Thyroid hormone receptors in a human hepatoma cell line: multiple receptor forms on isoelectric focusing.
The nuclear thyroid hormone receptors isolated from cultured human hepatoma cells (Hep G2) were characterized and compared with those from cultured human fibroblasts and rat liver. The Hep G2 nuclear thyroid hormone receptors had an affinity constant (Ka) of 2.1 X 10(10) M-1 and maximal binding capacity (MBC) of 21.0 fmol/100 micrograms DNA for T3 in assays performed on isolated nuclei. 16% of nuclear receptors were released into the media during incubation and had the same Ka. Salt-extracted receptors had a Ka of 1.8 X 10(10) M-1 and MBC of 0.1 pmol/mg protein for T3. Density gradient sedimentation and gel filtration chromatography revealed a sedimentation coefficient of 3.4 S and Stokes radius of 34 A. From these values, a molecular weight of 49,000 and total frictional ratio (f/f0) of 1.4 were calculated, suggesting an asymmetrical shape of the receptor molecule. Heat inactivation occurred with t1/2 of 28.1, 18.0, and 7.9 min at 38, 43, 45 degrees C, respectively. Isoelectric focusing (IEF) of Hep G2 nuclear receptors demonstrated T3 binding proteins at pH 5.3-5.5, 5.7, and 5.9. Evidence that these are nuclear thyroid hormone receptors includes the following: Triiodothyroacetic acid was the most potent competitor of [125I]T3 binding to these proteins followed by L-T3, and L-T4. Cytosolic protein, human serum, and fetal calf serum failed to show the same T3 binding proteins. Ka of these proteins measured by T3 displacement was 1.1-3.2 X 10(9) M-1. Human fibroblast nuclear extract showed similar T3 binding pattern in IEF, except for a slight difference in pI of an acidic band.(ABSTRACT TRUNCATED AT 250 WORDS)
D001667:Binding, Competitive; D006528:Carcinoma, Hepatocellular / Q000032:analysis* / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008099:Liver / Q000032:analysis* / Q000378:metabolism; D008113:Liver Neoplasms; D008970:Molecular Weight; D009994:Osmolar Concentration; D011988:Receptors, Thyroid Hormone / Q000032:analysis* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014284:Triiodothyronine / Q000378:metabolism
D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine