title: 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.
authors: Thyroid Autoimmune Disease Unit, Cedars-Sinai Research Institute and University of California, Los Angeles School of Medicine, Los Angeles, California 90048.
authors: Chun-Rong
authors: CR
authors: Chen
authors: Thyroid Autoimmune Disease Unit, Cedars-Sinai Research Institute and University of California, Los Angeles School of Medicine, Los Angeles, California 90048.
authors: Larry M
authors: LM
authors: Salazar
authors: Thyroid Autoimmune Disease Unit, Cedars-Sinai Research Institute and University of California, Los Angeles School of Medicine, Los Angeles, California 90048.
authors: Sandra M
authors: SM
authors: McLachlan
authors: Thyroid Autoimmune Disease Unit, Cedars-Sinai Research Institute and University of California, Los Angeles School of Medicine, Los Angeles, California 90048.
authors: Basil
authors: B
authors: Rapoport
mesh_terms: D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D018076:DNA, Complementary; D006801:Humans; D007313:Insecta; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D010446:Peptide Fragments; D011989:Receptors, Thyrotropin