Abstract
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-ERG, containing the wild-type ligand-binding domain derived from the cDNA HEG0, and VP16-GAL-ERV, 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-ERV by rendering it hormone-dependent for DNA binding and more strongly dependent on estradiol for transactivation compared with VP16-GAL-ERG. Glycerol gradient analyses and chemical cross-linking/coimmunoprecipitation showed that, unlike VP16-GAL-ERG, VP16-GAL-ERV 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-ERG does not interact with hsp90 in vitro. Hormone binding studies showed that the ligand-binding domain of VP16-GAL-ERV 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.
Highlights
§ Chercheur-boursier of the Fonds de la Recherche en Santedu Quebec
It has been suggested that ligand binding induces a conformational change in the estrogen receptor (ER) ligand-binding domain that releases hsp90, exposing those regions on steroid receptors required for homodimerization, nuclear localization, and DNA binding
Analysis of hsp90-ER interactions has been complicated by the overlap in domains D and E between regions of the receptor required for DNA binding, homodimerization, and nuclear localization and those required for interaction with hsp90 [1]
Summary
§ Chercheur-boursier of the Fonds de la Recherche en Santedu Quebec. To whom correspondence should be addressed: Dept. of Physiology, McIntyre Medical Sciences Bldg., McGill University, 3655 Drummond St., Montreal, Quebec H3G 1Y6, Canada. Region D contains amino acids required for stable DNA binding [16] and has been shown to stabilize interactions with accessory proteins [1]. It has been suggested that ligand binding induces a conformational change in the ER ligand-binding domain that releases hsp, exposing those regions on steroid receptors required for homodimerization, nuclear localization, and DNA binding. Analysis of hsp90-ER interactions has been complicated by the overlap in domains D and E between regions of the receptor required for DNA binding, homodimerization, and nuclear localization and those required for interaction with hsp90 [1]. Transactivation potential was increased by replacing the N-terminal AF-1 region with the strong acidic activator VP16, derived from herpes simplex virus, creating VP16-GAL-ER chimeras These chimeras do not interact stably with hsp in
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