REGULATION OF GLUCOCORTICOID RECEPTOR FUNCTION BY CAMP

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The overall goal is to achieve a greater understanding of the mechanisms by which glucocorticoid hormones and hormones which act through cAMP regulate lymphoid viability and growth. Both steroids and cyclic nucleotide have the demonstrated capacity to suppress the immune system as a part of an organism's response to stress. The primary transducers for these agents are known to be a gene- regulating receptor and a protein kinase, but the immediate functions that they affect are only partially understood. Evidence of crossregulation is presented, showing that cAMP has the ability to promote an increase of glucocorticoid binding capacity in a murine lymphoma line, WEHI-7. Two possible mechanisms are proposed: 1) Conversion of a cryptic pool of receptors into an active form; 2) Increased synthesis of receptor protein. The involvement of receptor modification vs. synthesis will be studied using 2D-gel electrophoresis, thioredoxin assays, density gradient analysis and molecular probes for synthesis of receptor protein and messenger RNA. At certain stages of T-cell differentiation, glucocorticoids and cyclic nucleotide can elicit a cytolytic response. This behavior has provided a basis for the use of steroids in treatment of certain forms of leukemia. The cytolytic response has also presented a tool for selection of resistant variants containing altered glucocorticoid receptors and cyclic AMP-dependent protein kinase. This proposal deals with the discovery and characterization of a "second generation" of steroid/cyclic nucleotide resistant variants. It was found that ac initial selection for resistance to cAMP in the murine lymphoma WEHI-7 acts as a permissive step towards selection of steroid resistance at s high frequency. The results indicate that the variants which have been obtained represent new forms of steroid resistance, possibly involving functions that activate the glucocorticoid receptor into a steroid binding state. Characterization of these variant cell lines is proposed in an attempt to identify the altered functions which lead to the loss of functional receptor.