FOLDING OF THE RECOMBINANT HUMAN THYROTROPIN (TSH) RECEPTOR EXTRACELLULAR DOMAIN - IDENTIFICATION OF FOLDED MONOMERIC AND TETRAMERIC COMPLEXES THAT BIND TSH RECEPTOR AUTOANTIBODIES

We have analyzed protein folding and disulfide bond formation in the e xtracellular domain of the human TSH receptor (hTSHR-ecd) expressed in Escherichia cobi. This domain, which begins at the amino-terminus and ends at residue 415, is a major autoantigen in human autoimmune thyro id disease. Refolding of reduced and denatured hTSHR-ecd occurred in p olyacrylamide gels treated with 0.25 M KCl for visualization of protei n bands. Under conditions of partial renaturation, at least three form s of the hTSHR-ecd were resolved by reelectrophoresis using sodium dod ecyl sulfate-polyacrylamide gel electrophoresis: 1) unfolded monomers, 2) folded monomers, and 3) tetramers. Disulfide bond formation was im plicated in both folding and tetramerization, as reduction of these fo rms produced only unfolded monomers. A natural variant of the hTSHR (v 1.3), sharing 231 N-terminal amino acids with hTSHR-ecd, farmed folded monomers and dimers on renaturation, but not tetramers, implicating o ne or more of the five cysteine residues residing between positions 23 1-415 in the association of dimers into tetramers. Binding of three di fferent sources of hTSHR antibodies to these various forms of the hTSH R-ecd was assessed by immunoblotting using: 1) murine monoclonal antib odies (MAbs) generated against hTSHR-ecd, 2) rabbit polyclonal antiser a generated against overlapping synthetic peptides spanning residues 3 7-71 of the hTSHR-ecd, and 3) human immunoglobulin G from patients wit h Graves' disease and detectable hTSHR-Ab. One of the MAbs, shown to r ecognize residues 21-35, and the rabbit polyclonal antibodies bound to all three forms of the hTSHR-ecd. Some of the htSHR autoantibodies bo und predominantly to the monomeric forms of the htSHR, but autoantibod ies were also identified that recognized tetrameric hTSHR-ecd. These d ata demonstrate that hTSHR-Abs recognize differing nonlinear and linea r epitopes in the hTSHR-ecd and provide a methodology that should be u seful for further defining the structural requirements for folding of this functionally and immunologically important domain of the hTSHR.