Proteomic identification of glucocorticoid receptor interacting proteins.
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.
Hedman
Erik
E
Department of Biosciences and Nutrition, Division of Medical Nutrition, Karolinska Institutet, Novum, S-141 86 Stockholm, Sweden. erik.hedman@mednut.ki.se
Widén
Christina
C
Asadi
Abolfazl
A
Dinnetz
Ingrid
I
Schröder
Wolfgang P
WP
Gustafsson
Jan-Ake
JA
Wikström
Ann-Charlotte
AC
D000818:Animals; D000911:Antibodies, Monoclonal; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008024:Ligands; D008099:Liver / Q000378:metabolism; D025941:Protein Interaction Mapping; D020543:Proteome / Q000378:metabolism*; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000819:agonists / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000911:Antibodies, Monoclonal; D008024:Ligands; D020543:Proteome; D011965:Receptors, Glucocorticoid