INITIAL INTRACELLULAR EVENTS OF STEROID HORMONE ACTION

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The objective of this project is to define the initial, intracellular events of glucocorticoid hormone action, and steroid hormone action in general. The first step of steroid binding to the intracellular receptor molecule is followed by activation of the receptor-steroid complex to a DNA-binding and nuclear-binding species and then binding of activated complexes to those nuclear acceptor sites involved in the regulation of transcription of specific genes. A combination of techniques have been used to examine the crucial first step for glucocorticoid steroids. Biochemistry, affinity labeling, and proteolysis have been used to define a steroid binding core element of the receptor which contains most, if not all, of the amino acids required for specific steroid binding along with the ability to bind heat shock protein 90. Metabolic labeling and immunochemistry revealed that posttranslational glycosylation does not appear to be required for the steroid binding activity of the receptor. Molecular biology, used to create point mutations of the 3 cysteines in the steroid binding core element, has yielded "super" receptors with increased binding affinity and specificity for glucocorticoids. Such "super" receptor may prove to be of use in future genetic engineering treatment of patients with defective receptors and/or steroid biosynthesis. Finally, a rational examination of the steroid binding domains of all the steroid receptors for the presence of potential vicinal dithiols permitted us to devise a protocol involving pretreatment with sodium arsenite that specifically inactivates only glucocorticoid receptors. This procedure should be of great assistance in the quantitation of progesterone and mineral-ocorticoid receptors. Future studies concerning the molecular details of steroid-receptor interactions should provide further information which will aid in understanding the control of steroid-regulated gene transcription and the treatment of patients.