Deleting the Redundant TSH Receptor C-Peptide Region Permits Generation of the Conformationally Intact Extracellular Domain by Insect Cells

Abstract

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.

Autoantibody and hormone activation of the thyrotropin G protein-coupled receptor

“…To simplify purification of intact receptors for structural studies, we generated a TSHR construct devoid of the C-peptide, which could be purified as a single intact receptor construct. In agreement with prior studies demonstrating that this region is not required for proper receptor folding and signaling 14 , removal of the Cpeptide did not affect TSH or TSI activation of Gs signaling (Supplementary Fig. 1).…”

“…TSHR is naturally proteolytically cleaved within the extracellular domain leading to removal of residues 317 to 366, a region termed the "C-peptide" 13 . While the physiological relevance of Cpeptide excision remains unclear, previous biochemical studies have demonstrated that cleavage in this domain leads to receptor instability 14 . To simplify purification of intact receptors for structural studies, we generated a TSHR construct devoid of the C-peptide, which could be purified as a single intact receptor construct.…”

Basis for antibody- and hormone-mediated activation of TSHR in Graves’ disease

“…TSHR used in our studies contains a deletion of residues 317-366 in the hinge region, which is naturally removed from matured TSHR 15 . It has been shown that the region of residues 317-366 is not required for TSHR folding and signaling, and its deletion increases TSHR stability 15,16 . In addition, a TSHR constitutively active mutation, S281I, was also introduced to enhance the assembly of the TSH-TSHR-Gs complex 6,17 .…”