From the Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030
Received for publication, August 22, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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Protein disulfide isomerase (PDI) is a folding
assistant of the eukaryotic endoplasmic reticulum, but it also binds
the hormones, estradiol, and
3,3',5-triiodo-L-thyronine (T3).
Hormone binding could be at discrete hormone binding sites, or it could
be a nonphysiological consequence of binding site(s) that are involved
in the interaction PDI with its peptide and protein substrates.
Equilibrium dialysis, fluorescent hydrophobic probe binding
(4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid
(bis-ANS)), competition binding, and enzyme activity assays reveal that the hormone binding sites are distinct from the
peptide/protein binding sites. PDI has one estradiol binding site with
modest affinity (2.1 ± 0.5 µM). There are two
binding sites with comparable affinity for T3 (4.3 ± 1.4 µM). One of these overlaps the estradiol site,
whereas the other binds the hydrophobic probe, bis-ANS. Neither estradiol nor T3 inhibit the catalytic or chaperone
activity of PDI. Although the affinity of PDI for the hormones
estradiol and T3 is modest, the high local concentration of
PDI in the endoplasmic reticulum (>200 µM) would drive
hormone binding and result in the association of a substantial fraction
(>90%) of the hormones in the cell with PDI. High capacity, low
affinity hormone sites may function to buffer hormone concentration in
the cell and allow tight, specific binding to the true receptor while
preserving a reasonable number of hormone molecules in the very small
volume of the cellular environment.
Protein disulfide isomerase
(PDI)1 is a versatile and
multi-functional folding assistant of the endoplasmic reticulum (1-6). Its essential function in eukaryotes (7) is as a catalyst of disulfide
rearrangements (isomerase activity) during oxidative protein folding
(ER). There are two remarkable facts about PDI. It is a very abundant
protein in the cell; its concentration in the ER lumen has been
estimated to be 100-200 µM (8). The other remarkable
fact about PDI is its uncanny ability to bind things. Ligands range
from peptide/protein substrates to hormones and calcium.
Peptide/protein interactions are essential for effective catalysis of
disulfide formation and isomerization. PDI interacts covalently with
substrate cysteines (9), but it also binds a variety of peptide/protein
substrates (10). There are few strict sequence/structure requirements
for binding (11, 12), although there is some effect of sequence on
reactivity with PDI (13). PDI activity can be inhibited by a number of
peptides (11), including the cyclic peptide antibiotic, bacitracin (14, 15). A tripeptide affinity probe, which originally identified PDI as a
glycosylation site binding protein (16) localized one peptide binding
site near the C terminus of PDI (12), but deletion of the C-terminal
binding site has little effect on the oxidase and isomerase activities
(17). Binding of radiolabeled peptides to various fragments of PDI has
recently suggested that multiple sites exist that may interact with
various peptides/proteins (10) and that a distinct structural domain
(b') that is N-terminal to one of the two active sites contributes
significantly to peptide binding.
Noncovalent interactions with protein substrates can suppress substrate
aggregation during protein refolding. The chaperone activity of PDI is
observed with proteins that form disulfides (18) and proteins that do
not (19-21). By interacting with multiple sites on aggregation-prone
substrates, PDI can facilitate substrate aggregation under certain
conditions, a behavior we have termed anti-chaperone activity (18, 20,
22).
In addition to unfolded protein substrates, PDI associates tightly with
some folded proteins. It serves as the In addition to peptides and proteins, PDI binds hormones. It was
identified by photoaffinity labeling as a thyroid-hormone binding
protein (26). Displacement of radiolabeled T3 from PDI purified from liver suggested a high affinity for T3 (27).
However, Guthapfel et al. (28) have recently shown that the
high affinity binding of T3 (Kd = 21 nM) has very low capacity (0.002 mol of T3/mol
of PDI), implying that high affinity binding is due to an impurity
rather than PDI itself. PDI also binds The relationships between the hormone and peptide/protein binding sites
are not at all clear. The interactions of PDI with hormone ligands
might be due to specific binding sites, or it may be a reflection of
nonspecific binding site(s) that are normally involved in PDI/substrate
interactions. There have been no systematic attempts to determine the
number of binding sites for individual ligands, nor have there been any
attempts to examine overlap in the interaction sites. We have used
competitive ligand binding of
4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid
(bis-ANS), radiolabeled E2, and T3 along with
various unlabeled peptide inhibitors to probe the various interactions
these ligands make with PDI. The results show that PDI has multiple
binding sites for hormones. Binding occurs at two different hormone
binding sites. Both sites bind T3, whereas only one of them
interacts with estradiol. These sites are distinct from peptide/protein
binding sites. Thus, the hormone binding capacity of PDI appears to be
a discrete function of the molecule and not coupled to its catalytic
activity in disulfide isomerization. Because of its high local
concentration in the ER, PDI could serve as an intracellular reservoir
for these hormones despite its relatively low affinity. Based on the
relative concentrations and affinities of PDI and the estrogen
receptor, more than 90% of the intracellular T3 and
estradiol would be expected to be bound to PDI. This high capacity
intracellular reservoir would tend to raise the cellular levels of
hormone that are necessary to cause receptor occupancy, even when the
estrogen and T3 receptors have nM affinity for hormones.
Materials--
Recombinant rat PDI was expressed and purified as
described previously (34). The concentration of PDI monomers (55 kDa) was determined by absorbance at 280 nm (E0.1%= 0.94 cm Fluorescence Measurements--
Measurements were made in 50 mM HEPES-NaOH, pH 7.0, containing 10 mM DTT at
25 °C using various concentrations of PDI and bis-ANS.
The excitation wavelength was 370 nm, and emission was scanned from 425 to 575 nm using a PerkinElmer LS-5 fluorescence spectrometer.
Thermal Aggregation Assay--
ADH aggregation upon thermal
denaturation was followed by measuring the apparent absorbance at 360 nm caused by light scattering because of increasing turbidity (20).
Temperature was controlled at 40 °C using a circulating water bath
connected to a Beckman DU 7500 spectrophotometer. Initiation of the
assay was by addition of ADH (final concentration, 11 µM
dimer) to a 50 mM HEPES-NaOH buffer, pH 7.0, containing 10 mM DTT and PDI at various concentrations. The apparent
velocity of aggregation (A360/min) was measured
after any lag as the maximum change in absorbance with time.
Isomerase Activity Assay--
Oxidative refolding of RNase in
the presence and absence of inhibitors was followed by an established
continuous assay (8) where the formation of native RNase was measured
spectrophotometrically by monitoring hydrolysis of cCMP, an RNase
substrate, at 296 nm. Reduced, denatured RNase (final concentration, 8 µM) was added to PDI (1.4 µM) in a buffer
containing 4.5 mM cCMP, 1 mM glutathione, 0.2 mM glutathione disulfide, 2 mM EDTA, 100 mM Tris-HCl, pH 8. RNase was reduced and denatured by
overnight incubation at 10 mg/ml in denaturing buffer (140 mM DTT, 2 mM EDTA, 6 M GdnHCl, 100 mM Tris-HCl, pH 8). Denaturing buffer was exchanged for
0.1% acetic acid using a Bio-Gel P6 spin column. Reduced, denatured RNase concentration was determined by absorbance at 280 nm using an
extinction coefficient of 9300 cm Equilibrium Dialysis--
Microdialysis cells from Bel-Art
Products were used with two 100 µl cavities separated by 12-14 kDa
molecular mass cut-off dialysis membrane (Spectra/Por).
Experiments were performed at 25 °C and incubated for 18-24 h,
sufficient time to ensure equilibrium as determined by following the
time course for approach to equilibrium in control experiments.
Dialysis buffer contained 10 mM DTT, 10 mM
NaCl, and 50 mM HEPES-NaOH, pH 7.0. Bis-ANS
concentration after equilibrium dialysis was determined by absorbance
at 385 nm. [3H]E2 and 125[I]T3
concentrations were determined by scintillation counting calibrated
against standard concentrations of [3H]E2 and
125[I]T3. There was no detectable degradation
of PDI during equilibration as detected by SDS-polyacrylamide gel
electrophoresis analysis.
Ligand Binding--
PDI binds a diverse set of ligands (Fig.
1), interacting with unfolded and folded
proteins, E2, T3, peptide-like antibiotics such as
bacitracin, and peptides such as oxidized insulin chain B (InB).
Despite this promiscuity in interacting with potential ligands, the
relationships among these sites have not previously been determined.
PDI has been shown to bind hydrophobic ligands such as
Nonspecific, hydrophobic surfaces and/or binding sites can often be
detected by fluorescence changes on binding the environmentally sensitive fluorescent probe, bis-ANS (40, 41). When bound to
hydrophobic sites, bis-ANS exhibits enhanced fluorescence
and a characteristic blue shift in the emission maximum. PDI binds bis-ANS with µM affinity, and the fluorescence
of the bound probe is enhanced and blue-shifted (~30 nm shift in
maximum emission), indicating that the bis-ANS is binding to
a hydrophobic site(s) on the protein (Fig.
3 and Table I). The fluorescence
intensity of bis-ANS bound to PDI was determined by
titrating a limited amount of bis-ANS (0.5 µM)
with increasing concentrations of PDI (0-20 µM) (40).
When calibrated in this way, the fluorescence intensity observed at
excess, saturating bis-ANS suggests the binding of 0.9 ± 0.2 mol of bis-ANS/mol of PDI (Table I). Calcium, yet
another PDI ligand (31), enhances the fluorescence of PDI-bound bis-ANS by ~1.6-fold with half-maximal enhancement
occurring at 0.9 ± 0.1 mM calcium (data not shown).
Magnesium, which like calcium affects PDI-chaperone/anti-chaperone
activity, has a similar effect, but sodium chloride does not.
Competition experiments show that T3 inhibits
bis-ANS binding (Fig. 4). The
maximum concentration of T3 is limited by solubility; however, extrapolation by fitting the data to a hyperbola suggests that
T3 displaces (>87%) bis-ANS fluorescence with
half-maximal inhibition occurring at 69 ± 5 µM at a
bis-ANS concentration of 10 µM. None of the
other known PDI ligands compete for this site; neither E2 (up to 64 µM), InB (up to 250 µM) nor bacitracin (up to 500 µM) significantly affects bis-ANS
binding to PDI. Thus, PDI has one hydrophobic site that binds
bis-ANS or T3, and the other ligands must bind
at different sites.
Equilibrium dialysis shows that there are two T3 binding
sites (Fig. 2). Although one of these also binds bis-ANS,
the other accommodates E2. Of all the ligands tested, only
T3 affects E2 binding (Fig.
5). Half-maximal inhibition occurs at
8.5 ± 3.2 µM T3, and T3 can
displace E2 completely ( Effects of Ligands on PDI Catalytic Activity--
E2 inhibits the
PDI-catalyzed refolding of reduced RNase; however, the inhibition in
only partial, and saturating E2 inhibits the isomerase activity by only
half (Fig. 6). Although T3
and E2 bind to the same site, T3 has no significant effect on PDI activity up to a concentration of 70 µM. Guthapfel
et al. (28) reported that T3 also partially
inhibits PDI with a Ki of 1.3 µM;
however, under very similar conditions, we repeatedly failed to observe
significant (±10%) inhibition of the isomerase activity. InB has
previously been shown to inhibit the isomerase activity with an
inhibition constant of 230 ± 40 µM (11).
Bacitracin, at concentrations that have previously been reported to
inhibit PDI (42), does inhibit PDI-catalyzed RNase refolding. However, at the same concentrations bacitracin also inhibits spontaneous RNase A
refolding in the absence of PDI to the same extent (data not shown).
The effects of bacitracin on PDI activity may reflect its interactions
with the RNase substrate and/or its interaction with PDI.
Effect of Ligands on PDI Chaperone and Anti-chaperone
Activities--
At high concentrations, PDI inhibits the aggregation
of unfolded proteins. This chaperone activity, which has been
attributed to the interaction of PDI and the unfolded protein substrate
(8, 19, 43), is observed during the folding of proteins that have disulfides as well as proteins that do not form disulfides. Under certain conditions and with certain substrate proteins, PDI exhibits what has been termed anti-chaperone activity. When it functions in its
anti-chaperone mode, PDI facilitates rather than inhibits substrate
aggregation (18, 20). We have suggested that the chaperone/anti-chaperone functions of PDI result from multiple, noncovalent interactions between PDI and an aggregation-prone substrate
(20). These interactions cross-link substrate aggregates until the PDI
concentration is sufficiently high to inhibit cross-linking, much like
the inhibition of classical immunoprecipitation under conditions of
antibody excess. Both chaperone and anti-chaperone activity are
observed during the thermal aggregation of ADH (20). At lower
concentrations of PDI, increasing PDI enhances substrate aggregation,
but at higher PDI concentrations, increasing PDI inhibits substrate
aggregation. At concentrations that should result in significant
occupancy of their binding sites, neither E2 nor T3 have
significant effects on the chaperone or anti-chaperone activity (Fig.
7). These ligands also have no effect on
PDI chaperone activity using citrate synthase as a substrate (20) (data
not shown). Both InB and bis-ANS inhibit ADH aggregation in
the absence of PDI, so that their effect on PDI chaperone and
anti-chaperone activities could not be determined. By contrast,
relatively low concentrations of bacitracin inhibited the chaperone
activity without having a large effect on substrate aggregation in the absence of PDI (Fig. 7).
PDI has been repeatedly isolated from cells based on its
propensity to bind a surprisingly diverse group of ligands. It is the
PDI, like some other folding assistants (47), must interact with a
large number of different substrates with few recognizable sequence
cues except for a small preference for peptides that contain cysteine
(10, 11). Recently, Winther and colleagues (13) found that peptide
sequence can affect the rate at which PDI reacts with peptide
disulfides with some preference for a sequence that has the features,
small/helix breaker-Cys-X-hydrophobic/basic-hydrophobic. Peptide
binding sites have been difficult to localize on PDI. Early indications
with a photo-affinity label suggested that a peptide binding site was
located near the C terminus (12). Deletion of this region has little
effect on isomerase activity, but it does eliminate chaperone activity
(17). Klappa et al. (10) have recently found that interaction between
PDI and peptides involves a single domain (b'), but other sites are
involved in the binding of large, protein substrates. Thus, multiple
sites with limited specificity are involved in the interaction of PDI with its protein substrates.
The competition binding experiments reported here show that PDI has
multiple ligand binding sites (Fig. 8),
some of which display overlapping specificity. However, the sites
involved in hormone binding are distinct from those involved in peptide
and substrate binding. Peptides and peptide analogs do not displace hormones from their binding sites, and the binding of hormones is
noncompetitive with protein substrate. The partial inhibition by E2
means that protein substrates can still interact with PDI, even when E2
is bound. The lack of an observable effect on chaperone/anti-chaperone behavior also argues that the hormone sites are not utilized during the
interaction between PDI and its unfolded protein substrates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of prolyl
4-hydroxylase (23) and as a subunit of the microsomal triglyceride-transfer complex (24). The level of PDI present in the ER
is considerably higher than either of these complexes, suggesting that
PDI is involved in the folding, stability, or assembly of the complex
and in its retention in the ER through the C-terminal KDEL signal of
PDI (25).
-estradiol (E2), and although
the number of estrogen binding sites has not been determined, estrogen
has been reported to inhibit isomerase activity (29). A short segment
of the PDI sequence exhibits some similarity to the estrogen receptor,
but this sequence does not appear to constitute an independent
structural domain (30). Finally, like many ER-resident proteins, PDI
binds calcium with low affinity (2-5 mM) and high capacity
(19 mol Ca2+/mol protein) (31). Calcium affects the
chaperone activity of PDI (20) but has no influence on the isomerase
function (20, 33).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 ml/mg) (35). bis-ANS was obtained from
Molecular Probes, and its concentration was determined by absorbance at
385 nm using an extinction coefficient of 16,790 cm
1
M
1 (36). E2, T3, DTT,
HEPES, equine liver alcohol dehydrogenase (ADH), bacitracin,
insulin chain B (oxidized, sulfonated), calcium chloride, and magnesium
chloride were purchased from Sigma. ADH concentration was determined by
absorbance at 280 nm using an extinction coefficient of 0.455 cm
1 ml/mg (37). Bacitracin is a mixture of multiple
peptides, with the prominent species being bacitracin-A (38, 39). The
molecular weight of bacitracin-A (1421) was used for estimating
bacitracin concentration. Tritiated E2 (2,4,6,7-[3H]E2;
specific activity, 84 Ci/mmol) and labeled T3
(L-5-[125I]T3, 230 Ci/mmol) were
from PerkinElmer Life Sciences with purity checked by reverse phase
high pressure liquid chromatography.
1
M
1.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-estradiol
(27) and T3 (28); however, the numbers of E2 and
T3 binding sites have not been determined. Equilibrium dialysis using purified, recombinant rat PDI (Fig.
2) shows that there is a single E2
binding site but two T3 sites, both of which have modest
affinity (Table I). Individual
Kd values cannot be resolved for the two
T3 binding sites, suggesting that the Kd
values are similar.
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Fig. 1.
PDI ligand structures. Top
panel, bis-ANS; middle panel,
T3; bottom panel, E2. The structures were drawn
using CS Chemdraw Pro, v3.1.
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Fig. 2.
Binding of T3 and
-estradiol to PDI. A, estradiol. 2 µM PDI was incubated with
2,4,6,7-3H(N)-estradiol in 50 mM HEPES, pH 7.0, 10 mM NaCl, and 10 mM DTT at 25 °C. The
binding is characterized by a Kd of 2.1 ± 0.5 µM with a maximum occupancy of 0.8 ± 0.1 sites
(solid curve). B, T3. PDI (4 µM) was equilibrated with
[125I]T3 50 mM HEPES, pH 7.0, 10 mM NaCl, and 10 mM DTT at 25 °C. The binding
is characterized by a Kd of 4.3 ± 1.4 µM with a maximum occupancy of 2.3 ± 0.2 sites
(solid curve).
Binding of hydrophobic ligands to PDI
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Fig. 3.
Fluorescence of bis-ANS in
the presence of PDI. 1 µM PDI was titrated with
increasing concentrations of bis-ANS in 50 mM
HEPES, pH 7.0, and 10 mM DTT at 25 °C. A,
emission spectra (excitation 370 nm) with increasing bis-ANS
concentrations (solid curves) and the emission spectrum of
bis-ANS in the absence of PDI (dotted curve).
B, fluorescence intensity at 489 nm (excitation at 370 nm)
as a function of total bis-ANS concentration. The
solid curve is a hyperbola drawn with parameters as shown in
Table I. The fluorescence intensity corresponds to the binding of
0.9 ± 0.2 bis-ANS molecules with a
Kd of 3 ± 0.3 µM (solid
curve).
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Fig. 4.
Displacement of bis-ANS by
other PDI ligands. The decrease in fluorescence intensity of
bis-ANS (10 µM) bound to 1 µM PDI in 50 mM HEPES, pH 7.0, and 10 mM DTT at 25 °C was
measured as a function of the concentration of other PDI ligands. 100%
corresponds to the fluorescence in the absence of added ligand. ,
T3;
, bacitracin;
, E2;
, InB.
95%). By contrast, neither
bis-ANS, bacitracin, nor InB has any effect on E2 binding. T3 displaces both E2 and bis-ANS from their
binding sites, but E2 and bis-ANS do not compete with each
other. This also implies that there must be at least two independent
binding sites for T3, consistent with the observed
stoichiometry of T3 binding.
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Fig. 5.
Effect of ligands on E2 binding. 2 µM PDI was incubated with
2,4,6,7-3H(N)-estradiol in 50 mM HEPES, pH 7.0, 10 mM NaCl, and 10 mM DTT at 25 °C. Various
concentrations of either T3 ( ), bacitracin (
), bis-ANS (
), or
InB (
) were added to constant concentrations of PDI and E2.
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Fig. 6.
Effect of ligands on PDI isomerase
activity. Various concentrations of either bis-ANS ( ) or E2
(
) were added PDI during the RNase A refolding assay. The
solid line represents effects of bis-ANS. Curves are drawn
using hyperbolic expressions and parameters (Table I) determined by
nonlinear least squares fitting. The PDI activity is shown relative to
that observed in the absence of ligands.
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Fig. 7.
Bacitracin affects the chaperone and
anti-chaperone activity of PDI. The aggregation of ADH (11 µM native dimer) was observed by turbidity (absorbance)
measurements at 360 nm during the thermal denaturation (40 °C) in
the presence of PDI alone ( ), PDI and 5 µM bacitracin
(
), or PDI and 15 µM bacitracin (
). Buffer was 10 mM DTT, 50 mM HEPES-NaOH, pH 7. Aggregation
rates are normalized so that the rate of ADH alone has a value of 1. Curves are drawn, not fitted. Data with 1 or 2 µM
bacitracin were not significantly different from PDI alone.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of prolyl hydroxylase (23) and a subunit of an ER
triglyceride transferase (24), although its catalytic activity is not
required for the function of these complexes (25). PDI was also
identified in a screen for proteins that interact with a potential
glycosylation binding site present on a small peptide photo-affinity
label (44), although it was subsequently found that PDI does not
participate directly in glycosylation (45). In a search for thyroid
hormone binding proteins, PDI was identified through photoaffinity
labeling as a major T3 binding protein (46).
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Fig. 8.
Model for hydrophobic binding sites of
PDI. Competition binding experiments and stoichiometry
measurements indicate that there are two hormone binding sites on PDI.
One site binds T3 or E2. The other site binds the
hydrophobic probe, bis-ANS, or another molecule of
T3. Thus, there are two sites that bind T3.
Ligands that bind to either of these sites are not displaced by the
binding of peptides.
Previous measurements of T3 and E2 binding to PDI have been performed by displacement of a small amount of labeled hormone by unlabeled competitor. Affinities estimated in this way suggested that PDI binds both hormones with nM affinity (26, 29), although lower affinity sites were observed as backgrounds. Guthapfel et al. (28) has recently shown that high affinity (nM) T3 binding is not stoichiometric (<0.01 mol hormone/mol PDI), implying that tight binding is from impurities rather than from PDI itself. By contrast, the low affinity (µM) binding we observe for T3 and E2 (Table I) is stoichiometric. Although PDI does not exhibit high affinity for these hormone ligands, it does have a limited number of reasonably specific sites for their interaction that do not interfere significantly with the catalytic or chaperone activities of the molecule. Thus, hormones are not binding to sites that interact with protein substrates.
With dissociation constants of 2-4 µM (Table I), it
would be tempting to conclude that hormone binding by PDI does not have any physiological relevance because the Kd values
for hormone binding to their cellular receptors are in the low
pM range (Kd = 10-300 pM)
(48-50). However, the local concentration of PDI in the ER is quite
high, estimated at ~200 µM (51). Thus, the binding of
hormone to PDI will be driven by the high, local concentration of PDI
despite the low Kd value. A specific example
illustrates how the presence of a high capacity, low affinity reservoir
will affect the location of hormone (Fig.
9). With a Kd for
hormone binding to PDI of 2 µM and a concentration of 200 µM PDI, simple equilibrium binding tells us that ratio [HR-PDI]/[HR]free will be 100, and 99% of the E2 in
the endoplasmic reticulum will be bound to PDI. If the ER represents
10% of the cell volume, then about 90% of the intracellular E2 would
be bound to PDI at any given free concentration of hormone. At a free
cytosolic concentration of hormone (10 pM) that is
sufficient to half-saturate the estrogen receptor, hormone binding to
PDI would require a total intracellular concentration of 100 pM to generate a free hormone concentration of 10 pM. The total concentration of hormone bound to PDI would
be ~90 pM, whereas the free concentration of hormone
would be 10 pM. With a high capacity reservoir for hormone binding, the concentration of hormone necessary to generate a physiological response would be significantly higher than the Kd for equilibrium hormone binding. Physiological
concentrations of T3 range from about 10 pM in
serum (52) to 75 pM in brain cytosol (53). The
physiological E2 concentrations are larger, from 0.2-2 nM
in human serum (54), to 0.4 µM in placenta (55), and up
to 3-5 µM in developing follicles in the ovary (56). All
of these are significantly higher than the Kd
measured in vitro (48-50), suggesting that there must be
some mechanism for modulating the response of hormone receptor to
physiological concentrations of hormone.
|
The function of high capacity reservoirs of low affinity may be to
balance the need for high specificity and regulation. Tight binding is
essential for high specificity and for discriminating between similar
structures. For very high affinity receptors, such as the estrogen and
T3 receptors, cell volume may limit the number of molecules
present in a given cell. For example, with a cell volume of 6000 µ3 (32), at a concentration of 10 pM, there
would be only 40 molecules of hormone/cell. A stochastic fluctuation in
the distribution of hormone molecules in the cells would raise the
possibility that some cells would not have any hormone molecules. By
increasing the number of molecules in the cell by 10-fold or more, a
low affinity, high capacity reservoir of hormone binding could
significantly increase the number of hormone molecules/cell and
decrease the probability that a cell would, by chance, find itself
without hormone when a response was warranted.
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ACKNOWLEDGEMENTS |
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We thank Dr. John Putkey for use of his fluorimeter and advice and Jeff Lawton for assistance with scintillation counting.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM40379.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Biological Sciences, University of Texas
at El Paso, 500 West University Ave., Biology Bldg., Rm. B416, El Paso,
TX 79968.
§ To whom correspondence should be addressed. Tel.: 713-798-5880; Fax: 713-796-9438; E-mail: hgilbert@bcm.tmc.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007670200
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ABBREVIATIONS |
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The abbreviations used are:
PDI, protein
disulfide isomerase;
bis-ANS, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid;
T3, 3,3',5-triiodo-L-thyronine;
ER, endoplasmic reticulum;
DTT, dithiothreitol;
E2, 17-estradiol;
ADH, horse liver alcohol
dehydrogenase;
InB, insulin chain B.
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REFERENCES |
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