The long term objective of this proposal is to study the mechanism of steroid hormone action at the molecular level. After hormone binding occurs, steroid-recptor complexes are transformed in a temperature-dependent manner from a form that does not bind to DNA to a form that bind to DNA with affinity. This transformation of the receptor is a necessary step in the pathway by which these hormones regulate gene expression. The goal of this work is to understand the nature of this transformation process as it occurs with the glucocorticoid-receptor complex. Steroids of the glucocorticoid class are used in the therapy of a wide variety of diseases and understanding the mechanism by which these hormones cause their receptor-mediated effects constitutes a fundamental problem in molecular endocrinology. During the past three years we developed methods to directly demonstrate that the glucocorticoid receptor in mouse L cells is a phosphoprotein. In this proposal, I present a series of experiments utilizing covalent labeling of the L cell receptor protein with 32P or with (3H)dexamethasone 21-mesylate followed by specific immunoadsorption with anti-receptor antibody to study the structure of the transformed and untransformed receptor and the way in which covalent modification may be involved in defining the DNA-binding state.
Specific aims are: 1) to determine how many phosphates are present on the 98K steriod-binding protein and to map the phosphopeptides produced by complete protease digestion; 2) to determine whether the association of the steriod-receptor complex with DNA following cell-free transformation and the association of the receptor with nuclei after transformation in the intact cell is related (as our preliminary studies suggest) to the extent of receptor phosphorylation; 3) to determine if protein phosphatases and an endogenous membrane-bound, receptor-inactivating enzyme are able to convert non-DNA-binding phosphorylated receptors to a DNA-binding state; 4) to determine by direct assay if phosphatase-mediated inactivation of steroid-binding capacity in L cell cytosol is accompanied by receptor dephosphorylation; 5) to purify a protein kinase from L cell cytosol that phosphorylates the receptor and determine if phosphorylation of the DNA-binding form of the receptor converts it to a non-DNA-binding form.
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