The Mysterious Universe of the TSH Receptor
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ABSTRACT:
SUBMITTER: Vieira I
PROVIDER: S-EPMC9315062 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Studies on the TSH receptor (TSHR) have numerous practical applications in vitro and in vivo. For example human monoclonal autoantibodies (MAbs) to the TSHR are useful reagents for in vitro diagnostics. Measurement of TSHR autoantibodies (TRAbs) is helpful in diagnosis and management of autoimmune thyroid disease. Currently available highly sensitive and specific assays to measure TRAbs use the human TSHR MAb M22 instead of the TSH. Furthermore, preparations of the human TSHR MAb M22 are useful as the World Health Organisation International Standard for thyroid stimulating antibody and for calibration of the assays for measuring TRAbs. Preparations of thermostabilised TSHR extracellular domain have recently become available and this is likely to have an impact on improvements in specificity testing for TRAb assays. In addition the stable TSHR preparations have practical application for specific immunoadsorption of patient serum TRAbs. Human TSHR MAbs also have promising prospects as new therapeutics. Autoantibodies with TSHR antagonistic activities are "natural" inhibitors of TSHR stimulation and are expected to be helpful in controlling TSHR activity in patients with Graves' disease, Graves' ophthalmopathy and thyroid cancer.
| S-EPMC7266850 | biostudies-literature
Project description: Not available
| S-EPMC3868806 | biostudies-literature
Project description:Thyroid-stimulating hormone (TSH)-induced reduction in ligand binding affinity (negative cooperativity) requires TSH receptor (TSHR) homodimerization, the latter involving primarily the transmembrane domain (TMD) but with the extracellular domain (ECD) also contributing to this association. To test the role of the TMD in negative cooperativity, we studied the TSHR ECD tethered to the cell surface by a glycosylphosphatidylinositol (GPI) anchor that multimerizes despite the absence of the TMD. Using the infinite ligand dilution approach, we confirmed that TSH increased the rate of dissociation (k(off)) of prebound (125)I-TSH from CHO cells expressing the TSH holoreceptor. Such negative cooperativity did not occur with TSHR ECD-GPI-expressing cells. However, even in the absence of added TSH, (125)I-TSH dissociated much more rapidly from the TSHR ECD-GPI than from the TSH holoreceptor. This phenomenon, suggesting a lower TSH affinity for the former, was surprising because both the TSHR ECD and TSH holoreceptor contain the entire TSH-binding site, and the TSH binding affinities for both receptor forms should, theoretically, be identical. In ligand competition studies, we observed that the TSH binding affinity for the TSHR ECD-GPI was significantly lower than that for the TSH holoreceptor. Further evidence for a difference in ligand binding kinetics for the TSH holoreceptor and TSHR ECD-GPI was obtained upon comparison of the TSH K(d) values for these two receptor forms at 4 °C versus room temperature. Our data provide the first evidence that the wild-type TSHR TMD influences ligand binding affinity for the ECD, possibly by altering the conformation of the closely associated hinge region that contributes to the TSH-binding site.
| S-EPMC3057818 | biostudies-literature
Project description:Autoimmune thyroid-stimulating antibodies are activating the thyrotropin receptor (TSHR) in both the thyroid and the eye, but different molecular mechanisms are induced in both organs, leading to Graves' disease (GD) and Graves' orbitopathy (GO), respectively. Therapy with anti-thyroid drugs to reduce hyperthyroidism (GD) by suppressing the biosynthesis of thyroid hormones has only an indirect effect on GO, since it does not causally address pathogenic TSHR activation itself. GO is thus very difficult to treat. The activated TSHR but also the cross-interacting insulin-like growth factor 1 receptor (IGF-1R) contribute to this issue. The TSHR is a heptahelical G-protein-coupled receptor, whereas the IGF-1R is a receptor tyrosine kinase. Despite these fundamental structural differences, both receptors are phosphorylated by G-protein receptor kinases, which enables β-arrestin binding. Arrestins mediate receptor internalization and also activate the mitogen-activated protein kinase pathway. Moreover, emerging results suggest that arrestin plays a critical role in the cross-interaction of the TSHR and the IGF-1R either in their common signaling pathway and/or during an indirect or potential TSHR/IGF-1R interaction. In this review, novel pharmacological strategies with allosteric small-molecule modulators to treat GO and GD on the level of the TSHR and/or the TSHR/IGF-1R cross-interaction will be discussed. Moreover, monoclonal antibody approaches targeting the TSHR or the IGF-1R and thereby preventing activation of either receptor will be presented. Another chapter addresses the immunomodulation to treat GO using TSHR-derived peptides targeting the human leukocyte antigen DR isotope (HLA-DR), which is a feasible approach to tackle GO, since HLA-DR and TSHR are overexpressed in orbital tissues of GO patients.
| S-EPMC7802447 | biostudies-literature
Project description:The TSH receptor (TSHR) ectodomain comprises a tubular leucine-rich repeat domain (LRD) and a hinge [or signaling specificity domain (SSD)]. TSH binds to both the LRD and SSD, leading to signal transduction by the transmembrane domain. The SSD structure and spatial orientation to the other components are unknown. We exploited a fortuitous observation to obtain mechanistic insight into the relationship between TSH binding and signal transduction. A mouse TSHR cDNA generated by PCR was found to express a receptor with poor TSH-induced cAMP generation despite normal TSH binding. Progressive reversion to wild-type of six mutations revealed E251K in the LRD to be critical for reduced signal transduction in both mouse and human TSHR. An I286F substitution in the SSD had a much weaker effect and was additive with E251K. To our knowledge, there are no previous examples of specific amino acid mutations in the TSHR LRD that dissociate TSH binding from TSHR signal transduction. To prevent flailing of the TSHR LRD, its position vis-à-vis the SSD must be stabilized by multiple amino acid interactions. The present data suggest that TSHR residue E251 is one of these residues involved in stabilizing the LRD relative to the SSD, thereby enabling ligand binding to transduce a signal by the latter. That the E251K mutation can reduce signal transduction despite high-affinity TSH binding comparable with the wild-type TSHR provides mechanistic insight into the coupling between ligand binding and receptor activation.
| S-EPMC2851189 | biostudies-literature
Project description:Thyroid-associated ophthalmopathy (TAO) is a vexing and undertreated ocular component of Graves disease in which orbital tissues undergo extensive remodelling. My colleagues and I have introduced the concept that fibrocytes expressing the haematopoietic cell antigen CD34 (CD34(+) fibrocytes), which are precursor cells of bone-marrow-derived monocyte lineage, express the TSH receptor (TSHR). These cells also produce several other proteins whose expression was traditionally thought to be restricted to the thyroid gland. TSHR-expressing fibrocytes in which the receptor is activated by its ligand generate extremely high levels of several inflammatory cytokines. Acting in concert with TSHR, the insulin-like growth factor 1 receptor (IGF-1R) expressed by orbital fibroblasts and fibrocytes seems to be necessary for TSHR-dependent cytokine production, as anti-IGF-1R blocking antibodies attenuate these proinflammatory actions of TSH. Furthermore, circulating fibrocytes are highly abundant in patients with TAO and seem to infiltrate orbital connective tissues, where they might transition to CD34(+) fibroblasts. My research group has postulated that the infiltration of fibrocytes into the orbit, their unique biosynthetic repertoire and their proinflammatory and profibrotic phenotype account for the characteristic properties exhibited by orbital connective tissues that underlie susceptibility to TAO. These insights, which have emerged in the past few years, might be of use in therapeutically targeting pathogenic orbit-infiltrating fibrocytes selectively by utilizing novel biologic agents that interfere with TSHR and IGF-1R signalling.
| S-EPMC4687015 | biostudies-literature
Project description:PurposeThe thyroid-stimulating hormone receptor (TSHR) is the target autoantigen for TSHR-stimulating autoantibodies in Graves' disease. The TSHR is composed of: a leucine-rich repeat domain (LRD), a hinge region or cleavage domain (CD) and a transmembrane domain (TMD). The binding arrangements between the TSHR LRD and the thyroid-stimulating autoantibody M22 or TSH have become available from the crystal structure of the TSHR LRD-M22 complex and a comparative model of the TSHR LRD in complex with TSH, respectively. However, the mechanism by which the TMD of the TSHR and the other glycoprotein hormone receptors (GPHRs) becomes activated is unknown.MethodsWe have generated comparative models of the structures of the inactive (TMD_In) and active (TMD_Ac) conformations of the TSHR, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) TMDs. The structures of TMD_Ac and TMD_In were obtained using class A GPCR crystal structures for which fully active and inactive conformations were available.ResultsMost conserved motifs observed in GPCR TMDs are also observed in the amino acid sequences of GPHR TMDs. Furthermore, most GPCR TMD conserved helix distortions are observed in our models of the structures of GPHR TMDs. Analysis of these structures has allowed us to propose a mechanism for activation of GPHR TMDs.ConclusionsInsight into the mechanism of activation of the TSHR by both TSH and TSHR autoantibodies is likely to be useful in the development of new treatments for Graves' disease.
| S-EPMC5136658 | biostudies-literature
Project description:Pathological activation of the thyroid-stimulating hormone receptor (TSHR) is caused by thyroid-stimulating antibodies in patients with Graves' disease (GD) or by somatic and rare genomic mutations that enhance constitutive activation of the receptor influencing both G protein and non-G protein signaling. Potential selective small molecule antagonists represent novel therapeutic compounds for abrogation of such abnormal TSHR signaling. In this study, we describe the identification and in vitro characterization of a novel small molecule antagonist by high-throughput screening (HTS). The identification of the TSHR antagonist was performed using a transcription-based TSH-inhibition bioassay. TSHR-expressing CHO cells, which also expressed a luciferase-tagged CRE response element, were optimized using bovine TSH as the activator, in a 384 well plate format, which had a Z score of 0.3-0.6. Using this HTS assay, we screened a diverse library of ~80,000 compounds at a final concentration of 16.7 μM. The selection criteria for a positive hit were based on a mean signal threshold of ≥50% inhibition of control TSH stimulation. The screening resulted in 450 positive hits giving a hit ratio of 0.56%. A secondary confirmation screen against TSH and forskolin - a post receptor activator of adenylyl cyclase - confirmed one TSHR-specific candidate antagonist molecule (named VA-K-14). This lead molecule had an IC50 of 12.3 μM and a unique chemical structure. A parallel analysis for cell viability indicated that the lead inhibitor was non-cytotoxic at its effective concentrations. In silico docking studies performed using a TSHR transmembrane model showed the hydrophobic contact locations and the possible mode of inhibition of TSHR signaling. Furthermore, this molecule was capable of inhibiting TSHR stimulation by GD patient sera and monoclonal-stimulating TSHR antibodies. In conclusion, we report the identification of a novel small molecule TSHR inhibitor, which has the potential to be developed as a therapeutic antagonist for abrogation of TSHR signaling by TSHR autoantibodies in GD.
| S-EPMC5037132 | biostudies-literature
Project description:Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.
| S-EPMC3434159 | biostudies-literature
Project description:G protein coupled receptors (GPCRs) can lead to G protein and non-G protein initiated signals. By virtue of its structural property, the TSH receptor (TSHR) has a unique ability to engage different G proteins making it highly amenable to selective signaling. In this study, we describe the identification and characterization of a novel small molecule agonist to the TSHR which induces primary engagement with Gαq/11. To identify allosteric modulators inducing selective signaling of the TSHR we used a transcriptional-based luciferase assay system with CHO-TSHR cells stably expressing response elements (CRE, NFAT, SRF, or SRE) that were capable of measuring signals emanating from the coupling of Gαs , Gαq/11, Gβγ, and Gα12/13, respectively. Using this system, TSH activated Gαs , Gαq/11, and Gα12/13 but not Gβγ. On screening a library of 50K molecules at 0.1,1.0 and 10 μM, we identified a novel Gq/11 agonist (named MSq1) which activated Gq/11 mediated NFAT-luciferase >4 fold above baseline and had an EC50= 8.3 × 10-9 M with only minor induction of Gαs and cAMP. Furthermore, MSq1 is chemically and structurally distinct from any of the previously reported TSHR agonist molecules. Docking studies using a TSHR transmembrane domain (TMD) model indicated that MSq1 had contact points on helices H1, H2, H3, and H7 in the hydrophobic pocket of the TMD and also with the extracellular loops. On co-treatment with TSH, MSq1 suppressed TSH-induced proliferation of thyrocytes in a dose-dependent manner but lacked the intrinsic ability to influence basal thyrocyte proliferation. This unexpected inhibitory property of MSq1 could be blocked in the presence of a PKC inhibitor resulting in derepressing TSH induced protein kinase A (PKA) signals and resulting in the induction of proliferation. Thus, the inhibitory effect of MSq1 on proliferation resided in its capacity to overtly activate protein kinase C (PKC) which in turn suppressed the proliferative signal induced by activation of the predomiant cAMP-PKA pathway of the TSHR. Treatment of rat thyroid cells (FRTL5) with MSq1 did not show any upregulation of gene expression of the key thyroid specific markers such as thyroglobulin(Tg), thyroid peroxidase (Tpo), sodium iodide symporter (Nis), and the TSH receptor (Tshr) further suggesting lack of involvement of MSq1 and Gαq/11 activation with cellular differentation. In summary, we identified and characterized a novel Gαq/11 agonist molecule acting at the TSHR and which showed a marked anti-proliferative ability. Hence, Gq biased activation of the TSHR is capable of ameliorating the proliferative signals from its orthosteric ligand and may offer a therapeutic option for thyroid growth modulation.
| S-EPMC7333667 | biostudies-literature