Glucocorticoid receptor function : interactions, mutants and ligand responses

Abstract

The protein that is investigated in this thesis is the glucocorticoid receptor (GR), which belongs to the nuclear hormone receptor superfamily of ligand activated transcription factors. The nuclear receptors share three conserved structural domains, the N-terminal transactivating domain, the central DNA-binding domain and the C-terminal ligand-binding domain.

Glucocorticoids are well known for their anti-inflammatory and apoptotic effects and are therefore used as treatment for a multitude of diseases including, asthma, rheumatoid arthritis, ulcerative colitis and leukemia s. GR mediates the effects of glucocorticoids not only by the activation and repression of specific target genes that play important roles in several physiological processes such as metabolism, cell proliferation and inflammatory and immune responses but also through protein-protein interactions with other signalling pathways, referred to as cross-talk mechanisms.

In the first study, we described a method to investigate new GR interacting proteins on a large scale using two dimensional gel electrophoresis in combination with MALDI-TOF/TOF mass spectrometry. We found 27 novel potentially important proteins that interacted with the GR receptor complex. Our data suggests that those interactions are variable depending on the presence or absence of glucocorticoids and that they also are present in different GR multiprotein complexes of different composition, indicating the existence of new GR cross-talk mechanisms.

In the second study, we characterized the interaction between GR and FMS-like tyrosine kinase 3 (Flt3). We showed that the DNA-binding domain of GR is sufficient for the Flt3 interaction. Addition of Flt3 ligand also proved to be necessary for potentiation of glucocorticoid dependent transcription.

The data presented in the third study aimed to detect glucocorticoid regulated genes from blood samples within a short time frame. We also measured the amount of GR in different subpopulations of peripheral blood leucocytes with flow cytometry. Together these results serve as a starting point for a quick determination of glucocorticoid responsiveness in patients.

In the fourth study, we characterized the functional properties of the two GR mutations, R477H and G679S. We showed that both R477H and G679S have a dominant negative effect on wild-type GR and that R477H has impaired DNA-binding which explains the severe clinical phenotypes of cortisol resistance that are associated with these mutations.