To understand the molecular mechanism(s) by which thyroid hormone 3,3',5- triiodo-L-thyronine (T3) promotes growth, differentiation and development, the structure and activity of the cellular thyroid hormone binding proteins have been studied. I. Molecular interaction of T3 with thyroid hormone nuclear receptor (TR). Using three monoclonal antibodies and immunocytochemistry, TR expressed and purified from E. coli was shown to be structurally identical to the endogenous TR. To determine the effect of domains A/B, C and D on the binding of T3 to domain E, a series of deletion mutants of h-TR(beta1) were expressed and purified from E. coli. Analysis of binding of these mutants to T3 and its analogs indicated that domain E alone does not bind T3. D domain is essential for T3 binding and C domain has modulation activity. In contrast, A/B domain is not required for T3 binding. Deletion analysis further showed that the last eight carboxyl amino acids are essential for T3 binding. The T3 binding site(s) in TR was covalently labeled with underivatized [3',5'-125I]-L-thyroxine (T4). The amino acids labeled with [125I]T4 were located in two tryptic peptides. The apparent molecular weight of the labeled peptides suggested that one of the two peptides could be the C-terminal peptide. These results are consistent with the deletion analysis in which the critical importance of the last eight C-terminal amino acids was demonstrated. II. Regulation of cytosolic thyroid hormone binding protein. The human p58-M2 isolated from A431 cells is a monomer of pyruvate kinase, subtype M2 (PKM2). The effects of T3 on enzymatic activity was evaluated using the active p58-M2 expressed and purified from E. coli. T3 inhibited the kinase activity of p58-M2 in a dose-dependent manner. However, it has no effect on its association to the tetrameric PKM2. Fructose- 1,6-bisphosphate activated the conversion of p58-M2 to PKM2 in vitro. This mode of regulation also occurs in vivo. Dissociation of tetrameric PKM2 to the monomeric p58-M2 is a novel mechanism by which glycolysis shuts off in responding to glucose deprivation. The recombinant monomer of isoenzyme PKMI was also found to bind T3 with affinity and specifity. Thus, cytosolic T3 binding proteins belong to a multigene family probably functioning in a tissue-specific and developmentally-regulated manner.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Intramural Research (Z01)
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Division of Cancer Biology and Diagnosis
United States
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