Thyroid hormones (TH) are of central importance for thermogenesis, energy homeostasis and metabolism. Here, we will discuss these aspects by focussing on the physiological aspects of TH-dependent regulation in response to cold exposure and fasting, which will be compared to alterations in primary hyperthyroidism and hypothyroidism. In particular, we will summarise current knowledge on regional thyroid hormone status in the central nervous system (CNS) and in peripheral cells. In contrast to hyperthyroidism and hypothyroidism, where parallel changes are observed, local alterations in the CNS differ to peripheral compartments when induced by cold exposure or fasting. Cold exposure is associated with low hypothalamic TH concentrations but increased TH levels in the periphery. Fasting results in a reversed TH pattern. Primary hypothyroidism and hyperthyroidism disrupt these fine-tuned adaptive mechanisms and both, the hypothalamus and the periphery, will have the same TH status. These important mechanisms need to be considered when discussing thyroid hormone replacement and other therapeutical interventions to modulate TH status.
K Alexander Iwen, Rebecca Oelkrug, and Georg Brabant
KJ Starkey, A Janezic, G Jones, N Jordan, G Baker, and M Ludgate
The thyrotrophin receptor (TSHR) provides an autoantigenic link between the thyroid and orbit in Graves' (GD) and thyroid eye diseases (TED). We measured TSHR transcripts in different fat depots to determine whether TSHR expression levels are influenced by the autoimmune/inflammatory process and/or thyroid hormone status, using quantitative real-time PCR. Nine intact or fractionated adipose samples, from patients with GD and/or TED, were analysed ex vivo. Eight expressed the TSHR, at levels approaching the thyroid, and one was at the limit of detection. Thirteen/fifteen orbital and abdominal fat samples from patients free of GD and TED, measured ex vivo, were negative for TSHR transcripts and two were at the limit of detection. All preadipocyte samples induced to differentiate in vitro expressed the TSHR. To investigate the influence of thyroid hormone status on adipose TSHR expression, we induced hyper- and hypothyroidism in BALBc mice by administering tri-iodothyronine and propylthiouracil respectively. In euthyroid animals, whole fat samples were at the limit of detection and were not altered by thyroid hormone status. The results show that adipose TSHR expression ex vivo indicates adipogenesis in progress in vivo and is associated with the autoimmune/inflammatory process in GD and TED but is not restricted to the orbit or influenced by thyroid hormone status.
GS Seetharamaiah, S Kaithamana, RK Desai, and BS Prabhakar
Expression of large quantities of conformationally intact thyrotropin receptor (TSHR) is essential to understand the structure-function relationship of the receptor. We expressed three different constructs of full-length human TSHR in insect cells: (a) a TSHR cDNA lacking signal sequence (TSHR-ns), (b) a TSHR cDNA containing human TSHR signal sequence (TSHR-hs) and (c) a TSHR cDNA with baculovirus envelope protein encoded signal sequence gp-67 (TSHR-gp). No unique protein band, corresponding to any of these recombinant proteins, was visible upon Coomassie Blue staining after SDS-PAGE. However, Western blot using TSHR specific monoclonal antibody showed unique bands around 80, 100 and 100 kDa in TSHR-ns, TSHR-hs and TSHR-gp virus infected insect cells respectively. All three full-length TSHR proteins could neutralize the TSH binding inhibitory immunoglobulin (TBII) activity from sera of experimental animals. However, only glycosylated proteins (TSHR-hs and TSHR-gp) neutralized the TBII activity of sera from autoimmune thyroid patients, confirming the importance of glycosylation for patient autoantibody reactivity. Expression levels of full-length TSHR proteins were much lower than the levels of similarly produced corresponding ectodomains of TSHR proteins. Southern blot and Northern blot analyses showed that DNA and RNA levels in full-length TSHR virus infected insect cells were comparable to the levels found in cells infected with viruses encoding only the ectodomain of TSHR. These data suggest that full-length TSHR expression is very low and is regulated at the translational level.
S Costagliola, L Alcalde, J Ruf, G Vassart, and M Ludgate
The availability of high affinity antibodies to the human TSH receptor (TSHR) would help in defining its functional domains, but this requires the production of pure receptor as immunogen. We have expressed the extracellular domain (ECD) of the TSHR (residues 21–414) as a fusion protein with maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cRl vector. The major protein in an electrophoretically separated, crude bacterial lysate had a molecular mass of 89 kDa, in agreement with the size predicted for the MBP-ECD fusion product. Its identity was confirmed by Western blotting in which it was recognized by two polyclonal antibodies to synthetic peptides of the TSHR and an anti-MBP. Following purification on an amylose column, 15 mg pure MBP-ECD per litre of culture were produced, which was 5% of the total bacterial protein. Following extensive dialysis in a buffer which produces slight denaturation, MBP-ECD was cleaved with factor Xa. The identity of each protein was confirmed by Western blotting.
To investigate the possibility of using the fusion protein as an immunogen we produced rabbit polyclonal antibodies to the ECD which were able to produce immunofluorescent staining of Chinese hamster ovary cells that expressed the TSHR, and revealed a protein of 95 kDa in Western blots of the same cells, in addition to a protein of 55 kDa. Only the protein of 55 kDa was detected in Western blots of human thyroid membranes. Subsequently, immunoglobulins from mice immunized with MBP-ECD were shown to contain TSH-binding inhibiting activity and to inhibit TSH-mediated cyclic AMP production; these mice had a lower serum thyroxine level when compared with mice immunized with the MBP—β galactosidase fusion protein MBP-GAL.
The study shows the feasibility of using recombinant TSHR expressed in E. coli (i) to produce antibodies which recognize the native receptor and thus could be applied to studies of TSHR expression (e.g. in thyroid tumours), (ii) to establish animal models of autoimmune hypothyroidism and (iii) as the starting material in denaturation and refolding experiments which may help in defining structure—function relationships.
R Núñez Miguel, J Sanders, D Y Chirgadze, J Furmaniak, and B Rees Smith
The TSH receptor (TSHR) ligands M22 (a thyroid stimulating human monoclonal antibody) and TSH, bind to the concave surface of the leucine rich repeats domain (LRD) of the TSHR and here, we show that M22 mimics closely the binding of TSH. We compared interactions produced by M22 with the TSHR in the M22–TSHR crystal structure (2.55 Å resolution) and produced by TSH with the TSHR in a TSH–TSHR comparative model. The crystal structure of the TSHR and a comparative model of TSH based on the crystal structure of FSH were used as components to build the TSH–TSHR model. This model was built based on the FSH–FSH receptor structure (2.9 Å) and then the structure of the TSHR in the model was replaced by the TSHR crystal structure. The analysis shows that M22 light chain mimics the TSHβ chain in its interaction with TSHR LRD, while M22 heavy chain mimics the interactions of the TSHα chain. The M22–TSHR complex contains a greater number of hydrogen bonds and salt bridges and fewer hydrophobic interactions than the TSH–TSHR complex, consistent with a higher M22 binding affinity. Furthermore, the surface area formed by TSHR residues N208, Q235, R255, and N256 has been identified as a candidate target region for small molecules which might selectively block binding of autoantibodies to the TSHR.
Jennifer Miller-Gallacher, Paul Sanders, Stuart Young, Andrew Sullivan, Stuart Baker, Samuel C Reddington, Matthew Clue, Katarzyna Kabelis, Jill Clark, Jane Wilmot, Daniel Thomas, Monika Chlebowska, Francesca Cole, Emily Pearson, Emma Roberts, Matthew Holly, Michele Evans, Ricardo Núñez Miguel, Michael Powell, Jane Sanders, Jadwiga Furmaniak, and Bernard Rees Smith
The crystal structures of the thyroid-stimulating hormone receptor (TSHR) leucine-rich repeat domain (amino acids 22–260; TSHR260) in complex with a stimulating human monoclonal autoantibody (M22TM) and in complex with a blocking human autoantibody (K1-70™) have been solved. However, attempts to purify and crystallise free TSHR260, that is not bound to an autoantibody, have been unsuccessful due to the poor stability of free TSHR260. We now describe a TSHR260 mutant that has been stabilised by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55TM, which is approximately 900 times more thermostable than wild-type TSHR260. These six mutations did not affect the binding of human TSHR monoclonal autoantibodies or patient serum TSHR autoantibodies to the TSHR260. Furthermore, the response of full-length TSHR to stimulation by TSH or human TSHR monoclonal autoantibodies was not affected by the six mutations. Thermostable TSHR260-JMG55TM has been purified and crystallised without ligand and the structure solved at 2.83 Å resolution. This is the first reported structure of a glycoprotein hormone receptor crystallised without ligand. The unbound TSHR260-JMG55TM structure and the M22 and K1-70 bound TSHR260 structures are remarkably similar except for small changes in side chain conformations. This suggests that neither the mutations nor the binding of M22TM or K1-70TM change the rigid leucine-rich repeat domain structure of TSHR260. The solved TSHR260-JMG55TM structure provides a rationale as to why the six mutations have a thermostabilising effect and provides helpful guidelines for thermostabilisation strategies of other soluble protein domains.
Ricardo Núñez Miguel, Jane Sanders, Paul Sanders, Stuart Young, Jill Clark, Katarzyna Kabelis, Jane Wilmot, Michele Evans, Emma Roberts, Xiaoling Hu, Jadwiga Furmaniak, and Bernard Rees Smith
Binding of a new thyroid-stimulating human monoclonal autoantibody (MAb) K1–18 to the TSH receptor (TSHR) leucine-rich domain (LRD) was predicted using charge–charge interaction mapping based on unique complementarities between the TSHR in interactions with the thyroid-stimulating human MAb M22 or the thyroid-blocking human MAb K1–70. The interactions of K1–18 with the TSHR LRD were compared with the interactions in the crystal structures of the M22–TSHR LRD and K1–70–TSHR LRD complexes. Furthermore, the predicted position of K1–18 on the TSHR was validated by the effects of TSHR mutations on the stimulating activity of K1–18. A similar approach was adopted for predicting binding of a mouse thyroid-blocking MAb RSR-B2 to the TSHR. K1–18 is predicted to bind to the TSHR LRD in a similar way as TSH and M22. The binding analysis suggests that K1–18 light chain (LC) mimics binding of the TSH-α chain and the heavy chain (HC) mimics binding of the TSH-β chain. By contrast, M22 HC mimics the interactions of TSH-α while M22 LC mimics TSH-β in interactions with the TSHR. The observed interactions in the M22–TSHR LRD and K1–70–TSHR LRD complexes (crystal structures) with TSH–TSHR LRD (comparative model) and K1–18–TSHR LRD (predictive binding) suggest that K1–18 and M22 interactions with the receptor may reflect interaction of thyroid-stimulating autoantibodies in general. Furthermore, K1–70 and RSR-B2 interactions with the TSHR LRD may reflect binding of TSHR-blocking autoantibodies in general. Interactions involving the C-terminal part of the TSHR LRD may be important for receptor activation by autoantibodies.
Michelle Mohyi and Terry J Smith
Thyroid-associated ophthalmopathy (TAO) is a vexing and poorly understood autoimmune process involving the upper face and tissues surrounding the eyes. In TAO, the orbit can become inflamed and undergo substantial remodeling that is disfiguring and can lead to loss of vision. There are currently no approved medical therapies for TAO, the consequence of its uncertain pathogenic nature. It usually presents as a component of the syndrome known as Graves’ disease where loss of immune tolerance to the thyrotropin receptor (TSHR) results in the generation of activating antibodies against that protein and hyperthyroidism. The role for TSHR and these antibodies in the development of TAO is considerably less well established. We have reported over the past 2 decades evidence that the insulin-like growth factorI receptor (IGF1R) may also participate in the pathogenesis of TAO. Activating antibodies against IGF1R have been detected in patients with GD. The actions of these antibodies initiate signaling in orbital fibroblasts from patients with the disease. Further, we have identified a functional and physical interaction between TSHR and IGF1R. Importantly, it appears that signaling initiated from either receptor can be attenuated by inhibiting the activity of IGF1R. These findings underpin the rationale for therapeutically targeting IGF1R in active TAO. A recently completed therapeutic trial of teprotumumab, a human IGF1R inhibiting antibody, in patients with moderate to severe, active TAO, indicates the potential effectiveness and safety of the drug. It is possible that other autoimmune diseases might also benefit from this treatment strategy.
Y Oda, J Sanders, S Roberts, M Maruyama, R Kato, M Perez, VB Petersen, N Wedlock, J Furmaniak, and B Rees Smith
We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR. The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated. The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35S-labelled full-length TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125I-TSH/TSHR complexes, inhibition of 125I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface. Fab fragments of monoclonal antibodies were isolated, labelled with 125I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation. Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10(7) M(-1). Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells. The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactively-labelled probes in analysis of the TSH receptor. The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.
Paul Sanders, Stuart Young, Jane Sanders, Katarzyna Kabelis, Stuart Baker, Andrew Sullivan, Michele Evans, Jill Clark, Jane Wilmot, Xiaoling Hu, Emma Roberts, Michael Powell, Ricardo Núñez Miguel, Jadwiga Furmaniak, and Bernard Rees Smith
A complex of the TSH receptor extracellular domain (amino acids 22–260; TSHR260) bound to a blocking-type human monoclonal autoantibody (K1-70) was purified, crystallised and the structure solved at 1.9 Å resolution. K1-70 Fab binds to the concave surface of the TSHR leucine-rich domain (LRD) forming a large interface (2565 Å2) with an extensive network of ionic, polar and hydrophobic interactions. Mutation of TSHR or K1-70 residues showing strong interactions in the solved structure influenced the activity of K1-70, indicating that the binding detail observed in the complex reflects interactions of K1-70 with intact, functionally active TSHR. Unbound K1-70 Fab was prepared and crystallised to 2.22 Å resolution. Virtually no movement was observed in the atoms of K1-70 residues on the binding interface compared with unbound K1-70, consistent with ‘lock and key’ binding. The binding arrangements in the TSHR260–K1-70 Fab complex are similar to previously observed for the TSHR260–M22 Fab complex; however, K1-70 clasps the concave surface of the TSHR LRD in approximately the opposite orientation (rotated 155°) to M22. The blocking autoantibody K1-70 binds more N-terminally on the TSHR concave surface than either the stimulating autoantibody M22 or the hormone TSH, and this may reflect its different functional activity. The structure of TSHR260 in the TSHR260–K1-70 and TSHR260–M22 complexes show a root mean square deviation on all Cα atoms of only 0.51 Å. These high-resolution crystal structures provide a foundation for developing new strategies to understand and control TSHR activation and the autoimmune response to the TSHR.