Effects of TSH Receptor Mutations on Binding and Biological Activity of Monoclonal Antibodies and TSH

Abstract

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.

Thyroid stimulating autoantibody M22 mimics TSH binding to the TSH receptor leucine rich domain: a comparative structural study of protein–protein interactions

“…Finally, TSHR Y185 has a face to side aromatic interaction with M22 LC Y50 and a hydrogen bond with M22LC Y49. This aromatic patch is likely to play an important role in the stabilization of the complex and this is consistent with TSHR Y185A causing considerable reduction in M22 binding affinity and stimulating activity (20-40% of the wild-type; Sanders et al 2006).…”

“…These intramolecular interactions are likely to reduce the extent of the intermolecular hydrogen bonds. In mutation experiments, TSHR R80A showed similar porcine TSH binding and cyclic AMP response to porcine TSH as the wild-type TSHR while change of charge mutation (TSHR R80D) caused a small reduction in response to stimulation by porcine TSH (80-100% of the wildtype; Sanders et al 2006) most likely due to structure modification. By contrast, TSHR mutations R80A and R80D had marked effects on the ability of the TSHR to respond to stimulation by M22 (!20% relative to the wild-type; Sanders et al 2006).…”

“…4C). Substitutions at position E107 in TSHR cause a marked reduction in M22 stimulation (!20% relative to the wild-type; Sanders et al 2006), probably due to the disruption of this hydrogen bond. Indeed, solvent inaccessible main chain NH groups are rarely found without a hydrogen bond partner at protein-protein interfaces or in protein cores (Baker & Hubbard 1984).…”

“…The E107A mutation caused a small reduction in stimulation by porcine TSH (60-80% of the wild-type; Sanders et al 2006) probably due to structure modification. The TSHR E107R mutation resulted in a more marked reduction in stimulation by porcine TSH (20-40% of the wild-type; Sanders et al 2006). In this TSHR E107R mutation, the longer side-chain of the new arginine is likely to be positioned closer to TSHa K91 resulting in an unfavourable charge-charge interaction.…”

“…The importance of this aromatic interaction is highlighted by the TSHR Y82A mutation associated with decrease of response to M22 stimulation (60-80% of the wild-type; Sanders et al 2006). TSHR K129 is involved in strong interactions with M22 but does not interact with TSH.…”