The broad objectives of the proposed research are to understand at molecular and cellular levels the physiological actions of glucocorticoids, particularly those on cells of the immune system. These actions account for widespread clinical applications of glucocorticoids as antiinflammatory and immunosuppressive agents and in treatment of lymphocytic leukemias and lymphomas. Studies will be undertaken to elucidate (1) the structure, generation and function of various phosphorylated states of glucocorticoid receptors in cells; (2) mechanisms of glucocorticoid inhibition of lymphokine production. (1) As shown in the P. I.'s laboratory with WEHI-7 mouse thymoma cells, the average number of phosphates per receptor steroid-binding protein is about 3 without hormone, increases to about 5 with hormone, and decreases to 3 or less in the saltunextractable nuclear fraction. """"""""Null receptors"""""""", formed in ATP-depleted cells, lose one phosphate. Partial proteolysis reveals phosphoserines in the steroid-binding and N-terminal domains, but not in the DNA-binding domain.
The Specific Aims are to: (a) Identify, by studies of the kinetics of phosphorylation, the receptor species that are substrates for phosphorylation. Initial results suggest the substrate for hormone-induced phosphorylation is the activated receptor. (b) Locate the phosphoaminoacids by partial sequencing of phosphopeptides obtained by HPLC analysis of limit tryptic digests. Preliminary HPLC analysis with unliganded receptors reveals two major phosphopeptides. (c) Analyze the influence of phosphorylation on biological activity using site-directed mutagenesis and kinase and phosphatase inhibitors. (d) Identify kinases that phosphorylate the receptor, using kinase-deficient cells, kinase and phosphatase inhibitors, and synthetic peptide substrates. Evidence so far indicates A-kinase is not involved. (2) Glucocorticoid immunosuppression may be due mainly to inhibition of cytokine production, which for interleukin-2 (IL-2) and gamma-interferon is associated with lowered mRNA levels. Nuclear runoff and mRNA stability assays will first establish if the lower levels reflect decreased transcription rates and/or mRNA stability. Present results suggest mRNA stability is not affected. Detailed mechanisms of these actions will be investigated by mutational and footprint analysis of the regulatory regions of the IL-2 gene.
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