title: Probing structural variability at the N terminus of the TSH receptor with a murine monoclonal antibody that distinguishes between two receptor conformational forms.
abstract: Despite elucidation of the crystal structure of M22, a human thyroid-stimulating autoantibody (TSAb) bound to the TSH receptor (TSHR) leucine-rich repeat domain (LRD), the mechanism by which TSAs activate the TSHR and cause Graves' disease remains unknown. A nonstimulatory murine monoclonal antibody, 3BD10, and TSAb interact with the LRD N-terminal cysteine cluster and reciprocally distinguish between two different LRD conformational forms. To study this remarkable phenomenon, we investigated properties of 3BD10, which has a linear epitopic component. By synthetic peptide ELISA, we identified 3BD10 binding to TSHR amino acids E34, E35, and D36 within TSHR cysteine-bonded loop 2 (C31-C41), which includes R38, the most N-terminal contact residue of TSAb M22. On flow cytometry, despite not contributing to the 3BD10 and M22 epitopes, chimeric substitution (but not deletion) of TSHR cysteine-bonded loop 1 (C24-C29) eliminated 3BD10 binding to the TSHR ectodomain (ECD) expressed on the cell surface, as found previously for TSAb including M22. Furthermore, 3BD10 did not recognize all cell surface TSHR ECDs, consistent with recognition of only one conformational receptor form. Reversion to wild-type of small components of the loop 1 chimeric substitution partially restored 3BD10 binding to the TSHR-ECD but not to synthetic peptides tested by ELISA. Molecular modeling supports the concept that modification of TSHR C-bonded loop 1 influences loop 2 conformation as well as LRD residues further downstream. In conclusion, the present study with mouse monoclonal antibody 3BD10 confirms TSHR conformational heterogeneity and suggests that the N-terminal cysteine cluster may contribute to this structural variability.
authors.affiliation: Thyroid Autoimmune Disease Unit, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, CA 90048, USA.
authors.forename: Sepehr
authors.initials: S
authors.lastname: Hamidi
authors.forename: Chun-Rong
authors.initials: CR
authors.lastname: Chen
authors.forename: Ramachandran
authors.initials: R
authors.lastname: Murali
authors.forename: Sandra M
authors.initials: SM
authors.lastname: McLachlan
authors.forename: Basil
authors.initials: B
authors.lastname: Rapoport
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D018828:Immunoglobulins, Thyroid-Stimulating / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin