The TSH receptor (TSHR) A-subunit is more effective than the holoreceptor in inducing thyroid-stimulating antibodies (TSAb) that cause Graves' disease. A puzzling phenomenon is that 2 recombinant, eukaryotic forms of A-subunits (residues 22-289), termed active and inactive, are recognized mutually exclusively by pathogenic TSAb and mouse monoclonal antibody 3BD10, respectively. Understanding the structural difference between these TSHR A-subunit forms could provide insight into Graves' disease pathogenesis. The 3-dimensional structure of the active A-subunit (in complex with a human TSAb Fab, M22) is known, but the structural difference with inactive A-subunits is unknown. We solved the 3BD10 Fab 3-dimensional crystal structure. Guided by prior knowledge of a portion of its epitope, 3BD10 docked in silico with the known active TSHR-289 monomeric structure. Because both TSAb and 3BD10 recognize the active TSHR A-subunit monomer, this form of the molecule can be excluded as the basis for the active-inactive dichotomy, suggesting, instead a role for A-subunit quaternary structure. Indeed, in silico analysis revealed that M22, but not 3BD10, bound to a TSHR-289 trimer. In contrast, 3BD10, but not M22, bound to a TSHR-289 dimer. The validity of these models is supported experimentally by the temperature-dependent balance between active and inactive TSHR-289. In summary, we provide evidence for a structural basis to explain the conformational heterogeneity of TSHR A-subunits (TSHR-289). The pathophysiologic importance of these findings is that affinity maturation of pathogenic TSAb in Graves' disease is likely to involve a trimer of the shed TSHR A-subunit.

The TSH receptor (TSHR) hinge region, the least well understood component, bridges the leucine-rich repeat and transmembrane domains. We report data on clusters of hinge charged residues the mutation of which to Ala is compatible with cell surface expression and normal, or near normal, TSH binding affinity yet with a relative reduction in receptor activation. Mutation to Ala of E409 at the junction with the transmembrane domain was the most potent in uncoupling TSH binding and signal transduction (~22-fold less sensitive than the wild-type TSHR) and was unique among the residues studied in reducing both the amplitude and the sensitivity of the ligand-induced signal. Unexpectedly, a dual E409A/D410A mutation partially corrected the major suppressive effect of TSHR-E409A. The combined Ala substitution of a cluster of positively charged hinge residues (K287, K290, K291, R293; termed "K3R1") synergistically reduced sensitivity to TSH stimulation approximately 21-fold without altering the TSH binding affinity. Simultaneous Ala substitutions of a cluster of acidic hinge residues D392, E394, and D395 (termed "DE392-5A") partially uncoupled TSH binding from signal transduction (4.4-fold reduction in sensitivity), less than for E409A and K3R1A. Remarkably, the combination of the K3R1A and DE392-5A mutations was not additive but ameliorated the major uncoupling effect of K3R1A. This lack of additivity suggests that these two clusters contribute to a common signaling pathway. In summary, we identify several TSHR hinge residues involved in signal transmission. Our data support the concept that the hinge regions of the TSHR (and other glycoprotein hormone receptors) act as surrogate ligands for receptor activation.

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.

The TSH receptor (TSHR) extracellular domain (ECD) comprises a N-terminal leucine-rich repeat domain and an hinge region (HR), the latter contributing to ligand binding and critical for receptor activation. The crystal structure of the leucine-rich repeat domain component has been solved, but previous attempts to generate conformationally intact complete ECD or the isolated HR component for structural analysis have failed. The TSHR HR contains a C-peptide segment that is removed during spontaneous TSHR intramolecular cleavage into disulfide linked A- and B-subunits. We hypothesized that deletion of the redundant C-peptide would overcome the obstacle to generating conformationally intact TSHR ECD protein. Indeed, lacking the C-peptide region, the TSHR ECD (termed ECD-D1) and the isolated HR (termed HR-D1) were secreted into medium of insect cells infected with baculoviruses coding for these modified proteins. The identities of TSHR ECD-D1 and HR-D1 were confirmed by ELISA and immunoblotting using TSHR-specific monoclonal antibodies. The TSHR-ECD-D1 in conditioned medium was folded correctly, as demonstrated by its ability to inhibit radiolabeled TSH binding to the TSH holoreceptor. The TSHR ECD-D1 purification was accomplished in a single step using a TSHR monoclonal antibody affinity column, whereas the HR-D1 required a multistep protocol with a low yield. In conclusion, we report a novel approach to generate the TSHR ECD, as well as the isolated HR in insect cells, the former in sufficient amounts for structural studies. However, such studies will require previous complexing of the ECD with a ligand such as TSH or a thyroid-stimulating antibody.