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

Abstract

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.

Phosphorylation of Steroid Hormone Receptors*

“…For example, several transcription factors become hyperphosphorylated on stimulation, and much indirect evidence points to regulation of both receptors and transcription factors by kinases and phosphatases, with cycling between different phosphorylated states. Functions of receptors that are regulated by phosphorylation are February, 1992 STEROID HORMONE RECEPTOR PHOSPHORYLATION 123 only beginning to be investigated. With transcription factors a substantial body of information is already available, and functions that appear to be thus regulated include dimerization, interactions with other proteins, binding to DNA, nuclear-cytoplasmic localization, and transcriptional activity.…”

“…Treatment of the receptors with heat or alkaline phosphatase promoted increased DNA-cellulose binding and a change in the elution profile toward more basic forms (123). Microheterogeneity was also determined by isoelectric focusing under nondenaturing conditions, and a shift toward more basic forms was observed after activation (124).…”

The Ah receptor and the mechanism of dioxin toxicity

“…For example, phosphorylation has been suggested to explain the presence of several isoforms of the glucocorticoid receptor, although processes other than phosphorylation (e.g. acylation, prenylation) could be responsible for their charge differences (Gruol & Wolfe, 1989;Smith et al, 1986). Interestingly, studies with the Ah receptor in crude preparations indicate that it does not rely on phosphorylation for ligand binding.…”

Transformation of glucocorticoid receptors bound to the antagonist RU 486: effects of alkaline phosphatase

“…The dissociation of glucocorticoid receptors from the heat shock protein complex into the smaller 4S form appears to be a necessary but not a sufficient change to generate a fully activated receptor. Reports from several laboratories have shown that receptor transformation is a multistep process and that release from the 8-9S complex does not result in the complete conversion of receptor into a protein that can bind DNA (Schmidt et al, 1985;Harmon et al, 1988; Ben-Or & Chrambach, 1988; Gruol & Wolfe, 1989). Moreover, the transition may be dependent upon cellular activities that are not inherent to the receptor protein itself.…”