Nuclear factors specifically favor thyroid hormone binding to c‐ErbAα1 protein (thyroid hormone receptor α) over‐expressed in E. coli

Abstract

A recombinant rat thyroid hormone receptor ~ (TR~ or c-ErbA~l) was produced in E. coli as a non-mutated, nonfusioned protein and obtained as an efficient DNA and T3 binding protein that could he easily handled in a buffer-soluble state (rec-TR~). It was found that nuclear extracts (NE) added to rec-TR~ markedly amplified not only DNA binding, which has been well documented, but also T3 binding (increased binding site concentration), which has not yet been reported. This T3 binding amplifying effect on rec-TR~ occurs at low NE protein concentrations that produce no or minimal endogenous TR with respect to rec-TR, while similar concentrations of other proteins (e.g. ovalbumin or cytosol) only moderately enhanced T3 binding. The T3 binding amplifying nuclear factors, which are partly heatlabile, appeared as necessary auxiliaries in the analyses of partialiy purified rec-TR~. A protective effect of NE against a loss of affinity for T3 under the action of antibodies directed to certain sequences in the TR~ D domain suggests that nuclear factors help rec-TR~ to acquire and/or stabilize a conformation that allows the high affinity T3 binding. The nature of this nuclear amplifying factor is still unknown: RXR~ which, produced in vitro, could amplify binding of the rec-TR~ to a DNA thyroid response element, was unable to display such a rescue of high affinity binding sites.

A Variant Form of the Nuclear Triiodothyronine Receptor c-ErbAα1 Plays a Direct Role in Regulation of Mitochondrial RNA Synthesis

“…T3 binding assays. p43 affinity for T3 (K a ) was measured in saturation experiments by using 125 I-labeled T3 (3.3 mCi/g; NEN Life Science Products) according to the method of Daadi et al (8). Nonspecific binding was assessed in simultaneous assays in which a 1 M concentration of cold T3 was added.…”

p28, a truncated form of TRα1 regulates mitochondrial physiology

“…p28 affinity for T3 (Ka) was measured in saturation experiments using [ 12 5I]‐T3 (3.3 mCi/μg; NEN Life Science Products) according to Daadi et al [15]. Non‐specific binding was assessed in simultaneous assays in which 1 μM of cold T3 was added.…”