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

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-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.In the absence of hormone, the estrogen receptor (ER), 1 similar to all steroid receptors, has been shown to form heteromeric complexes with accessory proteins both in vivo and in vitro (1-10). Among these accessory proteins are at least two members of the heat shock protein (hsp) family, hsp90 and hsp70, as well as members of the immunophilin family and several other receptor-associated proteins. It is thought that ligand-free steroid receptors interact directly with hsp90, stabilizing the receptor in an inactive state.The ER is a member of a large family of nuclear receptors and, as such, is a hormone-dependent transcriptional regulator. The ER has been subdivided into six functionally distinct domains on the basis of sequence homology between receptors from different species (see Fig. 1) (11,12). The highly conserved DNA-binding domain, encoded by region C, is composed of two zinc finger motifs, each with four conserved cysteine residues (13,14). In addition to amino acids controlling site-specific DNA binding, region C contains a dimerization interface (15). Region D contains amino acids required ...

Differential SERM Effects on Corepressor Binding Dictate ERα Activity in Vivo

“…The SERMs tamoxifen and raloxifene allowed some residual activity that stems from AF-1 (24), but ICI allowed none. ER␣G400V, which exhibits reduced constitutive activity (49,50), showed similar activity to ER␣ in the presence of each ligand. In parallel, ER␣537X showed three differences from wild type ER␣.…”

Hsf-1 and POB1 Induce Drug Sensitivity and Apoptosis by Inhibiting Ralbp1

“…However, complex formation between all three proteins had not been previously demonstrated. In the present studies, we addressed the question of whether a trimeric complex exists between these three proteins by using protein cross-linking and immunoprecipitation using the established method of Aumais et al (30) (Fig. 2).…”

“…Protein Cross-linking and Immunoprecipitation-The protein cross-linking and immunoprecipitation assay was performed according to the method of Aumais et al (30). Briefly, purified recombinant Ralbp1, POB1, and Hsf-1 proteins (10 g each) were cross-linked by incubation with 0.1 mM N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP; from Sigma) in a total volume of 0.5 ml in 10 mM sodium phosphate buffer, pH 7.4, for 30 min.…”

Functional Protein Interactions in Steroid Receptor-Chaperone Complexes

“…Indeed, this may form part of the mechanism that allows the Hsp90 chaperone complex to restrict transactivation of receptor in the absence of hormone (Picard, 2006). In comparison to GR, studies have revealed that ER is less reliant on Hsp90 regulatory control over its hormone-dependent function (Picard et al, 1990), allowing the ER LBD to mediate dimerization in the absence of hormone in vivo (Aumais et al, 1997). ER homodimer formation in the LBD is mediated through helix 10, thus differing in configuration to that of GR (Bledsoe et al, 2002).…”

“…ER homodimer formation in the LBD is mediated through helix 10, thus differing in configuration to that of GR (Bledsoe et al, 2002). It is of interest that for ER , substitution of a valine residue for Gly400, also within the helix 5-6 loop of the ER LBD, induces a conformational change that destabilizes the receptor LBD, promoting a stronger, more stable association with Hsp90, similar to that for GR and rendering receptor transactivation more hormone-dependent (Aumais et al, 1997).…”