A molecular analysis of the human estrogen receptor

Abstract

The human myometrial estrogen receptor, isolated in buffer containing diisopropylftuorophosphate (DFP) to inhibit proteolytic activity, was partially purified by ammonium sulfate fractionation. Sucrose gradient analysis in dilute Tris buffer, pH 7.5, without KCl indicates that the cytoplasmic estrogen receptor sediments as an 8 S, 5 S and 4 S estrogen-binding protein (EBP). Isolation of the estrogen receptor without DFP yields predominantly a proteolytic fragment of the receptor which sediments as a 3 S EBP in gradients having low salt or high salt concentrations, and has a molecular weight of 30,000 to 40,000. The nuclear form of the estrogen receptor has a sedimentation coefficient of 3.8 ± 0.04 S in sucrose gradients containing high salt concentrations, a molecular Stokes radius of 38.5 ± 0.9 A ̊ , and a molecular weight of 60,000 to 70,000, values similar to the cytoplasmic form of the receptor. The cytoplasmic estrogen receptor shows a temperature-enhanced activation as indicated by its increased affinity for isolated uterine nuclei. In contrast to the activated estrogen receptor from the rat or calf uterus, the human uterus does not produce a 5 S EBP (a dimer of the 4 S EBP with another macromolecule) in high salt concentration at 0–4°C. In sucrose gradients containing 0.15 M KCl, centrifugation at 0, 6, 12, 20, and 25°C indicates that the human estrogen receptor undergoes a temperature dependent increases in its sedimentation coefficient from 4 S to 5 S and a transformation similar to that of the rat or calf estrogen receptor, but it is more readily dissociated by high salt or low temperature; the preferred form of the human estrogen receptor under more physiological conditions is the 5 S EBP dimer.