Cytosolic 3,5,3'-Triiodo-L-Thyronine (T3)-Binding Protein (CTBP) Regulation of Nuclear T3Binding: Evidence for the Presence of T3-CTBP Complex-Binding Sites in Nuclei

Abstract

Effect of cytosolic 3,5,3'-triiodo-L-thyronine (T₃)-binding protein (CTBP) on [¹²⁵I]T₃ binding to nuclei was investigated in vitro. CTBP and nuclei were prepared from rat kidney. CTBP was inactivated by incubating with charcoal and activated by incubating with NADPH or with NADP and dithiothreitol. Two complexes of CTBP and T₃ [CTBP-T₃(NADPH) and CTBP-T₃(NADP)] were separately prepared, and the functions of these complexes were estimated. [¹²⁵I]T₃ binding to nuclei was not influenced by the inactive form of CTBP. NADP or NADPH alone did not modify [¹²⁶I]T₃ binding to the nuclei. However, the binding was markedly inhibited by NADPH in the presence of the inactive form of CTBP, but it was not inhibited by NADP in the presence of the inactive form of CTBP. The ability of nuclei to bind [¹²⁵I]T₃ was markedly diminished by pretreatment of the nuclei with 10^-8 M unlabeled T<._>3. The diminished activity was not modified by adding NADPH or NADP. However, [¹²⁵I]T₃ bound to these nuclei in the presence of NADP and the inactive form of CTBP. Binding of [¹²⁵I]T₃ to these nuclei was not observed in the presence of NADPH and the inactive form of CTBP. When the nuclei that had previously been saturated with 10^-6 M unlabeled T₃ were incubated with [¹²⁵I]T₃-CTBP(NADP) complex, radioactivity bound to the nuclei. The binding of radioactivity, however, was not observed when these nuclei were incubated with [¹²⁵I]T₃. The [¹²⁶I]T₃-CTBP(NADPH) complex did not bind to these nuclei. When the nuclei that had previously been treated with T₃- CTBP(NADP) complex were incubated with [¹²⁶I]T₃, radioactivity bound to the nuclei. The binding of radioactivity, however, was not observed when these nuclei were incubated with [¹²⁵I] T₃-CTBP(NADP) complex. The [¹²⁵I]T₃-CTBP(NADPH) complex did not bind to these nuclei. These results suggested that 1) CTBP activated by NADP plays a role as a carrier protein for T₃ from cytoplasm to nucleus; 2) there are binding sites for T₃-CTBP(NADP) complex in rat kidney nuclei; and 3) these binding sites are different from nuclear T₃ receptors. (Endocrinology124: 2851-2856, 1989)