Experiments related to the functions, activities and regulation of the glucocorticoid receptor and projected in this proposal which combines two previous grants into a single application. The structure of the purified unactivated/nontransformed 310-320 kDa heterologous receptor complex will be determined with particular emphasis of the nature of the non-steroid receptor protein(s) and RNA. The 24 kDa component of unactivated receptor complex which has not been identified and is phosphorylated in vivo will be isolated and characterized. The synthesis of the heterologous unactivated complex will be attempted using purified activated receptor, purified 90 kDa heat shock protein, purified modulator and purified RNA as a means to confirm occurrence of various components. Polyclonal antibodies will be produced to synthetic peptides (approximately 15 mer) synthesized with reference to the rat receptor cDNA. These antibodies will be used to confirm topology of active sites and to determine the locus of the epitope of 3A6 monoclonal antibody and the existence of other potential activities, such as ATP binding. Ability of such antibodies to cross-react with native versus denatured receptor will provide clues to the 3-dimensional array of the receptor polypeptide. These antibodies will be used in indirect immunofluorescence with intact cells fixed on microscope slides to determine the physiological location of the receptor under various conditions. Preliminary evidence indicates that the homogeneous receptor can be phosphorylated by purified protein kinase C. This will be investigated thoroughly and other protein kinase will be tested for this capacity. Biological experiments in cell culture will be used to extend this observation to learn about cellular control of receptor functions. The genetically pure glucocorticoid receptor will be expressed in large quantity in an expression vector in order to assess its functions and for structural studies as well as use in reassembling the 320 kDa complex.
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