Characterization of the thyrotropin binding pocket.

Area of Science:

• Endocrinology and Immunology
• Molecular and Cellular Biology

Background:

• The thyrotropin receptor (TSHR) is a critical G protein-coupled receptor involved in thyroid hormone regulation.
• Understanding TSHR's interaction with thyrotropin (TSH) is essential for diagnosing and treating thyroid disorders.

Purpose of the Study:

• To generate and characterize a panel of monoclonal antibodies (mAbs) against the TSHR.
• To identify specific TSHR epitopes involved in TSH binding.
• To elucidate the structural basis of the TSH-TSHR interaction.

Main Methods:

• Immunization strategies were employed to produce 138 mAbs against the TSHR.
• Epitope mapping was performed to identify linear and conformational epitopes.
• Inhibition assays using labeled TSH and various mAb formats (Fab, IgG) were conducted to assess TSH binding disruption.

Main Results:

• mAbs recognized epitopes across the TSHR extracellular domain, including linear and conformational sites.
• Five TSHR regions (amino acids [aa] 32-41, 36-42, 246-260, 277-296, and 381-385) were identified as binding sites for inhibitory mAbs.
• mAbs targeting aa 246-260, 277-296, and 381-385 effectively inhibited TSH binding in both Fab and IgG formats, indicating their direct involvement in the binding pocket.
• mAbs targeting N-terminal regions (aa 32-41, 36-42) showed reduced inhibition with Fab fragments, suggesting a lesser role in direct TSH binding.

Conclusions:

• Three discontinuous TSHR regions (aa 246-260, 277-296, and 381-385) assemble to form a complex TSH binding pocket.
• These critical TSH binding regions are highly conserved across different species, highlighting their functional importance.
• The generated mAbs provide valuable tools for further research into TSHR structure-function relationships and thyroid disease mechanisms.