Document Detail


Probing structural variability at the N terminus of the TSH receptor with a murine monoclonal antibody that distinguishes between two receptor conformational forms.
MedLine Citation:
PMID:  23183178     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Sepehr Hamidi; Chun-Rong Chen; Ramachandran Murali; Sandra M McLachlan; Basil Rapoport
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2012-11-26
Journal Detail:
Title:  Endocrinology     Volume:  154     ISSN:  1945-7170     ISO Abbreviation:  Endocrinology     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2012-12-25     Completed Date:  2013-02-22     Revised Date:  2014-01-09    
Medline Journal Info:
Nlm Unique ID:  0375040     Medline TA:  Endocrinology     Country:  United States    
Other Details:
Languages:  eng     Pagination:  562-71     Citation Subset:  AIM; IM    
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Antibodies, Monoclonal / metabolism*
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Immunoglobulins, Thyroid-Stimulating / metabolism*
Mice
Molecular Sequence Data
Protein Binding
Protein Conformation
Protein Structure, Secondary
Receptors, Thyrotropin / chemistry*,  metabolism*
Grant Support
ID/Acronym/Agency:
DK 19289/DK/NIDDK NIH HHS; R01 DK019289/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/Antibodies, Monoclonal; 0/Immunoglobulins, Thyroid-Stimulating; 0/Receptors, Thyrotropin
Comments/Corrections

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