Characterization of the 4 S and 5 S Forms of the Estradiol Receptor Protein and Their Interaction with Deoxyribonucleic Acid

The cytoplasmic 4 S receptor protein that specifically binds the steroid hormone 17β-estradiol has been studied in soluble extracts of rat uterine tissue at physiological ionic strengths. After labeling the protein with [3H]estradiol, two related interactions of the receptor with other macromolecular components have been detected and characterized by DNA-cellulose chromatography and sucrose gradient sedimentation. In a reaction requiring the presence of both hormone and DNA-cellulose, the receptor binds a second subunit (Subunit X), increasing its sedimentation rate from 4 S to 5 S. Concomitantly, this 5 S complex binds to DNA. From gel permeation and sedimentation behavior, the molecular weights of the 4 S and 5 S receptor proteins are estimated as 60,000 and 105,000, respectively. Although the strong tendency of these receptors to adsorb to surfaces necessitates the use of carrier proteins as protective agents, essentially identical results have been obtained whether bovine serum albumin, lysozyme, or insulin is used for this purpose. These interactions proceed rapidly at high temperatures, but only slowly at 4°. At 4°, the 4 S to 5 S conversion is second order in extract concentration and rate-limiting, and the 5 S form of receptor binds rapidly to DNA. At low ionic strength, the 4 S form of the receptor can also bind to DNA-cellulose, but it elutes at 0.21 m NaCl while the 5 S receptor remains bound. The X subunit of the 5 S receptor does not itself bind estradiol; it appears to be capable of binding to DNA independently of the receptor, and is present in both target and non-target tissues of the rat. These in vitro results parallel the hormone- and temperature-dependent 4 S to 5 S conversion and nuclear migration observed for the estradiol receptor protein in vivo. We suggest that this nuclear migration results simply from the increased DNA affinity of the 5 S form of receptor, and that the true nuclear binding sites for this receptor are therefore at least partially composed of DNA.