AN ESSENTIAL ROLE OF DOMAIN-D IN THE HORMONE-BINDING ACTIVITY OF HUMAN BETA-1 THYROID-HORMONE NUCLEAR RECEPTOR

By analogy with steroid receptors, human placental thyroid hormone nuclear receptor (hTR-beta-1) could be divided into four functional domains: A/B (Met1-Leu101), C (Cys102-Ala170), D (Thr171-Lys237), and E (Arg238-Asp456). The E domain was thought to bind thyroid hormone. To evaluate whether domain E alone is sufficient to bind T3 or requires the presence of other domains for functional T3-binding activity, a series of deletion mutants was constructed. The mutants were expressed in Escherichia coli, and the expressed proteins were purified. Analysis of the T3-binding affinity and analog specificity of the purified truncated hTR-beta-1 indicated that domain E alone did not have T3-binding activity. Extension of the amino-terminal sequence of domain E to include part of domain D yielded a mutant (Lys201-Asp456) with a K(a) for T3 of 0.5 +/- 0.2 x 10(9) M-1. Further extension to include the entire domain D (Met169-Asp456) yielded a mutant with T3-binding activity with a K(a) of 0.8 +/- 0.1 x 10(9) M-1. Further extension of the amino-terminal sequence to include domain C increased the affinity for T3 by nearly 2-fold (K(a) = 1.5 +/- 0.4 x 10(9) M-1). The K(a) for the wild-type hTR-beta-1 is 1.5 +/- 0.2 x 10(9) M-1. Furthermore, mutant (Met169-Asp456) binds to 3',5',3-triiodo-L-thyropropionic acid, D-T3, L-T4, and L-T3 with 307%, 37%, 7%, and 0.1%, respectively, of the activity of L-T3. This order of analog affinity is similar to that of the wild-type hTR-beta-1. These results indicate that domain D is essential for hormone-binding activity. In contrast, the A/B domain is not required for T3-binding activity. Domain C has modulation activity for domains D and E. Deletion of the last eight carboxyl amino acids completely abolishes the T3-binding activity of the mutant (Met169-Asp456). Thus, domain D is essential for domain E to function as a hormone-binding domain.