As a means of identifying functional regions of the TSH receptor (TSHr), we immunized four rabbits with recombinant extracellular TSHr (ETSHr) protein and systematically evaluated their antibody response. The antibody response was characterized by testing serial serum samples for immunoglobulin G (IgG) against ETSHr protein and 26 synthetic peptides which span the entire ETSHr. Sera were also tested for their ability to block TSH binding to native TSHr. All four rabbits developed high serum IgG titers (<1:100, 000) to ETSHr. None of the rabbits developed significant IgG titers against 11 of the peptides, but each showed persistent high titers against several of the others. After multiple inoculations of antigen, sera from 3 rabbits showed significant ability to block TSH binding. Based on the ability of peptides to reverse this blocking activity, we identified 3 regions of the TSHr (i.e. amino acids 292-311, 367-386, and 397-415) through which antibodies can block TSH binding. Moreover, antibodies purified on either peptide 292-311 or peptide 367-386 affinity columns could block both TSH binding and TSH-mediated activation of thyroid cells in culture. These studies show ETSHr protein is sufficient to induce production of functionally relevant antibodies. Furthermore, we have identified several sites on the TSHr through which antibodies can inhibit TSH binding, thus leading to identification of several potential TSH-binding regions.
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