The goals of our research are to achieve an understanding of the mechanisms by which glucocorticoid receptor molecules mediate hormonal information in target tissues. We propose to isolate, purify and characterize the rat lymphocyte glucocorticoid receptor, using classical differential chromatography, affinity labeling, and immunological procedures. Purified glucocorticoid receptors will be used further for two purposes. 1) We will produce monoclonal antibodies to both dissociable steroid receptor complexes and steroid affinity labeled receptors. These antibodies will subsequently be utilized to further characterize the glucocorticoid receptor and provide structural information regarding its physical properties. 2) Purified receptors will also be utilized to study in detail its presumptive interaction with P32 pyridoxal phosphate. We will characterize the number, the affinity and the localization of pyridoxal phosphate binding sites on the glucocorticoid receptors. Finally we propose a series of definitive experiments to elucidate the mechanisms by which pyridoxal phosphate interacts with specific sites on the glucocorticoid receptor to inhibit DNA binding. These investigations should further our knowledge of steroid receptor structure and determine whether pyridoxal phosphate interaction with receptor is specific, and of possible physiological importance. In accomplishing these goals we hope to advance current knowledge of the mechanism of hormone action.
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