Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor

Abstract

Graves disease is directly caused by thyroid-stimulating autoantibodies (TSAb’s) that activate the thyrotropin receptor (TSHR). We observed upon flow cytometry using intact cells that a mouse mAb (3BD10) recognized the TSHR ectodomain with a glycosidylphosphatidylinositol (ECD-GPI) anchor approximately tenfold better than the same ectodomain on the wild-type TSHR, despite the far higher level of expression of the latter. The 3BD10 epitope contains the N-terminal cysteine cluster critical for TSAb action. Consequently, we hypothesized and confirmed that TSAb (but not thyrotropin-blocking autoantibodies [TBAb’s]) also poorly recognize the wild-type TSHR relative to the ECD-GPI. Despite poor recognition by TSAb of the holoreceptor, soluble TSHR A subunits (known to be shed from surface TSHR) fully neutralized autoantibody-binding activity. These data indicate that the epitope(s) for TSAb’s, but not for TBAb’s, are partially sterically hindered on the holoreceptor by the plasma membrane, the serpentine region of the TSHR, or by TSHR dimerization. However, the TSAb epitope on the soluble A subunit is freely accessible. This observation, as well as other evidence, supports the concept that A subunit shedding either initiates or amplifies the autoimmune response to the TSHR, thereby causing Graves disease in genetically susceptible individuals

Adenovirus encoding the thyrotropin receptor A-subunit improves the efficacy of dendritic cell-induced Graves' hyperthyroidism in mice

“…This approach developed from the observations suggesting that the TSHR A-subunit, rather than the full-length receptor, appears to be the primary autoantigen initiating and/or enhancing induction of TSAb [6]. We now demonstrate that a higher disease incidence is achieved by injecting DCs infected with adenovirus expressing the TSHR A-subunit (AdTSHR289) than adenovirus expressing the full-length TSHR [3].…”

“…Recently, it was observed that TSAb in human sera bind preferentially to the TSHR ectodomain tethered to the cell membrane by a glycosylphosphatidylinositol-anchor versus the same ectodomain in the wild-type TSHR [6]. These findings raised the possibility that the shed A-subunit, rather than the full-length receptor, was the autoantigen responsible for the induction of TSAb.…”

Receptor Oligomerization as a Process Modulating Cellular Semiotics

“…For instance, binding of auto-antibodies against the ectodomain of the TSH receptor, as it occurs in the Grave's disease, may lead to enhancement of the receptor constitutive activity, even in the absence of TSH (Chazenbalk et al 2002). Similarly, the occasional occurrence of certain mutational changes in b 2 -adrenergic receptors may render the receptor tertiary structure more unstable and therefore more readily adjustable to constitutively active states (Wilbanks et al 2002).…”

Residues 34–39 in the Thyrotropin Receptor are not the Target of Autoantibodies from Sera of Patients with Graves' Disease

“…Results of many studies pointed to conformational nature of the epitopes (8 -10). The study of Rapoport's group, involving mutagenesis of four cysteines in positions 24, 29, 31, and 41, has presented evidence that disulfide bonds between C 41 and either C 29 or C 31, but not C 24, are important for the TSHR autoantibodies' stimulatory function (8,9). The cysteine-rich amino terminus of the TSHR was also the immunodominant linear antibody epitope in a murine model of Graves' hyperthyroidism (11).…”