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. These events include steroid binding to the intracellular receptor molecule, activation of the receptor-steroid complex to a DNA-binding and nuclear-binding species, and 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 first two steps for glucocorticoid steroids. Studies with the affinity label dexamethasone mesylate (Dex-Mes) and the thiol-specific reagent methyl methanethiolsulfonate (MMTS) have revealed the involvement of two thiol groups in steroid binding. Contrary to the current dogma, we found that reduced thiols are not absolutely required for binding to glucocorticoid receptors if the steric bulk of the oxidized thiols is small. We raised polyclonal antibodies against a defined epitope near the DNA-binding domain of the receptor. These antibodies recognized only activated complexes, thus providing direct evidence for a topological change in the receptor resulting from activation. Thiol reagents were also used to establish, for the first time, functional heterogeneity among activated complexes. Thus MMTS inhibited the DNA binding of one sub-class of complexes while arsenite, which is specific for vicinal dithiols, blocked the DNA binding of the other sub-class. Furthermore, one sub-class of complexes undergoes a two-step activation process in which the association of a small molecular weight (700-3000 daltons) factor is required for DNA binding. Further studies using these techniques along with molecular biology should provide new information about steroid hormone control of gene transcription at a molecular level.