Glucocorticoid receptor-interacting proteins

Glucocorticoids (cortisol and derivatives) are vital steroid hormones produced in the adrenal cortex that are essential for the proper functioning of numerous organ systems under conditions of stress. Synthetic glucocorticoids are a class of frequently applied drugs that exploit the immunosuppressive, antiinflammatory and apoptosis-inducing function of these steroid hormones to treat conditions such as rheumatoid arthritis, asthma bronchiale and leukemia. Glucocorticoids act via the glucocorticoid receptor, a protein that in the absence of hormone is part of a cytoplasmic multiprotein complex. Upon ligand binding, it translocates to the nucleus where it acts as a transcription factor to activate or silence specific genes. When genes were identified that were obviously regulated by glucocorticoids without direct binding of the receptor to DNA, it became clear that part of receptor functioning was mediated by direct interaction with other proteins involved in transcription regulation. Over time, numerous transcription-related interaction partners were discovered. At the same time, intensive efforts were directed towards identifying the components and understanding the function of the cytoplasmic multiprotein complex retaining the unliganded receptor. In contrast, less attention was given to potential other interactions "away from DNA", probably because there was little reason to expect them. Recent reports, e. g. on an interaction with the tumor suppressor p53, have highlighted the potential functional significance of such interactions. The main intent of this project is a systematic search for further cytoplasmic interaction partners. We will use a recently developed modification of the yeast two-hybrid technique, as the conventional screening method cannot be used for proteins that, like the glucocorticoid receptor, contain transcription activating domains. The ultimate goal of studying the molecular mechanisms of glucocorticoid receptor function is to identify target molecules for novel pharmacological approaches in the therapy of inflammation, unwanted immune reactions and leukemia.

Cortisone, or rather its active form hydrocortisone or cortisol, is being produced by the adrenal cortex and enables our body to cope with various forms of stress. In addition to being a vital hormone, cortisol and its synthetic derivatives are also one of the most successful classes of medical drugs, which are widely used to treat allergies, rheumatic diseases and leukemia. Virtually every cell in our body reacts to glucocorticoids, which helps to explain the range of unwanted side effects accompanying protracted therapeutic use. To reduce side effects, it would be desirable to develop drugs that adress only part of the broad spectrum of glucocorticoid effects. While much has been learned about the molecular effects of glucocorticoids, which seem to involve mainly gene regulation by the intracellularly located glucocorticoid receptor, we need to learn a lot more to approach that goal. The main aim of this research project was to identify cellular proteins that interact directly with the receptor. This objective was achieved: about 100 interacting protein fragments were isolated. Of the list of identified interactants, many proteins will have to be investigated more closely, but considerable progress has been made on one protein of the zinc finger family. It is located in the nucleus, while the glucocorticoid receptor in the absence of glucocorticoids is cytoplasmic. Upon binding its ligand, the receptor shuttles to the nucleus. According to our findings, the zinc finger protein seems to very efficiently recruit the receptor to distinct subnuclear structures. After 12 hours of glucocorticoid treatment, the glucocorticoid receptor colocates with the zinc finger protein at exactly the same spots of a grainy appearance. The result of this cooperation is not yet clear. The zinc finger protein contains a so-called KRAB domain, which can contribute to the silencing of genes by chromatin condensation. We are currently searching for genes that might be regulated by the zinc finger protein. If successful, we will then assess a potential glucocorticoid effect on this regulation. Another part of our work was devoted to aspects of the gene encoding the glucocorticoid receptor, specifically to the sites where transcription of the messenger RNA initiates. There is no single first exon; instead, several gene segments are being used as alternative first exons which are all spliced to the identical second exon. In collaboration with the group of Reinhard Kofler, we were able to contribute to our understanding of glucocorticoid receptor expression by identifying two additional, yet unknown alternative first exons.