Insight into Thyroid-Stimulating Autoantibody Interaction with the Thyrotropin Receptor N-Terminus Based on Mutagenesis and Re-Evaluation of Ambiguity in This Region of the Receptor Crystal Structure

Abstract

Background: Thyroid-stimulating autoantibodies (TSAb) bind to the thyrotropin receptor (TSHR) extracellular domain, or ectodomain (ECD), comprising a leucine-rich repeat domain (LRD) linked by a hinge region to the transmembrane domain (TMD). The LRD (residues 22-260; signal peptide 1-21) contains two disulfide-bonded loops at its N-terminus. In the crystal structure of the isolated LRD complexed with human TSAb monoclonal antibody (mAb) M22, N-terminal disulfide loop 1 (residues 22-30) could not be determined because of crystal disorder. Nevertheless, present crystal structure data are interpreted to exclude a role for the LRD N-terminal disulfide loops in the TSAb epitope(s), contradicting prior functional evidence of a role for these loops in TSAb function. Materials and Methods: To re-examine this issue we studied two cell types expressing the TSHR with the extreme Nterminal loop 1 (residues 22-30) deleted: the TSHR ECD lacking the TMD and tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor, and the TSH holoreceptor containing the TMD. Because TSAb including M22 ''see'' the holoreceptor poorly relative to the TSHR ECD-GPI, we used the latter to examine the effect of deleting residues 22-30 on M22 binding by flow cytometry and the holoreceptor to test the effect of this deletion on the functional response to M22. Results: Deletion of TSHR N-terminal loop 1 (residues 22-30) reduced the number of TSHR-ECD-GPI recognized by M22 relative to two TSHR mAb with epitopes far downstream of the LRD N-terminal loops. Relative to control mAb 2C11, M22 recognized only 60.4% of cell surface receptors (p ¼ 0.02). In contrast to M22 binding to TSHR-ECD-GPI, in functional studies with the TSH holoreceptor, M22 stimulation of cAMP generation was unaltered by the loop 1 deletion. Conclusions: Our data support the concept that TSAb interact with the cysteine-rich N-terminus of the TSHR. Comparison of crystal structures of the same TSHR LRD in complex with TSAb M22 or blocking antibody K1-70 helps reconcile contradictory viewpoints. A difference between M22 interaction with the identical TSHR N-terminus expressed on the TSHR-ECD-GPI and holoreceptor suggests that crystallization of the TSHR LRD-M22 complex may not provide a complete understanding of the functional TSAb epitope(s) in Graves' disease.

Antibody Protection Reveals Extended Epitopes on the Human TSH Receptor

“…It has also been shown that a tyrosine residue located downstream of the C terminus cystein cluster in this region is mandatory for high affinity TSH binding and activation of the receptor [37] Studies from our laboratory have shown that neutral antibodies directed to the cleaved region (316–366) are capable of activating the receptor leading to non-cAMP dependent signaling [30] . In agreement with these studies, mutation analysis of the hinge region of the receptor demonstrated an extended hormone binding site that is also involved in receptor activation [29] , [38] . Mutation studies within the LRD itself have shown that such binding sites can vary, suggesting subtle differences in the amino acids that make contact with the different stimulators and blockers.…”

“…The blocking antibody epitopes, when compared to the stimulating epitopes, showed binding to a shorter amino terminus segment of the receptor and a larger region of the hinge region encompassing the known critical residue 255 [20] , [29] which has been shown to be needed for normal receptor signaling. On comparing the blocker to the stimulating antibodies we saw some common regions but also several unique regions to the TSHR blocker ( Figure 6B ).…”

Blocking type TSH receptor antibodies

“…For example, it has been proposed [ 45 , 46 ] that the TSHR N-terminal region (aa 22–41) is part of a highly conformational epitope for thyroid stimulating autoantibodies. However, a more recent study using a mutated TSHR, with the first 30 amino acids removed, confirmed the binding arrangements observed in the TSHR260-M22 crystal structure, i.e., that the stimulating antibody M22 does not interact with the extreme N terminus (aa 22–34) of the TSHR [ 47 ]. The N terminus of the TSHR most likely acts as a protective N-terminal cap aiding stability, preventing degradation and keeping the LRD in its correct conformation [ 48 , 49 ].…”

TSH receptor autoantibody immunoassay in patients with Graves' disease: Improvement of diagnostic accuracy over different generations of methods. Systematic review and meta-analysis

“…In recent years there has been significant progress in elucidating the TSH receptor structure and the functional activities of TRAb [3] and in developing advanced techniques for their measurement [4]. After the Adams' historical discovery of TRAb (at that time called first long-acting thyroid stimulator -LATS) as a cause of hyperthyroidism [5] and identification of LATS as an immunoglobulin [6], until the early 1970s the only available methods for detection of TRAb were in vivo cell-based bioassays [7].…”