A Conserved Proline in the hsp90 Binding Region of the Glucocorticoid Receptor Is Required for hsp90 Heterocomplex Stabilization and Receptor Signaling

Abstract

Studies of hsp90 in yeast have supported the notion that this chaperone plays a critical role in signaling by steroid receptors. One limitation to these studies is that yeast expressing hsp90 mutants may also be deficient in fundamental cellular functions of the chaperone required for steroid-dependent induction of transcription. In this work, we have prepared mutants of the glucocorticoid receptor (GR) that permit analysis of hsp90 binding and transcriptional activity in cells with normal chaperone function. Our previous data supported a model in which hsp90 binds to the receptor steroid binding domain according to a two-site model. By amino acid mutagenesis of these two sites, we have now generated three receptor mutants and analyzed their function. Upon their translation in vitro, all three mutants interacted with hsp90 similarly to the wild-type receptor. However, one mutant, P643A (GRo), was of particular interest because, although it showed normal steroid binding and transformation to a glucocorticoid response element-specific DNA binding form, it was remarkably deficient in nuclear translocation and transcriptional function at 37°C. Furthermore, GRo⅐hsp90 heterocomplexes formed in vivo or assembled under cell-free conditions were much less stable than wildtype GR⅐hsp90 heterocomplexes. Our results demonstrate that Pro-643 plays a critical role in both stabilizing the receptor⅐hsp90 complex and in permitting an efficient nuclear translocation and, thus, support the concept that the chaperone is an integral component of the steroid-receptor signaling pathway.The untransformed form of steroid receptors, such as the glucocorticoid receptor (GR), 1 exists in cells as a large (9 S) heteromeric complex containing the 90-kDa heat-shock protein (hsp90) (1-5), p23, and one of several immunophilins (for review of receptor heterocomplex structure, see Refs. 6 -8). Upon steroid binding, hsp90 dissociates from the original multicomponent structure (3, 9), and the steroid-binding protein is transformed to its nuclear, DNA binding, and transcriptionally active form (9, 10). Given the correlation between hsp90 association and inactivity of the receptor, an inhibitory role for this association has been typically postulated (1, 2, 11). In apparent contradiction with the inhibitory model of hsp90, genetic analysis in yeast expressing low levels of hsp90 (12) or hsp90 point mutants (13,14) indicates that hsp90 is required for steroid receptor signaling in vivo. Moreover, the drug geldanamycin, a benzoquinone ansamycin, specifically binds to hsp90 and, by interference with critical steps in the hsp90-assisted folding, causes significant functional defects in the substrate proteins for hsp90. In the case of GR, geldanamycin suppresses the steroid binding (15, 16), nuclear translocation (16), and transcriptional functions (15, 17) of the receptor. Also, the results of experiments where both the GR and the mineralocorticoid receptor are expressed in bacteria (18, 19) support a permissive role of hsp90. Although affinity-purified ...

Cpr6 and Cpr7, Two Closely Related Hsp90-associated Immunophilins from Saccharomyces cerevisiae, Differ in Their Functional Properties

“…One may speculate that the PPIase activity of Cpr7 may have evolved for special substrates. It is interesting to note that a conserved proline of glucocorticoid receptor is required for the stabilization of Hsp90 heterocomplexes and receptor signaling (57).…”

Unliganded and hormone-bound glucocorticoid receptors interact with distinct hydrophobic sites in the Hsp90 C-terminal domain

“…Mutational analyses and peptide-competition studies revealed that formation of apo-GR-Hsp90 heterocomplexes that are hormone-binding competent depends on several GR subregions that cluster around the ligand-binding pocket and on three regions within the Hsp90 middle and C-terminal domain (CTD) (5,(14)(15)(16). Because these Hsp90 domains are also engaged in interactions with cochaperones, it has been uncertain whether these regions contain direct binding sites for GR.…”

Definition of Protein Kinase Sequence Motifs That Trigger High Affinity Binding of Hsp90 and Cdc37

“…Despite over a decade of intense study, the basis for the recognition by Hsp90 of its diverse clients remains one of the primary mysteries in the field. Studies using site-directed and deletion mutagenesis (5)(6)(7)(8)(9)(10)(11) have identified potential sites within steroid hormone receptors and protein kinases that may be recognized by Hsp90. However, although a recent study has indicted that point mutations generated in the Cdk4 kinase (12) affect its interaction with Hsp90, no common primary, secondary, and/or tertiary structural motifs have been defined that may be responsible for Hsp90 client recognition.…”

“…[1][2][3][4]. Current models suggest that co-chaperones may confer specificity to Hsp90 facilitated protein folding by also interacting with client targets (5,13,14). Cdc37 is one such Hsp90 co-chaperone that interacts with immature forms of Hsp90-dependent kinases (reviewed in Ref.…”