Identification of an important thyrotrophin binding site on the human thyrotrophin receptor using monoclonal antibodies

A thyrotrophin (TSH) binding site has been identified on the extracellular domain of the human thyrotrophin receptor (hTSHR) using monoclonal antibodi es that recognise the native hTSHR. These antibodies were produced by immun ising BALB/c mice with denatured recombinant material, selected by their re action with recombinant hTSHR expressed on heterologous cell lines using fl ow cytofluorimetric analysis, and characterised by immunoblotting and immun oprecipitation. The epitopes the monoclonal antibodies recognise were deter mined using multiple overlapping synthetic peptides. All of the antibodies reacted with epitopes within the region 335-390; these epitopes must be acc essible on the external surface of the native hTSHR. None of the antibodies stimulated cAMP production of recombinant hTSHR cell lines. The epitopes o f two antibodies (residues 337-342 and 355-358) are in the small peptide th ought to be removed by proteolytic processing of hTSHR. A further five diff erent antibodies (determined from their variable region sequences) all reac ted with residues 381-384 emphasising the immunogenicity of this region. Th e functional importance of residues 381-384 as a TSH binding site was shown by the fact that some of these monoclonal antibodies caused inhibition of radiolabelled TSH binding of 80-90% at 1 mu g/ml and greater than 50% inhib ition at 0.1 mu g/ml (0.65 nM-i.e. comparable in effectiveness with TSH its elf). Residues 381-384 may form part of the target regions recognised by in hibitory autoantibodies found in Graves' disease. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.