Heat-induced degradation of overexpressed glucocorticoid receptor Separate protective roles of hsp90 and hsp70.
The glucocorticoid receptor (GR) occurs in cells in the form of a hormone-responsive complex (HRC) with hsp90. The HRC is dynamic, with hsp90 constantly directing disassembly, and hsp70, assisted by hsp90, driving reassembly. WCL2 cells stably overexpress GR to an extent that reduces the excess of hsp90 and hsp70 over GR by about 10-fold, compared to the ratio in HeLa cells. Yet the half-lives of the HRC in WCL2 and HeLa cells are comparable. As a result, the rate of assembly in WCL2 is overwhelmed by accumulation of the non-hormone-binding form of GR in its complex with hsp70 and hsp90. This form comprised some 50% of total GR in WCL2 cells. When the cells were heated to 44 degrees C, the hormone-binding activity and solubility of GR fell in parallel, and the receptor formed heavy aggregates by sequestering large amounts of hsp70. About 40% of this aggregated receptor was degraded in cells recovering at 37 degrees C in the presence of cycloheximide. Concentration of GR protein increased with increasing induction of hsp70 following exposure to 41-44 degrees C. However, balance between hormone-binding and inert forms of GR could shift in either direction in response to the increase or decrease of hsp90 induction, depending on the temperature. Suppression of degradation following re-exposure of the cells to 44 degrees C correlated better with induction of hsp90 than hsp70. We infer that sequestration of hsp70 by heat-unfolded receptor is the primary factor opposing degradation, while induction of hsp90 acts to further suppress degradation by accelerating HRC assembly.
Siriani
Despina
D
Molecular Endocrinology Programme, Institute of Biological Research and Biotechnology, The National Hellenic Research Foundation, 48 Vas. Constantinou Ave, 11635 Athens, Greece.
Mitsiou
Dimitra J
DJ
Alexis
Michael N
MN
D000818:Animals; D002460:Cell Line; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D013816:Thermodynamics; D014162:Transfection
D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins