STRUCTURE OF THE CARBOXY-TERMINAL REGION OF THYROID-HORMONE NUCLEAR RECEPTORS AND ITS POSSIBLE ROLE IN HORMONE-DEPENDENT INTERMOLECULAR INTERACTIONS

The thyroid hormone nuclear receptors (TRs) are ligand-dependent trans cription factors. To understand the molecular basis of ligand-dependen t transactivation, we studied the structure of their carboxyterminal a ctivation domain. We analyzed the structures of the peptides derived f rom the C-terminal sequences of human TR subtypes beta 1 (h-TR beta 1) and alpha 1 (h-TR alpha 1) and a human TR mutant, PV, by circular dic hroism (CD). Mutant PV has a C-terminal frameshift mutation and does n ot bind to the thyroid hormone, 3,3',5-triiodo-L-thyronine (T-3) Analy ses of the secondary structures of the peptides by CD indicate that fi ve amino acids, EVFED, are part of an amphipathic alpha-helix which is required to maintain the structural integrity of the hormone binding domain. A monoclonal antibody, C4 (mAb C4), which recognizes both h-TR beta 1 and h-TR alpha 1 was developed. Using a series of truncated mu tants and synthetic peptides, we mapped the epitope of mAb C4 to the c onserved C-terminal amino acids, EVFED. Analysis of the binding data i ndicates that binding of T-3 to either h-TR beta 1 or h-TR alpha 1 was competitively inhibited by mAb C4. Deletion of C-terminal amino acids including EVFED led to a total loss of T-3 binding activity. Thus, pa rt of the T-3 binding site is located in this five amino acid segment. T-3 may transduce its hormonal signal to the transcriptional machiner y via interaction with EVFED at the C-terminus of TRs.