Characterization of the Thyrotropin Binding Pocket

Abstract

A panel of monoclonal antibodies (mAbs) to the thyrotropin receptor (TSHR) was prepared using three different immunization strategies. The mAbs obtained (n = 138) reacted with linear epitopes covering most of the TSHR extracellular domain and with conformational epitopes. mAbs that bound to five different regions of the TSHR (amino acids [aa] 32-41, aa 36-42, aa 246-260, aa 277-296, and aa 381-385) were able to inhibit (125)I-labeled thyrotropin (TSH) binding to solubilized TSHR preparations. Fab and immunoglobulin G (IgG) preparations were similarly effective inhibitors for mAbs reactive with aa 246-260, aa 277-291 and aa 381-385 suggesting that these three regions of the TSHR are involved in TSH binding. In contrast mAbs reactive with aa 32-41 and aa 36-42 were not effective at inhibiting TSH binding when Fab preparations were used, suggesting that these N terminal regions of the TSHR were less critical for TSH binding. Our studies suggest that three distinct and discontinuous regions of the TSHR (aa 246-260 and 277-296 on the TSHR A subunit) and aa 381-385 (on the TSHR B subunit) fold together to form a complex TSH binding pocket. Alignment of the aa sequences of these three regions in TSHRs from different species indicates that they are highly conserved.

Animal Models of Graves' Hyperthyroidism

“…In contrast, an attempt to induce Graves' disease using conventional immunization method with conformationally intact full-length TSH receptor protein and complete Freund adjuvant (CFA) failed [33]. These discrepant data can possibly be best explained by the recent novel finding that TSAbs in sera from Graves' patients preferentially recognized the free A subunit rather than the two-subunit receptor or the full-length single polypeptide receptor, indicating that the free A subunit may be the better autoantigen than the other two forms of the receptor [34].…”

“…In the successful mouse models mentioned above [7][8][9][10][11][12], the free A subunit very likely sheds from the full-length TSH receptor expressed in vivo and can be recognized as autoantigen to TSAb. In contrast, it is unlikely that the full-length receptor used for the conventional immunization [33] undergoes cleavage. This hypothesis was reinforced by the fact that our two models were optimized with AdTSHR289 as mentioned above.…”

Differences in TSH Receptor Binding and Thyroid-stimulating Properties between TSH and Graves' IgG. Slowly-acting TSH Receptor Antibody Moieties in Graves' Sera Affect Assay Data

“…The antigen was further located to the TSH receptor (TSHR), and in vitro assay systems of TSHR antibody (TRAb) were developed for both receptor-binding and thyroid-stimulating activities [4][5][6]. After the cloning of TSHR cDNA [7,8], numerous efforts have been made to elucidate the binding sites of TSH as well as TRAb [9][10][11][12][13][14][15]. However, little has been known until now about the binding mechanisms of TSH and TRAb to TSHR [16].…”

TSH signalling and cancer

“…The ectodomain consists mainly of 9 leucine rich repeats (LRR) with interconnecting loops important for receptor structure and activation (35). Precise delineation of the TSH binding pocket of the receptor has been made through deletion-mutation analysis and a panel of antibodies (36,37). The β subunit contains the 7 TM domains joined by extracellular loops (ECL) and intracellular loops (ICL), and interacts selectively with G proteins when the TSHR is activated (34,38).…”