The long-term objective of this research is to extend our understanding of the molecular mechanisms involved in the actions of glucocorticoids on their target tissues. Glucocorticoids are widely used in the treatment of cancer and inflammatory diseases and characterizing the mechanisms by which their receptors regulate gene expression constitutes a fundamental problem in molecular pharmacology.
The aims of this proposal focus on determining the role of receptor phosphorylation in glucocorticoid hormone action. The glucocorticoid receptor is a phosphoprotein and we have mutated the cDNA for the mouse receptor at all seven phosphorylation sites. We have analyzed the transcriptional activity of mutant receptors at the mouse mammary tumor virus promoter in transient transfections and we find a significant 22% reduction compared to wild-type receptor activity. In cells stably transfected with mutant receptors, we observe a 40% reduction in receptor activity at this promoter. Additionally, induction of the endogenous glucocorticoid-responsive gene for glutamine synthetase by mutant receptors is reduced by 25%. As the effect of receptor phosphorylation on transactivation may be promoter-specific, we will extend this analysis to other endogenous glucocorticoid- regulated genes and to minimal GRE promoter constructs containing defined sites for other transcriptional factors. The first set of experiments will examine the effect of phosphorylation site mutations on the regulation of the endogenous glucocorticoid- responsive genes for mouse nerve growth factor and sgk. The mRNA for nerve growth factor is reduced and the mRNA for sgk is induced by glucocorticoids. Our studies will also determine if receptor phosphorylation is involved in the down-regulation of receptor mRNA and the turnover of receptor protein. The induction of responsive genes by certain steroid hormones is potentiated by activation of cellular phosphorylation cascades and by stress. A third series of experiments will evaluate the contribution of receptor phosphorylation in coupling extracellular signaling pathways with steroid receptor signal transduction to the nucleus. There is some evidence that receptor phosphorylation may regulate the intracellular trafficking of the glucocorticoid receptor. A fourth set of experiments will determine if phosphorylation modulates receptor recycling and reutilization. A fifth set of experiments will identify the remaining phosphorylation site on the receptor and examine its contribution to receptor activities.
