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Department of Pharmacology (W.B.P.), The University of Michigan Medical School, Ann Arbor, Michigan 48109; and Department of Biochemistry and Molecular Biology (D.O.T.), Mayo Graduate School, Rochester, Minnesota 55905
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Abstract |
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 Top Abstract I. IntroductionII. 9S ReceptorsIII. Receptor TransformationIV. Molybdate Stabilization of...V. Purification of Untransformed...VI. Role of hsp90...VII. Other Proteins Recovered...VIII. The Receptor Heterocomplex...IX. Other Proteins That...X. SummaryReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. 9S ReceptorsIII. Receptor TransformationIV. Molybdate Stabilization of...V. Purification of Untransformed...VI. Role of hsp90...VII. Other Proteins Recovered...VIII. The Receptor Heterocomplex...IX. Other Proteins That...X. SummaryReferences |
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The study of the supramolecular receptor structures has led to the observation that the chaperone proteins exist in cytosols as multiprotein heterocomplexes independent of their association with receptors. These hsp heterocomplexes appear to act as protein-folding machines that assemble heterocomplexes between the hsps and the receptors, as well as with some protein kinases involved in signal transduction from membrane receptors. Recently, immunopurified hsp heterocomplexes have been used to refold denatured luciferase to the catalytically active state. It now seems likely that the study of the nuclear receptor heterocomplex assembly mechanism will yield very basic information regarding more general protein-folding reactions in the cytoplasm.
The subject of nuclear receptor-hsp interactions has been reviewed briefly at various times during the past 10 yr (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). This review is intended as a comprehensive treatment of all work published through September of 1996. We begin with some of the earliest observations on nuclear receptor structure and the stabilization and purification of receptor complexes so that readers may appreciate the background leading to the discovery of receptor-hsp interactions. A number of assumptions about receptor behavior that were based on cytosolic steroid-binding studies performed during the 1960s and 1970s require revision in light of our knowledge of these receptor-hsp interactions. We will note these revisions as we review the background observations leading to the discovery of receptor heterocomplexes and the current state of our understanding regarding the cell-free mechanism of heterocomplex assembly. We do not review here the extensive literature on hsps or immunophilins or other protein-folding systems; appropriate reviews of these subjects are indicated in the text. However, we will discuss some interactions between hsps and nonreceptor proteins that may contribute to a more general understanding of hsp function.
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II. 9S Receptors |
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TopAbstractI. IntroductionII. 9S ReceptorsIII. Receptor TransformationIV. Molybdate Stabilization of...V. Purification of Untransformed...VI. Role of hsp90...VII. Other Proteins Recovered...VIII. The Receptor Heterocomplex...IX. Other Proteins That...X. SummaryReferences |
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In 1965, Noteboom and Gorski (15) demonstrated that injection of
[3H]estradiol to immature rats was followed by
accumulation of steroid in both the nuclear-myofibrillar and cytosolic
fractions of the rat uterus. The [3H]estradiol was bound
in the nuclear fraction in a stereospecific manner, and it was released
by digestion with trypsin, suggesting that the "estrophile" [a
term coined by Jensen (16) to avoid objections to the name
"receptor"] was a protein. The work of Noteboom and Gorski (15)
initiated the study of steroid receptors in cell-free systems, but
nevertheless, attempts to demonstrate a single intracellular estradiol
binding species (e.g. by molecular seive chromatography) had
been unsuccessful. At this time, Toft and Gorski (17) prepared a
105,000 x g cytosolic fraction from uteri of rats
injected with [3H]estradiol and centrifuged the cytosol
components through a gradient of 5–20% sucrose. The estradiol
radioactivity sedimented in a nearly symmetrical peak at 9.5S, and,
like the [3H]estradiol binding studied previously in the
uterine nuclear myofibrillar fractions, the cytosolic estradiol binding
recovered at 
9S was stereospecific and eliminated by proteases (17).
Shortly thereafter, Toft et al. (18) demonstrated that
addition of [3H]estradiol directly to cytosol prepared
from uteri of untreated rats yielded a 9S complex that appeared
identical to the complex obtained after in vivo hormone
administration. This publication represents the first direct
demonstration of hormone or drug binding to a receptor protein in a
cell-free system.
The publication of the sucrose gradient technique triggered well over
100 papers that appeared throughout the next decade and established the
existence of large (9S) and small (4S) forms of all the steroid
receptors. These studies were summarized in several early reviews
(19, 20, 21, 22, 23, 24), and a recent comprehensive review by Jensen (25) may be
consulted for more detail and complete references to primary sources.
It is our goal in the remainder of this section to focus primarily on
the observations in steroid receptor biochemistry that logically led to
the discovery of receptor-hsp heterocomplexes. In various publications,
the large receptor complex has been assigned sedimentation values
between 8S and 10S, but to simplify the text, we will refer to the
receptor heterocomplex as 9S. We will use the word
transformation to refer to the conversion of 9S
non-DNA-binding receptor to the 4S DNA-binding form. The term
activation will refer specifically to the conversion of
receptors from a form that does not bind steroid to a steroid-binding
form.
A. Estrogen receptors (ERs)
The discovery of the 9S form of the cytosolic ER was rapidly
confirmed by other laboratories (26, 27, 28, 29). Importantly, it was found
that treatment of cytosol with 0.3 M KCl (followed by
subsequent centrifugation on a sucrose gradient containing KCl)
"transformed" the cytosolic ER from a 9S to a 4S form (27, 28, 29). The
discovery of this salt effect gave rise to the notion that the 9S ER
was an oligomer that could be dissociated to a 4S state, a dissociation
that occurred regardless of whether the receptor was free or bound with
estrogen (28). Because dialysis of KCl-treated cytosol followed by
sucrose gradient centrifugation in the absence of salt yielded
predominantly a larger form of the ER, it was thought that the 9S to 4S
transformation was reversible (27, 29). In a more direct approach, it
was reported (20, 28) that the 4S peak of
[3H]estrogen-bound ER isolated from a KCl-containing
gradient could be recovered as 9S upon recentrifugation in the absence
of salt. This assumption of a "reversible equilibrium" between the
9S and 4S forms of the receptor remained an important component of
models describing steroid hormone action for many years. However, these
reversibility studies were compromised by poor receptor recovery and
heterogeneous aggregation, and it is now known that the notion of
simple reversible receptor forms was incorrect. Recent experiments
demonstrate (see Section VIII) that the large heterocomplex
form of the steroid receptors is reconstituted from the 4S monomeric
form only via an ATP-dependent protein-folding process. The 9S form is
not an oligomer of 4S units; rather, it is a receptor-hsp
heterocomplex.
It was known from early autoradiography (30) and cell fractionation (15) studies that, after in vivo injection, the majority of [3H]estrogen in the uterine cells was associated with nuclei. Jensen and co-workers (31) were able to solubilize the ER from uterine nuclei by repeated extraction with 0.3 M KCl and show that the salt-extracted nuclear receptor sedimented at 5S in sucrose gradients containing 0.4 M KCl. In 1968, the laboratories of Jensen (32) and Gorski (19, 33) demonstrated that the nuclear 5S ER was generated in a temperature-dependent manner at the expense of the cytosolic 9S ER complex. Incubation of the uterine cell nuclear fraction with [3H]estradiol did not yield a steroid-receptor complex, and it was only when nuclei were exposed to cytosol containing the [3H]estradiol-bound 9S complex that subsequent extraction with salt yielded the 5S receptor. These critical studies led to a very useful model of the initial events in steroid hormone action in which it was envisioned that the steroid first binds to a 9S form of the ER that is recovered in the cytosolic fraction upon cell rupture. This 9S complex was present in uterine cells before binding of steroid and was presumed to be an inactive form of the receptor. Steroid binding then, in some way, facilitated the temperature-dependent conversion of the biologically inactive 9S receptor to the biologically active form that could bind tightly to nuclei and was extractable as a 5S unit. This model is known as the "two-step" model of steroid action (32).
For a while, it was unclear whether the 5S salt-extracted nuclear ER
was the same as the 4S form produced by salt dissociation of the 9S
cytosolic ER (29, 34). However, Jensen et al. (35)
demonstrated, by sedimentation in salt-containing sucrose gradients,
that the nuclear 5S receptor was clearly larger than the 4S subunit of
the 9S cytosolic ER. Subsequently, it was shown in cell-free systems
that estrogens promote the temperature-dependent conversion of the 4S
cytosolic ER to the 5S form (36, 37, 38). At this time, it was thought by
many [but not all (38)] investigators that the 4S form of the ER did
not have DNA-binding activity and that the 5S ER was the DNA-binding
form. Laboratories studying ERs then focused on the 4S to 5S
conversion, considering this step, rather than the conversion from the
9S to the 4S form, to be the "receptor transformation" event (22).
Physical studies by the Notides’ laboratory (39, 40) showed that the conversion of 4S to 5S ER was a dimerization reaction, most likely a homodimerization (41). Kumar and Chambon (42) subsequently established that the ER binds to its response element in the DNA as a ligand-induced homodimer. The exceptionally strong dimerization site located within the HBD of the ER (43) accounts for the unique ability of the ER among the steroid receptors to maintain a homodimer in the presence of salt. Although it was clear that heat transformation of cytosolic ER complexes to a DNA-binding form occurs before receptor dimerization (44, 45), the strong dimerization property of the ERs led investigators of estrogen action to focus on the equally critical receptor dimerization event rather than pursuing the structure and properties of the 9S precursor. Thus, although the 9S complex was discovered with the ER, the subsequent fundamental observations regarding receptor heterocomplex structure and function were made in laboratories studying progesterone and glucocorticoid receptors.
B. Progesterone receptors (PRs)
In 1970, PRs in cytosols prepared from chicken oviduct (46) and
guinea pig uterus (47) were shown to migrate as both large (9S) and
small (4S) species in low-ionic strength sucrose gradients and, in both
cases, the large species was converted to 4S at high-ionic strength. In
rabbit uterine cytosol, the 4S species was observed in castrate
animals, but both 9S and 4S species could be visualized after estrogen
treatment (48). In some reports, only a 4S species of PR was observed
in rat or rabbit uterine cytosol under low salt conditions (49, 50),
giving the impression that the 9S PR complex may dissociate more
readily than the 9S ER complex. Nevertheless, the chicken oviduct PR
has proven to be an exceptionally useful system for study of the 9S
steroid receptor heterocomplex. Because large amounts of PR protein are
produced in the oviduct of the estrogen-primed chick, it became
possible, once a procedure for stabilizing the 9S complex was
established, to immunoadsorb large amounts of receptor and identify
coadsorbed proteins by direct staining of bands resolved on denaturing
gels. The coadsorbed proteins were present in sufficient quantity to
permit their identification by amino acid sequencing (9). The same
sequence of events that had been described for the ER — binding of
steroid to a cytosolic receptor, followed by temperature-dependent
transformation of the receptor to a DNA-binding form, and subsequent
association of receptor with nuclei — was demonstrated for the chick
oviduct PR, both in tissue slices and under cell-free conditions (51, 52).
C. Androgen receptors (ARs)
The first indications that the two-step model might apply in a
general way to other nuclear receptors came from studies of androgen
binding in the rat prostate. Mainwaring (53) was the first to
demonstrate a 9S form of the AR in rat prostate cytosols, an
observation confirmed by the Baulieu laboratory in both rat prostate
(54) and muscle (55) cytosols, with salt dissociation to a 4S form
being demonstrated in both cases. Selective retention of radiolabeled
androgen by prostatic cell nuclei had been demonstrated in 1968
(56, 57, 58), and, in 1969, Fang et al. (59) showed that binding
to the nuclear fraction required the presence of cytosol. The
progression of AR from cytosolic 9S to salt-extractable nuclear 4S in a
cell-free system was then published by Mainwaring and Peterken (60),
confirming the general progression of events outlined in the two-step
model.
D. Glucocorticoid receptors (GRs)
GRs were first identified in rat thymic lymphocyte cytosol by the
Munck laboratory in 1966 (61), with detailed studies being published by
the laboratories of Munck (62) and Schaumburg (63) in 1968. However,
physical studies of the receptor awaited the introduction of
tritium-labeled high affinity binders, such as dexamethasone and
triamcinolone acetonide. Baxter and Tomkins (64) were the first to
report that GRs were present as 9S complexes that dissociate to 4S in
rat hepatoma cell (HTC) cytosol in the presence of 0.5 M
KCl. The observation was rapidly confirmed for GR in cytosols prepared
from rat liver (65, 66), brain (67), and mammary gland (68). It was
determined that incubation of whole cells at 37 C with glucocorticoid
resulted in the majority of the specifically bound hormone being
recovered in the nuclear fraction (69, 70, 71) and that the increase in
specifically bound steroid in the nucleus was accounted for by the loss
of specific binding from the cytosolic fraction (72). As with the other
steroid receptor systems, it was shown under cell-free conditions that
isolated nuclei did not bind steroid, but if cytosol containing
steroid-bound receptors was warmed and then incubated with nuclei, the
specifically bound steroid became associated with nuclei (70, 71, 73, 74, 75, 76). Thus, by 1973, the two-step model had been extended to
glucocorticoid hormone action.
Although it was known that cytosolic GR existed in a 9S complex (64, 65, 66, 67, 68) and that the temperature-transformed GR extracted from nuclei with salt was 4S (76) [owing to the fact that the homodimer must be stabilized by cross-linking to be detected as a 6S entity (77)], investigators in glucocorticoid hormone action did not focus on the dissociation of the 9S complex as the transformation event. Rather, they focused on the other half of the coin, i.e. the acquisition of DNA/nuclear binding activity (71, 72, 73, 74, 75, 76, 78, 79), an obviously critical change for a receptor with a nuclear site of action. Indeed, in one rather influential report, it was maintained that both the untransformed and the heat-transformed, steroid-bound GR sedimented at 9S in low-salt gradients and at 4S in high salt (80). The suggestion was that the 9S form was observed only in media at unphysiologically low ionic strengths and that in vivo the GR exists predominantly in the 4S form (80). In mechanistic studies of GR transformation published in the late 1970s, the process was thought to involve solely a 4S receptor that could reversibly exist in a non-DNA-binding or a DNA-binding form (44, 81).
The 9S form of the GR is much less stable in cytosol preparations than the 9S forms of the ER and PR. Despite the instability of the complex and the focus of most laboratories on the acquisition of DNA-binding activity upon transformation, GR systems have been very useful in defining the composition and function of the components of the 9S complex. Actually, the instability of the 9S GR complex turned out to be an advantage. As we now know, the GR must be bound to hsp90 for the HBD to be in a high-affinity steroid-binding conformation (82). Dissociation of hsp90 from the unliganded receptor is reflected by a simultaneous loss in cytosolic steroid-binding activity (82). It was the study of this instability of cytosolic glucocorticoid-binding activity that led to the discovery of agents that inhibit the loss of steroid-binding activity, most notably molybdate and some other transition metal oxyanions (83, 84). Molybdate stabilizes the interaction of hsp90 with steroid receptors, and it permitted the purification of the untransformed PR and GR and identification of the hsp90 component of the 9S complex. A second feature of the GR that has made it particularly useful in the study of the heterocomplex is that it is essentially ubiquitous. Thus, it is present in a wide variety of cultured cell lines, some of which contain relatively high glucocorticoid-binding activity. The systems that have been especially useful are mouse L cells, which have a naturally high level of glucocorticoid-binding activity (85), and WCL2 cells, which are Chinese hamster ovary (CHO) cells that have been engineered to overexpress the mouse GR (86).
E. Mineralocorticoid receptors (MRs)
In 1963, Edelman et al. (87) first showed that
[3H]aldosterone localized to nuclei in cells of the toad
bladder. In 1970, Edelman’s laboratory (88) demonstrated that, after
administration of [3H]aldosterone to rats,
stereospecifically bound steroid could be extracted from a renal
chromatin fraction in a 4S form. Subsequent studies showed that a 9S
complex was present in cytosols (89, 90), and from the time-course of
9S and 4S generation, it was deduced that the 9S cytosolic receptor was
likely a precursor of the nuclear 4S form (89), a conclusion that
agreed with in vitro data showing salt-mediated
transformation of 9S receptor to the 4S form (89, 91). Unliganded MRs
in cytosols rapidly lose their steroid- binding activity (92). It is
now known that these receptors must be also associated with hsp90 to
have a steroid-binding site (93), and the lability of cytosolic
steroid-binding activity is due to rapid dissociation of the 9S MR to
4S. The binding of the MR to hsp90 is the least stable among the
mammalian steroid receptors.
F. Dioxin receptors (DRs)
A variety of halogenated hydrocarbons bind to a receptor that
mediates the induction of aryl hydrocarbon hydroxylase, a
cytochrome-P-450-mediated monooxygenase involved in the metabolism of
many xenobiotics. This receptor is called the Ah receptor,
or more commonly, the dioxin receptor (for review, see Refs. 94 and
95). The DR differs from the steroid receptors in several respects,
including the fact that its DNA-binding domain (DBD) contains a basic
helix-loop-helix rather than a double-zinc finger structure.
Nevertheless, dioxin receptors possess a number of physical properties
in common with steroid receptors. After dioxin administration, for
example, mouse hepatic receptors were found to accumulate in nuclei,
and there was a concomitant decrease in specific dioxin-binding
capacity in the cytosol (96). As with the steroid receptors, the
conversion from a cytosolic to a nuclear receptor was both
dioxin-dependent and temperature-dependent (97, 98). Thus, the model
for dioxin receptors as it originally developed was identical to the
two-step model proposed for steroid receptors (99, 100). The dioxin
receptor in cytosols from several species was shown to be 9S and to
dissociate to 4S in the presence of salt (98, 101). Indeed, the general
properties of the glucocorticoid and dioxin receptors in rat hepatic
cytosol were identical (102). However, it became apparent that the
mouse dioxin receptor differs from those of rat and other species in
that the 9S complex is highly resistant to salt-mediated dissociation
(102, 103). This unique salt stability appears to be a property of the
mouse receptor protein itself (104), and the binding of the mouse
dioxin receptor to hsp90 is the most stable of all receptors examined
to date. The dioxin receptor differs from the steroid receptors in
that, after ligand-mediated dissociation from hsp90, it forms a
heterodimer with the ARNT protein rather than dimerizing with itself
(95).
G. Antheridiol receptors
Sexual reproduction in the eukaryotic filamentous fungus
Achlya ambisexualis is regulated by steroid pheromones. One
of the steroids, antheridiol, is released from female cells and induces
development and differentiation of the male sex organs or antheridia.
Binding studies revealed the presence of an antheridiol receptor in
cytosol prepared from A. ambisexualis (105). Under low salt
conditions in the presence of molybdate, this receptor is 9S (105, 106). Molybdate stabilization is required to visualize the 9S form and
in the presence of salt, with or without molybdate, the receptor is 4S.
Of all the steroid receptors, the binding of the antheridiol receptor
to hsp90 is the least stable, and the actual composition of the
receptor complex remains unknown. Nevertheless, the system proved to be
highly useful because it led to the purification of Achlya
hsp90, which was used to prepare the very broad spectrum AC88
monoclonal antibody (IgG) that has been widely used in studies of hsp90
structure and function (107).
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III. Receptor Transformation |
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TopAbstractI. IntroductionII. 9S ReceptorsIII. Receptor TransformationIV. Molybdate Stabilization of...V. Purification of Untransformed...VI. Role of hsp90...VII. Other Proteins Recovered...VIII. The Receptor Heterocomplex...IX. Other Proteins That...X. SummaryReferences |
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However, as various laboratories began to explore a variety of artifactual (i.e. non-hormone-dependent) methods of transforming the receptor and as different criteria were used to define transformation (e.g. 9S dissociation, ER dimerization, DNA binding, polyanion binding, nuclear binding, loss of negative charge), the study of cell-free transformation appeared to be illogical and, to some investigators, possibly irrelevant to biological activity. As Grody et al. (110) stated in a review published in 1982, "The remarkably dissimilar characteristics of transformation among different steroid receptors discussed above have so far resisted attempts at incorporation into a universal mechanistic model and have led to much confusion in the field." Nevertheless, with the exception of the differing views regarding the importance of 9S receptor dissociation vs. receptor dimerization cited above (Section II.A), certain common themes emerged, and they are summarized in reviews written before the discovery of the receptor-hsp90 complex (110, 111, 112).
A. Transformed receptors bind polyanions
Initially, investigators focused on the ability of transformed
cytosolic receptors to bind to nuclei, but a variety of other assays
for receptor transformation were soon published. The finding that
treatment of nuclei with DNAse released transformed receptors (71, 73, 113) or prevented receptor binding to nuclei in a cell-free
translocation assay (114) was consistent with DNA being at least part
of the nuclear binding site. It was then determined that transformed
cytosolic steroid receptors bound to chromatin preparations (115) and
to purified DNA from any source, either in solution (71, 116) or
immobilized to cellulose (38, 76, 117, 118). Indeed, it was shown that
the receptors are transformed to a state that binds polyanions in
general (78), and assays of receptor transformation were developed
based on binding to phosphocellulose (119, 120), ATP-Sepharose (121, 122), and carboxymethyl-Sephadex (78, 123). Although assay of
transformation using DNA-cellulose did not yield as high a percentage
of receptors binding as simultaneous assays using nuclei (124), the
DNA- and nuclear-binding activities paralleled each other, and
conversion of the receptor to a general polyanion-binding state was
accepted as a valid qualitative assay for receptor transformation. It
was clear that any biologically meaningful interactions with specific
DNA sequences required for gene activation would have to be detected
above this background of nonspecific binding (118, 125). In the
mid-1970s, many of the major laboratories in the field of steroid
hormone action began gene transfer experiments to identify the hormone
response elements required for gene regulation and then performed
direct binding studies with partially purified receptors and hormone
response elements (for reviews, see Refs. 126–130). This highly
productive emphasis on the function of the transformed receptor was
accompanied by somewhat diminished interest in the mechanism of
receptor transformation itself.
The polyanionic binding property of transformed vs. untransformed receptors greatly facilitated purification of the transformed receptors, but similar purification could not be achieved with the untransformed state because the various conditions of dilution, salt, etc., employed in conventional protein purification protocols themselves promote transformation. The Gustafsson laboratory (131) used hormone- and temperature-dependent transformation to the DNA-binding state as the initial enrichment step in a protocol that yielded highly purified transformed GR (131). This purified GR was then used in a collaboration between the laboratories of Yamamoto and Gustafsson (132) to demonstrate for the first time the specific binding in vitro of a receptor to a DNA fragment whose transcription is regulated by hormone in vivo. Importantly, the Gustafsson laboratory performed limited proteolysis studies of both transformed cytosolic GR (133, 134) and the highly purified transformed GR (135, 136) that quite accurately defined the HBDs and DBDs of the receptor several years before receptor DNAs were cloned (see Ref. 137 for review). These limited proteolysis techniques proved to be very useful in later studies localizing the hsp90-binding region on molybdate-stabilized untransformed receptors (138, 139).
B. Artifactual transforming conditions
1. Salt transformation.
Cytosolic receptors can be transformed
by a variety of means. As noted above, salt causes dissociation of the
non-DNA-binding, 9S complex to the DNA-binding, 4S monomer (27, 28, 29, 46, 47, 54, 55, 64, 65, 66, 67, 68, 91, 98, 101, 102, 103, 105, 106). In the 1970s, the
heteromeric nature of the 9S complex had not been established, and it
was thought by many investigators that salt promoted dissociation of a
9S receptor homotetramer to the monomer (140). It is now clear,
however, that salt disrupts the binding of the receptor to hsp90 (82, 141, 142, 143) and that dissociation from hsp90 is accompanied by
concomitant and proportional generation of the DNA-binding state (144).
The disruption of the heterocomplex by salt indicates that ionic bonds
contribute significantly to the receptor-hsp90 interaction. In
contrast, binding of hsp70 to peptides and proteins in general is
determined by hydrophobic residues (for review, see 145 , and
complexes between steroid receptors and hsp70 are not disrupted by salt
(141, 142, 143).
In some early studies, salt-mediated GR transformation, like temperature-mediated transformation, was thought to be hormone-dependent (78). At that time, antireceptor antibodies had not been produced, and transformation had to be assayed by centrifuging [3H]steroid-bound receptors in density gradients or by binding them to DNA or nuclei. Because salt causes hsp90 to dissociate from the GR and because hsp90-free GR cannot bind steroid (82), one cannot treat the unliganded GR with salt and then bind steroid and assay for transformation. Now that antibodies are available for detection of DNA-bound receptors by Western blotting, we know that salt treatment of unliganded receptors causes transformation to the DNA-binding state.
2. Precipitation with ammonium sulfate.
DeSombre et
al. (146) first showed that precipitation of cytosolic ER with
ammonium sulfate at 25% of saturation transformed the receptor, with
transformation in this case being measured by dimerization to the 5S
form. Transformation by ammonium sulfate was estrogen-independent and
occurred at 0 C. Similar results were obtained with chick oviduct PR
(52) and rat hepatic GR (78), with transformation being assayed by
nuclear binding. Transformation by ammonium sulfate is now well
understood. In the absence of a stabilizing agent, such as molybdate,
ammonium sulfate dissociates the receptors from hsp90 and the
transformed receptor is precipitated (at about 30% of saturation),
yielding significant receptor purification (147, 148). Even though
precipitation with 30% ammonium sulfate eliminates the hormone-binding
activity of the unliganded GR (147), it has been shown by Western
blotting that the hormone-free receptor is transformed to a state that
binds to DNA-cellulose (148).
3. Transformation by dilution, gel filtration, and dialysis.
Dilution, gel fitration, and dialysis all transform cytosolic
receptors. These procedures reduce the concentration of, or eliminate,
a small heat-stable cytosolic factor that, like molybdate, stabilizes
the 9S receptor complex. Dilution was first shown by Higgins et
al. (74) to transform GRs in HTC cell cytosol, and subsequent work
by Milgrom’s laboratory (149) demonstrated quite clearly that dilution
acted by decreasing the concentration of a cytosolic inhibitor of
transformation. The inhibitor was described as a small (mol wt <
500), heat-stable molecule (149). Similarly, Litwack and his colleagues
(150, 151) showed that passage of cytosol through small molecular seive
columns transformed the GR by removing a low molecular weight
component. This factor clearly inhibits the transformation process and
not subsequent binding of the transformed receptor to DNA (149, 152).
The heat-stable fraction of cytosol was also shown to inhibit
conversion of the GR to the more positively charged species
characteristic of the DNA-binding state (153). Both dilution and gel
filtration of cytosol facilitated the transformation of progesterone
receptors (154) and elimination of a low molecular weight cytosolic
inhibitor by dialysis was shown to transform GRs, ERs, and ARs (155).
Two laboratories maintained an interest in purifying and characterizing this small, heat-stable, transformation inhibitor (for review, see 156 . Bodine and Litwack (157, 158) purified an active factor from rat liver, and physical analysis of the active fraction suggested that the inhibitor was a novel ether aminophosphoglyceride. Because the inhibitor stabilized the untransformed state of the cytosolic receptor, it was thought to possibly modulate receptor action and was called "modulator" (151, 156). The inhibitor (modulator) was subsequently separated into two isoforms that appear to interact synergistically in stabilizing untransformed receptors in cytosol (159). The proposed ether-linked aminophosphoglycerides have not yet been synthesized and shown to be biologically active; thus, their chemical structures and activities have not been proven. The purified modulator preparation has been shown to stimulate protein kinase C activity in vitro (160), suggesting more general effects or perhaps multiple activities in the preparation.
While studying conditions that would prevent inactivation of glucocorticoid-binding activity in cytosol, Pratt and colleagues (161, 162) found a small heat-stable, cytosolic factor that stabilized unoccupied GRs. This ubiquitous, dialyzable, anionic factor was subsequently found to inhibit receptor transformation to the DNA-binding state (163), and it was shown by a direct technique to stabilize hsp90 binding to the GR (164). The factor was stable to ashing and bound to a metal-chelating resin, leading to the conclusion that it was a metal anion (164). The factor produced the same effects on GR physical properties and function as molybdate (164), and, when the inhibitor was extensively purified from the heat-stable components of rat liver cytosol on a column matrix with high resolving properties for metal anions, it was found to coelute with the cytosolic molybdenum (165). This purified endogenous cytosolic metal anion was shown to inhibit hsp90 dissociation from the GR (165).
Although the Litwack and Pratt groups arrived at different conclusions regarding the chemical nature of the transformation inhibitor(s), both showed that their purified inhibitor preparations produced the same effects on the cytosolic GR as molybdate (156, 158, 164). Dilution, gel filtration, and dialysis of cytosol could facilitate receptor transformation by reducing the concentration of both aminophosphoglycerides and metal anions, and it is entirely possible that there are multiple factors of different chemical composition having molybdate-like effects on receptors. It has been found, however, that rapid passage of cytosol through the Chelex-100 metal-chelating resin also makes the GR unstable, facilitating both dissociation of hsp90 and simultaneous receptor transformation to the DNA-binding state (144). This chelation method of artifactual transformation should be selective for removal of metals, perhaps arguing that the endogenous cytosolic inhibitor of prime interest is a metal anion. In any event, both laboratories have proposed that molybdate may exert its stabilizing effects on the untransformed receptor heterocomplex by occupying the binding site of the endogenous transformation inhibitor (158, 164). As we will describe below (see Section IV.C.2), this binding site is probably located on hsp90, rather than on the receptor.
4. Other transforming conditions.
Bailly et al.
(81) first showed that elevating cytosolic pH promoted GR
transformation. It is now known that elevation of cytosol pH from 7.2
to 8.2 promotes dissociation of the GR from hsp90 (144). One can
speculate that a titration of charged amino acids involved in the
receptor-hsp90 interaction may be responsible for this mode of
transformation, but the mechanism for the pH effect is really not
known. The polyanion heparin has been reported to dissociate 9S
receptor complexes (166, 167, 168), but again the mechanism of the effect is
not clear. In isolated reports, a variety of small molecules, such as
nucleotides (169, 170, 171) and methylxanthines (172), as well as some
enzymes, such as an alkaline phosphatase (173) and an uncharacterized
endogenous transforming enzyme (174), have been reported to promote
receptor transformation (for review, see 112 . However, these
methods have not yet proven to be helpful in deriving mechanistic
models of the transformation event.
C. Models of cytosolic receptor transformation
As noted in reviews of steroid receptor transformation published
in the early 1980s (110, 140), diverse mechanisms had been proposed,
including a conformational change in the receptor without a change in
mass (44, 79), receptor dimerization (39, 40, 41), dissociation of a
receptor oligomer (167, 175, 176, 177, 178, 179), dissociation of macromolecular or
low molecular weight inhibitors (149, 152, 155, 162), receptor
dephosphorylation (162, 173), and receptor proteolysis (174). The
latter two possibilities, receptor dephosphorylation and partial
proteolysis, were eliminated by subsequent experiments (180, 181, 182).
Given the variety of conditions for bringing about transformation and
the variety of ways of assaying transformation, it is not surprising
that some reviewers (110) concluded "... most likely, there is no
single mechanistic explanation for all these findings, and it is
probably futile to insist on one."
As early as 1976, however, Atger and Milgrom (79) had published a
careful study of the kinetics and thermodynamics of GR transformation
that imposed important limits to be met as mechanistic models were
refined. They showed that transformation was of apparent first order
and that the free energy of thermodynamic activation was much higher
(
G* = 21.3 kcal) than expected for an enzymatic process. They noted
that high positive values for enthalpy (H* = 31.4 kcal) and entropy
(S* = 34 cal/degree) were similar to those described for protein
denaturation, suggesting breakage of noncovalent bonds during
transformation. These kinetic and thermodynamic data for GR
transformation were consistent with either a dissociation model or a
model involving a change in conformation without a change in mass.
Milgrom and his colleagues (79, 81, 111) developed the strong impression that GR transformation proceeded until there was an equilibrium between transformed and nontransformed receptors, and they concluded that transformation consisted of a simple change in conformation of a 4S receptor molecule induced by the hormone. We now know that the assumption of an equilibrium was incorrect. During the transformation process, the HBD of the steroid receptor undergoes a change in its folding state, and the process is reversed only by a complex protein-folding reaction involving hsp90, hsp70, and other proteins (Section VIII). Thus, the steroid does not act as an allosteric modifier of receptor structure as was proposed in a number of early papers from the Tomkins laboratory (72, 183, 184). Despite the fact that Milgrom’s equilibrium model of transformation turned out to be wrong, his kinetic and thermodynamic data were entirely consistent with a model for cytosolic receptor transformation in which steroid binding promotes a temperature-dependent dissociation of a 9S receptor-hsp90 complex (109, 141, 142, 144, 148, 185, 186).
Atger and Milgrom (79) made another observation of considerable
potential importance. When they examined the energy changes that
accompany the binding of the hormone to the receptor and the energy
changes that accompany the subsequent receptor transformation at 25 C,
they found that binding of steroid to the receptor requires a moderate
thermodynamic activation energy (G* = +10.6 kcal), but the complex
corresponds to a markedly lower level of free energy (G = -11.3
kcal). A high energy of activation (G* = +21.3 kcal) is required for
receptor transformation, but the transformed receptor is at a level of
free energy similar to that of the untransformed receptor (G =
-0.24 kcal). Thus, they concluded that the overall reaction is driven
mainly by binding of hormone to the receptor, which is accompanied by a
large variation in free energy. We now know that only the hsp90-bound
HBD of the GR is in the high-affinity steroid binding conformation, and
that an important energy barrier that must be overcome in
transformation of cytosolic receptor is provided by the noncovalent
bonds responsible for the protein-protein interaction between the
receptor and hsp90. It is now established that hsp90 is a component of
the chaperone system responsible for folding of the HBD, and it is
thought that the steroid receptors are anomalous with respect to other
proteins in that an intermediate state in the folding process is
maintained in the form of the relatively stable 9S receptor-hsp90
complex. The receptor interaction with hsp90 can be imagined as
trapping the HBD in a partially unfolded state and thereby trapping
some of its inherent folding energy (187). Once it occupies the
steroid-binding pocket, the steroid favors the naturally folded
conformation of the HBD, and in a sense, the hormone utilizes the
trapped potential energy of spontaneous folding to convert the receptor
to its active state by releasing it from hsp90.
The observations of the Milgrom laboratory (44, 78, 79, 81) and most of the other observations cited above that led to various models of transformation were made with cytosolic receptors. However, the process of receptor transformation in the intact cell is undoubtedly more complex. It is highly unlikely, for example, that unliganded receptors are diffusing in Brownian fashion through the cytoplasm or within the nuclear space as 9S particles. Thus, the 9S form is most likely a minimal or "core" unit derived from a state of the receptor that is retained in one compartment or the other by association with cell structures until it binds hormone and is transformed (188). As we will describe later, several proteins in addition to hsp90 and hsp70 are present in immunoadsorbed, untransformed steroid receptor complexes (141, 142, 188), and all of these proteins are not necessarily retained in the receptor heterocomplex during gradient centrifugation. Some 9S components, such as hsp70 and a 60-kDa stress-related protein (189), are tightly bound to receptors during the folding of the HBD, but then they can cycle out of the folded complex (190, 191). Also, the receptor heterocomplex in the cell is probably dynamic in the sense that receptors are being folded and unfolded continuously, much as simultaneous folding and unfolding occurs in the cell- free systems for receptor heterocomplex assembly with hsp90 (192). Because hormone is required for steroid receptors to occupy their response elements in the genome (108), it seems that hormone-mediated transformation must, in some way, be coupled with receptor movement to these elements. This is obviously the case for receptors that are in the cytoplasm before hormone binding, but some movement through space is likely required also for receptors that have functional nuclear localization signals (NLSs) in their hormone-free state and thus await the hormone at nuclear sites of retention.
There seems little doubt but that models of receptor transformation based on purely cytosolic observations will be simplistic. However, it is only through examining hormone-mediated dissociation of more purified receptor heterocomplexes and through studying the reversal of this transformation with purified hsp chaperone systems that we will eventually develop a correct molecular model describing how the steroid drives receptor transformation. As will be described later, in more complex systems where receptors are being continuously folded and unfolded, the binding of steroid has a second effect in that it blocks reformation of the heterocomplex because the hsp chaperone system forms a stable heterocomplex only with the unliganded HBD (193, 194). Thus, in the intact cell, binding of hormone probably drives receptor transformation much as it does in cytosolic systems, but additionally, hormone binding stabilizes the HBD in a folded state such that transformation is not readily reversed by the chaperone system (192, 193, 194).
D. Physiological relevance of receptor transformation before the
discovery of hsp90 binding
In reviewing potential mechanisms of transformation before the
discovery of hsp90 association with receptors, Grody et al.
(110) considered the possibility that transformation was an in
vitro artifact — "Finally we must consider the very real
possibility that the reason no satisfactory mechanism has been found to
explain transformation is that none exists in vivo.
Transformation may be purely an in vitro phenomenon, brought
about by the artifactual association of various proteins, enzymes, or
other factors with receptors as a result of cell disruption and cytosol
preparation procedures." Even now, there are laboratories that
consider the 9S untransformed receptor-hsp90 complex to be such an
artifact of tissue processing (195). The in vivo validity of
the receptor-hsp90 heteroligomer will be considered later, but it is
useful to review here the evidence for physiological relevance of
receptor transformation that existed by the early 1980s.
Initial interest in receptor transformation was driven by the role played by hormone in temperature-dependent nuclear binding of receptors in both intact cells and cell-free systems. As early as 1972, Jensen’s laboratory (196) had published evidence that transformation in vitro converted the ER to a form that was biochemically active when mixed with nuclei. It was thought unlikely that the hormone-dependent transformation event examined in cytosol could be irrelevant to the physiological action of the hormone in the intact cell. Importantly, Munck and Foley (197) demonstrated directly that GRs underwent the same transformation change in the intact cell as in cytosol. To do this, they used the observation that the more acidic, non-DNA-binding, untransformed receptor can be separated from the more positively charged, DNA-binding, transformed species on diethylaminoethyl (DEAE) columns eluted with a phosphate buffer gradient (123, 153). Using suspensions of rat thymocytes, Munck and Foley (197) found that a rapid shift from untransformed to transformed state occurred with a half-time of <2.5 min. After injection of [3H]triamcinolone acetonide to rats, Markovic and Litwack (198) demonstrated a similar shift in DEAE elution behavior of GR recovered in hepatic cytosol, with a longer half-time (10–15 min) for transformation, probably reflecting a delay due to steroid distribution after injection.
The argument for the physiological relevance of receptor transformation was buttressed considerably when Munck’s laboratory (177) exposed intact cells to glucocorticoid at 37 C and ruptured them in buffer containing sodium molybdate, which blocks any subsequent receptor transformation during cytosol preparation. They clearly showed that hormone-dependent and temperature-dependent transformation of the GR from 9S to 4S occurred under physiological conditions in the cell. Miyabe and Harrison (199) showed that transformation (assayed by DEAE chromatography) occurred in a ligand-dependent manner in intact mouse pituitary tumor cells and showed that the extent of nuclear binding was proportional to the degree of receptor transformation.
In 1983, Raaka and Samuels (176) performed a very illuminating study in which they used both a dense amino acid-labeling technique and molybdate stabilization during cell rupture to show that the GR in hormone-free GH1 rat pituitary tumor cells is 9S and is converted in the presence of hormone to 4S cytosolic and nuclear-bound forms. Because the 4S cytosolic and nuclear forms decreased upon hormone withdrawal while the 9S form increased in a manner that is not dependent upon new protein synthesis, they assumed that there was an equilibrium between the 9S and 4S forms in the cell. The observations of Raaka and Samuels (176) provided strong support for the importance of hormone-dependent receptor transformation in the intact cell, but their assumptions that 1) the 9S form is a receptor tetramer, 2) the 9S and 4S forms are in an equilibrium, and 3) the steroid acts as an allosteric modifier [a model originally proposed by Samuels and Tomkins in 1970 (183)] that shifts the equilibrium to the 4S form have proven to be incorrect.
When the steroid receptors were cloned, it was possible to directly correlate biological properties of mutant receptors with their recovery as the 9S or 4S forms. Analysis of receptors produced after transfection of hormone-free cells with mutant human GR and ER cDNAs demonstrated that steroid-inducible forms of the receptor were recovered in molybdate-stabilized cytosols entirely as 9S complexes, whereas mutant receptors with constitutive activity were recovered only in the 4S form (200, 201, 202). These observations provided strong support for the argument that the 9S, non-DNA-binding form is derived from the physiologically inactive state of the receptor that is transformed by the steroid in the cell and that the 4S, DNA-binding form is derived from receptor that is active in transcriptional enhancement.
Gorski and his colleagues (203, 204, 205) raised a direct challenge to the physiological significance of the two-step model of steroid hormone action that should be mentioned here. Their rejection of the model is largely based on the observation that ERs were found to lie within the nuclei of hormone-free cells (206, 207), an observation that has been repeated for several steroid receptors in several cell systems (for review, see 5 . The nuclear localization of an unliganded receptor obviously eliminates the requirement for a spatial translocation of the receptor from the cytoplasm to the nucleus after transformation, but as Jensen notes (208), "where the receptor is actually located has no direct bearing on the validity of the two-step mechanism." As noted by Schrader (209), "there are still two identifiable states of a steroid receptor an active state and an inactive one." In that hormone binding and transformation can be differentiated as distinct and sequential processes independent of receptor translocation, the two-step model is retained.
On the other hand, Gorski and Hansen (205) have rejected the transformation event itself, feeling that "... changes in the receptor induced by warming to 25–30 C (‘transformation’ or second step) are probably artifacts." This rejection of receptor transformation also reflects a conclusion derived by Hansen and Gorski (210, 211) from their studies of the changes in the aqueous two-phase partitioning behavior of the ER that occur with steroid binding and/or heating in vitro. They concluded that the major change in partitioning behavior, and thus physical properties of the ER, took place on ligand binding and not with the heating step. A major problem with this work is that transformation was not independently assayed by conversion of receptors from 9S to 4S or by acquisition of DNA-binding activity. Indeed, it was assumed that the "unoccupied ER monomer" was untransformed, yet it has been established by others that, like other steroid receptors, the 4S ER monomer is already transformed (44, 45). However, the work of Hansen and Gorski (210, 211) brings up an important point that is still not answered, which is whether the greatest conformational change in the receptor HBD occurs with steroid binding or with the subsequent temperature-dependent transformation step.
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IV. Molybdate Stabilization of Receptors |
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TopAbstractI. IntroductionII. 9S ReceptorsIII. Receptor TransformationIV. Molybdate Stabilization of...V. Purification of Untransformed...VI. Role of hsp90...VII. Other Proteins Recovered...VIII. The Receptor Heterocomplex...IX. Other Proteins That...X. SummaryReferences |
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A. Stabilization of steroid-binding activity
1. GR instability.
Rees and Bell (218) were the first to
demonstrate that one component of GR inactivation was due to disulfide
bond formation that could be prevented by reducing agents such as
dithiothreitol. Granberg and Ballard (219) then showed that addition of
dithiothreitol-activated glucocorticoid-binding activity in cytosols
prepared from rat tissues that had low endogenous reducing capacity. A
series of investigations by the Pratt laboratory identified the
endogenous cytosolic reducing activity as the thiol-disulfide exchange
factor thioredoxin (163, 220, 221), and it is now established that the
steroid-binding activity of the GR is inactivated by redox conditions
that promote intramolecular disulfide bond formation (222, 223).
A recent series of studies from Simons’ laboratory has demonstrated that steroid-binding activity of the GR is inactivated by the formation of disulfide bonds between cysteine SH groups that are vicinally spaced in the HBD when it is bound to hsp90 (222, 224, 225, 226). The untransformed, unliganded GR has been cleaved with trypsin to a 16-kDa fragment of the HBD that is bound to hsp90 (139) and has steroid-binding activity (227). The 16-kDa fragment contains three cysteines, of which any two can form an intramolecular disulfide (226). This cysteine cluster appears to lie in a portion of the steroid-binding pocket that is critical for binding the D ring of the steroid (228, 229), and a variety of observations indicate that a short region of the HBD containing this thiol cluster directly contacts hsp90 in the untransformed GR heterocomplex (139, 201, 230).
We now know that there are two requirements for the GR to bind steroid: 1) the redox conditions must be such that the thiols in the cluster are not oxidized to form intramolecular disulfide bonds, and 2) the receptor must be bound to hsp90. In retrospect, we appreciate that the lability of glucocorticoid-binding activity in cytosols was due both to disulfide bond formation and hsp90 dissociation. In 1979, it was only known that two distinct processes were responsible for inactivating glucocorticoid-binding activity, one that was reversed by dithiothreitol and another that was inhibited by molybdate (162, 231). The vicinal thiol groups are unique to the GR, and the vicinal thiol-selective agent arsenite selectively inactivates the steroid- binding activity of the GR vs. the MR (232). The lability of steroid-binding activity of the MR in cytosol appears to reflect its less stable interaction with hsp90. Detailed information on GR thiols and steroid binding activity is available in a recent review (233).
2. The phosphorylation hypothesis leads to the discovery of
molybdate stabilization.
Even in cytosols with good reducing
activity, the steroid-binding activity of the unliganded GR was rapidly
inactivated at room temperature, and essentially all steroid-binding
experiments were performed at 0–4 C. By the early 1970s, Munck and his
colleagues (62, 70, 214) had shown that the ability of intact
thymocytes to bind glucocorticoids was energy-dependent, seeming to be
related to cellular ATP content. Other investigators then reported that
exposure of mouse fibroblasts (234), thymic lymphocytes (235), or chick
embryo retina (236) to the metabolic inhibitor dinitrophenol resulted
in loss of glucocorticoid-binding capacity. When the metabolic blockade
was removed, steroid-binding activity returned, and the return was
unaffected by inhibition of protein synthesis (214, 234, 236, 237).
Wheeler et al. (237) confirmed the very tight association
between the cellular ATP level and steroid-binding activity achieved in
cytosols. These observations led to the speculation that the GR might
be a phosphoprotein, with ATP promoting its phosphorylation to a
steroid-binding form (70, 235). Following up on this notion, Nielsen
et al. (238) reported that incubation of L cell cytosol with
highly purified alkaline phosphatase inactivated the steroid-binding
activity of the unliganded GR in a manner that was clearly related to
the dephosphorylating activity of the enzyme.
This report by Nielsen et al. (238) really led people to think that the steroid receptors might be phosphoproteins and that steroid binding might be regulated by phosphorylation. It was subsequently shown that the steroid receptors are phosphoproteins, and the study of steroid receptor phosphorylation has become a subfield of nuclear receptor research [see Orti et al. (239) for review]. It is now known that the GR is not phosphorylated in the HBD and the GR phosphorylation state does not affect its steroid-binding activity. Nevertheless, in the mid 1970s, two approaches were taken to address the possibility that the lability of cytosolic glucocorticoid-binding activity was due to a dephosphorylation — one approach used phosphatase inhibitors to stabilize receptors and the other approach focused on ATP-dependent generation of steroid-binding activity.
In the first approach, Nielsen et al. (83, 84) looked for
the presence of an enzyme activity in cells that would inactivate the
steroid-binding activity of unliganded GR. The 27,000–100,000 x
g particulate fraction of mouse L cells, rat thymocytes, and
rat liver was found to contain an enzyme(s) that inactivated
glucocorticoid-binding activity when mixed with cytosol containing
steroid-free GR. The GR-inactivating activity was extracted from the
membrane fraction, partially purified (83), and several phosphatase
inhibitors were tested for their ability to inhibit receptor
inactivation. Ishii and Aronow (217) had previously found that certain
glucose metabolites stabilized glucocorticoid-binding activity in L
cell cytosol, the most effective of these inhibitors being
glucose-1-phosphate. Glucose-1-phosphate had been reported to be a
phosphatase inhibitor, and Nielsen et al. (83, 84) found
that both it and another phosphatase inhibitor, fluoride, had a
moderate ability to inhibit the GR-inactivating activity extracted from
their particulate fraction. Molybdate, however, produced a profound
inhibition of receptor inactivation. Nielson et al. (83, 84)
also found that the three phosphatase inhibitors inhibited GR
inactivation when cytosol was incubated at elevated temperature without
the addition of the inactivating activity from the particulate
fraction. In L cell cytosol, for example, the half-time for
inactivation of steroid-binding activity at 25 C went from 30–60 min
to more than 1 day, and the half-time at 37 C went from 2 min to
2 h when 10 mM molybdate was present (83). When this
work was published in 1977, it was thought that fluoride and molybdate
were inhibiting a phosphatase(s) that was inactivating the GR.
In the second approach to the phosphorylation/dephosphorylation model, Sando et al. (240) obtained ATP-dependent reactivation of steroid-binding activity in L cell cytosol. The GR was first inactivated by incubating L cell cytosol at 25 C, molybdate was then added to prevent further inactivation, ATP was added, and the incubation was continued to permit regeneration of steroid-binding activity. Reactivation of 40–70% of the receptors was obtained by an ATP/Mg2+-dependent and temperature-dependent process. When combined with the phosphatase inhibitor data, these results led Sando et al. (240) to conclude that regeneration of steroid-binding activity was due to a phosphorylation process, most likely phosphorylation of the receptor polypeptide itself.
Inasmuch as this conclusion was ultimately found to be wrong, why did Sando et al. (240) observe ATP-dependent regeneration of steroid-binding activity? It has now been shown that L cell cytosol contains the same hsp90/hsp70-based chaperone activity as the well studied system in reticulocyte lysate that has been used to form steroid receptor·hsp90 heterocomplexes (241). When immunopurified, hsp90-free GR is incubated with GR-free L cell cytosol, receptors are reassociated with hsp90, and steroid binding is regenerated (241). It is highly likely, therefore, that Sando et al. (240) were generating steroid-binding activity due to ATP-dependent chaperoning of the GR by the hsp90/hsp70-based protein chaperone system.
3. Molybdate stabilization is a physical effect.
Molybdate
stabilization of steroid-binding activity was reported for GRs in a
variety of cytosol preparations (162, 168, 231, 237, 242, 243, 244, 245) and two
other transition metal oxyanions, tungstate and vanadate, were also
found to be active (237, 242). The steroid-binding activity of PRs
(246, 247, 248, 249, 250, 251) ERs (248, 252), ARs (245, 253, 254), MRs (255), and DRs
(256) was also stabilized. Because the transition metal oxyanions
stabilized receptors to inactivation by nonenzymatic means, such as
exposure to salt, heparin, or ammonium sulfate (147, 168, 231, 242), it
rapidly became clear that the metal oxyanions were not acting as
phosphatase inhibitors.
Because fluoride was a less effective stabilizer than molybdate in the original reports (83, 84), its activity was not widely tested, although Grody et al. (247) showed that fluoride inhibited thermal inactivation of the PR. Much later, Housley (257) showed that receptor stabilization by fluoride requires aluminum, which in the earlier studies (83, 84, 247) was derived from the water and glassware. The requirement for aluminum is reminiscent of fluoride effects on G protein regulation of adenylyl cyclase, where the effect is also due to aluminum fluoride (258). Like molybdate, aluminum fluoride was found to stabilize the GR to inactivation by ammonium sulfate precipitation and gel filtration as well as to thermal inactivation (257). Moreover, like the transition metal oxyanions, aluminum fluoride inhibited GR transformation (assayed by DNA binding), and it was shown by a direct method to inhibit dissociation of hsp90 from the receptor (257).
By 1981, it was clear that molybdate was not acting as a phosphatase inhibitor to stabilize steroid receptors to thermal inactivation, and we know now that molybdate and fluoride both act by stabilizing the receptor·hsp90 complex. We also know that GR phosphorylation is not required for steroid-binding activity, yet addition of alkaline phosphatase to L cell cytosol inactivates steroid-binding activity (238, 257, 259, 260). How could this be? The answer lies in the fact that redox conditions change when cytosol is incubated with alkaline phosphatase.
Housley et al. (259) showed that the presence of both molybdate and dithiothreitol completely stabilized the steroid-binding activity of the GR when cytosol was incubated with alkaline phosphatase. In the absence of the reducing agent, steroid-binding activity was eliminated during the incubation with enzyme. If molybdate was present during the enzyme digestion, the GR could be fully reactivated by addition of dithiothreitol, but if molybdate was not present, there was no reactivation (259). At the time the work was performed, it was proposed that a conformation of the receptor, which was determined by its phosphorylation, stabilized vicinal thiol residues to maintain the receptor in its active steroid-binding form, with dephosphorylation of the receptor promoting its oxidation (259, 260). The notion was that molybdate interacted with receptor to replace the conformational effect of the phosphate moiety(ies) (260). The valid explanation, however, is quite different.
We now know that unsupplemented cytosol (i.e. no added reducing agent) from L cells or rat liver has relatively stable steroid-binding activity at room temperature because it maintains a high level of NADPH and consequently of reduced thioredoxin via the NADPH-dependent thioredoxin reductase reaction (220, 221). When cytosol is incubated with alkaline phosphatase, NADPH is inactivated, thioredoxin acumulates in the oxidized form, and disulfide bond formation in the vicinal thiol cluster of the GR HBD is not reversed by thiol-disulfide exchange. Thus, the receptor is inactivated due to its oxidation. If molybdate is present during the incubation with phosphatase, the GR·hsp90 complex is preserved and the steroid-binding activity can be restored, either by addition of dithiothreitol at 0 C (259) or by a short incubation at 25 C with added NADPH to regenerate the active reduced form of thioredoxin (P. R. Housley and W. B. Pratt, unpublished observation).
During the 1970s (i.e. before the development of anti-receptor antibodies and site-specific affinity labels), only a limited number of approaches were available to investigators studying the biochemistry of steroid receptors, and all of the approaches required the identification of the receptor through its steroid-binding property. As discussed above, a variety of salts, enzymes, and other compounds were added to cytosols to identify conditions that would inhibit or promote receptor transformation, and a similar phenomenological approach was taken to determine what would inactivate or stabilize steroid-binding activity. Incubation of cytosols with proteases, nucleases, sialidases, phosphatases, and phospholipases were reported at one time or another to affect receptor size, transformation, or steroid-binding activity (112, 140, 260, 261, 262, 263). In retrospect, it is quite easy to see how artifacts could lead to inappropriate conclusions regarding receptor composition and structure. The phosphatase studies are one example that led to a major advance, despite the fact that the hypothesis and conclusion were wrong. Some of these observations simply led to misunderstanding. Despite the fact that artifacts were generated and erroneous conclusions were drawn, it was becoming clear that most conditions that inactivated the steroid-binding activity of the cytosolic GR also promoted its transformation and that the two processes had a common inhibitor, molybdate (242, 264).
B. Inhibition of transformation
In 1979, Nishigori and Toft (246, 265) first reported inhibition
of receptor transformation by molybdate. Both thermal and salt
transformation of the cytosolic PR were inhibited by molybdate, and
transformation was inhibited regardless of whether it was assayed by
receptor binding to ATP-Sepharose or by shift in receptor sedimention
from 9S to 4S. The availability of an effective inhibitor of
transformation was of broad interest and, during the next few years,
many papers were published showing inhibition of GR (147, 168, 173, 242, 245, 264, 266, 267, 268, 269, 270), PR (167, 175, 246, 247, 249, 250, 251, 267, 268, 271), ER (245, 252, 267, 268, 272, 273, 274), AR (245, 267, 275), MR (276),
and DR (256, 277) transformation in cytosol. Molybdate inhibited
receptor transformation caused by salt, heparin, or ammonium sulfate
precipitation, as well as thermal transformation. Tungstate and
vanadate were also active (242, 265, 271, 278). The effect of molybdate
was reversible, and it was effective only when added before
transformation; that is, addition of molybdate after transformation
neither promoted formation of 9S receptor from the 4S form nor affected
DNA binding by the transformed receptor.
1. Molybdate — the physiologically relevant artifact.
The
profound ability of molybdate to preserve all of the steroid receptors
and the DR in similar 9S complexes of Mr 300,000–330,000
suggested that this large structure might be essential to receptor
function (268). Because molybdate inhibition of GR transformation in
cytosol was correlated with maintenance of the 9S form of the receptor
and transformation was accompanied by generation of 4S receptor,
several investigators proposed that receptor transformation represents
the dissociation of an oligomeric protein (176, 177, 178, 179). At that time, it
was not known whether the molybdate-stabilized species was a receptor
tetramer (140) or a receptor heterocomplex.
Raaka et al. (279) directly tested the transformation-inhibiting effect of molybdate added to intact cells. Cells treated with molybdate had more of the 9S form and less of the 4S form after glucocorticoid treatment than control cells. Treatment of the intact cells with molybdate also reduced nuclear accumulation of GR and ER after steroid treatment. This study on molybdate effects in intact cells supported the proposal (see Section III.D) that molybdate inhibited a steroid-dependent change in receptor state that was related to hormone action.
2. Physical properties of molybdate-stabilized, untransformed
receptors.
Molybdate was shown to stabilize steroid receptors
against dissociation during chromatrographic procedures of long
duration (147, 269, 273, 280), and this stabilization provided a much
clearer definition of the Stokes’ radii and sedimentation coefficients
and consequently more accurate calculation of the molecular weights of
the complexes (102, 103, 140, 147, 175, 178, 179, 268, 273, 276, 277, 280, 281). Much was learned from the effect of molybdate on GR behavior
during DEAE-cellulose chromatography. When cytosol containing
untransformed GR is adsorbed to a DEAE-cellulose column and eluted with
phosphate buffer, most of the receptor is eluted at about 0.25
M salt, which is characteristic of the untransformed state
of the receptor (153, 282). In contrast, elution with KCl in a Tris
buffer yields the more positively charged state characteristic of the
transformed receptor, which elutes at much lower salt (123). The
presence of molybdate in both the loading buffer and eluting buffer
completely prevented transformation during the running of the KCl
gradient, with the GR eluting from DEAE-cellulose in a well defined
high salt peak that yielded a 33-fold purification of the untransformed
receptor (147). It was later shown that the molybdate-stabilized,
untransformed GR coelutes with hsp90 (148). hsp90 is quite acidic and
when molybdate is present to stabilize the GR·hsp90 complex the
charge of the hsp90 dominates and determines the overall behavior of
the complex (148). When cytosol is submitted to ammonium sulfate
fractionation in the presence of molybdate, the GR is precipitated out
in the 45–55% range of saturation, whereas the bulk of the hsp90 is
precipitated at 50–70% and transformed receptor at 20–30% of
saturation (147, 148). Thus, the salting-out properties of the
molybdate-stabilized, untransformed GR are more like those of hsp90
than those of the GR polypeptide, but they are not the same as the
salting-out properties of hsp90.
C. Mechanism of molybdate stabilization
1. The thiol model.
Little is known about how molybdate
stabilizes receptor·hsp90 complexes or even the ligand(s) with which
it interacts. In 1979, it was proposed that molybdate may stabilize
through an interaction with thiol groups on the receptor (242, 265).
Molybdate has a well known avidity for sulfur (283), and it has been
shown to interact with the
