Unliganded and hormone-bound glucocorticoidreceptors interact with distinct hydrophobicsites in the Hsp90 C-terminal domainL. Fang, D. Ricketson, L. Getubig, and B. Darimont*

Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229

Communicated by Keith R. Yamamoto, University of California, San Francisco, San Francisco, CA, October 17, 2006 (received for review August 6, 2006)

Unlike most chaperones, heat-shock protein 90 (Hsp90) interactswith a select group of ‘‘client proteins’’ that regulate essentialbiological processes. Little is known about how Hsp90 recognizesand binds these proteins. The glucocorticoid receptor (GR) is a wellcharacterized Hsp90 client protein, whose hormone binding,nuclear-cytoplasmic trafficking, and transcriptional activity areregulated by Hsp90. Here, we provide evidence that unligandedand hormone-bound GR interact with two distinct, solvent-exposed hydrophobic sites in the Hsp90 C-terminal domain thatcontain the sequences ‘‘MxxIM’’ (HM10) and ‘‘L/MxxIL’’ (HM9). Ourresults indicate that binding of Hsp90 HM10 to unliganded GRstabilizes the unliganded ligand-binding pocket of GR indirectly bypromoting an intramolecular interaction between the C-terminal�-helix (H12) and a solvent-exposed hydrophobic groove in the GRligand binding domain. In the presence of hormone, Hsp90 appearsto bind the hydrophobic groove of GR directly by mimicking theinteractions of GR with transcriptional coactivators. The identifiedinteractions provide insights into the mechanisms that enableHsp90 to regulate the activity of both unliganded and hormone-bound GR and to sharpen the cellular response to hormone.

binding sites � heat-shock protein 90 � steroid hormone receptors

Hsp90 is an unusual chaperone that assists the folding andfunction of a restricted yet diverse set of structurally distinct

regulatory proteins involved in cell cycle regulation, DNAprocessing, intercellular communication, protein trafficking, andprotein turnover (1). Hsp90 is essential for the viability ofeukaryotic cells and has become a prime therapeutic target fora variety of human diseases (2).

Among the best studied client proteins of Hsp90 are steroidhormone receptors (SRs), such as the glucocorticoid receptor(GR), which belong to the large nuclear receptor (NR) family(3). NRs are hormone-activated transcription factors that bindhormones through a ligand-binding pocket that is deeply buriedwithin the ligand-binding domain (LBD) (4). High-affinity hor-mone binding by SRs requires the interaction of the LBD withHsp90 and associated cochaperones (5). The unliganded (apo)-GR-Hsp90 heterocomplex has been extensively studied andreconstituted by using purified proteins (6). These studies re-vealed that binding of Hsp90 to apo-GR is ATP-dependent andinitially mediated by the Hsp90 cochaperone Hop and a GR-bound Hsp70 assembly complex. In a later step, Hsp90 binds GRdirectly, and the GR-Hsp90 heterocomplex becomes hormone-binding competent. This transition is accompanied by the ATP-dependent replacement of Hop, Hsp70, and Hsp70 cochaper-ones by the Hsp90 cochaperone p23 and one of severaltetratricopeptide repeat domain proteins (e.g., FKBP51). TheHsp90 inhibitor geldanamycin (GA) prevents the direct but notthe Hsp70- and Hop-mediated interaction between GR andHsp90 and triggers the proteolytic degradation of GR (7).

In addition to assisting hormone binding, Hsp90 also regulatessignaling events initiated by hormone-bound GR (holo-GR)(8–9). Hormone binding by GR induces the replacement of theGR-Hsp90 heterocomplex component FKBP51 by FKBP52,

whose interaction with the motor protein dynein has beenimplicated in the nuclear import of holo-GR (9–10). Hsp90 andp23 are present at glucocorticoid response elements (GREs) andregulate the interaction of GR with GREs and transcriptionalcoregulators (11–13).

Despite many efforts, little is known about the structuralfeatures of GR and Hsp90 required for their interaction. Mu-tational analyses and peptide-competition studies revealed thatformation of apo-GR-Hsp90 heterocomplexes that are hor-mone-binding competent depends on several GR subregionsthat cluster around the ligand-binding pocket and on threeregions within the Hsp90 middle and C-terminal domain (CTD)(5, 14–16). Because these Hsp90 domains are also engaged ininteractions with cochaperones, it has been uncertain whetherthese regions contain direct binding sites for GR. The bindingsites of holo-GR and Hsp90 have yet to be characterized.

Because ectopically expressed GR assembles with yeast Hsp90and cochaperones into hormone-responsive GR-Hsp90 hetero-complexes, yeast has been a very useful model system to studythe composition and function of these complexes (17). Using afunctional screen in yeast, we have identified four rat GR LBDmutants (Y616N, F620S, M622T, and M770I), which are lessdependent on Hsp90 but bind hormone with similar affinity asWT GR (D.R., U. Hostick, L.F., K. Yamamoto, and B.D.,unpublished work). The amino acids that are replaced in theseGR mutants belong to a conserved allosteric network thattransmits hormone-induced structural changes in the buriedhormone-binding pocket to a solvent-exposed hydrophobicgroove in the GR LBD, which interacts with transcriptionalcoregulators (18–20) (Fig. 1). Because this mechanism shouldoperate in both directions, these mutants suggested that Hsp90might stabilize the ligand binding pocket of apo-GR throughinteractions with the hydrophobic groove. The goal of this studywas to test this hypothesis.

ResultsIdentification of Potential Hydrophobic Groove-Binding Sites inHsp90. Transcriptional coregulators bind the hydrophobic grooveof NRs through amphipathic �-helices that contain the sequenceLxxLL (x, any amino acid) (21). In the case of GR, the leucineresidues of this sequence can be replaced by other hydrophobicresidues with only minor effects on the affinity of coregulatorsfor GR (see Fig. 7, which is published as supporting informationon the PNAS web site). Hypothesizing that Hsp90 interacts with

Author contributions: L.F., D.R., and B.D. designed research; L.F., D.R., L.G., and B.D.performed research; L.F., D.R., and B.D. analyzed data; and L.F., D.R., and B.D. wrote thepaper.

The authors declare no conflict of interest.

Abbreviations: cort, corticosterone; CTD, C-terminal domain; D, 2% glucose; dex, dexa-methasone; GA, geldanamycin; GR, glucocorticoid receptor; LBD, ligand-binding domain;NR, nuclear receptor; S, minimal medium; SR, steroid hormone receptor.

*To whom correspondence should be addressed. E-mail: bead@molbio.uoregon.edu.

© 2006 by The National Academy of Sciences of the USA

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GR by mimicking the binding of transcriptional coregulatorswith the hydrophobic groove in the GR LBD, we identifiedeleven ‘‘hxxhh’’ sequence motifs (called HM1,. . . , HM11; h,hydrophobic amino acid; x, any amino acid) within the middleand C-terminal domains of Hsp90 and replaced the hydrophobicresidues of each motif by alanines (Fig. 2A). Introduced into ayeast strain (YBD100) in which expression of endogenous Hsp90can be silenced, four of the resulting Hsp90 HM mutants(HM2m, HM9m, HM10m, and HM11m) did not or only mod-estly impair growth but significantly reduced the response of GRto hormone (Fig. 2 B and C; the phenotypes of the other mutantsis shown in Fig. 8, which is published as supporting informationon the PNAS web site). Immunoblot analysis revealed that allHsp90 HM mutants are expressed, although the expressionlevels of HM9m and HM11m were �30% lower than WT Hsp90(Fig. 2D). Suggesting that these Hsp90 mutants can be recruitedby GR, we found that all four Hsp90 HM mutants coimmuno-precipitated with apo-GR (Fig. 2E). However, in the case ofHM2m and HM10m, these interactions were at least partiallydue to nonspecific interactions with protein A/G agarose-boundantibodies. Moreover, these experiments did not resolve whetherthe interactions of GR with these Hsp90 mutants are direct ormediated by Hsp70. Yeast-expressing HM2m and HM10mshowed a temperature-sensitive phenotype, suggesting that thestructure of these mutants might be altered (Fig. 2B). Whereastrypsin digests with purified proteins at 25°C and 30°C confirmeddifferences in the structure of HM2m, in these studies as well asin unfolding experiments, WT Hsp90 and HM10m did notdisplay any differences (see Fig. 9, which is published as sup-porting information on the PNAS web site).

HM9 and HM10 Have the Structural Features of Potential GR-BindingSites. Based on the recently solved structure of full-length yeastHsp90 (22), the HM2 sequence is part of a buried �-sheet in theHsp90 middle domain, and HM11 is buried within the CTDhomodimer interface (Fig. 3A). Hence, these sequences arelikely not engaged in direct interactions with GR. In contrast,most of the hydrophobic residues of HM9 (I557 and L558) andHM10 (M589, I592, and M593) are solvent exposed and part ofhydrophobic surfaces in the Hsp90 CTD that could be engagedin protein–protein interactions (Fig. 3C). HM9 resides in �-helix1 (H1) and HM10 in �-helix 2 (H2) of the Hsp90 CTD. Becauseof the location and flexibility of H2, it has already been suggestedthat this �-helix plays a role in client protein binding (23).

In the case of transcriptional coregulators, sequences amino-

or carboxy-terminal to the conserved LxxLL motifs modulate theaffinity and specificity for NRs (24). Supporting the role of HM9and HM10 as potential GR-binding sites, Hsp90 residues adja-cent to HM9 (K552, A553, G559, and D560) and HM10 (W585and F586) are also solvent-exposed and homologous to theSR-binding site of p160 coactivators (NR-box 3) and to H12 ofGR and other NRs, respectively (Fig. 3B and C). P160 coacti-vators bind the hydrophobic groove of agonist-bound GR. H12interacts with the hydrophobic groove of GR bound to the mixedagonist/antagonist Ru486 (19).

HM9 and HM10 Differ in Their Interactions with apo- and holo-GR.Motivated by these sequence similarities, we next determinedwhether the Hsp90 HM9- and HM10-binding sites imitate theinteractions of coactivators or H12 of GR with the GR LBD. Tothis end, we studied the effects of peptides representing Hsp90H1 (pHM9), H2, (pHM10), and NR-binding site (NR-box) 3 ofthe p160 coactivator GRIP1 (pGRIP1) on the interaction of H12with the apo-GR LBD, the ability of apo-GR to bind hormone,and the interaction of GRIP1 with hormone-bound GR (Fig. 4).

The NR H12 is a structural switch that can bind the core LBDat different positions (4). In the presence of the agonist dexa-methasone (dex), the GR H12 occupies a position that facilitatesthe binding of coactivators to the hydrophobic groove, whereasin Ru486-bound GR, H12 docks within the hydrophobic grooveof GR and prevents the interaction of this groove with coacti-vators (18–19) (Fig. 4B). These GR LBD conformations can bedistinguished by their different sensitivity to trypsin (25). Al-though unliganded GR is readily digested by trypsin, dex- andRu486-bound GR display distinct trypsin-resistant LBD frag-ments that result from differences in the accessibility of K781 atthe C terminus of H12 (25) (Fig. 4C). To determine whether

Fig. 2. Functional analysis of hydrophobic hxxhh sequences in Hsp90. (A)Identified hxxhh sequences (HM1,. . . , HM11) in the Hsp90 middle domain andCTD (yeast HSP82 numbering). Alanine replacements of selected hydrophobicresidues (bold) within these sequences yielded the Hsp90 HM mutants (HM1m,. . . , HM11m). (B and C) Duplication times (B) and �-galactosidase activity (C)of yeast (YBD100) expressing GR and WT Hsp90, HM2m, HM9m, HM10m, orHM11m. Shown are the averages and standard deviations of at least threeindependent experiments performed in duplicate. (D) Immunoblot analysis ofHsp90 (WT/mutants) expression levels in YBD100 grown in the presence ofeither vehicle (ethanol, �H) or 10 �M cort (�H). Equal loading and transferwas monitored by Ponceau red staining of the immunoblot (data not shown).(E) Coimmunoprecipitation of Hsp90 and GR in yeast expressing either WTHsp90 or the Hsp90 HM mutants HM2m, HM9m, HM10m, and HM11m. GR-Hsp90 heterocomplexes were immunoprecipitated with either a GR-specificantibody (FiGR) or unspecific IgGs (negative control) and monitored by im-munoblot analysis. To reduce unspecific binding, in the case of HM10m, theamount of yeast lysates in the coimmunoprecipitation reactions was titrated.

Fig. 1. Identification of the hydrophobic groove of GR as a potentialHsp90-binding site. In the NR LBD, an allosteric network (green) transmitshormone-induced structural changes within the buried ligand-binding pocket(dark gray) into changes in a solvent-exposed hydrophobic groove (yellow)that interacts with amphipathic �-helices of coregulators (red) (18–19). Wefound that, in GR, replacements of residues within this allosteric network alterthe dependence of GR on Hsp90 (D.R., U. Hostick, L.F., K. Yamamoto, and B.D.,unpublished work, indicating that Hsp90 may stabilize the ligand-bindingpocket of apo-GR through interactions with this groove.

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Hsp90 affects the interaction of H12 with the core GR LBD, weanalyzed the trypsin sensitivity of GR in the absence andpresence of the pHM10 and pHM9 peptides. We found that inthe presence of pHM10, apo-GR showed similar trypsin-resistant fragments to Ru486-bound GR (Fig. 4C). pHM10 hadno effect on the ability of trypsin to cleave Ru486-bound GR,which demonstrates that the decreased sensitivity of apo-GR to

trypsin is not caused by changes in the activity or specificity oftrypsin (Fig. 4C). In contrast to pHM10, pHM9 slightly reducedthe activity of trypsin but did not alter the trypsin-cleavagepattern of apo- or Ru486-bound GR (Fig. 4C). These resultssuggested that pHM10 stabilizes apo-GR in a conformation thatis similar to that of Ru486-bound GR.

The interaction of coactivators with the hydrophobic groove ofdex-bound GR reduces the mobility of H12 and the release ofhormone. Consequently, in hormone-binding experiments,pGRIP1 increased binding of dex to GR by �70% (Fig. 4D).pHM9 also enhanced hormone binding (by 50%), whereas amutant pHM9 peptide (pHM9m), in which the hydrophobicLxxIL motif has been replaced by ‘‘AxxAA,’’ had no effect (Fig.4D). Contrary to pHM9, pHM10 reduced hormone binding byGR by �25%. The ability of coactivators to bind to the hydro-phobic groove depends on an ionic interaction of E773 in GRH12 with the backbone of the coactivator LxxLL motif (18).Consequently, replacement of E773 by arginine impaired thestabilizing effect of pGRIP1 on GR hormone binding (Fig. 4D).Indicating that binding of pHM9 to the hydrophobic grooverequires similar interactions, the E773R mutation also signifi-cantly curtailed the stabilizing effect of HM9 on GR hormonebinding but did not affect the destabilizing effect of pHM10.These results indicate that HM9 interacts with dex-bound GR bymimicking the interactions of coactivators with the hydrophobicgroove of GR. Consistent with this conclusion, we found that theGRIP1 NR-box 3 and the Hsp90 HM9 peptides, but not themutant HM9 or the HM10 peptides, are able to compete with aGST-GRIP1 fusion protein for binding to dex-bound GR (Fig.4E). However, whereas 30 �M pGRIP1 released �85% ofGRIP1-bound GR, in these experiments 300 �M pHM9 wasrequired to release about half of this amount (40%). Hence, theaffinity of the HM9 peptide for hormone-bound GR is likely inthe upper �M range. In conclusion, these in vitro assays indicatedthat Hsp90 HM10 binds apo-GR and stabilizes GR in a confor-mation similar to that of Ru486-bound GR, whereas the Hsp90HM9 mimics the interaction of the coactivator LxxLL motifswith holo-GR.

Replacement of HM10 and Treatment with GA Have Similar Effects onthe Expression, Nuclear Import, and Activity of GR in Yeast. TheHsp90 inhibitor GA blocks the transition from Hop- and Hsp70-mediated interactions to direct interactions between GR andHsp90, which arrests the GR-Hsp90 heterocomplex in a hor-mone-binding-incompetent state and ultimately triggers degra-dation of GR by the proteasome (7, 26). If the Hsp90 HM10sequence is necessary for direct binding of apo-GR, we expectthat HM10m yeast have similar phenotypes to yeast treated with

Fig. 3. HM9 and HM10 are potential GR-binding sites. (A) Based on the structure of full-length yeast Hsp90 (22), HM9 (‘‘L/MxxIL’’; red) resides in the Hsp90 CTD�-helix H1, HM10 (‘‘MxxIM’’; orange) in H2 and HM11 (LxxLL; black) in H4. (B) Sequence comparisons of HM9 and HM10 of human (h) and yeast (y) Hsp90 withthe NR-binding sites (NR boxes) of the p160 coactivator GRIP1 as well as with the �-helix H12 of selected SRs, respectively. MR, mineralcorticoid receptor; PR,progesterone receptor; AR, androgen receptor. Corresponding residues in Hsp90 and GRIP1 or H12 are colored. (C) Solvent accessibility of HM9, HM10, and HM11in the full-length yeast Hsp90 dimer (22) [CTD, blue; N-terminal (N) and middle (M) domains, gray]. L558 and I557 of HM9 (red) form a solvent-exposedhydrophobic surface that is extended by Y647 (violet; H4) and flanked by K522, A553, G559, and D560 (purple). The solvent-exposed hydrophobic surface formedby the HM10 residues I592, M593, and M589 (all orange) is flanked by F583, W583 (both yellow), and Q596 (brown). HM11 is buried within the homodimerinterface.

Fig. 4. HM9 and HM10 differ in their interactions with apo- and holo-GR. (A)Peptides representing NR-box 3 of the p160 coactivator GRIP1 and Hsp90 H1(pHM9), H2 (pHM10), and a mutant H1 (pHM9m) (sequences correspond tohHsp90� CTD). The hxxhh motifs are labeled (*). (B) Hormone-induced struc-tural changes in the GR LBD based on structural studies (18, 19). In Ru486-bound GR, H12 binds within the hydrophobic groove and blocks the interac-tion of GR with coactivators. In dex-bound GR, H12 forms one wall of thehydrophobic groove and stabilizes binding of coactivators to this groove. (C)The conformational changes shown in B alter the accessibility of K781 at theC terminus of H12 to trypsin. A trypsin-resistant GR LBD fragment that ischaracteristic for Ru486-bound GR is labeled (*). Autoradiogram of apo- andRu486-bound GR (WT and K781N mutant) after incubation with trypsin (0, 75,150, or 300 �g/ml) in the presence of vehicle (H2O), pHM10, or pHM9 (300 �Meach). (D) Binding of 3H-dex (4 nM; subsaturation) to in vitro-expressed GR (WTor E773R) � pGRIP1, pHM9, pHM9m, or pHM10 (300 �M each). Data werenormalized with respect to dex bound in the absence of peptides. (E) Inter-action of GR with GST or GST-GRIP1 (amino acid 563-1121) (15 �M) � pGRIP1(30 �M), pHM9, pHM9m, or pHM10 (300 �M each). The fraction of GR boundto GST-GRIP1 [% Input] was quantified by using PhosphorImaging. The resultsshown in D and E represent the averages and standard deviations of at leastfour independent experiments.

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GA. As shown in Fig. 2C, the Hsp90 mutant HM10m stronglyimpaired the transcriptional activity of GR. Moreover, immu-noblot analyses revealed that like GA, the presence of HM10m,but not any of the other Hsp90 mutants, reduces the expressionlevels of apo-GR (Fig. 5A). In contrast to apo-GR, the expres-sion of proteins that are not Hsp90 clients (e.g., the vacuolarH�-ATPase component Vma2) were unaffected by HM10m(data not shown).

Another function of the interaction of Hsp90 with GR is toprevent nuclear import of apo-GR. In the presence of Hsp90,WT and HM11m nuclear import of GFP-GR was hormone-dependent, whereas in �25% of yeast expressing HM9m GR waspredominantly nuclear in the absence of hormone (Fig. 5B). Inmost HM10m yeast, nuclear GFP-GR staining was below thelevel of detection both in the absence and presence of hormone(Fig. 5B). Similar to GA-treated yeast and mammalian cells thatexpress a GFP-GR fusion protein, we found that, in HM10myeast, GR often displayed a ‘‘punctate’’ localization pattern thatis likely formed by partially degraded and aggregated GFP-GRfusion proteins (27) (Fig. 5B). Consistent with this interpreta-tion, immunoblot analysis of these cells using a GFP antibodyshowed high levels of GFP-domain-containing GR fragmentsbut low levels of full-length GFP-GR fusion protein (data notshown). Demonstrating that this phenotype is specific forHM10m and not a general consequence of impaired Hsp90activity, in yeast expressing HM2m, GR was constitutivelynuclear and not aggregated (data not shown). In control exper-iments, we determined that fusion of GFP to either the N or Cterminus of GR did not affect the functional interactions of GRwith Hsp90 (data not shown). These results are consistent withthe role of HM10 as a direct binding site of apo-GR.

DiscussionIt is only by understanding the nature of the Hsp90–clientprotein complex that we can begin to comprehend the functionalroles of this unusual chaperone. In this study, we provideevidence that Hsp90 interacts with apo- and holo-GR throughtwo distinct binding sites in the Hsp90 CTD. The interaction ofapo-GR with Hsp90 appears to be mediated by the Hsp90 �-helix2, which contains the HM10 ‘‘MxxIM’’ sequence as well as othersolvent-exposed hydrophobic residues whose contributions tothe interaction of Hsp90 with GR remain to be investigated. H2has sequence homologies to H12 at the C terminus of the SRLBD, which regulates the interactions of SRs with coregulators.We found that binding of an H2 peptide (pHM10) stabilizes

apo-GR in a conformation in which H12 docks within thehydrophobic groove at the surface of the GR LBD. Thisconformation appears to be similar to that of GR bound to theantagonist Ru486, which explains previous observations indi-cating that Ru486 stabilizes GR-Hsp90 heterocomplexes (19,28). These results indicate that Hsp90 interacts with a definedsurface in the apo-GR LBD and stabilizes the unligandedligand-binding pocket indirectly by promoting the interaction ofH12 with the hydrophobic groove. These observations imply thatthe differences in the dependence of apo-SRs on Hsp90 maycorrelate with the ability of these receptors to adopt thisconformation.

The similar in vivo phenotypes of HM10m yeast and yeasttreated with GA suggest that the Hsp90 mutant HM10m trapsthe GR-Hsp90 complex in a premature, hormone-binding-incompetent state in which interactions between GR and Hsp90are mediated by Hsp70 and Hop and are not stabilized by p23and immunophilins. The indirect recruitment of Hsp90 byHsp70-primed apo-GR may enable Hsp90 to overcome the lowaffinity of the Hsp90 HM10-binding site for GR (upper �Mrange) allowing the direct interaction between Hsp90 and GR toremain dynamic. In turn, a dynamic Hsp90-GR interaction wouldlikely relieve inhibition of hormone access to the ligand-bindingpocket by Hsp90. These predictions could be tested by analyzingthe interactions of HM10m and of the other Hsp90 mutants withcochaperones involved in the assembly and maturation of GR-Hsp90 heterocomplexes.

Binding of hormone to the GR LBD enables H12 to replaceHsp90 and to provide contacts that stabilize the interaction oftranscriptional coactivators with the hydrophobic groove. Ourbinding studies demonstrated that these hormone-induced struc-tural changes in GR also facilitate interaction of GR with theHsp90 HM9-binding site (Fig. 6A). Hence, hormone binding byGR might transfer GR from the Hsp90 HM10 to the HM9-binding site. In the crystal structure of yeast Hsp90, HM9 andHM10 are in close proximity to each other, suggesting that thistransfer might be accomplished without the dissociation of theGR-Hsp90 heterocomplex. Because HM9 is near the binding siteof the immunophilins FKBP51 and FKBP52 at the Hsp90 Cterminus, transfer of GR from HM10 to HM9 might trigger theobserved hormone-induced exchange of the immunophilinsFKBP51 by FKBP52 (10). Moreover, the ability of the Hsp90

Fig. 5. Expression of HM10m and treatment of yeast with GA result in similarphenotypes. (A) Immunoblot analysis of GR protein levels in yeast (YBD100)expressing Hsp90 WT, HM9m, HM10m, or HM11m in the presence of eithervehicle (ethanol, �H) or 10 �M cort (�H). Equal loading and transfer wasmonitored by Ponceau red staining of the immunoblot (data not shown). (B)Subcellular localization of GFP-GR in live yeast expressing Hsp90 WT, HM9m,HM10m, or HM11m. The vacuole and the nucleus of yeast cells are identifiedin one of the Hsp90 WT images. In the case of HM9m (�H), �25% of yeast cellsdisplayed nuclear GFP staining. All other phenotypes are representative of�90% of cells of the respective cultures (evaluated cells, n � 200).

Fig. 6. Proposed interactions of Hsp90 with apo- and holo-GR. (A) In apo-GR,H12 is flexible and can adopt various positions. By binding apo-GR at a positionthat, in holo-GR, is occupied by �-helix H12 of GR, Hsp90 HM10 foces GR H12to dock within the hydrophobic groove, which stabilizes the unligandedligand-binding pocket. Upon hormone binding, GR H12 replaces Hsp90 HM10and provides the contacts that enable coactivators or Hsp90 HM9 to bindwithin the hydrophobic groove. (B) Location of known Hsp90 mutations thataffect the formation of functional GR-Hsp90 heterocomplexes (black) (22,29–30). An amphipathic loop that is bound by kinases is shown in green (31).

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HM9-binding site to compete with the coactivator LxxLL motifsfor binding to the hydrophobic groove provides a potentialexplanation for the observed ability of Hsp90 to inhibit thetranscriptional activity of GR (11, 13). These interactions ofHsp90 with apo- and holo-GR appear to optimize the transitionbetween inactive and active states of GR, resulting in a sharp-ening of the cellular response to hormone.

Although the yeast system is very well established and hasbeen extensively used to characterize the activity of apo-GR-Hsp90 heterocomplexes, little is known about the functionalroles of holo-GR-Hsp90 heterocomplexes in yeast. Moreover,some mechanisms that regulate the nuclear translocation andtranscriptional activity of GR in mammalian cells are not usedin yeast. In particular, yeast lack most of the known coactivatorsthat interact with the hydrophobic groove in the GR LBD.Therefore, further studies on the interaction of GR with theseputative Hsp90 interaction surfaces and evaluation of the con-tributions of these interactions to the cellular response tohormone need to be conducted in mammalian cells. Because thephenotypes of the Hsp90 HM mutants are not dominant nega-tive, these studies require mammalian cell lines that allow theselective expression of Hsp90 mutants. Selective down-regulation of endogenous Hsp90s by RNA interference could bea possible strategy to obtain these lines. However, because of theextraordinarily high cellular concentration of Hsp90, the highsequence conservation of Hsp90, and the importance of Hsp90for cell viability, the construction and functional analysis of theselines will not be easy.

Functional screens by others have identified various mutationsin the middle and C-terminal domain of Hsp90 that disrupt theformation or function of GR-Hsp90 heterocomplexes (29–30).Based on the location of the replaced residues in full-length yeastHsp90, some of these mutations likely impair the formation ofthe GR-Hsp90 heterocomplex indirectly by affecting either theN-terminal ATPase activity of Hsp90 or the recruitment of p23and other cochaperones (Fig. 6B). Several mutations (A587T,T525I, and R579K) replace residues at the base of the flexibleloop that contains HM10. Based on their location, it is unlikelythat these residues bind GR directly. However, they mightcontribute to the interaction of Hsp90 with GR by affecting theconformation of H2. One of the few solvent-exposed residuesidentified by these mutational analyses is E431, which is locatedin the middle domain (Fig. 6B). Replacement of E431 by lysineselectively impairs the interaction of Hsp90 with GR (29),providing further evidence that GR binds Hsp90 at the boundarybetween the middle and C-terminal domain of Hsp90. Thismutant also suggests that the interactions of GR with Hsp90 arenot restricted to the Hsp90 HM9- and HM10-binding sites butinvolve other contacts within Hsp90. Because of the dependenceon the Hsp70 assembly complex, the structural characterizationof GR-Hsp90 heterocomplexes is likely beyond reach in the nearfuture. However, the structural analysis of GR LBDs bound toHM9 or HM10 peptides may be feasible and could provide theentrance for the structural characterization of Hsp90–clientprotein interactions.

It has been shown that the kinase PKB/Akt binds a flexible,amphipathic loop in the Hsp90 middle domain that protrudesinto a cleft formed by the Hsp90 homodimer (Fig. 6B) (31).Because this kinase-binding site is in close proximity to HM10,kinases and SRs may bind to similar regions of Hsp90, which isconsistent with conclusions from previous mutational studies(30). Although it still remains to be shown experimentally, it islikely that this hydrophobic loop and the hydrophobic surfacesformed by HM9 and HM10 differ in their specificity for clientproteins and function as distinct client protein-binding sites.Indicating that HM9 and HM10 may interact with SRs selec-tively, we found that, in yeast, HM9m and HM10m have verylittle effect on the ability of the estrogen receptor to activate

transcription in response to hormone (L. Kelley and B.D.,unpublished observation). The interaction of Hsp90 with somany physiologically important client proteins has been a majorobstacle in the functional analysis and therapeutic manipulationof Hsp90. The identification of client-specific Hsp90-bindingsites would provide the means for a comprehensive functionaland structural analysis of Hsp90–client protein interactions andcould lead to novel rationales for the therapeutic targeting ofspecific Hsp90 client pathways.

Materials and MethodsYeast Strains and Transformations. YBD100 (PDR5::LEU2::GT3Z,HSC82::URA3, HSP82::GAL1-HSP82::LEU2) was generated bycrossing YNK410 (32) and 5CG2 (33). Yeast Hsp90 (WT andmutant Hsp82) and rat GR were expressed by using pRS316(pTCA) and pRS313 (pHCA) expression vectors and a constitutiveyeast glyceraldehyde-3-phosphate dehydrogenase (GPD) pro-moter. Transformation of YBD100 followed a standard lithiumacetate protocol. If not noted otherwise, YBD100 (GR, Hsp90)transformants were grown at 25°C in minimal medium (S) con-taining amino acids (-his, -ura, -leu, -trp) and 2% glucose (D).

Growth Assay. Saturated cultures of YBD100 (GR, Hsp90) werediluted 1:100 in SD-(his, ura, leu, trp) containing either 10 �Mcorticosterone (cort) or vehicle (ethanol). Cultures were incubatedin side-arm flasks at 25°C and 30°C, and their optical density(OD600) was monitored at 2-hour intervals until saturation.

�-Galactosidase Assays. Saturated cultures of YBD100 (GR,Hsp90) were diluted 1:50 in SD-(his, ura, leu, trp) containingeither vehicle (ethanol) or cort (10 nM-100 �M). Cultures wereincubated in 96-well microtiter plates at 25°C until they reachedOD600 0.6 (�16 h). �-galactosidase activity (vmax) was measuredas described (34).

Immunoblots. Saturated YBD100 (GR, Hsp90) cultures werediluted 1:100 in 25 ml of SD-(his, ura, leu, trp) containing eithervehicle (ethanol) or 10 �M cort. Cultures were incubated at 25°Cto an OD600 of 0.7–0.8 (�12 h), harvested by centrifugation(600 � g at room temperature for 5 min) and resuspended in 10mM Tris�HCl (pH 7.5), 50 mM NaCl, 10% glycerol, 2 mM DTT,0.2 mM PMSF, 1 mM EDTA (pH 7.5), and protease inhibitors(1 �g/ml leupeptin, pepstatin, and aprotinin, and 1 mM PMSFSigma, St. Louis, MO). Cells were lysed by vortexing withacid-washed glass beads (425–600 �m) (4°C for 40 min), celllysates were cleared twice by centrifugation (20,000 � g at 4°Cfor 15 min), and protein concentrations were quantified (proteinassay; Bio-Rad, Hercules, CA). Proteins (100 �g per lane) wereseparated by SDS/PAGE, and probed by using specific antibod-ies against GR (BUGR2; Abcam, Cambridge, U.K.), Hsp90 (giftfrom S. Lindquist, Massachusetts Institute of Technology, Cam-bridge, MA) or Vma2p (gift from T. Stevens, University ofOregon, Eugene, OR).

In Vitro Transcription-Translation. The rat GR mutants E773R andK781N were constructed by QuickChange PCR (Stratagene, LaJolla, CA). Cloning of pSG5 rat GR has been described (26). Asimilar cloning strategy yielded pSG5 E773R, pSG5 K781N, andpSP6T Hsp82. GR and yeast Hsp90 were expressed in theabsence or presence of L-[35S]methionine (PerkinElmer, Welle-sley, MA) by using a coupled reticulocyte lysate expression kit(TNT; Promega, Madison, WI). To obtain hormone-bound GR,10 �M hormone (cort, dex, and Ru486) were added duringsynthesis.

GR-Hsp90 Coimmunoprecipitations. Coimmunoprecipitation ofGR-Hsp90 heterocomplexes followed the procedures describedin ref. 35.

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Hormone Binding. Hormone-binding assays were performed as inref. 36, except that our reactions (40 �l) contained 10 �l of invitro-expressed GR (1–10 pmol), 0–300 �M coactivator or Hsp90peptides, and 2–50 nM [1,2,4,6,7-3H]dex (Amersham Pharmacia,Piscataway, NJ).

Limited Proteolysis. GRIP1 and Hsp90 peptides (for sequencessee Fig. 5A) were purchased from GenScript (www.genscript.com) and dissolved in water at a concentration of 10 mM. Invitro-expressed, 35S-labeled GR (WT or K781N) were incubatedwith Hsp90 HM9 or HM10 peptides (300 �M each) or vehicle(H2O) at 30°C for 30 min. Two microliters of these reactionswere digested with trypsin (0–300 �g/ml, Roche, Indianapolis,IN) in 20 mM Tris�HCl (pH 8.0), 0.1 M NaCl, and 10% glycerol(total volume 10 �l). Reactions were incubated on ice for 1 h andstopped by adding 2� SDS loading buffer. Proteolytic fragmentswere separated by PAGE and visualized by autoradiography.

Coactivator Peptide Competitions. GST and GST-GRIP1 wereexpressed, purified, and bound to glutathione agarose as de-scribed (20). For peptide competition, 12.5 �l of reticulocytelysate containing 500,000 counts of in vitro-expressed, 35S-labeled GR were incubated at 4°C for 2 h with 0–300 �Mpeptides and 20 �l of agarose-bound GST or GST-GRIP1 (15�M) in a total volume of 50 �l of 20 mM Tris�HCl (pH 8.0), 0.1M NaCl, 10% glycerol, 10 �M dex, 0.01% Nonidet P-40, 1 mM

DTT, 20 �g/ml BSA, 0.1 mM PMSF, and protease inhibitors(Complete; Roche). Agarose-bound proteins were collected bycentrifugation (1,000 � g for 1 min at 4°C), washed five timeswith 200 �l of binding buffer, eluted by boiling in 20 �l of SDSloading buffer, and analyzed by SDS/PAGE. Bound receptorswere quantified by PhosphorImaging (Molecular Dynamics,Sunnyvale, CA).

Nuclear-Cytoplasmic Localization. YBD100 were transformed withpTCA GFP-GR (kind gift of K. Yamamoto, University ofCalifornia, San Francisco, CA) and pHCA Hsp82 (WT/mutants)and grown as outlined before. Saturated cultures were diluted1:50 into SD-(his, ura, leu, trp) containing either cort (10 �M),GA (50 �M; Stressgen Bioreagents, Ann Arbor, MI), or vehicle(ethanol) and grown at 25°C to an OD600 0.5–1.0 (�12 h).Cultures (10 �l) were spotted on a coverslip, and localization ofGFP-GR was assessed by fluorescence microscopy (Axioplan 2;Zeiss, Thornwood, NY) by using a �100 oil-immersion lens.

We thank J. Morris, T. Kawamura, M. Muller, S. Noble, E. Cogan, R.Salvador, and J. Jacobson for help with the characterization of GR andHsp90 mutants; Drs. K. Yamamoto, T. Stevens, and S. Lindquist formaterials; and Drs. P. von Hippel, A. Berglund, T. Stevens, and I. Rogatskyfor critical comments on the manuscript. This work was supported byNational Institutes of Health Training Grant NIH T32 GM07759 (to D.R.),the Leukemia and Lymphoma Foundation (LSA3140–00) and PhilipMorris USA Inc. and by Philip Morris International. (to B.D.).

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