Thyroid cancer is the most common endocrine cancer, with differentiated thyroid cancers (DTCs) accounting for 95% of diagnoses. While most DTC patients are diagnosed and treated with radioiodine (RAI), up to 20% of DTC patients become RAI refractory (RAI-R). RAI-R patients have significantly reduced survival rates compared to patients who remain RAI-avid. This study explores [89Zr]Zr-TR1402 as a thyroid-stimulating hormone receptor (TSHR)-targeted PET radiopharmaceutical for DTC. [89Zr]Zr-TR1402 was synthesized with a molar activity of 25.9 MBq/nmol by conjugating recombinant human TSH (rhTSH) analogue TR1402 to chelator p-SCN-Bn-deferoxamine (DFO) in a molar ratio of 3:1 (DFO/TR1402) and radiolabeling with 89Zr (t1/2 = 78.4 h, β+ = 22.7%). As TSHR is absent in commonly available DTC-derived cell lines, TSHR was reintroduced via stable transduction by delivering a lentivirus containing the full-length coding region of the human TSHR gene. Receptor-mediated uptake of [89Zr]Zr-TR1402 was evaluated in vitro in stably transduced TSHR+ and wild-type TSHR- DTC cell lines. In vivo PET imaging was performed on Days 1-3 postinjection in male and female athymic nude mice bearing TSHR+ and TSHR- xenografts, along with ex vivo biodistribution on Day 3 postinjection. In vitro uptake of 1 nM [89Zr]Zr-TR1402 was significantly higher in TSHR+ THJ529T (P < 0.0001) and FTC133 (P < 0.01) cells than in TSHR- THJ529T and FTC133 cells. This uptake was shown to be specific in both TSHR+ THJ529T (P < 0.0001) and TSHR+ FTC133 (P < 0.0001) cells by blocking uptake with 250 nm DFO-TR1402. In vivo PET imaging showed accumulation of [89Zr]Zr-TR1402 in TSHR+ tumors, which was the highest on Day 1. In the male FTC133 xenograft model, ex vivo biodistribution confirmed a significant difference (P < 0.001) in uptake between FTC133+ (1.3 ± 0.1%ID/g) and FTC133- (0.8 ± 0.1%ID/g) tumors. A significant difference (P < 0.05) in uptake was also seen in the male THJ529T xenograft model between THJ529T+ (1.8 ± 0.6%ID/g) and THJ529T- (0.8 ± 0.4%ID/g) tumors. The in vitro and in vivo accumulation of [89Zr]Zr-TR1402 in TSHR-expressing DTC cell lines support the continued preclinical optimization of this approach.

Thyroid stimulating hormone (TSH), a glycoprotein synthesized and secreted from thyrotrophs of the pituitary gland, is composed of a glycoprotein hormone common alpha subunit (CGA) and a specific beta subunit (TSHB). The major biological function of TSH is to stimulate thyroidal follicles to synthesize and secrete thyroid hormones through activating its cognate receptor, the thyroid stimulating hormone receptor (TSHR). In the present study, polyclonal antisera against ricefield eel Tshb and Tshr were generated respectively, and the expression of Tshb and Tshr was examined at mRNA and protein levels. RT-PCR analysis showed that tshb mRNA was expressed mainly in the pituitary as well as in some extrapituitary tissues including the ovary and testis. Tshr mRNA was also expressed in a tissue-specific manner, with transcripts detected in tissues including the kidney, ovary, and testis. The immunoreactive Tshb signals in the pituitary were shown to be localized to the inner areas of adenohypophysis which are close to the neurohypophysis of adult ricefield eels. Tshb-immunoreatvie cells in the pituitary of ricefield eel larvae were firstly observed at hatching. The expression of immunoreactive Tshb and Cga was also detected in ricefield eel ovary and testis together with Tshr. In the ovary, immunoreactive Tshb, Cga, and Tshr were observed in oocytes and granulosa cells. In the testis, immunoreactive Tshb was mainly observed in Sertoli cells while immunoreactive Cga and Tshr were detected in germ cells as well as somatic cells. Results of the present study suggest that Tsh may be synthesized both in the ovary and testis locally, which may play paracrine and/or autocrine roles in gonadal development in ricefield eels.

OBJECTIVE: The study aimed to establish a mouse model of Graves\' disease (GD) with Graves\' orbitopathy (GO; GD + GO) that can represent the clinical disease characteristics.
METHODS: A eukaryotic expression plasmid of insulin-like growth factor 1 receptor (IGF-1R) α subunit (pcDNA3.1/IGF-1Rα) and a thyrotropin receptor (TSHR) A subunit plasmid (pcDNA3.1/TSHR-289) were injected in female BALB/c mice followed by immediate electroporation to induce a GD + GO model. Grouping was performed according to the frequency of injection (2- to 4-week intervals) and type of injected plasmids: T: pcDNA3.1/TSHR-289( +), I: pcDNA3.1/IGF-1Rα( +), or co-injection T + I: pcDNA3.1/TSHR-289( +) and pcDNA3.1/IGF-1Rα( +). Serum TSH, T4, TSAb, TSBAb, body weight, and blood glucose levels were evaluated. Thyroid 99mTcO4- imaging and retrobulbar magnetic resonance imaging (MRI) were performed, and bilateral eye muscle volumes were measured. Immunohistochemistry and hematoxylin-eosin staining were performed on the relevant tissues, and semi-quantitative analysis was performed.
RESULTS: A total of 60% of mice (3/5, one mouse died) in the T group developed GD + GO. In the T + I group, 83.3% of mice (5/6) developed GD + GO. Mice in the I group did not develop GD. Compared with the control group, serum T4, TSAb, and TSBAb of the mice in the GD + GO model groups were increased to varying degrees (P < 0.05), and serum TSH and body weight were significantly lower compared to the control group (P < 0.05). The thyroid uptake capacity of 99mTcO4- and the volume of eye muscle of mice in the GD + GO group were significantly higher compared to the control group (P < 0.05). The thyroid and retrobulbar muscles of these mice showed varying inflammatory infiltration and interstitial muscle edema. The severity of GD + GO in the co-injection group was not related to injection frequency; however, GD and ocular signs in co-injection mice were more severe compared to the T group.
CONCLUSIONS: We successfully induced a GD + GO mouse model by a repeated co-injection of pcDNA3.1/IGF-1Rα and pcDNA3.1/TSHR-289 plasmids. Injection of pcDNA3.1/IGF-1Rα alone failed to induce GD. Co-injection of two plasmids induced more severe GD + GO than pcDNA3.1/TSHR-289( +) alone.

Graves\' ophthalmopathy (GO) is an extrathyroidal manifestation of Graves\' disease, Orbital fibroblasts (OFs) are recognized as key players in GO pathogenesis, involved in orbital inflammation, tissue remodeling, and fibrosis. This study offers a primary exploration of cell behavior and characteristics on OFs from GO (GO-OFs), and compared to OFs from healthy control (HC-OFs). Results reveal that GO-OFs exhibit delayed migration from tissue fragments, while no significant difference in cell proliferation is observed between GO-OFs and HC-OFs. Aberrant expression pattern of surface proteins Thy-1, TSHR, and IGF-1R suggests shared autoantigens and pathways between GO and GD, contributing to inflammation and fibrosis. Investigations into cytokine responses unveil elevated secretion of hyaluronic acid (HA) and prostaglandin E2 (PGE2) in GO-OFs, emphasizing their role in tissue remodeling. These findings deepen our understanding of OFs in GO pathogenesis, offering potential therapeutic avenues.

The receptor for thyroid stimulating hormone (TSHR), a GPCR, is the primary antigen in autoimmune hyperthyroidism (Graves\' disease) caused by stimulating TSHR antibodies. While we have previously published a full length model of the TSHR, including its leucine rich domain (LRD), linker region (LR) and transmembrane domain (TMD), to date, only a partial LRD (aa 21-261) stabilized with TSHR autoantibodies has been crystallized. Recently, however, cryo-EM structures of the full-length TSHR have been published but they include only an incomplete LR. We have now utilized the cryo-EM models, added disulfide bonds to the LR and performed longer (3000 ns) molecular dynamic (MD) simulations to update our previous model of the entire full-length TSHR, with and without the presence of TSH ligand. As in our earlier work, the new model was embedded in a lipid membrane and was solvated with water and counterions. We found that the 3000 ns Molecular Dynamic simulations showed that the structure of the LRD and TMD were remarkably constant while the LR, known more commonly as the \"hinge region\", again showed significant flexibility, forming several transient secondary structural elements. Analysis of the new simulations permitted a detailed examination of the effect of TSH binding on the structure of the TSHR. We found a structure-stabilizing effect of TSH, including increased stability of the LR, which was clearly demonstrated by analyzing several intrinsic receptor properties including hydrogen bonding, fluctuation of the LRD orientation, and radius of gyration. In conclusion, we were able to quantify the flexibility of the TSHR and show its increased stability after TSH binding. These data indicated the important role of ligands in directing the signaling structure of a receptor.

The thyroid-stimulating hormone receptor (TSHR) transmembrane domain (TMD) is found in the plasma membrane and consists of lipids and water molecules. To understand the role of TSHR-associated water molecules, we used molecular dynamic simulations of the TMD and identified a network of putative receptor-associated transmembrane water channels. This result was confirmed with extended simulations of the full-length TSHR with and without TSH ligand binding. While the transport time observed in the simulations via the TSHR protein was slower than via the lipid bilayer itself, we found that significantly more water traversed via the TSHR than via the lipid bilayer, which more than doubled with the binding of TSH. Using rat thyroid cells (FRTL-5) and a calcein fluorescence technique, we measured cell volumes after blockade of aquaporins 1 and 4, the major thyroid cell water transporters. TSH showed a dose-dependent ability to influence water transport, and similar effects were observed with stimulating TSHR autoantibodies. Small molecule TSHR agonists, which are allosteric activators of the TMD, also enhanced water transport, illustrating the role of the TMD in this phenomenon. Furthermore, the water channel pathway was also mapped across 2 activation motifs within the TSHR TMD, suggesting how water movement may influence activation of the receptor. In pathophysiological conditions such as hypothyroidism and hyperthyroidism where TSH concentrations are highly variable, this action of TSH may greatly influence water movement in thyroid cells and many other extrathyroidal sites where the TSHR is expressed, thus affecting normal cellular function.

Graves\' disease (GD) is a prominent antibody-mediated autoimmune disorder characterized by stimulating antibodies (TRAb) that target the thyroid-stimulating hormone receptor (TSHR). Targeting and eliminating TRAb-producing B lymphocytes hold substantial therapeutic potential for GD. In this study, we engineered a novel chimeric antigen receptor T cell (CAR-T) therapy termed TSHR-CAR-T. This CAR-T construct incorporates the extracellular domain of the TSH receptor fused with the CD8 transmembrane and intracellular signal domain (4-1BB). TSHR-CAR-T cells demonstrated the ability to recognize and effectively eliminate TRAb-producing B lymphocytes both in vitro and in vivo. Leveraging this autoantigen-based chimeric receptor, our findings suggest that TSHR-CAR-T cells offer a promising and innovative immunotherapeutic approach for the treatment of antibody-mediated autoimmune diseases, including GD.

Congenital hypothyroidism (CH) is a common heterogeneous endocrine disorder. The thyroid-stimulating hormone receptor gene (TSHR) is one of the major candidate genes associated with CH. Studies have investigated the possible correlations between the specific clinical features and the presence of TSHR variants. However, only a few reports have focused on the long-term follow-up of patients with CH. Here we present a case of CH-associated TSHR p.Arg109Gln and p.Arg450His rare compound heterozygous variants, with a follow-up performed until adolescence. The patient had high serum TSH levels during newborn screening. Oral administration of levothyroxine (l-T4) was initiated at 1 month of age. The ultrasonogram revealed normal thyroid morphology and blood flow. Reduced uptake of I-123 and negative perchlorate test was observed. A small amount of l-T4 remained needed although l-T4 could be steadily reduced by puberty. The patient was diagnosed with orthotopic, nongoitrous, and permanent CH. He had no nonclassical TSH resistance. Patients with the TSHR p.Arg109Gln compound heterozygous variant exhibit permanent CH with high TSH levels and normal or slightly lower fT4 levels. In the future, genotype identification could help predict the long-term prognosis and reduce the requirement for detailed examinations. More case studies are needed to determine the relationship between genetic variants and clinical features in CH.

Autoantibodies against thyroid proteins are present in several thyroid diseases. Thyroid-stimulating hormone receptor (TSHR) is a G-protein-coupled receptor (GPCR) that binds to thyroid-stimulating hormone (TSH) and stimulates production of thyroxine (T4) and triiodothyronine (T3). When agonized by anti-TSHR autoantibodies, aberrant production of thyroid hormone can lead to Graves\' Disease (GD). In Hashimoto\'s thyroiditis (HT), anti-TSHR autoantibodies target the thyroid for immune attack. To better understand the role of anti-TSHR antibodies in thyroid disease, we generated a set of rat antimouse (m)TSHR monoclonal antibodies with a range of affinities, blocking of TSH, and agonist activity. These antibodies could be used to investigate the etiology and therapy of thyroid disease in mouse models and as building blocks in protein therapeutics that target the thyroid for treatment in either HT or GD.

OBJECTIVE: We investigated the clinical significance of thyroid-stimulating hormone receptor (TSHR) mutations detected in thyroid fine needle aspiration (FNA) specimens.
METHODS: The pathology archives at our institution were reviewed between 2018 and 2021 for indeterminate (Bethesda category III and IV) specimens with Thyroseq® analysis showing TSHR mutations.
RESULTS: A total of 2184 cases diagnosed as atypia/follicular lesion of undetermined significance (AUS/FLUS), and 2625 diagnosed as follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) were identified. A total of 1735 AUS/FLUS and 2339 SFN/FN underwent Thyroseq® analysis; 69 showed TSHR mutations (1.6%, 59 female, 10 male, average age: 55 years). Ten cases showed oncocytic features. Twelve patients underwent radionuclide scans within 1 year of FNA:11 were hyperfunctioning. Nodule size and TSH levels were weakly correlated. Twenty-two different TSHR mutations were identified (most common: M453T). A second mutation was found in five cases (EZH1 n = 2, and EIF1AX n = 3). The expression of sodium-iodide transporter (NIS) mRNA was in the range of 0.01%-62.43% out of all sequencing reads, and was elevated in 49 (71%) cases. Surgical pathology follow-up was available in five cases (all benign except one). On follow-up available for 38 cases (mean: 24 months; range: 7-47 months), 34 (89.5%) nodules remained stable and 3 (8%) increased in size.
CONCLUSIONS: TSHR mutations in thyroid FNA samples classified as indeterminate are rare, generally benign, and commonly associated with autonomy on scan if performed.