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.

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.

UNASSIGNED: Graves\' orbitopathy (GO) is a common autoimmune disease, but its specific pathogenesis has not been fully elucidated. MicroRNAs (miRNAs) possess an important regulatory function in the occurrence and development of autoimmune diseases. In the present study, we explored the potential role of miR-182 in the diagnosis of GO.
UNASSIGNED: The expression of miR-182, thyrotropin receptor (TSHR) and adipocytokines was ascertained by qRT-PCR assay. The triglyceride (TG) content was ascertained by ELISA assay. The lipid droplet content was identified by Oil Red O staining. The relationship between E74-like factor 3 (ELF3), miR-182 and TSHR was confirmed by ChIP, dual-luciferase reporter assay and RIP.
UNASSIGNED: The expression of miR-182 decreased, while TSHR increased, and adipocytokine (adiponectin, leptin, PPAR-γ, and AP2) levels were upregulated in preorbital adipose tissue of patients with GO and differential medium induced (DM-induced) 3T3-L1 cells. MiR-182 mimics inhibited adipocytokine expression, TG content and lipid droplets; however, miR-182 inhibitor had the opposite results. TSHR was a target gene of miR-182, and TSHR overexpression (oe-TSHR) reversed the effect of miR-182 mimics on adipogenic differentiation of 3T3-L1 by DM treatment. ELF3 transcription promoted miR-182 expression. Oe-ELF3 inhibited adipocytokine expression and reduced TG content and lipid droplets in DM-induced 3T3-L1 cells. MiR-182 inhibitor reversed the effect of oe-ELF3 on adipogenic differentiation in 3T3-L1.
UNASSIGNED: ELF3/miR-182/TSHR axis alleviated Graves\' orbitopathy by inhibiting adipogenic differentiation.

(GPCR)The receptor for TSH receptor (TSHR), a G protein coupled receptor (GPCR), is of particular interest as the primary antigen in autoimmune hyperthyroidism (Graves\' disease) caused by stimulating TSHR antibodies. To date, only one domain of the extracellular region of the TSHR has been crystallized. We have run a 1000 ns molecular dynamic simulation on a model of the entire TSHR generated by merging the extracellular region of the receptor, obtained using artificial intelligence, with our recent homology model of the transmembrane domain, embedded it in a lipid membrane and solvated it with water and counterions. The simulations showed that the structure of the transmembrane and leucine-rich domains were remarkably constant while the linker region (LR), known more commonly as the \'hinge region,\' showed significant flexibility, forming several transient secondary structural elements. Furthermore, the relative orientation of the leucine-rich domain with the rest of the receptor was also seen to be variable. These data suggest that this LR is an intrinsically disordered protein. Furthermore, preliminary data simulating the full TSHR model complexed with its ligand (TSH) showed that (a) there is a strong affinity between the LR and TSH ligand and (b) the association of the LR and the TSH ligand reduces the structural fluctuations in the LR. This full-length model illustrates the importance of the LR in responding to ligand binding and lays the foundation for studies of pathologic TSHR autoantibodies complexed with the TSHR to give further insight into their interaction with the flexible LR.

Proximity ligation assay (PLA) is a methodology that permits detection of protein-protein closeness, that is, proteins that are within 40 nanometers of each other, in cells or tissues at endogenous protein levels or after exogenous overexpression. It detects the protein(s) with high sensitivity and specificity because it employs a DNA hybridization step followed by DNA amplification. PLA has been used successfully with many types of proteins. In this methods paper, we will describe the workings of PLA and provide examples of its use to study TSH/IGF-1 receptor crosstalk in Graves\' orbital fibroblasts (GOFs) and TSH receptor homodimerization in primary cultures of human thyrocytes.

Biophysical studies have established that the thyrotropin (TSH) receptor (TSHR) undergoes posttranslational modifications including dimerization. Following our earlier simulation of a TSHR-transmembrane domain (TMD) monomer (called TSHR-TMD-TRIO) we have now proceeded with a molecular dynamics simulation (MD) of TSHR-TMD dimerization using this improved membrane-embedded model. The starting structure was the TMD protein with all extracellular and intracellular loops and internal waters, which was placed in the relative orientation of the model originally generated with Brownian dynamics. Furthermore, this model was embedded in a DPPC lipid bilayer further solvated with water and added salt. Data from the MD simulation studies showed that the dimeric subunits stayed in the same relative orientation and distance during the 1000 ns of study. Comparison of representative conformations of the individual monomers when dimerized with the conformations from the monomer simulation showed subtle differences as represented by the backbone root mean square deviations. Differences in the conformations of the ligand-binding sites, suggesting variable affinities for these \"hot spots,\" were also revealed by comparing the docking scores of 46 small-molecule ligands that included known TSHR agonists and antagonists as well as their derivatives. These data add further insight into the tendency of the TSHR-TMD to form dimeric and oligomeric structures and show that the differing conformations influence small-molecule binding sites within the TMD.

Determination of the full-length thyroid-stimulating hormone receptor (TSHR) structure by cryo-electron microscopy (cryo-EM) is described. The TSHR complexed with human monoclonal TSHR autoantibody K1-70™ (a powerful inhibitor of TSH action) was detergent solubilised, purified to homogeneity and analysed by cryo-EM. The structure (global resolution 3.3 Å) is a monomer with all three domains visible: leucine-rich domain (LRD), hinge region (HR) and transmembrane domain (TMD). The TSHR extracellular domain (ECD, composed of the LRD and HR) is positioned on top of the TMD extracellular surface. Extensive interactions between the TMD and ECD are observed in the structure, and their analysis provides an explanation of the effects of various TSHR mutations on TSHR constitutive activity and on ligand-induced activation. K1-70™ is seen to be well clear of the lipid bilayer. However, superimposition of M22™ (a human monoclonal TSHR autoantibody which is a powerful stimulator of the TSHR) on the cryo-EM structure shows that it would clash with the bilayer unless the TSHR HR rotates upwards as part of the M22™ binding process. This rotation could have an important role in TSHR stimulation by M22™ and as such provides an explanation as to why K1-70™ blocks the binding of TSH and M22™ without activating the receptor itself.

The thyroid-stimulating hormone receptor (TSHR) is a Class A G protein-coupled receptor (GPCR) that mediates signalling through the hypothalamic-pituitary-thyroid axis. Inappropriate activation of TSHR by autoantibodies or mutations, results in human disease such as Grave\'s disease and Hashimito\'s thyroiditis. Therefore, there is a need to develop novel therapeutics targeting the TSHR. Understanding the structure and mechanism of activation of this receptor would help elucidate the pathogenesis of disease and aid drug development. Here, we describe a method for the expression of the human TSHR in a mammalian cell line generated through a lentiviral expression system. The receptor is then purified by affinity chromatography in the ligand-free state and is suitable for structure determination by single-particle electron cryo-microscopy (cryo-EM).