Modulation of nuclear receptor function by interacting proteins

Sammanfattning: Nuclear receptors are a family of transcription factors involved in many biological processes. They represent key potentials as therapeutic targets for several clinical conditions such as diabetes, obesity, cardiovascular disease and cancer, because their activity is modulated by small molecules. Collectively these diseases represent a large market for prescription drugs. The nuclear receptor family consists of 48 members. The nuclear receptors have three conserved domains, the N-terminal transactivation domain, the central DNA binding domain and the C-terminal ligand binding domain. Nuclear receptors are ligand activated transcription factors that are not only regulated by small lipophilic ligands, but also by interaction with coactivators and corepressors as well as other binding proteins. Protein-protein interactions are essential in many biological pathways and are also attractive for drug discovery. In this thesis I elucidate the functional significance of some nuclear receptor interacting proteins. I have investigated the mechanism of the corepressor receptor interacting protein 140 (RIP140) for repression of glucocorticoid receptor (GR) and liver X receptor (LXR) and the functional significance of the novel interacting proteins polyamine-modulated factor 1 (PMF-1) and germinal center associated nuclear protein (GANP) in glucocorticoid signalling. RIP140 represses ligand activated nuclear receptors, such as GR and LXR. It is localized in small nuclear foci targeted by a 40 amino acid long sequence. Even though the foci targeting sequence in RIP140 overlaps with the binding site of the corepressor C-terminal binding protein (CtBP), interaction with CtBP is not essential for foci targeting. Upon coexpression of ligand activated GR or LXR with RIP140 is redistributed to larger foci distinct from RIP140 foci. The redistribution of RIP140/GR involves RIP140 repression domains and the DNA binding domain of GR. RIP140 repression domains include the C-terminal receptor interacting LXXLL motifs binding to GR and the interaction domain of the corepressor CtBP. The repression of RIP140/LXR involves the integrity of the C-terminal domain of RIP140 including the LXXML motif. Jointly, these results suggested that RIP140 represses in multiple ways including direct binding to ligand activated NR and the formation and redistribution of intranuclear repressive protein complexes. PMF-1 is a transcription factor induced by polyamines, which are important regulators of cell growth and cell death and are implicated in glucocorticoid induced apoptosis. We identified PMF-1 to functionally interact with RIP140 and GR. PMF-1 represses glucocorticoid induced GR activity and has an intrinsic repression activity, which could contribute to the repressive action. Although a physical interaction was observed between PMF-1 and RIP140, we found that PMF-1 does not further enhance RIP140 repressive effect. We identified GANP and the GANP splice variant MCM3 associated protein (MCM3AP) to bind to GR ligand binding domain. We found GANP to be a shuttling protein that shuttles between the nucleus and the cytoplasm, and contains nuclear localization and nuclear export signals. GANP and MCM3AP were initially reported as proteins binding to MCM3, which is a member of the MCM protein complex that is involved in initiating of DNA replication. We show that glucocorticoids regulate the chromatin loading of MCM3, inhibit DNA replication and arrest cells in the G1 phase of the cell cycle. We also show that MCM3AP counteracts the repressive activity of glucocorticoid on DNA replication. In conclusion we suggest that interaction between GR, GANP/MCM3AP and MCM3 could imply a new way for glucocorticoid regulation of cell proliferation.