Studies of glucocorticoid receptor interacting proteins

Abstract: The glucocorticoid receptor (GR) functions as a ligand dependent transcription factor. Nonliganded GR resides mainly in the cytoplasm bound to a complex containing heat shock proteins (hsp). Upon ligand binding, GR dissociates from the hsp-complex and translocates to the nucleus where it regulates the transcription of specific genes by binding to glueocorticoid response elements (GREs). Within the nucleus, GR is known to interact with other transcription factors via protein-protein contacts thereby affecting their transcriptional action via so called transcriptional cross-talk. GR is thought to be able to interact with other signaling pathways also in the cytoplasm although this is not as well studied as the transcriptional cross-talk in the nucleus. Therefore, we wanted to study GR interactions with proteins in the cytoplasm and to define conditions when specific GR-protein interactions occurred. We have developed a specific immunoaffinity chromatographic purification of GR to identify hitherto unknown cytosolic GR interacting proteins. Briefly, by using the antiGR monoclonal antibody (mAb) 250, GR is purified from the liver cytosol of adrenalectomized rats and peptide eluted. The elution of GR allows GR-interacting proteins to co-purify with the receptor as intact complexes. Using Western Blotting we have identified 14-3-3, Raf-1 and NF-kappaB and IkappaBalpha as GR co-purifying proteins in the rat liver cytosol. We identified Flt3 among the GR co-purifying proteins using Edman based N-terminal sequencing. Flt3 was found to interact with both the nonliganded and the liganded form of GR and the DNA-binding domain of GR is sufficient for Flt3 interaction as shown by GST-pulldown experiments using human GR and FIG. Using two-dimensional gel electrophoresis in combination with MALDI-TOF mass spectrometry, we have been able to identify 36 individual proteins, among which 28 are novel GR interacting proteins, for example: Major Vault Protein, TATA binding interacting protein 49 and glycoprotein PP63. Using Blue Native gel electrophoresis, we demonstrated that GR exists in several separate protein complexes. This study shows that GR interacts with a number of different proteins within rat liver cells. Proteins, regulating such a vast number of vital biological functions as GR does, perhaps need to integrate their functions with those of other proteins via proteinprotein interactions. We hypothesize that cytosolic GR, besides the well-known hspcomplex, also interacts with other specific proteins in dynamic multiprotein complexes, "receptosomes" which may form the biochemical basis for cross-talk between GR and other signaling pathways.