Affinity labeling of rat liver thyroid hormone nuclear receptor

Area of Science:

• Molecular Endocrinology
• Nuclear Receptor Research
• Biochemistry

Background:

• Thyroid hormones, thyroxine (T4) and triiodothyronine (T3), regulate crucial physiological processes.
• Understanding the structure and binding characteristics of the thyroid hormone receptor (TR) is vital for comprehending hormone action.

Purpose of the Study:

• To covalently label the rat liver nuclear thyroid hormone receptor with N-bromoacetyl derivatives of T3 and T4.
• To investigate the binding site interactions and structural properties of the TR using these labeled derivatives.

Main Methods:

• Covalent labeling of the rat liver nuclear TR with N-bromoacetyl-T3 and N-bromoacetyl-T4.
• Displacement binding assays using radiolabeled T3 and T4.
• Heat inactivation studies.
• High-pressure liquid chromatography (HPLC) for purification and behavior analysis.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Na-DodSO4-PAGE) for molecular weight determination.

Main Results:

• Displacement studies confirmed that T3, T4, and their bromoacetyl derivatives bind to the same site on the TR.
• Heat inactivation affected the binding of both natural ligands and labeled derivatives similarly.
• HPLC demonstrated consistent behavior of the TR regardless of ligand binding or labeling.
• Na-DodSO4-PAGE identified a single major radioactive component of 56,000 molecular weight in labeled receptors.
• Electrophoretic mobility studies suggested the TR is a single polypeptide chain.

Conclusions:

• The thyroid hormone receptor binds both T3 and T4 at the same site.
• The receptor appears to be a single polypeptide chain.
• Covalently labeled TRs can serve as markers for receptor purification.