(Received for publication, August 18, 1995)
From the
In order to attain competence to respond to hormone, certain steroid hormone receptors must be assembled into hetero-oligomeric aporeceptor complexes, containing Hsp90 and other proteins. Members of the Hsp90 gene family are highly conserved, strongly expressed, and required for viability in eukaryotic organisms. To elucidate the role of Hsp90 in the activity of steroid hormone receptors in vivo, four Hsp90 mutants, which cause defects in glucocorticoid receptor (GR) signaling, but support the viability of Saccharomyces cerevisiae, were previously isolated (Bohen, S. P., and Yamamoto, K. R.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11424-11428). In this study, I characterize the effects of the Hsp90 mutants on GR ligand response, ligand binding activity, and aporeceptor complex stability. The mutants fall into two classes. Three of the Hsp90 mutants cause defects in GR ligand binding in vivo and form aporeceptor complexes that are unstable in vitro, relative to those containing wild-type Hsp90. The other mutant affects GR signaling, but aporeceptor complexes with this mutant are not defective for ligand binding or stability. These findings indicate that the binding of Hsp90 to GR in the aporeceptor complex is insufficient to induce a high ligand affinity conformation, rather the high ligand affinity of GR requires a specific interaction with Hsp90, which is altered by certain Hsp90 mutants.
The 90-kDa heat shock protein (Hsp90) ()is a highly
conserved stress-induced protein that is abundantly expressed in almost
all cells under nonstress conditions and is required for viability in
eukaryotes. Biochemical analysis has shown that Hsp90 interacts with
various proteins, including signaling molecules(1) , other heat
shock proteins (2, 3) , and cytoskeletal
proteins(4, 5) , but the function of Hsp90 in these
complexes remains unclear. The physical association of Hsp90 with
steroid hormone
receptors(6, 7, 8, 9) , the
basic-helix-loop-helix dioxin receptor (DR)(10) , v-src family tyrosine kinases(1, 11) , the c-raf serine/threonine kinase(12) , and the
subunits
of G proteins(13) , together with genetic evidence that Hsp90
is involved in the function of the sevenless receptor tyrosine
kinase (14) has led to speculation that Hsp90 may play a common
role in the activity of diverse signaling systems. Genetic analysis in
yeast expressing low levels of Hsp90 (15, 16) or hsp90
point mutants (17, 18) indicates that Hsp90 is
required for steroid receptor and v-src signaling in
vivo. Furthermore, drugs that bind to Hsp90 and inhibit the
formation of v-src-Hsp90 complexes can reverse v-src transformation of mammalian cells(19) .
The role of Hsp90 in signal transduction is best characterized for steroid hormone receptors where Hsp90 appears to be required for the recognition of and response to ligand. In the absence of bound agonist, some of the steroid receptors, including the glucocorticoid receptor (GR), are components of aporeceptor complexes composed of certain nonreceptor proteins, minimally a dimer of Hsp90 and a monomer of hsp56/FKBP59(20, 21) . GR activation is a response to an increase in available ligand concentration that drives ligand binding to the receptor, as a component of the aporeceptor complex. The liganded aporeceptor then dissociates from the other components of the complex, resulting in receptor ``activation'' (for review, see Refs. 9 and 22); the activated receptor translocates into the nucleus, binds to specific sites on the chromosome, and modulates the transcriptional activity of target genes. Following ligand withdrawal, unliganded receptors are recycled into cytoplasmic aporeceptor complexes, reconstituting high ligand affinity, and restoring competence to undergo further rounds of activation(23) .
It
is the aporeceptor complex that recognizes and transduces the steroid
hormone signal. Several lines of evidence correlate GRHsp90
complex formation with competence to respond to ligand(9) . For
example, the high affinity ligand binding activity of the GR
aporeceptor complex is lost when Hsp90 is dissociated from GR in
vitro(24) , and reconstitution of the complex in
reticulocyte lysates restores high ligand affinity(25) . In
vivo, expression of decreased levels of Hsp90 in yeast results in
a decrease in the sensitivity of receptor to ligand(15) . In an
analogous situation, GR in rat epididymal cells, which lack Hsp90,
displays no ligand binding activity(26) . Correlations have
also been made in vitro between Hsp90 complex formation and
the ligand binding activities of the mineralocorticoid(27) ,
progesterone(28) , and dioxin receptors(29) . Notably,
however, analysis of a series of hsp90 point mutants shows that
GR
Hsp90 complex formation alone is not sufficient for normal
signal transduction(17) .
Four point mutants in HSP82, one of the yeast Hsp90 genes, were previously identified in a yeast genetic screen for decreased sensitivity of GR to ligand(17) . These mutants define two phenotypic classes based on their receptor specificity and on their effects on cell growth. One of the mutants, E431K, affects GR signaling, but alters neither the activities of mineralocorticoid, progesterone, and the estrogen receptor nor the growth rate or temperature sensitivity of yeast. In contrast, the other mutants, G313N, T525I, and A576T/R579K (a double mutant), affect all receptor types tested, result in temperature sensitivity for growth at 37 °C, and modestly decrease growth rates at permissive temperature. Interestingly, all of the hsp82 mutants seem to form complexes with GR in vivo, as assessed by coimmunoprecipitation with GR in yeast extracts.
There are several
mechanisms whereby hsp90 mutants that remain competent to associate
with steroid receptors may produce defects in receptor signaling. (i)
Mutant hsp90 may fail to induce a high ligand affinity conformation in
the receptor ligand binding domain, resulting in aporeceptor complexes
with decreased ability to bind ligand. (ii) Mutants may form
aporeceptor complexes with normal ligand binding activity, but mutant
hsp90 may not dissociate efficiently from the liganded aporeceptor.
(iii) Mutant hsp90 might form aporeceptor complexes that bind ligand
normally and efficiently dissociate from the liganded receptor, without
facilitating a further conformational change necessary to accomplish
transcriptional regulation. In previous experiments, efforts to
elucidate the role of Hsp90 in signaling were complicated by the fact
that association of Hsp90 with its target proteins was disrupted. Thus,
the hsp90 mutants presented a unique opportunity to gain new insight
into the role of Hsp90 in steroid receptor signal transduction through
biochemical analysis of the GRhsp90 interaction in defective
aporeceptor complexes.
To test whether F620S GR is an appropriate model for the role of Hsp90 in steroid receptor function, I first assessed the effects of the hsp82 mutants on the F620S GR ligand response (Fig. 1A). As with wild-type GR (Fig. 1B), the different hsp82 mutants produced a spectrum of signal transduction phenotypes; G313N resulted in a very severe decrease in dexamethasone-responsiveness, whereas T525I and A576T/R579K caused less severe signaling defects. The E431K mutant produced a subtle but significant decrease in the response of F620S GR. Thus, although F620S GR responds to lower concentrations of dexamethasone than does wild-type GR, signal transduction by wild-type and F620S GR was similarly affected by the hsp82 mutants, and, importantly, the individual mutants followed the same order of severity within the spectrum of mutant phenotypes (Fig. 1, compare A and B). These findings indicate that Hsp90 functions in a similar capacity in signaling by F620S and wild-type GR. Thus, F620S mutant GR provides a useful reagent for testing the effects of hsp82 mutants on the ligand binding activity of steroid receptors.
Figure 1:
Effects of hsp82
mutants on the response of GR to dexamethasone. A, response of
F620S GR; B, response of wild-type GR. Yeast cells
coexpressing wild-type or mutant hsp82 and F620S or wild-type GR and
containing a GR-inducible lacZ reporter were incubated overnight at
room temperature with the indicated concentrations of dexamethasone.
Cells were harvested, and -galactosidase activities were measured,
as described under ``Experimental Procedures.'' Data,
expressed in
-galactosidase units, represent the mean ±
range of two independent samples from a given experiment and are
representative of several experiments.
Figure 2:
Dexamethasone binding to GR in yeast
expressing wild-type versus mutant hsp82. Yeast cells
expressing F620S GR and wild-type or mutant hsp82 were preincubated
with FK506 for 2 h at room temperature (see ``Experimental
Procedures''), followed by a 2-h incubation with
[H]dexamethasone, with or without cold
dexamethasone competitor. Cells were washed 3 times with ice-cold PBS
plus 2% glucose, and counts bound were determined. Data represent the
mean ± range of specific counts bound at a given dexamethasone
concentration for two independent samples from a given experiment, and
are representative of several experiments. All data are normalized to
the A
of the
cultures.
In contrast to the other hsp82 mutants, dexamethasone binding activity in cells coexpressing E431K mutant hsp82 and F620S GR was identical to that in cells expressing the wild-type Hsp82 protein (Fig. 2). Although E431K produced the least severe ligand response defect (Fig. 1), comparison of the ligand response and in vivo dexamethasone binding defects of the other hsp82 mutants suggested that I would have detected a binding defect in the context of E431K if such a defect underlies the E431K ligand response phenotype. The lack of a ligand binding defect suggests that E431K and wild-type Hsp82 are capable of forming GR aporeceptor complexes of equivalent number and affinity. Thus, the E431K phenotype is consistent with the hypothesis that alterations in Hsp90 may affect a step in GR activation downstream of ligand binding; for example, E431K hsp82 might not dissociate efficiently from ligand-bound GR.
Figure 3: Stability of GR aporeceptor complexes. A, GR was immunoprecipitated under nondenaturing conditions from extracts of yeast cells expressing wild-type GR and wild-type or mutant hsp82. IPs were then analyzed for GR and hsp82 by immunoblotting. Note that the apparently decreased level of T525I hsp82 associated with GR in this blot (Fig. 3A, lane 4) is not a consistent finding; in most trials, coimmunoprecipitation of wild-type and mutant hsp82s with GR is comparable. B, relative stability of immunoprecipitated aporeceptor complexes containing wild-type versus mutant hsp82 was determined. Washed IPs were resuspended in 200 mM KCl at pH 7.5 and incubated at 0 °C for 2 h, and GR and hsp82 levels were determined by immunoblotting. All strains expressed wild-type GR and contained a lacZ reporter plasmid; numbers below the lanes correspond to the hsp82 mutant being expressed. Lane 1, wild-type; lane 2, G313N; lane 3, E431K; lane 4, T525I; lane 5, A576T/R579K. Data are from a single experiment and representative of three independent experiments.
Consistent with previous findings(17) , wild-type and mutant hsp82s efficiently coimmunoprecipitated with GR in yeast extracts (Fig. 3A). Interestingly, however, aporeceptor complexes containing G313N, T525I, or A576T/R579K mutant hsp82 were less stable to incubation with salt (Fig. 3B). Thus, under the conditions examined, these hsp82 mutants bind less tightly to GR than does wild-type hsp82. In contrast, complexes containing E431K mutant hsp82 did not appear to be significantly less stable. Similar results were obtained in coimmunoprecipitations of wild-type or mutant hsp82 with F620S GR (data not shown; the interaction of A576T/R579K hsp82 and F620S GR was not examined). Three of the hsp82 mutants display altered interactions with GR or with some component of the aporeceptor complex that is required for its stability. Thus, the GR ligand response and ligand binding defects caused by the hsp82 mutants are likely the direct result of these aberrant interactions.
In this report, I have examined the activity of GR and the properties of the GR aporeceptor complex in the context of hsp82 mutants that affect GR signal transduction in yeast. I demonstrated previously that these hsp82 mutants define two phenotypic classes(17) . Mutants in one class, composed of the G313N, T525I, and A576T/R579K hsp82 mutants, affect signaling by all steroid hormone receptors tested and cause decreased growth rates and temperature sensitivity; the second class of mutants, represented by E431K hsp82, affects GR signaling selectively, producing no defects in signaling by other steroid receptors or in cell growth. Interestingly, the mutants segregate into these same two classes on the basis of in vivo ligand binding activity and aporeceptor complex stability. The G313N, T525I, and A576T/R579K hsp82 mutants compromised GR ligand binding in vivo and formed aporeceptor complexes with decreased stability in vitro; in contrast, E431K caused no ligand binding defect in vivo and no significant decrease in aporeceptor complex stability in vitro. I shall consider the two mutant classes separately.
The GR signaling defect observed
in yeast expressing G313N, T525I, or A576T/R579K mutant hsp82 is
explained by decreased ligand binding activity. There are several
mechanisms whereby these mutants could alter their interaction with GR
to produce the observed phenotypes. Mutant hsp82 could abrogate the
formation of aporeceptor complexes, form a normal number of aporeceptor
complexes that are defective for ligand binding, result in decreased GR
protein levels, or disrupt GR ligand binding in vivo via one
of a myriad of potential indirect mechanisms. Several of these models
have been tested experimentally. For example, GR protein levels are
indistinguishable in strains expressing wild-type and mutant
hsp82(17) . Furthermore, the hsp82 mutant proteins
coimmunoprecipitate with GR from yeast extracts, suggesting that they
assemble into aporeceptor complexes in vivo (Fig. 3A and (17) ). (I have demonstrated
that the association of hsp82 with GR is specific (17) and that
GRhsp82 complexes form prior to cell lysis (data not shown).) In
principle, GR and hsp82 might associate passively and artifactually,
for example during chilling before lysis, but pulse-chase experiments
have demonstrated that GR
Hsp90 complexes are present in vivo in mammalian cells(38) , and aporeceptor complex assembly
in reticulocyte lysates requires ATP hydrolysis and moderate
temperature (25, 39) .) Finally, although it is
formally possible that the hsp82 mutants might indirectly affect
receptor signaling, the finding that mutant aporeceptor complexes are
less stable in yeast extracts is most consistent with signal
transduction defects resulting directly from an alteration in the
GR
Hsp90 interaction. Thus, although this class of hsp82 mutants
is competent to form aporeceptor complexes with GR, the defect in the
GR
mutant hsp82 interaction results in a corresponding defect in
aporeceptor ligand binding, thereby compromising GR signal transduction
and subsequent transcription activation.
In addition to the receptor, Hsp90 and hsp56/FKBP59, other proteins have been identified as components of aporeceptor complexes(40) , including hsp70, p60, which is a protein homologous to Sti1, p23(41) , and Ydj1, a yeast DnaJ-like protein(42) . It has been demonstrated that p23 (43) and hsp70 (44) are required for aporeceptor complex formation in reticulocyte lysates and that DnaJ plays a role in receptor function in vivo in yeast(42, 45) , but how these proteins affect receptor signaling is unknown. Cross-linking studies suggest that Hsp90 is the only protein interacting directly with GR in the aporeceptor complex(20) , and characterization of the interaction of Hsp90 with deletion mutants of GR indicates that the Hsp90 binds to GR within the region of the receptor that is responsible for recognizing ligand(46) ; these observations have led to the hypothesis that the interaction of Hsp90 directly with the GR ligand binding domain induces a high ligand affinity conformation in the receptor. The effects of hsp82 mutants on aporeceptor complex stability and ligand affinity may be the result of alterations in the interaction of Hsp82 with the ligand binding domain of GR directly or with other proteins in the aporeceptor complex that are important for its stability and function.
Given GR ligand
binding defects observed in vivo in cells expressing these
hsp82 mutants, I attempted to measure the dexamethasone binding
affinity of wild-type and mutant aporeceptor complexes in yeast
extracts. To my surprise the apparent affinity of receptor for ligand in vitro was not altered by the hsp82 mutants (data not
shown). This finding seems to contradict the in vivo binding
data; however, several observations compel me to conclude that the
problem probably lies in the in vitro binding assay itself. A
comparison of total GR levels, as determined by immunoblotting, versus ligand binding suggests that only about 2% of the
receptor expressed in yeast binds ligand in
vitro(36) . Furthermore, reduced expression of Hsp82 to 5%
of wild-type levels produces severe defects in GR signaling and ligand
binding in vivo, and Hsp82 does not coimmunoprecipitate with
GR in these extracts(15) , yet no defect in GR ligand binding
is observed in these extracts (data not shown). Hence, the residual
binding in vitro may represent either a subset of receptors
that attains a high affinity conformation without bound Hsp82 or a
small fraction of aporeceptor complexes that remains stably associated
in the extract. In either case, the in vitro binding assay
does not reflect the state of the GRhsp82 interaction in
vivo.
E431K mutant hsp82 affects signaling by F620S and wild-type GR, but no other phenotype of E431K hsp82 has been elicited genetically or biochemically. It is possible that E431K is mechanistically related to the other hsp82 mutants, resulting in aporeceptor complexes with compromised ligand binding activity but that the E431K defect is simply too subtle to be detected in the in vivo ligand binding assay. Alternatively, E431K may be mechanistically distinct and cause a defect in some aspect of GR signal transduction subsequent to ligand binding, such as Hsp90 dissociation or an as yet uncharacterized change in GR conformation that is mediated by Hsp90 and is required for GR to become competent for transcriptional regulation. In this case, E431K would represent a particularly useful reagent for the elucidation of Hsp90 function in receptor signaling subsequent to ligand binding.
The findings presented here yield several interesting insights into the role of Hsp90 in receptor signal transduction. It appears that aporeceptor complex formation alone is not sufficient for efficient ligand binding by steroid receptors; not surprisingly, Hsp90 must assume the proper conformation in these complexes to confer high ligand affinity to receptors. Although alteration of the Hsp90 interaction with target proteins has not been shown to regulate any system, it is interesting that changes in the interaction of Hsp90 with any of a number of target proteins may be used to regulate the activity of diverse signaling pathways. This proposal is given credence by the recent finding that drugs of the benzoquinone ansamycin family, which alter the activities of v-src and steroid receptors in vivo, bind to Hsp90 in vitro(19) . Analysis of E431K mutant hsp82 suggests that alterations in Hsp90 conformation may affect steps in signal transduction subsequent to ligand binding. Furthermore, such alterations in Hsp82 may be subtle enough to alter signaling specifically by particular Hsp82 target proteins.
Finally, it is intriguing to consider the origin of the steroid receptor-Hsp90 interaction. Members of the nuclear receptor family display a spectrum of dependences on Hsp90 for signaling(9) . For example, in contrast to GR, an interaction of Hsp90 with thyroid and retinoid receptors has not been demonstrated, and these receptors can bind their cognate ligands with high affinity as purified proteins (47) ; thus, it has been hypothesized that thyroid and retinoid receptors do not require Hsp90 for signaling. However, examination of signaling by these receptors in yeast expressing a low level of hsp82 demonstrates that these receptors are dependent on Hsp90 for normal ligand binding activity(48) . It seems likely that a transient interaction with Hsp90 is required sometime during or shortly after synthesis of these receptors to achieve a high ligand affinity conformation. GR, mineralocorticoid, and progesterone receptors may represent a subset of receptors whose intrinsic folding demands continuous interaction with Hsp90 to achieve a functional conformation. Such a strong dependence on Hsp90 may simply reflect an unstable conformation or the evolution of an additional potential for the regulation of these receptors. It is interesting to note that a similar interaction has arisen independently in DR(10) . DR is a basic-helix-loop-helix protein with no sequence homology to steroid hormone receptors. However, DR is a ligand regulated transcription factor that is dependent on bound Hsp90 to achieve high ligand affinity. Thus, the interaction of steroid receptors with Hsp90 may represent a common form of regulation of signaling systems by linking the proper folding of signal transduction proteins to a stable interaction with Hsp90.