THE AH RECEPTOR IS A SENSITIVE TARGET OF GELDANAMYCIN-INDUCED PROTEIN-TURNOVER

Geldanamycin (GA) binds directly to hsp90 and apparently disrupts cert ain hsp90 heterocomplexes. We have investigated the GA-hsp90 interacti on and its effect on other associated proteins. Incubation of 2-[I-125 ]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 v itro. 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 sensitivit y to GA exposure: AhR much greater than c-Raf-l > glucocorticoid recep tor > CDK4 much greater than p50. A series of hsp90 deletion mutants w ere used to map the domain that interacts with GA. Deletion of the fir st 221 amino acids in NH2-terminal domain resulted in loss of binding to solid-phase GA. Epitopes of monoclonal antibodies specific for hsp9 0 were also determined by direct immunoprecipitation with hsp90 mutant s. Results indicated that monoclonal antibodies 8D3 and 3G3 interact w ith hsp90 via the first 221 amino acids in NH2-terminal region, wherea s AC88 requires a COOH-terminal region between amino acids 661-677. (C ) 1997 Academic Press.