BINDING OF ANTITHYROTROPIN RECEPTOR AUTOANTIBODIES IN GRAVES-DISEASE SERUM TO NASCENT, IN-VITRO TRANSLATED THYROTROPIN RECEPTOR - ABILITY TO MAP EPITOPES RECOGNIZED BY ANTIBODIES

The binding of Graves' disease autoantibodies and xenogeneic antibodie s to the human TSH receptor (TSH-R) has been studied using receptor pr eparations generated in an in vitro transcription and translation reac tion. The complementary DNAs encoding for the full-length (764 amino a cids) and the extracellular region of TSH-R (amino acids 20-414, lacki ng the signal sequence) were used to generate the translated receptor antigen. Stable [S-35]methionine-labeled nascent protein for full-leng th and extracellular regions of TSH-R of approximate size 87 and 50 kD a, respectively, together with other smaller proteins were generated. The 87- and 50-kDa translated receptor proteins react by immunoprecipi tation analysis with monoclonal antibodies, polyclonal antisera, and u nfractionated Graves' disease serum containing autoantibody to TSH-R. The translated products of the extracellular TSH-R were examined in de tail. Using three well characterized murine monoclonal antibodies whos e epitopes encompass the amino-, central, and carboxyl-terminals of th e extracellular region of the receptor led to immunochemical identific ation of the smaller translated products to derive from internal methi onine start sites of TSH-R. These smaller, N-terminal-truncated transl ated proteins were also recognized by polyclonal antisera generated ag ainst recombinant TSH-R, thus allowing epitope mapping of some antibod ies. A large proportion of Graves' disease autoantibodies (>70%) bind to the translated extracellular region of TSH-R. This indicates that t he majority of pathogenic anti-TSH-R autoantibody binds the nascent tr anslated extracellular region of TSH-R, which is not influenced by the lack of glycosylation of the receptor.