Epitope analysis of the human thyrotropin (TSH) receptor using monoclonal antibodies.

A panel of thyrotropin (TSH) receptor (TSHR) monoclonal antibodies (mAbs), produced using highly purified Chinese hamster ovary (CHO) cell-produced TSHR, has been used to study TSHR structure. All 41 mAbs recognized full-length TSHR containing complex carbohydrate (120 kDa), and 40 mAbs recognized full-length precursor-containing high mannose sugars (100 kDa). The mAbs also recognized TSHR cleavage products with three types of reactivity: type 1 mAbs reacting with bands at 70 kDa and 58 kDa, type 2 with bands at 70 kDa and 52 kDa, and type 3 with bands at 52 kDa and 40 kDa. Deglycosylation studies showed that the 70-kDa and 58-kDa bands contained complex carbohydrate, whereas the 52-kDa and 40-kDa bands were unglycosylated. These results are consistent with TSHR cleavage occurring at two sites. Cleavage at both sites gives rise to glycosylated A subunit (58 kDa) corresponding to the extracellular domain of the receptor and nonglycosylated B subunit (40 kDa) corresponding to the C-terminal transmembrane domain. Cleavage only at site 1 gives rise to the 58-kDa A subunit and a large B subunit (52 kDa). Cleavage only at site 2 gives rise to a large A subunit (70 kDa) and the B subunit (40 kDa). Four of the mAbs inhibited 125I-labeled TSH binding to solubilized full-length TSHR. TSH binding was inhibited by (a) two type 3 mAbs reactive with the N-terminal region of the B subunit (epitopes between amino acids 381 and 385 and between 380 and 418, respectively) and (b) two type 2 mAbs reactive with epitopes on the A subunit (between amino acids 246 and 260). These results together with previous studies on the direct binding of TSH to the TSHR A subunit suggest that at least two distinct regions of the TSHR sequence, including one region on the A subunit and one region on the B subunit, fold together to form part of a complex TSH binding site.