Heat shock protein 70 is associated in substoichiometric amounts with the rat hepatic glucocorticoid receptor.
The 70-kDa heat shock protein (hsp70) has been shown to be an important participant in several intracellular events, including protein folding and trafficking. Hsp70 binds to many, if not all, proteins during their translation and maintains its association with some protein complexes as a subunit. We have examined the possibility that hsp70 may be associated with one or more forms of the rat hepatic glucocorticoid receptor (GR). Unliganded GR was immunoprecipitated from cytosol with the anti-GR monoclonal antibody BUGR2 and then subjected to western blotting. Both hsp70 and the 90-kDa heat shock protein (hsp90) were found to be specifically associated with the GR. Hsp70 was also bound to the liganded unactivated and activated (transformed) forms of the GR complex, while as expected, hsp90 was absent from the activated GR. Immunoprecipitation of cytosolic hsp70 with the anti-hsp70 monoclonal antibody N27 resulted in coprecipitation of GR. The components of the immunopurified GR were also analyzed by laser densitometry after polyacrylamide gel electrophoresis and Coomassie blue staining. These experiments revealed that hsp70 is bound to the GR in an approximate 1:5 ratio. Unactivated GR complexes isolated via a ligand affinity purification scheme contained hsp90 and trace amounts of hsp70. Collectively, these experiments demonstrate that hsp70 is specifically associated with several forms of the native rat hepatic GR, although its binding is substoichiometric. This is in direct contrast to hsp90, which binds as a dimeric subunit to the heteromeric unactivated GR complex.
Diehl
E E
EE
Department of Physiology and Biophysics, College of Medicine, University of Iowa, Iowa City 52242.
Schmidt
T J
TJ
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D003600:Cytosol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007163:Immunosorbent Techniques; D008099:Liver / Q000737:chemistry*; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D006360:Heat-Shock Proteins; D011965:Receptors, Glucocorticoid