Nuclear factors specifically favor thyroid hormone binding to c-ErbA alpha 1 protein (thyroid hormone receptor alpha) over-expressed in E. coli.
A recombinant rat thyroid hormone receptor alpha (TR alpha or c-ErbA alpha 1) was produced in E. coli as a non-mutated, nonfusioned protein and obtained as an efficient DNA and T3 binding protein that could be easily handled in a buffer-soluble state (rec-TR alpha). It was found that nuclear extracts (NE) added to rec-TR alpha 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 alpha 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-TR alpha. A protective effect of NE against a loss of affinity for T3 under the action of antibodies directed to certain sequences in the TR alpha D domain suggests that nuclear factors help rec-TR alpha 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 alpha which, produced in vitro, could amplify binding of the rec-TR alpha to a DNA thyroid response element, was unable to display such a rescue of high affinity binding sites.
Daadi
M
M
INSERM Unité 38, Biochimie Médicale, Faculté de Médecine, Marseille, France.
Lenoir
C
C
Dace
A
A
Bonne
J
J
Teboul
M
M
Planells
R
R
Torresani
J
J
D000818:Animals; D001685:Biological Factors / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D051379:Mice; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism*
D001685:Biological Factors; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014284:Triiodothyronine