Jane Sanders

Characterization of the thyrotropin binding pocket.

A panel of monoclonal antibodies (mAbs) to the thyrotropin receptor (TSHR) was prepared using three different immunization strategies. The mAbs obtained (n = 138) reacted with linear epitopes covering most of the TSHR extracellular domain and with conformational epitopes. mAbs that bound to five different regions of the TSHR (amino acids [aa] 32-41, aa 36-42, aa 246-260, aa 277-296, and aa 381-385) were able to inhibit (125)I-labeled thyrotropin (TSH) binding to solubilized TSHR preparations. Fab and immunoglobulin G (IgG) preparations were similarly effective inhibitors for mAbs reactive with aa 246-260, aa 277-291 and aa 381-385 suggesting that these three regions of the TSHR are involved in TSH binding. In contrast mAbs reactive with aa 32-41 and aa 36-42 were not effective at inhibiting TSH binding when Fab preparations were used, suggesting that these N terminal regions of the TSHR were less critical for TSH binding. Our studies suggest that three distinct and discontinuous regions of the TSHR (aa 246-260 and 277-296 on the TSHR A subunit) and aa 381-385 (on the TSHR B subunit) fold together to form a complex TSH binding pocket. Alignment of the aa sequences of these three regions in TSHRs from different species indicates that they are highly conserved.

Monoclonal autoantibodies to the TSH receptor, one with stimulating activity and one with blocking activity, obtained from the same blood sample

Objective  Patients who appear to have both stimulating and blocking TSHR autoantibodies in their sera have been described, but the two activities have not been separated and analysed. We now describe the isolation and detailed characterization of a blocking type TSHR monoclonal autoantibody and a stimulating type TSHR monoclonal autoantibody from a single sample of peripheral blood lymphocytes.

TSH Receptor Monoclonal Antibodies with Agonist, Antagonist, and Inverse Agonist Activities

Autoantibodies in autoimmune thyroid disease (AITD) bind to the TSH receptor (TSHR) and can act as either agonists, mimicking the biological activity of TSH, or as antagonists inhibiting the action of TSH. Furthermore, some antibodies with antagonist activity can also inhibit the constitutive activity of the TSHR, that is, act as inverse agonists. The production of animal TSHR monoclonal antibodies (MAbs) with the characteristics of patient autoantibodies and the isolation of human autoantibodies from patients with AITD has allowed us to analyze the interactions of these antibodies with the TSHR at the molecular level. In the case of animal MAbs, advances such as DNA immunization allowed the production of the first MAbs which showed the characteristics of human TSHR autoantibodies (TRAbs). Mouse MAbs (TSMAbs 1-3) and a hamster MAb (MS-1) were obtained that acted as TSHR agonists with the ability to stimulate cyclic AMP production in CHO cells expressing the TSHR. In addition, a mouse TSHR MAb (MAb-B2) that had the ability to act as an antagonist of TRAbs and TSH was isolated and characterized. Also, a mouse TSHR MAb that showed TSH antagonist and TSHR inverse agonist activity (CS-17) was described. Furthermore, a panel of human TRAbs has been obtained from the peripheral blood lymphocytes of patients with AITD and extensively characterized. These MAbs have all the characteristics of TRAbs and are active at ng/mL levels. To date, two human MAbs with TSHR agonist activity (M22 and K1-18), one human MAb with TSHR antagonist activity (K1-70) and one human MAb (5C9) with both TSHR antagonist and TSHR inverse agonist activity have been isolated. Early experiments showed that the binding sites for TSH and for TRAbs with thyroid stimulating or blocking activities were located on the extracellular domain of the TSHR. Extensive studies using TSHRs with single amino acid mutations identified TSHR residues that were important for binding and biological activity of TSHR MAbs (human and animal) and TSH. The structures of several TSHR MAb Fab fragments were solved by X-ray crystallography and provided details of the topography of the antigen binding sites of antibodies with either agonist or antagonist activity. Furthermore stable complexes of the leucine-rich repeat domain (LRD) of the TSHR with a human MAb (M22) with agonist activity and with a human MAb (K1-70) with antagonist activity have been produced and their structures solved by X-ray crystallography at 2.55 and 1.9Å resolution, respectively. Together these experiments have given detailed insights into the interactions of antibodies with different biological activities (agonist, antagonist, and inverse agonist) with the TSHR. Although the nature of ligand binding to the TSHR is now understood in some detail, it is far from clear how these initial interactions lead to functional effects on activation or inactivation of the receptor.

Autoantigens in the Autoimmune Endocrinopathies

Identification and characterization of specific autoantigens are integral parts of research into the understanding of the pathogenesis of autoimmune diseases. In the case of endocrine autoimmunity, the pituitary, thyroid, parathyroids, pancreatic islet cells, adrenals, and gonads can be subject to autoimmune attack, and many of the autoantigens involved have been identified and studied in detail (Table 1).

Similarities and differences in interactions of thyroid stimulating and blocking autoantibodies with the TSH receptor

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.

ORIGINAL ARTICLE: Monoclonal autoantibodies to the TSH receptor, one with stimulating activity and one with blocking activity, obtained from the same blood sample

Objective  Patients who appear to have both stimulating and blocking TSHR autoantibodies in their sera have been described, but the two activities have not been separated and analysed. We now describe the isolation and detailed characterization of a blocking type TSHR monoclonal autoantibody and a stimulating type TSHR monoclonal autoantibody from a single sample of peripheral blood lymphocytes.

Structure and activation of the TSH receptor transmembrane domain

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.

ORIGINAL ARTICLE: Monoclonal autoantibodies to the TSH receptor, one with stimulating activity and one with blocking activity, obtained from the same blood sample: Monoclonal autoantibodies to the TSH receptor

Objective  Patients who appear to have both stimulating and blocking TSHR autoantibodies in their sera have been described, but the two activities have not been separated and analysed. We now describe the isolation and detailed characterization of a blocking type TSHR monoclonal autoantibody and a stimulating type TSHR monoclonal autoantibody from a single sample of peripheral blood lymphocytes.