THYROTROPIN (TSH) INTERACTS WITH MULTIPLE DISCRETE REGIONS OF THE TSHRECEPTOR - POLYCLONAL RABBIT ANTIBODIES TO ONE OR MORE OF THESE REGIONS CAN INHIBIT TSH BINDING AND FUNCTION

As a means of identifying functional regions of the TSH receptor (TSHr ), we immunized four rabbits with recombinant extracellular TSHr (ETSH r) protein and systematically evaluated their antibody response. The a ntibody response was characterized by testing serial serum samples for immunoglobulin G (IgG) against ETSHr protein and 26 synthetic peptide s 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 per sistent high titers against several of the others. After multiple inoc ulations of antigen, sera from 3 rabbits showed significant ability to block TSH binding. Based on the ability of peptides to reverse this b locking activity, we identified 3 regions of the TSHr (i.e. amino acid s 292-311, 367-386, and 397-415) through which antibodies can block TS H 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 ET SHr protein is sufficient to induce production of functionally relevan t antibodies. Furthermore, we have identified several sites on the TSH r through which antibodies can inhibit TSH binding, thus leading to id entification of several potential TSH-binding regions.