Direct evidence that the glucocorticoid receptor binds to hsp90 at or near the termination of receptor translation in vitro.
We have translated the rat glucocorticoid receptor in both reticulocyte lysate and in wheat germ extract. Receptor synthesized in the reticulocyte lysate is immunoadsorbed by the 8D3 monoclonal antibody directed against the 90-kDa heat shock protein (hsp90) and it has a normal ability to bind glucocorticoid in a high affinity manner. Although the wheat germ extract synthesizes the full length receptor, the receptor is not immunoadsorbed by 8D3 and we cannot demonstrate high affinity steroid binding. Receptor synthesized by the reticulocyte lysate can be immunoadsorbed by antibody directed against hsp90 as soon as the translation product is full length, suggesting that the receptor becomes associated with hsp90 late during translation or immediately at the termination of translation. When newly synthesized receptor is bound with steroid and incubated at 25 degrees C, it is converted to a form that binds to DNA. This study provides direct evidence that association of hsp90 with the glucocorticoid receptor is a very early event and that the newly formed heteromeric receptor-hsp90 complex is fully competent to undergo transformation.
Dalman
F C
FC
Department of Pharmacology, University of Michigan, Ann Arbor 48109.
Bresnick
E H
EH
Patel
P D
PD
Perdew
G H
GH
Watson
S J
SJ
Pratt
W B
WB
D000327:Adsorption; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010443:Peptide Chain Termination, Translational; D010944:Plants / Q000378:metabolism; D014176:Protein Biosynthesis; D011817:Rabbits; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism; D014158:Transcription, Genetic
D006360:Heat-Shock Proteins; D011965:Receptors, Glucocorticoid