The proposed research aims at a better understanding of the mechanism of steroid hormone action at the molecular level. After hormone binding occurs, steroid-receptor complexes are transformed in a temperature-dependent manner to a form that associates with nuclear """"""""acceptor"""""""" sites involved in the regulation of gene expression. This transformation reaction can be studied in cytosol preparations by assaying the conversion of the steroid-receptor complex from a state that does not bind to nuclei or DNA to a stae that binds with high affinity to nuclei or DNA. The goal of this work is to understand the nature of this critical transformation process as it occurs with the glucocorticoid-receptor complex. Specifically, I am testing a model I published in 1979 (J. Biol. Chem. 254:4779, 1979) that is based on several studies from my laboratory investigating the role of receptor phosphorylation in the binding of glucocorticoids. In the model I propose that temperature-mediated transformation may be initiated by dephosphorylation of the steroid-bound receptor. The first four specific aims of the proposed work focus on using a 2 dimensional gel system to resolve receptor that has been covalently labeled with a radioactive probe and prepurified by a rapid affinity chromatography procedure or by antireceptor antibody. Receptor that is covalently labeled with [3H]steroid in the binding site will be used to localize the receptor on 2 D gels in an unequivocal manner. The rapid gel procedure will then be used to localize untransformed and transformed states of receptor that was labeled with 32P in intact L cells. Also, a radiolabeled probe that reacts covalently with ATP-binding sites will be used to detemine if an ATP-binding site on the receptor is exposed during transformation. Another series of experiments focuses on purifying and characterizing a protein that we have found to inhibit the binding of phosphatase-treated receptor to DNA. The goal here is to determine if this transformation protein normally exists in a complex with the untransformed receptor and blocks the DNA-binding site. For this purpose I will develop antibodies to the transformation protein and ask if they will react with the untransformed receptor complex but not with the transformed state.
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