Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain

Miller-Gallacher, Jennifer; Sanders, Paul G.; Young, Stuart; Sullivan, Andrew; Baker, Stuart; Reddington, Samuel C.; Clue, Matthew; Kabelis, Katarzyna; Clark, J. C. D.; Wilmot, Jane; Thomas, Daniel; Chlebowska, Monika; Cole, Francesca; Pearson, Emily; Roberts, Emma; Holly, Matthew; Evans, Michele K.; Miguel, Ricardo Núñez; Powell, Michael J.; Sanders, Jane; Furmaniak, Jadwiga; Smith, Bernard Rees

doi:10.1530/jme-18-0213

Cited by 10 publications

(10 citation statements)

References 45 publications

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“…However, when provided to hTSHR/NOD.NOD.H2 h4 mice, TSHR A-subunit protein enhanced the levels of pathogenic TSHR Abs. As mentioned above, the inactive (although native) form of TSHR A-subunits is not recognized by pathogenic TSHR Abs (10,39). The ability of this material to stimulate B cells specific for pathogenic Abs reflects the role of "original antigenic sin" [as discussed previously (40)].…”

Section: Discussionmentioning

confidence: 99%

Nanoparticles Bearing TSH Receptor Protein and a Tolerogenic Molecule Do Not Induce Immune Tolerance but Exacerbate Thyroid Autoimmunity in hTSHR/NOD.H2h4 Mice

McLachlan

Aliesky

Rapoport

2019

The Journal of Immunology

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Transgenic NOD.H2h4 mice that express the human (h) TSHR A-subunit in the thyroid gland spontaneously develop pathogenic TSHR autoantibodies resembling those in patients with Graves disease. Nanoparticles coupled to recombinant hTSHR A-subunit protein and a tolerogenic molecule (ligand for the endogenous aryl-hydrocarbon receptor; ITE) were injected i.p. four times at weekly intervals into hTSHR/NOD.H2h4 mice with the goal of blocking TSHR Ab development. Unexpectedly, in transgenic mice, injecting TSHR A-subunit–ITE nanoparticles (not ITE-nanoparticles or buffer) accelerated and enhanced the development of pathogenic TSHR Abs measured by inhibition of TSH binding to the TSHR. Nonpathogenic TSHR Abs (ELISA) were enhanced in transgenics and induced in wild-type littermates. Serendipitously, these findings have important implications for disease pathogenesis: development of Graves TSHR Abs is limited by the availability of A-subunit protein, which is shed from membrane bound TSHR, expressed at low levels in the thyroid. The enhanced TSHR Ab response following injected TSHR A-subunit protein-nanoparticles is reminiscent of the transient increase in pathogenic TSHR Abs following the release of thyroid autoantigens after radio-iodine therapy in Graves patients. However, in the hTSHR/NOD.H2h4 model, enhancement is specific for TSHR Abs, with Abs to thyroglobulin and thyroid peroxidase remaining unchanged. In conclusion, despite the inclusion of a tolerogenic molecule, injected nanoparticles coated with TSHR A-subunit protein enhanced and accelerated development of pathogenic TSHR Abs in hTSHR/NOD. NOD.H2h4. These findings emphasize the need for sufficient TSHR A-subunit protein to activate the immune system and the generation of stimulatory TSHR Abs in genetically predisposed individuals.

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Section: Discussionmentioning

confidence: 99%

Nanoparticles Bearing TSH Receptor Protein and a Tolerogenic Molecule Do Not Induce Immune Tolerance but Exacerbate Thyroid Autoimmunity in hTSHR/NOD.H2h4 Mice

McLachlan

Aliesky

Rapoport

2019

The Journal of Immunology

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“…The responses of the full length TSHR with the respective part of the sequence replaced by TSHR260-JMG55 to stimulation by TSH or M22 were similar to that of the wild type. Furthermore, the full length TSHR containing mutated TSHR260-JMG55 domain showed similar ability to wildtype TSHR to bind TSHR autoantibodies present in different Graves' patient sera [15].…”

Section: In Vitro Practical Applications Of Tshr Researchmentioning

confidence: 81%

“…The TSHR260-JMG55 was purified to homogeneity, deglycosylated and crystallised. The crystals diffracted to 2.83 Å resolution allowing for the structure of a ligand free TSHR260 to be solved by molecular replacement [15]. The solved TSHR260-JMG55 structure showed remarkable similarity to the crystal structures of the TSHR260 from TSHR260-M22 and TSHR260-K1-70 complexes.…”

Section: In Vitro Practical Applications Of Tshr Researchmentioning

confidence: 91%

“…The most thermostable mutations were then combined in a series of experiments until the most thermostable mutant containing six different mutations was obtained. This mutant was termed TSHR260-JMG55 and it is about 900 times more stable than the wild type TSHR260 [15]. The responses of the full length TSHR with the respective part of the sequence replaced by TSHR260-JMG55 to stimulation by TSH or M22 were similar to that of the wild type.…”

Section: In Vitro Practical Applications Of Tshr Researchmentioning

confidence: 99%

“…Furthermore, a long awaited mouse model of Graves' ophthalmopathy (GO) was finally described [14]. Very recently thermo-stable preparations of the TSHR LRD were obtained and for the first time the crystal structure of a ligand-free glycoprotein hormone receptor domain was solved (2.83 Å resolution) [15]. All of these considerable scientific achievements are leading to improvements in diagnosis, monitoring and management of patients with AITD.…”

Section: Introductionmentioning

confidence: 99%

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Practical applications of studies on the TSH receptor and TSH receptor autoantibodies

Furmaniak

Sanders

et al. 2020

Endocrine

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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.

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Computational model of the full-length TSH receptor

Mezei

Latif

Davies

2022

Preprint

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The receptor for thyroid stimulating hormone (TSHR), a 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 now generated a model of the entire TSHR 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 solvated it with water and counterions, and performed 1000ns Molecular Dynamic simulations on it. The simulations showed that the structure of the transmembrane and leucine-rich domains were remarkably constant while the linking 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 linker region is an intrinsically disordered protein (IDP). Furthermore, preliminary data simulating the full TSHR model complexed with its ligand (TSH) showed that (a) there is a strong affinity between the linker region and TSH ligand and (b) the association of the linker region and the TSH ligand reduces the structural fluctuations in the linker region. This full-length model illustrates the importance of the linker region 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 linker region.

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Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain

Cited by 10 publications

References 45 publications

Nanoparticles Bearing TSH Receptor Protein and a Tolerogenic Molecule Do Not Induce Immune Tolerance but Exacerbate Thyroid Autoimmunity in hTSHR/NOD.H2h4 Mice

Nanoparticles Bearing TSH Receptor Protein and a Tolerogenic Molecule Do Not Induce Immune Tolerance but Exacerbate Thyroid Autoimmunity in hTSHR/NOD.H2h4 Mice

Practical applications of studies on the TSH receptor and TSH receptor autoantibodies

Computational model of the full-length TSH receptor

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