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INTRODUCTION |
G protein-coupled receptors
(GPCR)1 respond to a wide
range of structurally diverse natural and synthetic ligands. The
majority of these ligands are classified as orthosteres. They compete
for binding with the natural ligand, and their binding site is
predicted to overlap with that of the natural ligand. Allosteric
modulation of ligand binding has been observed for several GPCRs.
Allosteric modulators include physiologically relevant ions as well as
small organic molecules. A large number of compounds allosterically increase binding affinity for muscarinic receptor antagonists. Allosteric regulation can be demonstrated in all five subtypes of
muscarinic receptors, but the m2 receptor appears to be the most
sensitive (1). Although most allosteric modulators for the muscarinic
receptors affect antagonist binding, allosteric modulation of agonist
binding has also been described (2).
In addition to the muscarinic receptor, allosteric compounds have been
identified for the A1 adenosine receptor. The
2-amino-3-benzoylthiophene, PD 81,723, has been shown to enhance
agonist binding and G protein coupling in a
Mg2+-dependent manner (3, 4).
The function of several Gi-coupled receptors has been shown
to be modulated by amilioride analogs and by Na+. This
effect has been particularly well characterized for the
2 adrenergic
receptor (5-7) and for the dopamine D2 (8) and D4 subtypes (9).
Na+ both uncouples the receptor from Gi and
reduces agonist affinity. A conserved Asp residue within the
cytoplasmic side of TM2 is responsible for the
Na+-sensitive binding of the
2A receptor (6) and the D4
dopamine receptor (9). This Na+ sensitivity may be
physiologically relevant, as relatively high local concentrations of
Na+ ions may accumulate at the cytoplasmic side of the
receptor after membrane depolarization.
Recently the function of the calcium receptor has been shown to be
positively modulated by certain L-amino acids (10, 11). This finding may be physiologically relevant as nutrient and
Ca2+ homeostasis may be coordinately regulated.
We recently reported that Zn2+ has complex effects on the
functional properties of the
2AR (12). Zn2+
binding to a high affinity site (IC50 ~5
µM) enhances agonist affinity, whereas Zn2+
binding to one or more low affinity sites (IC50 >500
µM) inhibits antagonist binding and yet slows antagonist
dissociation. To identify the Zn2+ binding site responsible
for the positive allosteric effect of Zn2+ on agonist
affinity, we mutated seven histidines located in intracellular and
extracellular hydrophilic sequences connecting transmembrane (TM)
domains. Mutation of His-269 dramatically reduced the effect of
Zn2+ on agonist affinity. Mutations of other histidines had
no effect. Further mutagenesis of residues predicted to be adjacent to
His-269 in the three-dimensional structure of the
2AR
revealed that Cys-265 and Glu-225 are also required to achieve the full
allosteric effect of Zn2+ on agonist binding. Our results
suggest that bridging of the cytoplasmic extensions of TM5 and TM6 by
Zn2+ facilitates agonist binding. These results are in
agreement with recent biophysical studies demonstrating that agonist
binding leads to movement of TM6 relative to TM5.
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EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis--
The cDNA sequence encoding
the human
2AR, epitope-tagged at the amino terminus with
a cleavable influenza-hemagglutinin signal sequence followed by the
FLAG epitope (IBI, New Haven, CT) and tagged with six histidines at the
carboxyl terminus, was used as a template for mutagenesis (13). This
cDNA was subcloned in baculovirus expression vector pACMP2 at
BamHI/EcoRI site. Mutations were generated by the
polymerase chain reaction-mediated mutagenesis using Pfu
polymerase (Stratagene). The polymerase-generated DNA fragments were
subcloned into pACMP2 plasmid, and the single mutants H93A, H172A,
H178A, H241A, H256A, H269A, H296A, and E225A and double mutants
E225A/H269A and E225A/C265A were confirmed by DNA sequencing and
restriction analysis. The construct C265A
2AR was a gift
from Dr. Charles Parnot.
Mammalian Cell Culture--
HEK293 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. Stable cell lines were obtained for wild-type
2AR (WT
2AR) and the mutants in the presence of 0.5 mg/ml G418.
Expression of the Receptors in SF9 Insect
Cells--
SF-h
2-6H and the mutant constructs in the
baculovirus expression vector pACMP2 were co-transfected with
linearized SapphireTM Baculovirus DNA into SF9 insect cells
using the Insectin Plus transfection kit (Invitrogen). The resulting
viruses were harvested 4-5 days and amplified for another 4-5 days to
obtain a high titer virus. SF9 insect cells were grown in suspension
culture in SF-900 II medium (Invitrogen) containing 5% fetal
calf serum (Gemini, Calabasas, CA) and 0.1 mg/ml gentamicin
(Invitrogen). For membrane preparation the cells were grown in 100-ml
cultures. Cells were infected with a 1:100 dilution of a high titer
virus stock at a density of 3.5-5.5 × 106 cells/ml
and harvested after 48 h by centrifugation for 10 min at 5000 × g. The resulting cell pellets were kept at
80 °C
until further use.
Membrane Preparation--
All of the membrane preparation steps
were done at 4 °C as described elsewhere (12). Cells were harvested
and washed once with phosphate-buffer saline, recentrifuged, and then
resuspended in lysis buffer (10 mM Tris-HCl, pH 7.5, with 1 mM EDTA) containing protease inhibitors (leupeptin and
benzamidine at a final concentration of 10 µg/ml) and lysed using 25 strokes of a Dounce homogenizer. Nuclei and unbroken cells were removed
by centrifugation (5 min at 500g). The supernatant was removed and
centrifuged at 40,000 × g for 30 min. The resulting
pellet was resuspended in 20 ml of 10 mM Tris-HCl, pH 7.5, buffer alone containing protease inhibitors and recentrifuged.
Membranes were resuspended at 0.5-1.5 mg/ml in binding buffer (75 mM Tris-HCl, pH 7.5) and stored at
80 °C until use.
Binding Assays--
All of the binding assays were done on SF9
membranes expressing either the WT
2AR or mutant
receptors. The saturation binding assays were done by incubating the
membranes (20-50 µg) with 10 different concentrations of the
antagonist [3H]DHA between 100 pM and 20 nM with or without 1 mM Zn(II). Competition assays were done by incubating the membranes with different
concentrations of agonist isoproterenol (10
9 to
10
3 M) with or without 20 µM
Zn(II). Zn2+ modified competition assays were also done
similar to competition assays expect that membranes were incubated with
different concentrations of Zn(II) (0.3-300 µM) in the
presence or absence of 100 nM isoproterenol. All of the
assays were performed for 1 h at room temperature with shaking at
230 rpm. Competition assays were carried out with 1 nM
[3H]DHA around the KD. Binding data
were analyzed by nonlinear regression analysis using Prism from
GraphPad Software, San Diego, CA. Inhibitory constant
(KI values) were calculated from
IC50 values using the Cheng-Prusoff equation:
KI = IC50/(1 + [ligand]/KD).
Dissociation Rate Kinetic Assay--
The effect of
Zn2+ on the rate of antagonist dissociation from
2AR was examined by measuring the
koff in the absence and presence of 1 mM Zn2+. Membranes were suspended in 75 mM Tris-HCl, pH 7.5, with 1 nM [3H]DHA for 30 min at room temperature (shaking at 230 rpm). At time zero, total binding was determined, and a saturating
amount of cold alprenolol (final 10
5 M) or
cold alprenolol (final 10
5 M) and
ZnCl2 (final 1 mM) was added to tubes
containing membranes and [3H]DHA. Bound
[3H]DHA was measured at 5-min intervals.
cAMP Accumulation--
The production of cAMP was determined by
adenylyl cyclase activation FlashPlate assay (PerkinElmer Life
Sciences), in which 96-well plates are coated with solid
scintillant to which anti-cyclic cAMP antibody has been bound. Briefly,
HEK293 cells expressing stable human
2AR and mutants
were detached, washed four times in 1× phosphate-buffered saline
without Ca2+/Mg2+, and then resuspended to a
density of ~2 × 106 cells/ml in stimulation buffer
(1× phosphate-buffered saline without calcium/magnesium, with 700 µM 3-isobutyl-1-methylxanthine, 0.1% protease-free
bovine serum albumin, and 0.09% chloroacetamide) from PerkinElmer Life
Sciences. Ligands (25 µl each) were diluted in Milli-Q water with
various concentrations and dispensed to the FlashPlate. Resuspended
whole cells (50 µl) were added to the ligand-loaded plate and
stimulated at 37 °C for 10 min before lysing cells with 100 µl of
Detection Buffer (Invitrogen) containing [125I]cAMP,
permeabilizer, and 0.09% sodium azide as provided by the manufacturer.
After 2 h of incubation at room temperature, radioactivity was
counted. To determine the concentrations of cAMP in the sample, cAMP
standards were run in the same plate and expressed as pmol/well. The
expression level of mutant
2ARs in stable HEK293 cell
lines was assessed using a single saturating concentration of
[3H]dihydroalprenelol (10 nM).
Miscellaneous--
Protein concentration was determined using
the Bio-Rad DC protein assay kit.
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RESULTS |
The Effect of Zn2+ on Binding of Agonist and
Antagonists to the
2AR--
To examine the effect of
Zn2+ on both agonist and antagonist binding to the
2AR, we performed a modified competition experiment in
which the
2AR is incubated with 1 nM
[3H]DHA and varying concentrations of Zn2+ in
the presence or absence of 100 nM isoproterenol. These
studies are also done with 10 µM GTP
S to eliminate the
effects of Gs on agonist binding. In the absence of
isoproterenol we observed a reduction of [3H]DHA binding
only at relatively high concentrations of Zn2+ (>500
µM). Agonist binding is more sensitive to
Zn2+. In the presence of increasing concentrations of
Zn2+, 100 nM isoproterenol becomes more
effective at displacing [3H]DHA (Fig.
1A). The maximal effect of
Zn2+ on isoproterenol affinity occurs at ~20
µM with an IC50 of 3.0 µM (12).
These effects of Zn2+ can also be observed in
more conventional saturation (Fig. 1B) and competition (Fig.
1C) binding studies. At 1 mM Zn2+ we
observe a decrease in Bmax and an increase in
the KD for [3H]DHA (Fig.
1B). In contrast, the affinity of the
2AR for
isoproterenol is enhanced in the presence of 20 µM
Zn2+ (Fig. 1C).

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Fig. 1.
Effect of Zn2+ on
antagonist and agonist binding in
WT 2AR expressed in SF9
membranes. A, inhibition of antagonist binding by
different concentrations of Zn2+ in the presence or absence
of 100 nM isoproterenol. The assays were performed as
described under "Experimental Procedures." B, saturation
binding of [3H]DHA (100 pM-10
nM) on membranes expressing WT 2AR receptor
(17 pmol/mg protein) in the presence or absence of 1 mM
Zn2+. Data were fit to a monophasic saturation hyperbolae.
C, effect of Zn2+ on agonist binding.
Isoproterenol competition assays were performed with 1 nM
[3H]DHA in the presence or absence of 20 µM
Zn2+. Data represent the mean of 2-4 experiments. Each
experiment was done in triplicates.
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Identification of the Allosteric Binding Site for
Zn2+--
Histidines are often involved in the formation
of Zn2+ binding sites in proteins (14, 15). We hypothesized
that the Zn2+ binding site responsible for the positive
allosteric effect on agonist binding to the
2AR would be
located in extracellular loops. However, mutations of histidines 93, 172, 178, and 296 (Fig. 2) had no effect
on the positive allosteric effect of Zn2+ (data not shown).
We therefore examined the role of cytoplasmic histidines
(His-241, His-256, and His-269) (Fig. 2). Of these, only H269A
exhibited a reduced response to Zn2+. The effect of
Zn2+ on both agonist and antagonist affinity is nearly
abolished in H269A (Fig. 3; Tables
I and II). Of interest, we found that
H269A mutation does not alter the effect of 1 mM
Zn2+ on the dissociation rate of [3H]DHA
(data not shown), indicating that the
site responsible for the positive allosteric effect of Zn2+
on agonist binding is different from the binding site responsible for
the effect of Zn2+ on the dissociate rate of
antagonists.

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Fig. 2.
Two-dimensional representation of human
2AR. The highlighted
residues have been mutated to identify putative Zn2+
binding sites.
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Fig. 3.
Effect of Zn2+ on
antagonist and agonist binding in
H269A 2AR expressed in SF9
membranes. A, inhibition of antagonist binding by
different concentrations of Zn2+ in the presence or absence
of 100 nM isoproterenol. The assays were performed as
described under "Experimental Procedures." B, saturation
binding of [3H]DHA (100 pM-10
nM) on membranes expressing H269A 2AR
receptor (4.4 pmol/mg protein) in the presence or absence of 1 mM Zn2+. Data were fitted to monophasic
saturation hyperbolae. C, effect of Zn2+
on agonist binding. Isoproterenol competition assays were performed
with 1 nM [3H]DHA in the presence or absence
of 20 µM Zn2+. Data represent the mean of
2-4 experiments, each done in triplicate.
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Table I
Binding properties of WT and 2AR mutants
Saturation experiments using [3H]DHA were performed on
membranes expressing 2AR or the mutants from SF9 membranes.
Values represent the means of two or more experiments ±S.E. Each
experiment was done in triplicate. All calculations were obtained using
the Graphpad software program.
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Table II
Pharmacological characterization of WT and mutant 2AR
receptors
Agonist binding properties of WT and mutants in the presence or absence
of 20 µM zinc are reported here. The competition assays
were performed as described under "Experimental Procedures" in the
presence of 10 µM GTP S. The results are reported as
KI (nM) and the
KI ratio (+zinc)/( zinc) is given. Data were
obtained from 2-5 experiments performed in triplicate.
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High affinity Zn2+ binding requires coordination by at
least two amino acids. In addition to histidine, cysteine, aspartate, and glutamate can contribute to the formation of a Zn2+
binding site (16, 17). We therefore used a three-dimensional model
based on rhodopsin (18) to identify cysteines as well as acidic
residues in proximity to His-269. This analysis identified the closest
candidates as Cys-265 on the cytoplasmic end of TM6 and Glu-225 on the
cytoplasmic end of TM5. Mutation of Glu-225 and Cys-265 to alanine had
no significant effect on either agonist or antagonist affinity in the
absence of Zn2+ (Tables I and II). However, both mutant
receptors had altered agonist and antagonist responses to
Zn2+. For both mutants there was a smaller decrease in
antagonist affinity in the presence of 1 mM
Zn2+ (Table I) and a smaller increase in agonist affinity
in the presence of 20 µM Zn2+ (Table II). The
effect of Zn2+ on agonist affinity is almost abolished for
the double mutant E225A/H269A and completely abolished when C265A is
combined with H269A (Tables I and II, and Fig.
4).

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Fig. 4.
Effect of Zn2+ on
agonist affinity for
C265A/H269A 2AR mutant expressed in
SF9 membranes. Isoproterenol competition assays were performed
with 1 nM [3H]DHA in the presence or absence
of 20 µM Zn2+. Data represent the mean ± S.D. of three independent experiments performed in triplicate.
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Zn2+ Augmentation of ISO-stimulated cAMP
Accumulation--
We previously reported that low concentrations of
Zn2+ enhance cAMP accumulation by submaximal concentrations
of isoproterenol in intact cells (12). To determine whether the
Zn2+ site formed by His-269, Cys-265, and Glu-225 is also
responsible for the effect of Zn2+ on cAMP accumulation, we
generated stable cell lines expressing the wild-type
2AR
as well as H269A, C265A, C265A/H2269A, and E225A/H269A. Fig.
5A shows the effect of
Zn2+ on cAMP accumulation in the presence and absence of
0.1 nM isoproterenol in nontransfected HEK293 cells and in
HEK293 cells stably expressing the wild-type
2AR.
Isoproterenol at 0.1 nM stimulates a submaximal cAMP
response that is augmented by 10 µM Zn2+. As
shown in Fig. 5B, this effect of Zn2+ was also
observed in all of the cell lines expressing mutant receptors.
Therefore, the Zn2+ binding site responsible for allosteric
modulation of agonist binding is not responsible for the effect of
Zn2+ on cAMP accumulation in intact cells.

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Fig. 5.
Zn2+ augmentation of
cAMP accumulation stimulated by a submaximal concentration of ISO.
A, effect of different concentrations of Zn2+ on
cAMP accumulation in the presence and absence of 0.1 nM ISO
in untransfected cells (UT) and cells expressing
WT 2AR. B, effect of different concentrations
of Zn2+ on cAMP accumulation in the presence of 0.1 nM ISO in cells stably expressing WT 2AR and
H269A, C265A, C265A/H2269A, and E225A/H269A. The expression of stable
WT 2AR and mutants are as follows (in pmol/mg protein):
WT 2AR, 14.9; H269A, 16.6; C265A, 19.7; C265A/H269A,
21.1; E225A/H269A, 26.7. Data represent the mean of three independent
experiments performed in triplicate.
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DISCUSSION |
Allosteric ligands have been identified for only a small number of
GPCRs including the M2 muscarinic receptor (19), the A1 adenosine
receptor (3), and the Ca2+-sensitive receptor (11).
Binding of the well characterized muscarinic allosteric ligand
gallamine has been mapped to extracellular loops 2 and 3 (20, 21). It
has been proposed that, when bound to gallamine, extracellular loops 2 and 3 may form a plug over the binding pocket, thereby slowing the
dissociation rate (22).
Another well characterized allosteric modulator of GPCR function is
ionized sodium. Sodium ions act as negative allosteric regulators for a
number of Gi-coupled receptors including the
2A
adrenergic receptor and the D2 and D4 dopamine receptor. In contrast to
the extracellular location of the allosteric binding site for
gallamine in muscarinic receptors, sodium sensitivity is mediated at
the cytoplasmic surface of the receptor. A highly conserved Asp at the
cytoplasmic end of TM2 has been shown to be critical for this effect of
sodium (6, 9).
Our mutagenesis studies localize the binding site responsible
for the positive allosteric effect of Zn2+ on agonist
affinity to the third intracellular loop. This Zn2+ binding
site consists of Cys-265 and His-269 on the cytoplasmic extension of
TM6 and Glu-225 on the cytoplasmic extension of TM5. This location is
of particular interest because recent biophysical studies from our
laboratory demonstrate that agonist binding is associated with a
movement of Cys-265 on TM6 relative to TM5 (23). These studies were
performed on purified
2AR labeled at Cys-265 with
fluorescein. Moreover, cysteine cross-linking studies on the M3
muscarinic receptor provide evidence for agonist-induced movement of
the cytoplasmic end of TM5 relative to TM6 (24). Thus, Zn2+
may form a bridge between TM5 and TM6. This bridge is likely to alter
the position of TM5 relative to TM6. On the basis of these
observations, we speculate that Zn2+ binding approximates
or stabilizes the conformational changes induced by agonists. It is of
interest that this Zn2+ binding site is not responsible for
the ability of Zn2+ to augment cAMP accumulation by
submaximal concentrations of isoproterenol (Fig. 5) or for the effect
of Zn2+ on antagonist dissociation (data not shown).
In conclusion, we have identified the Zn2+ binding site
responsible for the positive effect of Zn2+ ions on agonist
binding to the
2AR. Our results suggest that Zn2+ stabilizes an orientation of TM6 relative to TM5 that
favors agonist binding and inhibits antagonist binding. These results provide further evidence for the role of TM5 and TM6 in agonist-induced conformational changes.