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 antibodies that recognise the native hTSHR. These antibodies were produced by immunising BALB/c mice with denatured recombinant material, selected by their reaction with recombinant hTSHR expressed on heterologous cell lines using flow cytofluorimetric analysis, and characterised by immunoblotting and immunoprecipitation. The epitopes the monoclonal antibodies recognise were determined using multiple overlapping synthetic peptides. All of the antibodies reacted with epitopes within the region 335-390; these epitopes must be accessible on the external surface of the native hTSHR. None of the antibodies stimulated cAMP production of recombinant hTSHR cell lines. The epitopes of two antibodies (residues 337-342 and 355-358) are in the small peptide thought to be removed by proteolytic processing of hTSHR. A further five different antibodies (determined from their variable region sequences) all reacted with residues 381-384 emphasising the immunogenicity of this region. The 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 microg/ml and greater than 50% inhibition at 0.1 microg/ml (0.65 nM--i.e. comparable in effectiveness with TSH itself). Residues 381-384 may form part of the target regions recognised by inhibitory autoantibodies found in Graves' disease.
Shepherd
P S
PS
Department of Immunobiology, Guy's, King's and St Thomas' Hospitals Medical and Dental Schools, London, UK.
Da Costa
C R
CR
Cridland
J C
JC
Gilmore
K S
KS
Johnstone
A P
AP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D017931:DNA Primers / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013972:Thyrotropin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin; D000242:Cyclic AMP