Proteomic identification of glucocorticoid receptor interacting proteins

Proteomics. 2006 May;6(10):3114-26. doi: 10.1002/pmic.200500266.

Abstract

The glucocorticoid receptor (GR) acts as a ligand dependent transcription factor but can also cross talk with other signaling pathways via protein-protein interactions. In this paper we describe methods to study novel cytosolic GR interacting proteins, using mAb based immunoaffinity chromatography of GR from rat liver cytosol. Co-purifying proteins were identified by 2-DE in combination with MALDI-TOF-MS. Non-liganded/non-activated and in vitro liganded/activated GR, respectively, co-purifies with specific sets of proteins. Of these 34 were conclusively identified, seven have previously been reported to be part of the GR-complex, revealing 27 new possible interacting candidates for the GR-complex. Of the novel GR interacting proteins the major vault protein, TATA binding interacting protein 49a and glycoprotein PP63 were of special interest. Furthermore, using 2-D DIGE we show that the set of proteins interacting with non-liganded GR is distinctly different in protein amount compared to the proteins found with liganded/activated GR. This suggests the presence of different GR complexes in the cell, which was further substantiated by the finding of several separate GR native protein complexes, "GR-receptosomes", using blue native gel electrophoresis. Our findings suggest the existence of several new mechanisms for GR signaling and regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal
  • Cell Line, Tumor
  • Chromatography, Affinity
  • Cytosol / metabolism
  • Electrophoresis, Gel, Two-Dimensional
  • Electrophoresis, Polyacrylamide Gel
  • Immunoblotting
  • Ligands
  • Liver / metabolism
  • Protein Interaction Mapping*
  • Proteome / metabolism*
  • Rats
  • Receptors, Glucocorticoid / agonists
  • Receptors, Glucocorticoid / metabolism*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Antibodies, Monoclonal
  • Ligands
  • Proteome
  • Receptors, Glucocorticoid