Selective Interaction of hsp90 with an Estrogen Receptor Ligand-binding Domain Containing a Point Mutation

• Published in: The Journal of biological chemistry 1997-05, Vol.272 (18), p.12229-12235
• Main Authors: Aumais, J P, Lee, H S, Lin, R, White, J H
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; COS Cells; DNA, Complementary; DNA-Binding Proteins; Fungal Proteins - isolation & purification; Herpes Simplex Virus Protein Vmw65 - isolation & purification; HSP90 Heat-Shock Proteins - isolation & purification; HSP90 Heat-Shock Proteins - metabolism; Kinetics; Ligands; Luciferases; Mutagenesis, Site-Directed; Point Mutation; Receptors, Estrogen - chemistry; Receptors, Estrogen - isolation & purification; Receptors, Estrogen - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae Proteins; Transcription Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.18.12229

The 90-kDa heat shock protein (hsp90) has been implicated in modulating steroid receptor function in vitro and in vivo . Previous studies have suggested that hsp90 interacts with large portions of the estrogen receptor (ER) ligand-binding domain and sequences of the receptor required for stable DNA binding. To characterize the interaction of the ER ligand-binding domain with hsp90, we have compared the properties of chimeras created by coupling the ligand-binding domain to the constitutive transactivator VP16-GAL. Two types of chimeras were created: VP16-GAL-ER G , containing the wild-type ligand-binding domain derived from the cDNA HEG0, and VP16-GAL-ER V , containing the substitution G400V derived from the ligand-binding domain of the original ER cDNA isolate, HE0. The G400V mutation alters the physical properties of VP16-GAL-ER V by rendering it hormone-dependent for DNA binding and more strongly dependent on estradiol for transactivation compared with VP16-GAL-ER G . Glycerol gradient analyses and chemical cross-linking/coimmunoprecipitation showed that, unlike VP16-GAL-ER G , VP16-GAL-ER V formed stable complexes with hsp90 in vitro . These data show that hsp90 selectively recognizes the altered ER ligand-binding domain containing the G400V substitution and indicate that the wild-type ER ligand-binding domain of VP16-GAL-ER G does not interact with hsp90 in vitro . Hormone binding studies showed that the ligand-binding domain of VP16-GAL-ER V was destabilized by incubation in the presence of high concentrations of salt or in the absence of sodium molybdate, conditions that disrupt its interaction with hsp90. The ligand-binding domain of the Val-400 ER thus behaves similarly to that of the wild-type glucocorticoid receptor, which has previously been shown to interact with hsp90 in vitro . These results provide evidence for the action of hsp90 as a molecular chaperone by selectively recognizing destabilized proteins.