Glucocorticoids have widespread physiological functions and are used extensively in the treatment of inflammatory and immunological disorders and lymphoid cancers. Their actions all appear to be mediated by binding to glucocorticoid receptors (GRs) that function as ligand-dependent transcription factors. The hormone-receptor complexes form homodimers on glucocorticoid response elements (GREs) in certain genes, thereby regulating transcription of those genes. Protein-protein interactions are of fundamental importance to the regulation and function of the glucocorticoid receptor (GR). Through these interactions individual GRs form homodimers and interact with other transcription factors, such as Jun and CREB. Formation of such complexes allows the GR to potentially interact with the diacyl glycerol-protein kinase C signal transduction pathway (GR:Jun complexes) and the cAMP- dependent-protein kinase A pathway (GR:CREB complexes). The objective of this research is to understand at the molecular level the nature of these protein-protein interactions. Of particular interest is how interactions between the GR and other transcription factor may differ in solution compared to those formed on DNA, and the influence that phosphorylation has on these interactions. The PI has identified seven phosphorylated sites in the mouse GR. The N- terminal location of these sites suggests a possible role for phosphorylation in modulating protein-protein interactions, since deletion of the N-terminal region of the GR prevents detectable solution homodimer formation, and domains in the N-terminal are necessary for the GR repression of Jun-mediated transcription at a composite GRE. Changes in these critical functions could have important consequences in controlling gene expression. The proposed research evaluates cytosolic GR:Jun and GR:CREB complexes by determining the: (i) stoichiometry of each component in the complex, and the phosphorylation status of each component, (ii) specific contact areas between components, and (iii) the influence of mutating GR phosphorylated sites on equilibrium binding and kinetic constants of complex formation and interaction with specific DNA response elements. A parallel study will evaluate the nature of GR:Jun, GR:CREB, or GR:ARF complexes formed within the cell on the composite GRE from the glutamine synthetase gene by determining: (i) stoichiometry, (ii) contact areas, and (iii) the influence of phosphorylation on transcription from the composite GRE. This proposal also seeks to identify specific contact areas in the N- terminal domain responsible for stabilizing the GR homodimer and determine the influence of phosphorylation on homodimer formation and stability.
