Glucocorticoids influence numerous physiological parameters including the growth, differentiation and function of a wide variety of target tissues and because of these diverse effects are frequently used to treat hemolytic disorders and malignancies, pulmonary disease, and connective tissue disorders. Most of these effects are mediated at the cellular level by cytoplasmic glucocorticoid-receptors which must undergo an obligatory conformational change, termed """"""""activation"""""""", before they can be translocated to the nucleus or bind to DNA-cellulose. The basic long-term objective of this application is to elucidate the biochemical mechanism(s) of this """"""""activation"""""""" step and thus contribute to the present understanding of how receptors function to transmit steroidal signals within target cells. To investigate the in vitro regulation of activation the cytoplasmic unactivated complexes will be purified to near homogeneity by a three step procedure 1) affinity chromatography, 2) gel filtration, and 3) DEAE-cellulose chromatography. The first major aim is to investigate the in vitro regulation of activation of purified complexes by a variety of physical manipulations (including elevated temperature and increased pH) and biochemical probes (including pyridoxal 5 feet-phosphate, ATP and exogenous phosphatases) which are known to maximally activate unpurified complexes by unknown mechanism(s). Activtion in all proposed experients will be quantitates by the DNA-cellulose binding assay and by DEAE-cellulose chromatography. Reconstitution experiments will then be conducted to test the hypothesis that a cytoplasmic factor(s) may enhance or facilitate in vitro activation. This cytoplasmic """"""""activating factor"""""""" will be biochemically characterized and ultimately purified. The third major aim is to directly test the hypothesis that a relationship exists between glucocorticoid structure (hence receptor affinity) and the generation of activated complexes from purified unactivated complexes. A wide variety of tritiated steroids of different affinities will be used to elute receptors from the affinity gel and the rate of activation of the subsequently purified complexes as induced by physical manipulations, biochemical probes and partially purified activating factor will subsequently be studied. The fourth major aim is to investigate the potential kinase activity of the glucocorticoid receptor as determined by appropriate incubations of the purified unactivated complex with [Gamma-32P]-ATP. The substrate specificity and possible cyclic nucleotide activation of this potential kinase activity will also be investigated.
