Activation of thymocyte glucocorticoid receptors to the steroid binding form. The roles of reduction agents, ATP, and heat-stable factors.

Hydrogen peroxide and diamide inactivate the steroid-binding capacity of unoccupied glucocorticoid receptors in rat liver cytosol at 0 degrees C, and steroid-binding capacity is reactivated with dithiothreitol. Treatment of cytosol with peroxide or sodium molybdate, but not diamide, inhibits the irreversible inactivation (i.e., inactivation not reversed by dithiothreitol) of steroid-binding capacity that occurs when cytosol is incubated at 25 degrees C. Pretreatment of cytosol with the thiol derivatizing agent methyl methanethiosulfonate at 0 degrees C prevents the ability of peroxide, but not molybdate, to stabilize binding capacity at 25 degrees C. As derivatization of thiol groups prevents peroxide stabilization of steroid-binding capacity and as treatment with dithiothreitol reverses the effect, we propose that peroxide acts by promoting the formation of new disulfide linkages. The receptor in our rat liver cytosol preparations is present as three major degradation products of Mr 40,000, 52,000, and 72,000 in addition to the Mr 94,000 intact receptor. Like the intact receptor, these three forms exist in the presence of molybdate as an 8-9S complex, they bind glucocorticoid in a specific manner, and they copurify with the intact Mr 94,000 receptor on sequential phosphocellulose and DNA-cellulose chromatography. Despite the existence of receptor cleavage products, it is clear that peroxide does not stabilize steroid-binding capacity by inhibiting receptor cleavage.

Section: Hydrogen Peroxide Stabilizes Unoccupied Receptorssupporting

confidence: 87%

Hydrogen peroxide stabilizes the steroid-binding state of rat liver glucocorticoid receptors by promoting disulfide bond formation

Bresnick¹,

Sánchez²,

Harrison³

et al. 1988

“…protein, translocate to the nucleus, and eventually undergo productive interactions within a circumscribed set of genes could all potentially be controlled by modifications of the receptor's structure. For instance, there is considerable evidence that receptor sulfhydryl groups may play an important role in determining both steroidand DNA-binding capacities (Granberg & Ballard, 1977;Sando et al, 1979;Bodwell et al, 1984a,b;Tienrungroj et al, 1987). Covalent modification at these sites by sulfhydryl oxidizing reagents causes a loss 0006-2960/89/0428-2929$01.50/0 © 1989 American Chemical Society of receptor function.…”

mentioning

confidence: 99%

Transformed glucocorticoid receptors consist of multiple subspecies with differing capacities to bind DNA-cellulose

Gruol¹,

Wolfe²

1989

The glucocorticoid receptor can be transformed into a DNA-binding protein by a process that is both hormone and temperature dependent. We have used a modification of the conventional method of anion-exchange chromatography to separate and analyze a variety of receptor subspecies that result from this transition. One receptor form (peak A) was found to have a capacity to bind DNA-cellulose which was significantly greater than that of the other species. Under conditions of mild heating (15 degrees C), the relative abundance of peak A in the receptor population and the rate of receptor transformation were both increased as a result of incubating samples with alkaline phosphatase. The mechanism appears to involve the conversion of the more "acidic" forms into that of peak A. The results indicate that receptor transformation is a multistep process which may be promoted by the removal of phosphate from either the receptor or a receptor-bound regulatory factor.

“…Thus, other alterations in receptor structure including subunit dissociation, limited receptor proteolysis (Puca et al, 1972;Sica et al, 1976), and conformational change (Samuels & Tomkins, 1970;Rousseau et al, 1972;Bailly et al, 1980) have been invoked for the molecular mechanism of receptor activation. Any of these mechanism could also involve covalent modification of the receptor, for example, dephosphorylation (Sando et al, 1979;Barnett et al, 1980).…”

mentioning

confidence: 99%

Subunit dissociation as a possible mechanism of glucocorticoid receptor activation

Vedeckis¹

1983

153

For the elucidation of the mechanism of steroid hormone receptor activation, the hydrodynamic properties of the unactivated and activated forms of the nonproteolyzed glucocorticoid receptor from the mouse AtT-20 pituitary tumor cell line were determined. The unactivated, molybdate-stabilized receptor has the following properties: sedimentation coefficient = 9 S; Rs = 8.3 nm; Mr = 317 000; f/f0 = 1.70; axial ratio (prolate ellipsoid) = 14. The activated monomeric receptor has a sedimentation coefficient of 3.2 S, a Stokes radius of 6 nm, a molecular weight of 81 000, a frictional ratio of 1.93, and an axial ratio (prolate ellipsoid) of 18. A receptor species of intermediate size was detected when the analysis was performed in buffer containing both 0.3 M KCl and 20mM Na2MoO4. Its characteristics are as follows: sedimentation coefficient = 5 S; Rs = 8.3 nm; Mr = 176 000; f/f0 = 2.06; axial ratio (prolate ellipsoid) = 22. A preliminary study seemed to indicate that this is an activated form of the receptor. On the basis of the molecular weights, it is likely that the unactivated receptor is a tetramer of identical hormone-binding subunits (Mr = 81 000) while the intermediate form is a homodimer. Alternatively, non-hormone-binding components (receptor-binding factors) may be involved in forming the multimeric, nonactivated receptor complex. In either case, the dissociation of a multimeric, nonactivated receptor into subunits appears to be a possible mechanism of receptor activation. Finally, the addition of high concentrations of 1-thioglycerol promoted activation. Thus, sulfhydryl groups may be involved in receptor subunit interaction.