Novel Information on the Epitope of an Inverse Agonist Monoclonal Antibody Provides Insight into the Structure of the TSH Receptor

The TSH receptor (TSHR) comprises an extracellular leucine-rich domain (LRD) linked by a hinge region to the transmembrane domain (TMD). Insight into the orientation of these components to each other is required for understanding how ligands activate the receptor. We previously identified residue E251 at the LRD-hinge junction as contributing to coupling TSH binding with receptor activation. However, a single residue cannot stabilize the LRD-hinge unit. Therefore, based on the LRD crystal structure we selected for study four other potential LRD-hinge interface charged residues. Alanine substitutions of individual residues K244, E247, K250 and R255 (as well as previously known E251A) did not affect TSH binding or function. However, the cumulative mutation of these residues in varying permutations, primarily K250A and R255A when associated with E251A, partially uncoupled TSH binding and function. These data suggest that these three residues, spatially very close to each other at the LRD base, interact with the hinge region. Unexpectedly and most important, monoclonal antibody CS-17, a TSHR inverse agonist whose epitope straddles the LRD-hinge, was found to interact with residues K244 and E247 at the base of the convex LRD surface. These observations, together with the functional data, exclude residues K244 and E247 from the TSHR LRD-hinge interface. Further, for CS-17 accessibility to K244 and E247, the concave surface of the TSHR LRD must be tilted forwards towards the hinge region and plasma membrane. Overall, these data provide insight into the mechanism by which ligands either activate the TSHR or suppress its constitutive activity

Exceptional Hyperthyroidism and a Role for both Major Histocompatibility Class I and Class II Genes in a Murine Model of Graves' Disease

Autoimmune hyperthyroidism, Graves' disease, can be induced by immunizing susceptible strains of mice with adenovirus encoding the human thyrotropin receptor (TSHR) or its A-subunit. Studies in two small families of recombinant inbred strains showed that susceptibility to developing TSHR antibodies (measured by TSH binding inhibition, TBI) was linked to the MHC region whereas genes on different chromosomes contributed to hyperthyroidism. We have now investigated TSHR antibody production and hyperthyroidism induced by TSHR A-subunit adenovirus immunization of a larger family of strains (26 of the AXB and BXA strains). Analysis of the combined AXB and BXA families provided unexpected insight into several aspects of Graves' disease. First, extreme thyroid hyperplasia and hyperthyroidism in one remarkable strain, BXA13, reflected an inability to generate non-functional TSHR antibodies measured by ELISA. Although neutral TSHR antibodies have been detected in Graves' sera, pathogenic, functional TSHR antibodies in Graves' patients are undetectable by ELISA. Therefore, this strain immunized with A-subunit-adenovirus that generates only functional TSHR antibodies may provide an improved model for studies of induced Graves' disease. Second, our combined analysis of linkage data from this and previous work strengthens the evidence that gene variants in the immunoglobulin heavy chain V region contribute to generating thyroid stimulating antibodies. Third, a broad region that encompasses the MHC region on mouse chomosome 17 is linked to the development of TSHR antibodies (measured by TBI). Most importantly, unlike other strains, TBI linkage in the AXB and BXA families to MHC class I and class II genes provides an explanation for the unresolved class I/class II difference in humans

Constitutive Activation of the Thyroid-Stimulating Hormone Receptor (TSHR) by Mutating Ile691 in the Cytoplasmic Tail Segment

/inositol phosphate (IP) pathways, which stimulate thyroid hormone production and thyroid proliferation./IP signaling pathway./IP cascade

Distinct role of T helper Type 17 immune response for Graves\u27 hyperthyroidism in mice with different genetic backgrounds.

T helper type 17 (Th17) cells, a newly identified effector T-cell subset, have recently been shown to play a role in numerous autoimmune diseases, including iodine-induced autoimmune thyroiditis in non-obese diabetic (NOD)-H2(h4) mice, which had previously been thought Th1-dominant. We here studied the role of Th17 in Graves\u27 hyperthyroidism, another thyroid-specific autoimmune disease, in a mouse model. Two genetically distinct BALB/c and NOD-H2(h4) strains with intact or disrupted IL-17 genes (IL-17(+/+) or IL-17(-/-)) were immunized with adenovirus (Ad) expressing the thyrotropin receptor (TSHR) A-subunit (Ad-TSHR289). Both IL-17(+/+) and IL-17(-/-) mice developed anti-TSHR antibodies and hyperthyroidism at equally high frequencies on the BALB/c genetic background. In contrast, some IL-17(+/+), but none of IL-17(-/-), mice became hyperthyroid on the NOD-H2(h4) genetic background, indicating the crucial role of IL-17 for development of Graves\u27 hyperthyroidism in non-susceptible NOD-H2(h4), but not in susceptible BALB/c mice. In the T-cell recall assay, splenocytes and lymphocytes from the draining lymph nodes from either mouse strains, irrespective of IL-17 gene status, produced IFN-γ and IL-10 but not other cytokines including IL-17 in response to TSHR antigen. Thus, the functional significance of Th17 may not necessarily be predictable from cytokine expression patterns in splenocytes or inflammatory lesions. In conclusion, this is, to our knowledge, the first report showing that the role of Th17 cells for the pathogenesis of a certain autoimmune disease depends on the mouse genetic backgrounds

Evidence that the thyrotropin receptor protease is membrane-associated and is not within lipid rafts

The thyrotropin receptor (TSHR) cleaves to a variable extent within the ectodomain into a ligand-binding A subunit linked by disulfide bonds to the largely transmembrane B subunit. To obtain insight into this variability, we examined the extent of cleavage of TSHR ectodomains tethered to the plasma membrane by different means: (1) the wild-type, serpentine region, (2) a glycosylphosphatidylinositol (GPI) anchor, and (3) a single CD8alpha transmembrane region. For this purpose, we covalently cross-linked(125)I-TSH to the TSHR ectodomain expressed on the surface of intact cell monolayers. The extent of cleavage of the CD8alpha-tethered ectodomain was similar to the wild-type TSHR (approximately 50%) whereas the same ectodomain with a GPI anchor remained almost entirely (approximately 90%) uncleaved. These findings have three possible implications. First, differential cleavage of the TSHR ectodomain depending on its attachment to the plasma membrane suggests that the TSHR protease is membrane-associated and is not a soluble (secreted or shed) protease. Second, because GPI-anchored proteins (unlike CD8alpha) segregate in membrane lipid rafts, the TSHR protease appears not to be associated with lipid rafts. Finally, the similar extent of cleavage of the wild-type TSHR and the CD8alpha (not the GPI) tethered ectodomain supports the concept that the wild-type TSHR resides largely outside lipid rafts

Thyroid stimulation does not require antibodies with identical epitopes but does involve recognition of a critical conformation at the N terminus of the thyrotropin receptor A-subunit.

Whether monoclonal antibodies with thyroid-stimulating activity [thyroid-stimulating antibody/antibodies (TSAb)] from immunized animals are identical to human autoantibodies in Graves' disease is unknown. Here, we compared properties of a monoclonal hamster TSAb (MS-1) with human autoantibodies. The epitopes of neither MS-1 nor human autoantibodies can be determined by peptide scanning, indicating their conformational nature. A property of human TSAb is that their epitope is partially obscured on the TSH holoreceptor on the cell surface relative to the TSH receptor (TSHR) ectodomain tethered to the membrane by a glycosylphosphatidyl inositol anchor. On flow cytometry, as for human autoantibodies, MS-1 preferentially recognized the glycosylphosphatidyl inositol-anchored ectodomain vs. the TSH holoreceptor on Chinese hamster ovary cells. Also, as with human autoantibodies, only A-subunits with the active (but not the inactive) conformation adsorbed MS-1 binding activity. This difference localizes antibody binding to a cysteine-rich region at the TSHR N terminus. Remarkably, active TSHR A-subunit more effectively ( approximately 40-fold) neutralized human autoantibodies than it did MS-1. Therefore, MS-1 interacts less well than autoantibodies with the free A-subunit. In summary, we provide evidence that TSAb need not have identical epitopes. However, the TSAb epitope does appear to require involvement of the highly conformational N terminus of the A-subunit

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

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