine and recombinant human TSHR in two types of TBI assays: one using intact Chinese hamster ovary cells expressing the recombinant human TSHR on their surface, and the other using soluble receptors extracted from these cells with detergent. In the intact cell TBI assay, monolayers expressing large numbers of TSHR were less effective than cells expressing few receptors. These findings are consistent with the very low concentration of TSHR autoantibodies in serum. Binding of[ 125I]human TSH was about 5-fold lower than that of[ 125I]bovine TSH to the intact cells. Nevertheless, TBI values with the two ligands were similar for most sera. However, a few sera produced greater inhibition of human than of bovine TSH binding. In the solubilized human TSHR TBI assay, in contrast to the intact cell TBI assay, cells expressing very large number of TSHR were an excellent source for detergent extraction of soluble human TSHR, but only if the cells were extracted while still on the dish and not after scraping. A 10-cm diameter dish of cells provided TSHR for 100–200 replicate determinations when substituted for solubilized porcine TSHR in a commercial TBI kit. TBI values in serum from 30 individuals with suspected Graves’ disease correlated closely when tested with solubilized human and porcine TSHR (r = 0.954; P &lt; 0.001). However, 2 sera that were negative with the porcine TSHR were positive with the human TSHR. TBI and thyroid-stimulating activity in these sera correlated weakly regardless of whether the TBI used human or porcine TSHR. These findings open the way to a practical TBI assay using recombinant human TSHR.

Interference with thyrotropin receptor antibody determination by a spuriously occurring anti-bovine TSH antibody

1986 ◽  
Vol 112 (2) ◽  
pp. 197-203 ◽  
Author(s):  
Makoto Iitaka ◽  
Toshinori Tanikawa ◽  
Yoshiki Sakatsume ◽  
Morifumi Yanagisawa ◽  
Yoshihito Hara ◽  
...  
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Binding Activity ◽  
Tsh Receptor ◽  
Thyrotropin Receptor ◽  
Pepsin Digestion ◽  
Thyrotropin Receptor Antibody ◽  
Maximum Binding Capacity ◽  
Antibody Determination ◽  
Bovine Tsh

Abstract. Abnormally negative values of thyrotropin binding inhibitor immunoglobulin (TBII) were found in the sera from a patient with Graves' disease. This was due to the presence of potent bovine TSH (bTSH) binding activity in the sera. This activity was demonstrated to be in immunoglobulin G (IgG) with a λ light chain isotype, which was shown to have an affinity for bTSH with a Ka value of 3.5 × 1010 m−1 and a maximum binding capacity of 1.1 × 10−14m/mg IgG. F(ab')2 fragments obtained through pepsin digestion from the patient's IgG retained bTSH binding activity. [125I] bTSH binding to this IgG was inhibited by the TSH receptor. The inhibition was not completely competitive, suggesting the presence of different binding sites for this IgG and the TSH receptor on the TSH molecule. This IgG, however, could not bind labelled human TSH (hTSH). Since neither TSH nor other pituitary derivatives had ever been given to the patient, this bTSH binding activity was considered to be due to a spuriously occurring anti-bTSH antibody.

scholarly journals The Thyrotropin Receptor Hinge Region Is Not Simply a Scaffold for the Leucine-Rich Domain but Contributes to Ligand Binding and Signal Transduction

2008 ◽  
Vol 22 (5) ◽  
pp. 1171-1182 ◽  
Author(s):  
Yumiko Mizutori ◽  
Chun-Rong Chen ◽  
Sandra M. McLachlan ◽  
Basil Rapoport
Keyword(s):  
Signal Transduction ◽  
Ligand Binding ◽  
Point Mutations ◽  
Inverse Agonist ◽  
Transmembrane Domains ◽  
Hinge Region ◽  
Glycoprotein Hormone ◽  
Intact Cells ◽  
Rich Domain ◽  
Extracellular Loops

Abstract The glycoprotein hormone receptor hinge region connects the leucine-rich and transmembrane domains. The prevalent concept is that the hinge does not play a significant role in ligand binding and signal transduction. Portions of the hinge are redundant and can be deleted by mutagenesis or are absent in certain species. A minimal hinge will be more amenable to future investigation of its structure and function. We, therefore, combined and progressively extended previous deletions (Δ) in the TSH receptor (TSHR) hinge region (residues 277–418). TSHRΔ287–366, Δ287–371, Δ287–376, and Δ287–384 progressively lost their response to TSH stimulation of cAMP generation in intact cells, consistent with a progressive loss of TSH binding. The longest deletion (TSHRΔ287–384), reducing the hinge region from 141 to 43 amino acids, totally lost both functions. Surprisingly, however, with deletions extending from residues 371–384, constitutive (ligand-independent) activity increased severalfold, reversing the suppressive (inverse agonist) effect of the TSHR extracellular domain. TSHR-activating point mutations I486F and I568T in the first and second extracellular loops (especially the former) had reduced activity on a background of TSHRΔ287–371. In summary, our data support the concept that the TSHR hinge contributes significantly to ligand binding affinity and signal transduction. Residues within the hinge, particularly between positions 371–384, appear involved in ectodomain inverse agonist activity. In addition, the hinge is necessary for functionality of activating mutations in the first and second extracellular loops. Rather than being an inert linker between the leucine-rich and transmembrane domains, the TSHR hinge is a signaling-specificity domain.

scholarly journals Antigenic “Hot- Spots” on the TSH Receptor Hinge Region

2019 ◽  
Vol 9 ◽  
Author(s):  
Simeng Sun ◽  
Sarawut Summachiwakij ◽  
Ora Schneck ◽  
Syed A. Morshed ◽  
Risheng Ma ◽  
...  
Keyword(s):  
Hot Spots ◽  
Hinge Region ◽  
Tsh Receptor

TSH Receptor Antibody (TRAb) Assays Based on the Human Monoclonal Autoantibody M22 are more Sensitive than Bovine TSH Based Assays

2009 ◽  
Vol 42 (01) ◽  
pp. 65-69 ◽  
Author(s):  
K. Zöphel ◽  
D. Roggenbuck ◽  
P. von Landenberg ◽  
G. Wunderlich ◽  
T. Grüning ◽  
...  
Keyword(s):  
Tsh Receptor ◽  
Receptor Antibody ◽  
Tsh Receptor Antibody ◽  
Monoclonal Autoantibody ◽  
Bovine Tsh

Precise Determination of TSH Receptor Antibody Activity in Serum Containing Bovine TSH (bTSH) Binding Antibody by Absorption Using Denatured bTSH or Sheep FSH

Thyroid ◽  
1996 ◽  
Vol 6 (4) ◽  
pp. 295-299 ◽  
Author(s):  
TAKEHIRO INUI ◽  
TSUYOSHI KOUKI ◽  
HIDETOSHI OKABE ◽  
YUKIO OCHI ◽  
TAKASHI HACHIYA ◽  
...  
Keyword(s):  
Precise Determination ◽  
Tsh Receptor ◽  
Antibody Activity ◽  
Receptor Antibody ◽  
Tsh Receptor Antibody ◽  
Binding Antibody ◽  
Bovine Tsh
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