A recombinant rat thyroid hormone receptor α (TRα or c-ErbAα1) was produced in E. coli as a non-mutated,non-fusioned protein and obtained as an efficient DNA and T3 binding protein that could be easily handled in a buffer-soluble state (rec-TRα). It was found that nuclear extracts (NE) added to rec-TRα 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-TRα 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 heat-labile, appeared as necessary auxiliaries in the analyses of partially purified rec-TRα. A protective effect of NE against a loss of affinity for T3 under the action of antibodies directed to certain sequences in the TRα D domain suggests that nuclear factors help rec-TRα to acquire and/or stabilize a conformation that allows the high affinity T3 binding. The nature of this nuclear amplifying factor is still unknown: RXRα which, produced in vitro, could amplify binding of the rec-TRα to a DNA thyroid response element, was unable to display such a rescue of high affinity binding sites.
