Mapping the HSP90 binding region of the glucocorticoid receptor.
In animal cells, unliganded steroid receptors are complexed with a 90-kDa heat shock protein, HSP90; hormone binding by the receptor leads to the release of HSP90. We found that the 795-amino acid rat glucocorticoid receptor protein formed oligomeric complexes in vitro upon synthesis in rabbit reticulocyte lysates; these oligomers also dissociated in the presence of hormone. Similar complexes formed when X795, a receptor derivative containing only the C-terminal half (amino acids 407-795) of the protein, was translated in vitro. Moreover, X795 was co-immunoadsorbed from the reticulocyte lysates together with HSP90 by three different anti-HSP90 monoclonal antibodies, indicating that the in vitro translated receptor binds HSP90 and that the interaction occurs within the C-terminal half of the receptor. To localize the HSP90 binding region in greater detail, various deletion mutants of X795 were translated in vitro and assayed for oligomer formation and for co-immunoadsorption with HSP90. The results indicated that HSP90 interacted with the receptor within a subregion of the hormone binding domain, between amino acids 568 and 616. These findings are consistent with the proposal that HSP90 may participate in the mechanism of signal transduction by steroid receptors.
Howard
K J
KJ
Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio.
Holley
S J
SJ
Yamamoto
K R
KR
Distelhorst
C W
CW
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002460:Cell Line; D002850:Chromatography, Gel; D002872:Chromosome Deletion; D006360:Heat-Shock Proteins / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D014176:Protein Biosynthesis; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D006360:Heat-Shock Proteins; D011965:Receptors, Glucocorticoid