Abstract
The technique of aqueous two-phase partitioning has been used to study changes in estrogen receptor (ER) structure that occur upon ligand binding and/or heating in vitro. Studies with steroidal and nonsteroidal ligands indicate that the difference in partitioning properties between unoccupied and nontransformed ER is due to a ligand-induced change in this conformation of the protein. Furthermore, this conformational change is only partially induced by binding of 4-OH-tamoxifen. Although nontransformed 4-OH-tamoxifen complexes can be transformed by heat, there are significant differences in the transformation process for receptors bound to 4-OH-tamoxifen versus estrogenic ligands. A kinetic analysis of estrogen receptor transformation indicates that the process follows apparent first order kinetics, but is 2.5-fold slower for the 4-OH-tamoxifen-receptor complex. Direct heating of the unoccupied ER causes a significant change in receptor structure. Ligand binding to the heat-altered unoccupied receptor results in a further alteration of receptor structure. Experiments using polyethylene glycol palmitate indicate that the ligand-binding transition is associated with a reduction of the hydrophobic characteristics of the receptor. These results demonstrate that there are a number of independent conformational changes that occur within the monomeric ER steroid-binding subunit upon ligand binding and exposure to elevated temperature in vitro.
Highlights
The techniqueof aqueoustwo-phase partitioning haspresence of high salt concentrations.The molecular nature of been used to study changes in estrogen receptor (ER) the ligand-induced changes in estrogen receptor (ER’) strucstructure that occurupon ligand binding and/or heat- ture in vivo that are responsible for the biological activation ing in vitro
Ligand binding to the heat-alteretdionships between heat-induced changes in the properties of unoccupied receptor results in a further alteration of the monomeric estrogen-receptor complex, and a number of receptorstructure.Experimentsusingpolyethylene aggregation phenomena in which the receptor monomer can glycol palmitate indicate that the ligand-binding tran-participate (Notides and Nielsen, 1974; Notides et a!., 1981; sition is associated with a reductioofnthe hydrophobic see Sherman and Stevens, 1984, for review)
We demonstrated thatthe difference in partition coefficients at 0 'C and subsequent hydroxylapatite assay, as described (Hansen between unoccupied and nontransformed estrogen receptor and Gorski, 1985)
Summary
From the DeDartment of Biochemistr_v..College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wkconsin53706. The most extensively studied estrogen receptor transition(s) in vitro have been the changes in ER properties that accompany heating of the low temperature stabilized, nontransformed estrogen-receptor complex? Nontransformed ER refers to estrogen-receptor complexes formed in vitro by incubating unoccupied, cytosolic ER with ligand at 0 “C. Transition, we have 1) studied the partitioning properties of estrogen receptors bound to both steroidal and nonsteroidal estrogens as well as an antiestrogenic ligand, 2) used ATPP as thebasis for a kinetic analysisof the temperature-depend-. Cytosol and nontransformed estradiol-receptor complexes were quite different (Hansen and Gorski, 1985).In order to determine whether this difference inpartitioningproperties was due solely to addition of ligand to the ERsurface or was indicative containing the different receptor forms was partitioned to equilibrium in phase systems composed of 5.4%(w/w) dextran, 5.4% (w/w)PEG, and 0.1 M LipSol,buffered with TED, pH8. Binding of 4-OH-tamoxifen lowered the partition coefficient of the unoccupied receptor, thenontrans-
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