Learning from how antibodies interact with the TSH receptor
Terry F. Davies, Syed. Morshed & Rauf. Latif
Successful crystallization of TSHR residues 21260 has been reported (Sanders et al. Thyroid 2007 17 395) using a partial ectodomain (ECD) bound to the Fab fragment of a human stimulating TSHR monoclonal antibody (M22) and its conformational epitopes delineated. We have now studied antibody binding to the entire ECD (residues 1412) using epitope protection. In this approach, we first protected a highly purified ectodomain fragment with a variety of TSHR antibodies prior to enzyme digestion and the protected peptides were then delineated by mass spectrometry. There were 7 protected linear peptides spanning the TSHR-ECD. Five of these regions corresponded to peptides in leucine rich domains (LRD) but in addition, monoclonal thyroid stimulating antibodies contacted a region on the amino terminal of the TSHR and a protected epitope on the hinge region (residues 263293) which encompasses a highly conserved motif (SHCCAF) previously implicated in activation of the receptor. These data demonstrate that stimulating TSHR-Abs have epitopes not confined to the LRD but also incorporate epitopes not revealed in the available crystal structure and indicate the potential for such antibodies to activate the receptor by direct contact with the non-LRD regions of the TSHR. Only TSHR-Abs which bound to the LRD were able to activate Gαs and Gαq pathways and induce thyroid cell proliferation and activation. In contrast, neutral TSHR-Abs which bound only to the hinge region of the ectodomain activated only Gαq and in the absence of PKA activation induced thyroid cell apoptosis. These results indicate that the binding sites of TSHR-Abs determine the pattern of signal activation and the resulting transduction pathways involved in thyroid cell responses.