Molecular characterization and gene expression of thyrotropin receptor (TSHR) and a truncated TSHR-like in Senegalese sole.

Thyroid hormones (THs) play a key role in larval development, growth and metamorphosis in flatfish. Their synthesis is tightly regulated by the hypothalamic-pituitary-thyroid axis. Thyroid-stimulating hormone receptor (TSHR) is a key protein in the control of thyroid function stimulating TH synthesis after binding its ligand, the thyrotropin. In teleost fish, numerous reports have associated the TSHR with gametogenesis. However, little information about its role during larval development is available. In this study, we report the cloning of two different cDNAs with high similarity to TSHR. Phylogenetic analysis clustered both cDNAs separately. One of them (referred to TSHR) grouped with TSHR orthologs in tetrapods and teleost fish and possessed the three typical conserved domains and regulatory motifs. The second receptor (referred to as TSHRtr-like) represented a novel truncated cDNA bearing the extracellular and part of the transmembrane domain. TSHRtr-like orthologs were only found in teleosts, which suggests that it could have appeared after fish-specific 3R genome duplication. Expression profiles of both genes are analyzed in juvenile tissues and during larval development using a real-time PCR approach. In juvenile fish, TSHR and TSHRtr-like are expressed ubiquitously although transcript levels varied between organs. In both cases, the highest mRNAs levels are detected in brain. During larval development, both genes are expressed to a high level during the first stages (2-3days after hatching) reducing progressively their abundance in the whole larvae during metamorphosis. This reduction in mRNA abundance is more accentuated for the TSHRtr-like gene. To evaluate the possible regulation of both receptors by T4 during sole metamorphosis, larvae are exposed to the goitrogen thiourea (TU). Only TSHRtr-like modifies its expression, increasing its transcripts at 11days after treatment. Moreover, adding exogenous T4 hormone to TU-treated larvae restores the TSHRtr-like steady-state levels similar to the untreated control. Overall, these results demonstrate the existence of two thyrotropin receptors differentially regulated by THs in teleosts.

A sea lamprey glycoprotein hormone receptor similar with gnathostome thyrotropin hormone receptor.

The specificity of the vertebrate hypothalamic-pituitary-gonadal and hypothalamic-pituitary-thyroid axes is explained by the evolutionary refinement of the specificity of expression and selectivity of interaction between the glycoprotein hormones GpH (FSH, LH, and TSH) and their cognate receptors GpH-R (FSH-R, LH-R, and TSH-R). These two finely tuned signaling pathways evolved by gene duplication and functional divergence from an ancestral GpH/GpH-R pair. Comparative analysis of the protochordate and gnathostome endocrine systems suggests that this process took place prior or concomitantly with the emergence of the gnathostome lineage. Here, we report identification and characterization of a novel glycoprotein hormone receptor (lGpH-R II) in the Agnathan sea lamprey. This 781 residue protein was found approximately 43% identical with mammalian TSH-R and FSH-R representative sequences, and similarly with these two classes of mammalian receptors it is assembled from ten exons. A synthetic ligand containing the lamprey glycoprotein hormone beta-chain tethered upstream of a mammalian alpha-chain activated the lGpH-R II expressed in COS-7 cells but in a lesser extent than lGpH-R I. Molecular phylogenetic analysis of vertebrate GpH-R protein sequences suggests a closer relationship between lGpH-R II and gnathostome thyrotropin receptors. Overall, the presence and characteristics of the lamprey glycoprotein hormone receptors suggest existence of a primitive functionally overlapping glycoprotein hormone/glycoprotein hormone receptor system in this animal.

Cloning and functional characterization of a testicular TSH receptor cDNA from the African catfish (Clarias gariepinus).

A cDNA encoding a putative thyroid-stimulating hormone receptor (cfTSH-R) was cloned from the testis of the African catfish (Clarias gariepinus). The cfTSH-R showed the highest amino acid sequence identity with the TSH-Rs of other fish species. In addition, an insertion of approximately 50 amino acids, specific for the TSH-R subfamily, was also present in the carboxy terminus of the amino-terminal extracellular domain of the cfTSH-R. Next to the testis and thyroid follicles, abundant cfTSH-R expression was detected in cerebellum, brain, ovary, seminal vesicles and pituitary, while weaker expression was found in muscle, stomach, intestine, head-kidney, liver, kidney and heart. HEK-T 293 cells, transiently expressing the cfTSH-R, significantly increased intracellular cAMP levels in response to human TSH. Catfish LH, human choriogonadotropin and human FSH were also able to induce this cfTSH-R-mediated response, although with considerably lower efficiency than human TSH. These results indicated that a functional cfTSH-R had been cloned from the testis of African catfish.

Subtypes of anti-TSH receptor antibodies classified by various assays using CHO cells expressing wild-type or chimeric human TSH receptor.

To analyze the heterogeneity of anti-TSH receptor antibodies (TSHRAb), we measured serum TSH-binding inhibitory immunoglobulin (TBII), thyroid-stimulating antibody (TSAb), and thyroid stimulation blocking antibody (TSBAb) activities in 31 patients with positive TSHRAb, using CHO cells expressing wild-type TSHR (WT) or TSHR chimera (Mc2) wherein residues 90-165 were substituted by the LH/CG receptor. Using membranes from WT cells, we detected TBII activity in all 31 patients; 10 (32%), all with TSAb activity only, completely lost TBII activity using Mc2 membranes. TSAb activity was found in 26 sera using WT cells; 20 (77%) completely lost TSAb activity in Mc2 cells. Comparisons of TBII and TSAb activity in WT cells did not exhibit a strong positive correlation (r = 0.52). Of the 20 sera that completely lost TSAb activity in Mc2 cells, 10 retained some TBII activity in Mc2 cells. In each of the sera with retained TBII activity, TSAb activity was recovered in Mc2 cells using the conversion assay, which measures the conversion of a nonstimulating TSHRAb to a TSAb by the action of an anti-human IgG. Additionally, the TBII and conversion assay values in Mc2 cells exhibited a strong positive correlation (r = 0.86). Of the 31 sera, TSBAb was found in 7 samples, with no difference in WT and Mc2 cells. TBII activity was detected in all 7 sera with WT cells; TSAb activity in only 2. In the 5 sera with TSBAb but no TSAb activity, and with only a minimal or no decrease in TBII activity in Mc2 cell membranes, the in vitro conversion assay uncovered TSAb activity. Analyzing these data, we classify the sera into 5 groups containing multiple, different TSHR autoantibodies, including two different TSAbs, three different TBIIs, and one nonfunctional antibody. The heterogeneity of TBIIs as well as TSAbs provides a basis to explain the lack of correlation between TBII and TSAb activities in some past studies of Graves' sera.

[Pituitary glycoprotein hormone receptors]. / Les récepteurs des hormones glycoprotéiques hypophysaires.

Monoclonal antibodies have been raised against the porcine LH receptor and have allowed to clone the corresponding messenger RNA from testicular cells. The stricture of the LH receptor has been determined. It shows similarities but also differences with other G protein coupled receptors. Specially a large extracellular domain is specific of that new family of receptors. Variant forms of the LH receptor generated by alternative splicing and lacking transmembrane domain have been isolated. Immunochemical and immunocytochemical studies have been performed. Three different forms of the LH receptor are physiologically expressed: a mature 85kDa transmembrane species, a 68 kDa high mannose containing species corresponding to a precursor which accumulates inside the cells, and truncated 45-48kDa molecular weight species corresponding to the variant messenger RNAs identified during the cloning of the receptor. A novel zonation of the ovary has been described by immunocytochemical studies. Cross hybridization with the LH receptor clone allowed to isolate the related TSH receptor from human thyroid tissue. The human LH and FSH receptor genes have been localized to chromosome 2p21 and the TSH receptor gene to chromosome 14q31. The genes are very large (> 60 kbp) and have introns only within the 5' part encoding the extracellular domain of the receptor. Immunoelectron microscopic studies performed in Leydig cells and in stably transfected L cells have allowed to study intracellular traffic of the LH receptor. The same approach was used to study the transendothelial transfer of hCG in testicular microvasculature.

[LH and TSH receptors. A new family of G protein-coupled receptors]. / Récepteurs de la LH et de la TSH. Une nouvelle famille de récepteurs couplés à des protéines G.

Monoclonal antibodies have been raised against porcine LH receptor and allowed to clone the corresponding messenger RNA from testicular cells. Cross hybridisation with the LH receptor clone allowed to isolate a clone corresponding to the human TSH receptor from thyroids. The structure of both receptors have been determined. They show similarities but also differences to other G protein coupled receptors. In particular a large extracellular domain is specific of that new family of receptors. Variant forms of the LH receptor lacking transmembrane domains have been isolated. The obtention of monoclonal antibodies against both receptors allowed immunochemical and immunocytochemical studies to be performed. The human LH receptor gene have been localized to chromosome 2p21 and TSH receptor gene to chromosome 14q31. The complete organisation of the human TSH receptor gene has been determined.

Difference in species specificity of TSH receptor antibodies in Graves' disease and Hashimoto's thyroiditis.

UNLABELLED: TSH receptor antibodies have been detected in the sera of patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT). Since non-human thyroid tissue fractions or cells are used in the majority of assays, the species specificity of TSH receptor antibodies in GD or HT could be important. The species specificity was evaluated by means of an immunoprecipitation assay (IPA) using Triton X-100 solubilized TSH receptors prepared from human, porcine and rat thyroid as well as guinea pig fat cells (GPFC). In each assay the majority (4 or 5) of the 6 patients with GD were IPA-positive. In contrast, 9 out of 11 patients (82%) with HT had a positive human and rat IPA, while only 3 out of 11 (27% p less than 0.05) sera were positive in the porcine and GPFC assays. CONCLUSIONS: no species specificity of TSH receptor antibodies was detected for patients with GD; a selective species specificity for human and rat TSH receptors was found for HT sera. This suggests that TSH receptor antibodies in HT either recognize different determinants on the receptor than the antibodies in GD or are of lower affinity. Furthermore, the use of porcine thyroid tissue or GPFC may lead to an underestimation of the presence and level of TSH receptor antibodies in patients with HT.