Abstract
The TSH receptor (TSHR) hinge region was previously considered an inert scaffold connecting the leucine-rich ectodomain to the transmembrane region of the receptor. However, mutation studies have established the hinge region to be an extended hormone-binding site in addition to containing a region which is cleaved thus dividing the receptor into (A) and β (B) subunits. Furthermore, we have shown in-vitro that monoclonal antibodies directed to the cleaved part of the hinge region (often termed “neutral” antibodies) can induce thyroid cell apoptosis in the absence of cyclic AMP signaling. The demonstration of neutral antibodies in patients with Graves' disease suggests their potential involvement in disease pathology thus making the hinge a potentially important antigenic target. Here we examine the evolution of the antibody immune response to the entire TSHR hinge region (aa280–410) after intense immunization with full-length TSHR cDNA in a mouse (BALB/c) model in order to examine the immunogenicity of this critical receptor structure. We found that TSHR hinge region antibodies were detected in 95% of the immunized mice. The antibody responses were largely restricted to residues 352–410 covering three major epitopes and not merely confined to the cleaved portion. These data indicated the presence of novel antigenic “hotspots” within the carboxyl terminus of the hinge region and demonstrate that the hinge region of the TSHR contains an immunogenic pocket that is involved in the highly heterogeneous immune response to the TSHR. The presence of such TSHR antibodies suggests that they may play an active role in the immune repertoire marshaled against the TSHR and may influence the Graves' disease phenotype.
Highlights
The thyroid stimulating hormone receptor (TSHR) is the major regulator of thyroid gland function and a major autoantigen in Graves’ disease (GD) which is caused by stimulating TSH receptor (TSHR) autoantibodies inducing thyroid gland hyperactivity [1, 2]
A full-length TSHR plasmid cDNA muscle immunization model was used to characterize the antigenicity of the hinge region of the TSHR ECD to reveal novel antigenic “hot spots” within the hinge region of the human TSHR
The full-length TSHR was used as the immunogen since that is what is produced endogenously by the thyroid cell prior to posttranslational processing only a limited percentage of receptors may retain this complete structure at any one time because of subsequent receptor dimerization, cleavage, and ECD shedding [23, 28, 29]
Summary
The thyroid stimulating hormone receptor (TSHR) is the major regulator of thyroid gland function and a major autoantigen in Graves’ disease (GD) which is caused by stimulating TSHR autoantibodies inducing thyroid gland hyperactivity [1, 2]. Autoantibodies to the TSHR found in patients with Graves’ disease may be stimulating, blocking, or neutral based on their modulation of cyclic AMP signaling [3]. All stimulating and most blocking receptor antibodies bind conformationally to the leucine-rich repeat domain (LRD), part of the large ectodomain (ECD) of the TSHR while neutral antibodies recognize linear epitopes largely within the hinge region of the receptor which links the LRD to the transmembrane (TM) sequences. The crystal structure of the hinge region of the FSHR [11] has provided evidence of the structural features of this region which likely applies to all glycoprotein hormone receptors including the TSHR and has allowed homology modeling of the entire ectodomain [12] and extended our understanding on ligand displacement of specific hinge fragments
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