Human c-erb A protein expressed in Escherichia coli: changes in hydrophobicity upon thyroid hormone binding.
The human c-erb A beta gene sequence was inserted in an Escherichia coli expression vector plasmid. The E. coli cells transformed with this plasmid produced proteins with molecular masses of 52 and 50 kDa. These products bound 3,5,3'-triiodo-L-thyronine (T3) with an affinity constant of 4.3 x 10(9) liter/mol. The order of affinity for iodothyronine analogs was triiodothyroacetic acid greater than T3 greater than 3,5,3'-triiodo-D-thyronine greater than L-thyroxine. Affinity labeling experiments showed that the 50 kDa protein was covalently labeled with [125I]T3, and this was competed by triiodothyroacetic acid, T3, and L-thyroxine (from potent to weaker competitor). The c-erb A protein bound to calf thymus DNA-cellulose and the binding was inhibited by 0.3 M KCl or 10 mM pyridoxal 5'-phosphate. Aqueous two-phase partitioning studies showed that the c-erb A product became less hydrophobic upon T3 or triiodothyroacetic acid binding. The same finding was obtained when T3 bound to partially purified rat liver nuclear thyroid hormone receptor. However, thyroxine binding globulin became more hydrophobic upon T3 binding. Since the T3 molecule partitioned preferentially into the upper polyethylene glycol-rich phase, the alteration of partitioning behavior of thyroxine binding globulin was explained by a simple additive effect of T3. In contrast, the alteration of partitioning behavior of the c-erb A product or receptor reflected a conformational transition upon T3 binding. The c-erb A protein expressed in E. coli showed various characteristics similar to classical thyroid hormone receptor and may be useful in studying the structure and function of the thyroid hormone receptor.
Ichikawa
K
K
Department of Gerontology, Endocrinology, and Metabolism, Shinshu University School of Medicine, Matsumoto, Japan.
Hashizume
K
K
Furuta
S
S
Osumi
T
T
Miyamoto
T
T
Yamauchi
K
K
Takeda
T
T
Yamada
T
T
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone; D013974:Thyroxine / Q000378:metabolism; D014284:Triiodothyronine / Q000031:analogs & derivatives / Q000378:metabolism*
D011518:Proto-Oncogene Proteins; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; C010642:3,3',5-triiodothyroacetic acid; D004247:DNA; D013974:Thyroxine