Molecular interactions between the TSH receptor and a Thyroid-stimulating monoclonal autoantibody.

OBJECTIVE: To study the molecular interactions between the thyroid-stimulating hormone (TSH) receptor (TSHR) and a human thyroid-stimulating monoclonal autoantibody (M22). DESIGN: Amino acid mutations were introduced in the variable region gene sequences of M22 and the wild-type (WT) or mutated M22 Fab expressed in Escherichia coli. The ability of WT or mutated M22 Fab to inhibit binding of (125)I-TSH or (125)I-M22 to the TSHR and to stimulate cyclic adenosine monophosphate (AMP) production in Chinese hamster ovary cells expressing WT TSHRs was studied. Mutated TSHRs were also used in these studies in combination with WT or mutated M22 Fab to further identify interacting residues in the TSHR-M22 complex. MAIN OUTCOME: Out of 11 amino acid changes in the heavy chain (HC) of M22, 7 had an effect on M22 Fab biological activity, while in the case of 1 mutation the Fab was not expressed. In particular, stimulating activity of M22 Fab mutated at HC residues, D52, D54, and Y56 was markedly reduced. Mutation of M22 light chain (LC) D52 also reduced M22 Fab stimulating activity, while mutations at two further residues (LC D51 and LC D93) showed no effect. Reverse charge mutations at M22 HC D52 and TSHR R80 provided experimental evidence that these two residues interacted strongly with each other. CONCLUSION: Mutation of both the TSHR and M22 Fab has allowed identification of some residues critical for the receptor-autoantibody interaction. This approach should lead to detailed mapping of the amino acids important for M22 biological activity.

Crystal structure of the TSH receptor in complex with a thyroid-stimulating autoantibody.

OBJECTIVE: To analyze interactions between the thyroid-stimulating hormone receptor (TSHR) and a thyroid-stimulating human monoclonal autoantibody (M22) at the molecular level. DESIGN: A complex of part of the TSHR extracellular domain (amino acids 1-260; TSHR260) bound to M22 Fab was prepared and purified. Crystals suitable for X-ray diffraction analysis were obtained and the structure solved at 2.55 A resolution. MAIN OUTCOME: TSHR260 comprises of a curved helical tube and M22 Fab clasps its concave surface at 90 degrees to the tube length axis. The interface buried in the complex is large (2,500 A(2)) and an extensive network of ionic, polar, and hydrophobic bonding is involved in the interaction. There is virtually no movement in the atoms of M22 residues on the binding interface compared to unbound M22 consistent with "lock and key" binding. Mutation of residues showing strong interactions in the structure influenced M22 activity, indicating that the binding detail observed in the complex reflects interactions of M22 with intact, functionally active TSHR. The receptor-binding arrangements of the autoantibody are very similar to those reported for follicle-stimulating hormone (FSH) binding to the FSH receptor (amino acids 1-268) and consequently to those of TSH itself. CONCLUSIONS: It is remarkable that the thyroid-stimulating autoantibody shows almost identical receptor-binding features to TSH although the structures and origins of these two ligands are very different. Furthermore, our structure of the TSHR and its complex with M22 provide foundations for developing new strategies to understand and control both glycoprotein hormone receptor activation and the autoimmune response to the TSHR.

Effects of TSH receptor mutations on binding and biological activity of monoclonal antibodies and TSH.

The effects of an extensive series of mutations in the TSH receptor (TSHR) leucine-rich domain (LRD) on the ability of thyroid-stimulating monoclonal antibodies (TSMAbs) and TSH to bind to the receptor and stimulate cyclic AMP production in TSHR-transfected CHO cells has been investigated. In addition, the ability of a mouse monoclonal antibody with blocking (i.e., antagonist) activity (RSR-B2) to interact with mutated receptors has been studied. Several amino acids distributed along an extensive part of the concave surface of the LRD were found to be important for binding and stimulation by the thyroid-stimulating human MAb M22 but did not appear to be important for TSH binding and stimulation. Most of these amino acids important for M22 interactions were also found to be important for the stimulating activity of six different mouse TSMAbs and a hamster TSMAb. Furthermore, most of these same amino acids were important for stimulation by TSHR autoantibodies in a panel of sera from patients with Graves' disease. Amino acid R255 was the only residue found to be unimportant for TSH stimulation but critical for stimulation by all thyroid-stimulating antibodies tested (23 patient serum TSHR autoantibodies, M22, and all seven animal TSMAbs). About half the amino acids (all located in the N-terminal part of the LRD) found to be important for M22 activity were also important for the blocking activity of RSR-B2 and although the epitopes for the two MAbs overlap they are different. As the two MAbs have similar affinities, their epitope differences are probably responsible for their different activities. Overall our results indicate that different TSMAbs and different patient sera thyroid-stimulating autoantibodies interact with the same region of the TSHR, but there are subtle differences in the actual amino acids that make contact with the different stimulators.

A new assay for thyrotropin receptor autoantibodies.

A new procedure for measuring patient serum thyrotropin receptor (TSHR) autoantibodies is described in which the autoantibodies inhibit binding of a human monoclonal thyroid stimulating antibody M22 (labeled with biotin) to TSHR-coated enzyme-linked immunosorbent assay (ELISA) plate wells. In the assay, M22-biotin binding is detected by addition of streptavidin peroxidase. The M22 based assay was more sensitive than a similar ELISA based on inhibition of TSH-biotin binding to TSHR coated wells with 1 U/L of NIBSC 90/672 giving approximately 35% inhibition in the M22-based system compared to approximately 15% inhibition in the TSH-based ELISA. This had an important impact on the precision of the 2 assays with the M22-based ELISA showing an interassay coefficient of variation (CV) of 10% at 1 U/L whereas the TSH-based ELISA had an interassay CV of 20% at 1 U/L. Analysis of sera from 307 control subjects without a diagnosis of Graves' disease indicated that only 2 (0.65%) gave inhibition of M22 binding values of greater than 10% (11% and 12% inhibition). In the case of sera from 108 patients with Graves' disease (treated and untreated) 103 (95%) gave inhibition of M22 binding values of 14% or greater. Receiver operating characteristic (ROC) plot analysis showed that 100% specificity for TSHR autoantibody detection in Graves' disease was obtained at 95% sensitivity for the M22-based ELISA and 89% sensitivity for the TSH-biotin-based ELISA. Inhibition of M22 binding to the TSHR was closely correlated to inhibition of TSH binding in the 108 Graves' sera (r = 0.99). However, inhibition of M22 binding was almost always greater resulting in improved sensitivity and precision.