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 mole is followed by activation of the receptor-steroid complex to a DNA-binding and nuclear-binding species 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. Studies with the affinity label dexamethasone mesylate (Dex-Mes) and various proteases have defined a steroid-binding core fragment that is half the size of the previously acknowledged minimum steroid binding fragment. The sequence of this core fragment was four Thr-537 - Arg-673. Studies with arsenite (and Cd++), which specifically reacts with vicinal dithiols, and the affinity label Dex-Mes have confirmed the involvement of our previously proposed vicinal dithiol group in steroid binding to the glucocorticoid receptor. Further studies of proteolytic fragments of the receptor using these thiol-specific reagents, Dex-Mes, and a specific anti-receptor antibody, revealed that, of the 20 cysteines in the receptor, only the 3 closely spaced cysteines in the steroid binding core are candidates for being the vicinal dithiols. Further studies using these techniques, along with molecular biology should provide new information about steroid interactions with the receptor protein at a molecular information will be invaluable in understanding the determinants for glucocorticoid vs antiglucocorticoid hormone action at the level of gene transcription.