Interaction of thyroid hormone nuclear receptor with antibody: characterization of the thyroid hormone binding site.

To understand the structural basis in the hormone-dependent transcriptional regulation of human beta 1 thyroid hormone receptor (h-TR beta 1), we characterized the region which interacted with the thyroid hormone, 3,3',5-triiodo-L-thyronine (T3). Using the hormone binding domain of h-TR beta 1 (K206-D461) as an immunogen, we screened for monoclonal antibodies which inhibited the binding of T3 to h-TR beta 1. mAb C3, which recognized native h-TR beta 1, was obtained. Analyses of the binding data indicate that binding of T3 to h-TR beta 1 was competitively inhibited by mAb C3. Using a series of truncated mutants of h-TR beta 1 and synthetic peptides, we mapped the binding site of mAb C3 to the region of E248-V256. Thus, part of T3 binding site in h-TR beta 1 is in this nine-amino acid segment, which was shown by circular dichroism spectroscopy to be a random coil. Based on the proposed model of the hormone binding domain as an alpha/beta barrel, E248-V256 contains part of Loop 1 which is on the same side of the DNA binding domain. These results raise the possibility that Loop 1 could be in direct contact with the nearby DNA binding domain to affect the interaction of DNA binding domain with the T3 target genes.