The Human TSHß Subunit Proteins and Their Binding Sites on the TSH Receptor Using Molecular Dynamics Simulation.

To gain further insight into the binding of the normal and variant human TSHß subunits (TSHß and TSHßv), we modeled the 2 monomeric proteins and studied their interaction with the TSH receptor ectodomain (TSHR-ECD) using molecular dynamics simulation Furthermore, analyzed their bioactivity in vitro using recombinant proteins to confirm that such binding was physiologically relevant. Examining the interaction of TSHß and TSHßv with the TSHR-ECD model using molecular dynamic simulation revealed strong binding of these proteins to the receptor ECD. The specificity of TSHß and TSHßv binding to the TSHR-ECD was examined by analyzing the hydrogen-bonding residues of these subunits to the FSH receptor ECD, indicating the inability of these molecules to bind to the FSH receptors. Furthermore, the modelling suggests that TSHß and TSHßv proteins clasped the concave surface of the leucine rich region of the TSHR ECD in a similar way to the native TSH using dynamic hydrogen bonding. These mutually exclusive stable interactions between the subunits and ECD residues included some high-affinity contact sites corresponding to binding models of native TSH. Furthermore, we cloned TSHß and TSHßv proteins using the entire coding ORF and purified the flag-tagged proteins. The expressed TSHß subunit proteins retained bioactivity both in a coculture system as well as with immune-purified proteins. In summary, we showed that such interactions can result in a functional outcome and may exert physiological or pathophysiological effects in immune cells.

The Pathogenic TSH ß-subunit Variant C105Vfs114X Causes a Modified Signaling Profile at TSHR.

1) Background: Central congenital hypothyroidism (CCH) is a rare endocrine disorder that can be caused by mutations in the ß-subunit of thyrotropin (TSHB). The TSHB mutation C105Vfs114X leads to isolated thyroid-stimulating-hormone-(TSH)-deficiency and results in a severe phenotype. The aim of this study was to gain more insight into the underlying molecular mechanism and the functional effects of this mutation based on two assumptions: a) the three-dimensional (3D) structure of TSH should be modified with the C105V substitution, and/or b) whether the C-terminal modifications lead to signaling differences. 2) Methods: wild-type (WT) and different mutants of hTSH were generated in human embryonic kidney 293 cells (HEK293 cells) and TSH preparations were used to stimulate thyrotropin receptor (TSHR) stably transfected into follicular thyroid cancer cells (FTC133-TSHR cells) and transiently transfected into HEK293 cells. Functional characterization was performed by determination of Gs, mitogen activated protein kinase (MAPK) and Gq/11 activation. 3) Results: The patient mutation C105Vfs114X and further designed TSH mutants diminished cyclic adenosine monophosphate (cAMP) signaling activity. Surprisingly, MAPK signaling for all mutants was comparable to WT, while none of the mutants induced PLC activation. 4) Conclusion: We characterized the patient mutation C105Vfs114X concerning different signaling pathways. We identified a strong decrease of cAMP signaling induction and speculate that this could, in combination with diverse signaling regarding the other pathways, accounting for the patient's severe phenotype.

Conversion of TSH heterodimer to a single polypeptide chain increases bioactivity and longevity.

TSH is a dimeric glycoprotein hormone composed of a common α-subunit noncovalently linked to a hormone-specific ß-subunit. Previously, the TSH heterodimer was successfully converted to an active single-chain hormone by genetically fusing α and ß genes with [TSHß- carboxyl-terminal peptide (CTP)-α] or without (TSHß-α) the CTP of human chorionic gonadotropin ß-subunit as a linker. In the present study, TSH variants were expressed in Chinese hamster ovarian cells. The results indicated that TSHß-α single chain has the highest binding affinity to TSH receptor and the highest in vitro bioactivity. With regard to the in vivo bioactivity, all TSH variants increased the levels of T(4) in circulation after 2 and 4 h of treatment. However, the level of T(4) after treatment with TSH-wild type was significantly decreased after 6 and 8 h, compared with the levels after treatment with the other TSH variants. TSHß-α and TSHß-CTP-α single chains exhibited almost the same bioactivity after 8 h of treatment. Evaluating the half-life of TSH variants, TSHß-CTP-α single chain revealed the longest half-life in circulation, whereas TSH-wild type exhibited the shortest serum half-life. These findings indicate that TSH single-chain variants with or without CTP as a linker may display conformational structures that increase binding affinity and serum half-life, thereby, suggesting novel attitudes for engineering and constructing superagonists of TSH, which may be used for treating different conditions of defected thyroid gland activity. Other prominent potential clinical use of these variants is in a diagnostic test for metastasis and recurrence of thyroid cancer.

Thyrotropin in teleost fish.

Thyrotropin (TSH), a pituitary glycoprotein hormone that stimulates the thyroid gland, has been cloned and sequenced from over a dozen teleost fish species. Although TSH is established as a primary driver of systemic thyroid status in mammals, its importance in the regulation of fish thyroid function is still uncertain. We review recent studies indicating that TSH structure is highly conserved across species representing six teleost families. These studies have found TSH messenger RNA consistently expressed in teleost pituitary tissue, although ectopic expression, particularly in gonads, has also been observed. They have also provided evidence for negative feedback inhibition of TSH expression by thyroid hormones, as well as stimulation by hypothalamic peptides. Descriptive studies have found increased TSHbeta expression associated with life history events thought to be promoted by thyroid hormones. These results, coupled with the discovery of a G-protein coupled TSH receptor in several teleost species, supports an active and conserved role for TSH in the regulation of teleost thyroid function. The relative importance of central pathways in regulating thyroid hormone provision to targets and the identity of a proposed thyrotropin-inhibiting factor in teleost fish are still unanswered questions whose resolution will be facilitated by development of methods to measure circulating TSH and its secretion from the pituitary gland.

Bone marrow cells produce a novel TSHbeta splice variant that is upregulated in the thyroid following systemic virus infection.

Although cells of the immune system can produce thyroid-stimulating hormone (TSH), the significance of that remains unclear. Using 5' rapid amplification of cDNA ends (RACE), we show that mouse bone marrow (BM) cells produce a novel in-frame TSHbeta splice variant generated from a portion of intron 4 with all of the coding region of exon 5, but none of exon 4. The TSHbeta splice variant gene was expressed at low levels in the pituitary, but at high levels in the BM and the thyroid, and the protein was secreted from transfected Chinese hamster ovary (CHO) cells. Immunoprecipitation identified an 8 kDa product in lysates of CHO cells transfected with the novel TSHbeta construct, and a 17 kDa product in lysates of CHO cells transfected with the native TSHbeta construct. The splice variant TSHbeta protein elicited a cAMP response from FRTL-5 thyroid follicular cells and a mouse alveolar macrophage (AM) cell line. Expression of the TSHbeta splice variant, but not the native form of TSHbeta, was significantly upregulated in the thyroid during systemic virus infection. These studies characterize the first functional splice variant of TSHbeta, which may contribute to the metabolic regulation during immunological stress, and may offer a new perspective for understanding autoimmune thyroiditis.

Molecular cloning and sequence analysis of the cDNA encoding thyroid-stimulating hormone beta-subunit of common duck and mule duck pituitaries: in vitro regulation of steady-state TSHbeta mRNA level.

For better understanding of phylogenetic diversity and evolution of pituitary thyroid-stimulating hormone (TSH) in birds, we have cloned the cDNAs encoding TSH beta subunit (TSHbeta), by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) from two species of domestic ducks, common duck (Tsaiya duck and Pekin duck) (Anas platyrhynchos domesticus) and mule duck (hybrid of male muscovy duck Cairina moschata and female A. platyrhynchos domesticus). The nucleotide sequences of isolated TSHbeta cDNAs of the two species of ducks are identical, with each including 66 bp of 5'-untranslated region (UTR), 402 bp of coding region, and 82 bp 3'-UTR followed by 18 bp poly A tract. The deduced TSHbeta subunit of the ducks contains 134 amino acids consisting of a putative signal peptide of 19 amino acids and a putative mature protein of 115 amino acids. However, the TSHbetas of common duck and mule duck differ from the TSHbeta of muscovy duck in one amino acid at position 97 of the mature protein: isoleucine for common duck and mule duck, and valine for muscovy duck. Our findings thus demonstrate that inter-genus variation of TSHbeta exists in Family Anatidae, and that TSHbeta gene in the mule duck is preferentially transcribed from the maternal genome rather than from the paternal genome. TSHbeta mRNA expressions were investigated by culturing common duck pituitaries with various doses of hormones. Thyrotropin-releasing hormone (TRH) stimulated, while thyroid hormones, triiodothyronine (T(3)) and thyroxine (T(4)), inhibited the TSHbeta mRNA levels, in dose-related manners. The findings thus support that the mode of regulation of TSH gene expression in hypothalamo-pituitary-thyroid axis in birds is similar to that in mammals. Cortisol and corticosterone decreased the steady-state TSHbeta mRNA levels at the pituitary level, in a dose-related manner, the first-time demonstration in vertebrates. The results may suggest that glucocorticoids exert suppressive action directly at pituitary level in modulation of steady-state TSHbeta mRNA level.

An LXXLL motif in nuclear receptor corepressor mediates ligand-induced repression of the thyroid stimulating hormone-beta gene.

Nuclear receptor corepressor (N-CoR) regulates gene expression through interaction with DNA-bound nuclear receptors, recruiting multicomponent repressor complexes to the sites of target genes. We recently reported the presence of an LXXLL motif in N-CoR, and showed that this motif interacts in vitro and in vivo with retinoic acid receptor alpha (RARalpha) and thyroid hormone receptor beta (TRbeta). Transient transfection experiments now suggest that TRbeta and N-CoR act synergistically and may both be required for ligand-induced repression from the negative TR response element in the thyroid stimulating hormone-beta (TSHbeta) gene promoter. Mutation of the LXXLL motif in N-CoR abolished ligand-induced repression at this response element. Furthermore, in vitro binding of N-CoR to a complex between TRbeta and the negative TR response element was strictly ligand-dependent. We conclude that N-CoR and TRbeta cooperate in the regulation of the TSHbeta gene and that the ligand-dependent repression is mediated by the LXXLL motif in N-CoR.

Differential expression of thyroid-stimulating hormone beta subunit in gonads during sex reversal of orange-spotted and red-spotted groupers.

We have cloned and characterized the full-length cDNA encoding thyroid-stimulating hormone beta-subunit (TSHbeta) from orange-spotted grouper Epinephelus coioides. It contains 913 nucleotides with an open reading frame encoding 146 amino acids with a 20 amino acid signal peptide. The grouper mature TSHbeta has 75, 70, 61, 59, 41, 42 and 40% identities to that of rainbow trout, Atlantic salmon, zebrafish, European eel, chicken, mouse and human, respectively. RT-PCR analysis indicated that the TSHbeta mRNA was expressed abundantly not only in pituitary but also in gonads. A more interesting finding is to reveal the differential TSHbeta expressions between the ovaries and the transitional gonads or testes in natural individuals of orange-spotted grouper and red-spotted grouper Epinephelus akaara, and in artificial sex reversal individuals of red-spotted grouper induced by MT feeding. In situ hybridization localization provided direct evidence that the TSHbeta was transcribed in the germ cells. In the growing oocytes, the TSHbeta transcripts were concentrated on the ooplasm periphery. In testicular tissues, the intensively expressed TSHbeta cells were found to be spermatogonia and spermatocytes in the spermatogenic cysts. This is the first report of a TSHbeta expressed in the gonads of any vertebrates in addition to the expected expression in the pituitary, and it expresses more transcripts in the gonads during sex reversal or testis than in the ovaries both in E. coioides and E. akaara. Importantly, the TSHbeta identification in germ cells allows us to further investigate the functional roles and the molecular mechanisms in gametogenesis of groupers, especially in sex reversal and in spermatogenesis.

Cloning of a partial cDNA for Japanese quail thyroid-stimulating hormone and effects of methimazole on the thyroid and reproductive axes.

The purposes of this study were to develop a probe for the detection of thyroid-stimulating hormone (TSH) beta subunit mRNA, to validate the usefulness of that probe in measuring TSH, and to use it to investigate the effects of thyroid suppression on TSH and the reproductive axis in Japanese quail. The objectives of experiment 1 were to isolate and characterize a partial cDNA for quail TSH and validate a riboprobe transcribed from this cDNA. This riboprobe was then used to assess changes in TSHbeta mRNA levels in Japanese quail. We isolated a cDNA of 168 bp with 94% identity to the corresponding sequence in chicken TSHbeta. The transcribed riboprobe was shown to be pituitary gland specific, and differences in TSHbeta mRNA levels were detectable with 2.5 microg of total RNA in Northern blot analysis. In experiment 2, our objective was to determine if thyroid inhibition would result in a detectable change in TSHbeta mRNA and alterations in the pituitary luteinizing hormone (LH) or indices of gonadal function. We used adult, reproductively active, male Japanese quail on a long-day photoperiod. Treatment with a goitrogen, methimazole (MMI), increased (P < 0.05) thyroid gland and liver weights and decreased (P < 0.05) serum thyroxine (T4) concentrations compared to control birds. We detected increased TSHbeta mRNA in the pituitaries of MMI-treated birds compared to controls. There was no effect of MMI treatment on the reproductive variables measured, including LHbeta mRNA levels, serum androgen and estradiol concentrations, gonad weight, or cloacal gland area. Therefore, it appears that thyroid axis inhibition and the consequent increase in TSHbeta mRNA did not have direct effects on reproductive axis function in male Japanese quail.

Cloning of the gene for the thyrotropin beta subunit in the Japanese crested ibis, Nipponia nippon.

We isolated a putative gene for the thyrotropin beta subunit (TSHbeta) from two types of genomic libraries of the Japanese crested ibis, Nipponia nippon. Exon-intron structure was deduced by comparing the determined sequence with those of TSH beta cDNA of other birds. The deduced amino acid sequence shows extensive similarities to those of the other birds, which assures our assumption that the acquired nucleotide sequence represents the TSHbeta gene. The assembled genomic fragment is 4192 bp in size and consists of 1937 bp of putative 5' flanking region followed by exon-intron structure with three exons and two introns, similar to those observed in rat, human and goldfish counterparts. Locations of introns are also similar to those in mammals and goldfish. Comparison of the 5' flanking region of the ibis TSHbeta gene with those of mammals reveals that several regulatory sequences, such as negative thyroid hormone responsive element (nTRE), Pit-1 responsive element, and AP-1 responsive element, which were characterized in mammalian TSHbeta genes, are also found in the promoter region. This is the first report on the exon-intron structure and 5' flanking region of the TSHbeta gene in an avian species.