A Full Biological Response to Autoantibodies in Graves' Disease Requires a Disulfide-bonded Loop in the Thyrotropin Receptor N Terminus Homologous to a Laminin Epidermal Growth Factor-like Domain

Chun-Rong Chen,Kunihiko Tanaka,Gregorio D Chazenbalk,Sandra M Mclachlan,Basil Rapoport

doi:10.1074/jbc.m008001200

Chun-Rong Chen, Kunihiko Tanaka + Show 3 more

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https://doi.org/10.1074/jbc.m008001200

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Abstract

We observed amino acid homology between the cysteine-rich N terminus of the thyrotropin receptor (TSHR) ectodomain and epidermal growth factor-like repeats in the laminin gamma1 chain. Thyroid-stimulating autoantibodies (TSAb), the cause of Graves' disease, interact with this region of the TSHR in a manner critically dependent on antigen conformation. We studied the role of the cluster of four cysteine (Cys) residues in this region of the TSHR on the functional response to TSAb in Graves' patients' sera. As a benchmark we also studied TSH binding and action. Removal in various permutations of the four cysteines at TSHR positions 24, 29, 31, and 41 (signal peptide residues are 1-21) revealed Cys(41) to be the key residue for receptor expression. Forced pairing of Cys(41) with any one of the three upstream Cys residues was necessary for trafficking to the cell surface of a TSHR with high affinity TSH binding similar to the wild-type receptor. However, for a full biological response to TSAb, forced pairing of Cys(41) with Cys(29) or with Cys(31), but not with Cys(24), retained functional activity comparable with the wild-type TSHR. These data suggest that an N-terminal disulfide-bonded loop between Cys(41) and Cys(29) or its close neighbor Cys(31) comprises, in part, the highly conformational epitope for TSAb at the critical N terminus of the TSHR. Amino acid homology, as well as cysteine pairing similar to the laminin gamma1 chain epidermal growth factor-like repeat 11, suggests conformational similarity between the two molecules and raises the possibility of molecular mimicry in the pathogenesis of Graves' disease.

Highlights

The thyrotropin receptor (TSHR)1 is the target of the immune system in Graves’ disease, and autoantibodies directed against this antigen are the direct cause of clinical hyperthyroidism
Homology was found between TSHR amino acid residues 23– 43 and amino acid residues 979 –997 of the human laminin ␥1 chain [23]
From the known three-dimensional structure of EGF-like repeats in other regions of the laminin molecule [24, 25], we hypothesized that the critical conformational structure of the TSHR N-terminal region interaction with autoantibodies was unlikely to involve Cys24 but was more likely to involve disulfide bonding between Cys41 and Cys29 or, alternatively, between Cys41 and Cys31 (Fig. 1B)

Summary

Introduction

The thyrotropin receptor (TSHR) is the target of the immune system in Graves’ disease, and autoantibodies directed against this antigen are the direct cause of clinical hyperthyroidism (reviewed in Ref. 1). The poorly conserved TSHR N-terminal region extends from amino acid residues 22 to 56 (the signal peptide being residues 1–21) This short (35-residue) region contains four of the eleven Cys residues (Cys, Cys, Cys, and Cys41) in the large (397-residue) TSHR ectodomain, supporting the importance of secondary and tertiary structure in TSHR recognition by autoantibodies. Another indicator of the importance of conformation in the TSHR N terminus region is that TSHR autoantibodies and a mouse monoclonal antibody to this segment (3BD10; epitope within amino acid residues 25 to 51) interact reciprocally and exclusively with two different folded forms of the native molecule present in the same TSHR preparation [10]. An apparent paradox is that deletion or mutation of any of the potential partners of Cys (Cys, Cys, or Cys31) is reported to have no effect on ligand or autoantibody binding [8]

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