The Ah receptor is a sensitive target of geldanamycin-induced protein turnover.
Geldanamycin (GA) binds directly to hsp90 and apparently disrupts certain hsp90 heterocomplexes. We have investigated the GA-hsp90 interaction and its effect on other associated proteins. Incubation of 2-[125I]-iodo-3-azido-7,8-dibromo-p-dioxin-labeled Hepa 1c1c7 cytosol with GA-coupled beads revealed a stable association of Ah receptor (AhR)/hsp90 complex with GA. In addition, sucrose gradient sedimentation analysis demonstrated that GA does not disrupt the 9S Ah receptor complex in vitro. HeLa and Hepa 1c1c7 cells were subjected to a dose-response and time-course treatment with GA and the level of the AhR was determined. A 75% depletion in AhR levels was observed within an hour of exposure to 100 nM GA. The relative stability of other proteins that associate with hsp90 was determined with the following rank order of sensitivity to GA exposure: AhR >> c-Raf-1 > glucocorticoid receptor > CDK4 >> p50. A series of hsp90 deletion mutants were used to map the domain that interacts with GA. Deletion of the first 221 amino acids in NH2-terminal domain resulted in loss of binding to solid-phase GA. Epitopes of monoclonal antibodies specific for hsp90 were also determined by direct immunoprecipitation with hsp90 mutants. Results indicated that monoclonal antibodies 8D3 and 3G3 interact with hsp90 via the first 221 amino acids in NH2-terminal region, whereas AC88 requires a COOH-terminal region between amino acids 661-677.
Chen
H S
HS
Department of Veterinary Science, Pennsylvania State University, University Park 16802, USA.
Singh
S S
SS
Perdew
G H
GH
D000345:Affinity Labels; D000903:Antibiotics, Antineoplastic / Q000378:metabolism / Q000494:pharmacology*; D016227:Benzoquinones; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018841:HSP90 Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D047029:Lactams, Macrocyclic; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D014176:Protein Biosynthesis; D011809:Quinones / Q000378:metabolism / Q000494:pharmacology*; D018336:Receptors, Aryl Hydrocarbon / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D000903:Antibiotics, Antineoplastic; D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D011809:Quinones; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins; C001277:geldanamycin