From the NeuroScience PharmaBiotec Research Centre, Departments of
Medicinal Chemistry and ¶ Pharmacology, The Royal
Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen,
Denmark, and § ZymoGenetics Inc.,
Seattle, Washington 98102
Received for publication, August 9, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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The metabotropic glutamate receptors (mGluRs)
belong to family C of the G-protein-coupled receptor (GPCR)
superfamily. The receptors are characterized by having unusually long
amino-terminal domains (ATDs), to which agonist binding has been shown
to take place. Previously, we have constructed a molecular model of the ATD of mGluR1 based on a weak amino acid sequence similarity with a
bacterial periplasmic binding protein. The ATD consists of two globular
lobes, which are speculated to contract from an "open" to a
"closed" conformation following agonist binding. In the
present study, we have created a Zn2+ binding site in
mGluR1b by mutating the residue Lys260 to a histidine. Zinc
acts as a noncompetitive antagonist of agonist-induced IP accumulation
on the K260H mutant with an IC50 value of 2 µM. Alanine mutations of three potential "zinc
coligands" in proximity to the introduced histidine in K260H knock
out the ability of Zn2+ to antagonize the agonist-induced
response. Zn2+ binding to K260H does not appear to affect
the dimerization of the receptor. Instead, we propose that binding of
zinc has introduced a structural constraint in the ATD lobe, preventing
the formation of a "closed" conformation, and thus stabilizing a
more or less inactive "open" form of the ATD. This study presents
the first metal ion site constructed in a family C GPCR. Furthermore,
it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR and the loops connecting these. The findings offer valuable insight into the mechanism of ATD closure and family C receptor activation. Furthermore, the findings demonstrate that ATD regions other than those
participating in agonist binding could be potential targets for new
generations of ligands for this family of receptors.
(S)-Glutamic acid
(Glu)1 is the major
excitatory neurotransmitter in the central nervous system and plays an
essential role in a wide range of functions, such as brain development,
memory formation, and neurotoxicity (1). Excessive glutamatergic
signaling has been linked to the pathogenesis of certain psychiatric
and neurological disorders, including schizophrenia, epilepsy, and ischemia (1). Glu mediates its effects through two distinct types of
receptors: the ionotropic glutamate receptors and the metabotropic glutamate receptors (mGluRs). Whereas the ionotropic glutamate receptors form ligand-gated ion channels, which directly mediate electrical signaling of nerve cells, the G-protein-coupled mGluRs regulate signaling through stimulation or inhibition of intracellular metabolic cascades (1).
The mGluRs are members of family C of the G-protein-coupled receptor
(GPCR) superfamily, which also includes two With the exception of the orphan receptors in family C (5-7), all of
its members contain remarkably large extracellular amino-terminal domains (ATDs). The ATDs can be up to 600 amino acids long, and agonist
binding to the family C receptor has been demonstrated to take place to
this region (11-17). Weak amino acid sequence similarities exist
between these ATDs and a family of periplasmic binding proteins (PBPs)
involved in the transfer of nutrients into the cytoplasm in bacteria
(11). We have previously applied the crystal structure of one of these
PBPs, the leucine/isoleucine/valine-binding protein, as a template for
the construction of a tertiary molecular model of the ATD of mGluR1,
and similar models have been published for mGluR4, GABABR1,
and CaR (11, 18-20). According to these models, the ATD of the family
C receptor consists of two globular domains ("lobes") connected by
a hinge region. Based on the information obtained from crystal
structures of the PBPs, the ATD of the family C receptor is speculated
to exist in two conformations: an "open" inactivated and a
"closed" activated form (21). Agonist binding to the ATD is
believed to take place to a closed form of the region. How the
closure of ATD subsequently is translated into G-protein coupling and
effector enzyme activation or inhibition is at present poorly understood.
Two conserved serine/threonine residues throughout the PBPs and the
family C receptors have been shown to be important for agonist binding
to and/or activation of mGluR1, mGluR4, GABABR1, and CaR
(11, 16, 18, 19). Furthermore, an arginine residue conserved within the
mGluRs has been shown to be crucial for agonist binding to and/or
activation of mGluR1 and mGluR4, most likely through an ionic
interaction with the distal negatively charged group of the agonists
(18, 22). According to our model of the "open" ATD of mGluR1, the
three residues identified as key participants in agonist binding,
Arg78, Ser165, and Thr188, are
located in the same lobe of the ATD.
As an attempt to shed light on the contraction process of the ATD
following agonist binding, we have created a high affinity zinc binding
site in mGluR1 by mutating Lys260, located in the top of
the lobe opposite to the lobe containing the agonist binding residues,
to a histidine. The results of the study provide interesting
information regarding the ATD closure mechanism and the structural
flexibility within each ATD lobe.
Materials--
Culture media, serum, antibiotics, and buffers
for cell culture were obtained from Life Technologies (Paisley, UK).
[3H]Quisqualic acid ([3H]Quis) and
myo-[2-3H]inositol were purchased from Amersham Pharmacia
Biotech. (S)-4-Carboxyphenylglycine ((S)-4CPG)
and 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt) were purchased from Tocris (Bristol, UK).
Rabbit anti-mGluR1a polyclonal antibody and horseradish peroxidase-goat anti-rabbit IgG were purchased from Chemicon (Temecula, CA) and Zymed Laboratories Inc. (San Francisco, CA),
respectively. All other chemicals were obtained from Sigma. The pSI
vector was obtained from Promega (Madison, WI). The plasmids pmGluR1a,
rCaR-pRK5, and m5-pCD were generous gifts from Professor Shigetada
Nakanishi (Kyoto University, Japan), Professor Solomon H. Snyder (The
John Hopkins University School of Medicine, Baltimore, MD), and Dr. Mark R. Brann (ACADIA Pharmaceuticals, San Diego, CA), respectively. The tsA cells and the mGluR1a-CHO cell line were generous gifts from
Dr. Penelope S. V. Jones (University of California, San Diego, CA)
and professor Shigetada Nakanishi, respectively.
Subcloning of Receptors and Construction of Mutant
Receptors--
The subcloning of mGluR1b, mGluR1a, and CaR from their
respective vectors into the pSI vector have all been described
previously (16, 23). The point-mutated mGlu1b receptors
were constructed using the QuickChange mutagenesis kit according to the
manufacturer's instructions (Stratagene, La Jolla, CA). The
construction of mutant K260H mGluR1a-pSI for the Western blot
experiments were done by subcloning a region containing the
Lys260 Cell Culture--
tsA cells (a transformed HEK 293 cell line
(24)) were maintained at 37 °C in a humidified 5% CO2
incubator in Dulbecco's modified Eagle's medium supplemented with
penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% calf serum.
Inositol Phosphate (IP) Assay--
1 × 106 tsA
cells were split into a 10-cm tissue culture plate and transfected with
5 µg of plasmid the following day using Superfect as a DNA carrier
according to the protocol by the manufacturer (Qiagen, Hilden,
Germany). The day after transfection, the cells were split into a
poly-D-lysine-coated 24-well tissue culture plate in
inositol-free Dulbecco's modified Eagle's medium, supplemented with
penicillin (100 units/ml), streptomycin (100 µg/ml), 10% dialyzed
fetal calf serum, and 1 µCi/ml myo-[2-3H]inositol.
16-24 h later, the cells were washed with Hanks' balanced saline
solution (HBSS) and incubated at 37 °C for 10 min in HBSS supplemented with 0.9 mM CaCl2, 1.05 mM MgCl2, and 10 mM LiCl (and in
the antagonism experiments, various concentrations of ZnCl2
or 100 µM CPCCOEt). The buffer was removed, and the cells were incubated for 40 min in the same buffer containing various concentrations of Glu, a fixed Glu concentration and various
concentrations of ZnCl2 or 100 µM CPCCOEt, or
containing a fixed ZnCl2 concentration and various
concentrations of Glu. The reactions were stopped by exchanging the
buffer with 500 µl of ice-cold 20 mM formic acid, and
separation of total [3H]inositol phosphates was carried
out by ion exchange chromatography as described previously (23, 25,
26). The effect of ZnCl2 on tsA cells transfected with
CaR-pSI or m5-pCD was studied in the same manner, except that
CaCl2 and acetylcholine were used as agonists,
respectively. The effects of CuCl2 and CoCl2 on
cells transfected with wild type (WT) or K260H mGluR1b were studied using the same protocol as in the Zn2+ antagonism
experiments. Almost all IP experiments were performed in triplicate (a
few were performed in duplicate), and the results are given as
mean ± S.E. of at least three independent experiments.
[3H]Quis Binding Assays--
2 × 106 tsA cells were split into a 15-cm tissue culture plate
and transfected with 10 µg of plasmid the following day using Superfect as a DNA carrier according to the protocol by the
manufacturer (Qiagen, Hilden, Germany). The day after transfection, the
medium was changed to Dulbecco's modified Eagle's medium supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 10%
dialyzed fetal calf serum. The following day, the cells were suspended
in ice-cold 20 mM HEPES-NaOH, 10 mM EDTA (pH
7.4) and centrifuged at 50,000 × g at 4 °C for 20 min. The pellet was homogenized (using a Polytron homogenizer for
10 s) in ice-cold 20 mM HEPES-NaOH, 0.1 mM
EDTA (pH 7.4) and centrifuged at 50,000 × g at 4 °C
for 20 min. This step was performed twice. The membranes were
homogenized in assay buffer, 20 mM HEPES-NaOH, 2 mM MgCl2, 2 mM CaCl2
(pH 7.4), and protein concentrations were measured using Bradford Protein Assay with bovine serum albumin as standard (Bio-Rad). In the
saturation binding experiments with WT, K260H, and
K260H/H231A/E233A/E238A mGluR1b, 5-15 µg of membrane fractions were
incubated for 1 h at room temperature with various concentrations
of [3H]Quis in the absence or presence of 1 mM Glu (representing total and nonspecific binding,
respectively) in a total assay volume of 100 µl.
[3H]Quis was diluted with unlabeled
(S)-quisqualic acid at concentrations above 250 nM. Expression levels of other mutant receptors were estimated by measuring binding at a concentration of 250 nM
[3H]Quis in the absence and presence of 1 mM
Glu. To investigate whether Zn2+ was able to displace
[3H]Quis binding to WT or K260H mGluR1b, 50-100-µg
membrane fractions of cells transfected with the two receptors were
incubated for 1 h at room temperature with 30 nM
[3H]Quis in the absence or presence of 1 mM
Glu, 100 µM (S)-4CPG, 100 µM
CPCCOEt, or 100 µM ZnCl2. The assay mixture
was aspirated onto a GF/C filter (Whatman) and washed with 3 × 5 ml of ice-cold assay buffer, after which the filters were dried and
counted in a scintillation counter. The binding experiments were
performed in duplicate or triplicate at least three times.
Western Blots--
The rabbit anti-mGluR1a polyclonal antibody
used is directed toward the Glu1116-Leu1130
peptide in the C terminus of mGluR1a. This region does not exist in the
"short tailed" splice variant mGluR1b. Hence, we used WT mGluR1a
and the mutant K260H mGluR1a in the experiments. 1 × 106 tsA cells were split into a 10-cm tissue culture plate
and transfected with 5 µg of plasmid (WT mGluR1a-pSI, K260H
mGluR1a-pSI, or pSI) the following day using Superfect as a DNA carrier
according to the protocol by the manufacturer (Qiagen, Hilden,
Germany). The day after transfection, the medium was changed to
Dulbecco's modified Eagle's medium supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% dialyzed fetal calf
serum. The following day, the cells were washed twice in HBSS and
scraped into lysis buffer (2 mM HEPES, 2 mM
EDTA, containing protease inhibitors (CompleteTM, Roche, Switzerland)
and 100 mM iodoacetamide) and homogenized. After
centrifugation at 1000 × g for 5 min, the nuclear pellet was discarded, and membranes were harvested by centrifugation at
35,000 × g for 30 min. The membranes were homogenized
in HBSS containing 1% SDS and 100 mM iodoacetamide and
incubated for 30 min in the presence or absence of 300 mM
Data Analysis--
Data from Glu concentration-response
experiments were fitted to the following simple mass equation:
R = Rbasal + (Rmax/(1 + (EC50/[A]n))),
where [A] is the concentration of agonist, n is the Hill coefficient, and R is the response. Data from the
Zn2+ antagonism experiments were fitted to the following:
R = Rmax Effects of Zn2+ on WT mGluR1b--
Exposure of tsA
cells transiently transfected with WT mGluR1b to Zn2+ in
concentrations of 300 µM and above led to an inhibition
of agonist-induced IP accumulation with an IC50 value of
the metal ion of ~1 mM (Fig.
1). However, similar pharmacological
profiles were observed when two other phosholipase C-coupled receptors, CaR and the muscarinic acetylcholine receptor m5, were exposed to
Zn2+ (Fig. 1). The observation that zinc displays the same
apparent antagonistic effect at three different receptors strongly
suggests that the observed inhibition is of a nonspecific nature.
Zn2+ appears to have an inhibitory or a toxic effect on the
tsA cells. Alternatively, this ion may inhibit events downstream in the
signal transduction pathway of the receptors. In agreement with the
findings in this study, the group of Pin have also only observed a
minute decrease in the maximal response (induced by 1 mM
Glu) exerted by 100 µM ZnCl2 on
mGluR1.2 Since the
nonspecific effect of Zn2+ on receptor functionality in our
assay is negligible at concentrations below 300 µM, 100 µM was used as the maximal concentration of Zn2+, when testing the WT and mutant mGlu1b
receptors.
Effects of Zn2+ on mutant mGluR1bs--
In the
experiments of Zn2+ antagonism of Glu-induced receptor
activation, an agonist concentration of 1 mM Glu was used.
At this concentration, the WT and all mutant receptors were fully activated. In cells transfected with mutant K260H mGluR1b, where Lys260 had been substituted with a histidine, the
IC50 of Zn2+ was decreased 500-fold compared
with the "apparent" IC50 of this ion on the WT receptor
(Fig. 2 and Table
I). Zn2+ showed an affinity
on the mutant receptor independent of the agonist concentration used,
indicating a noncompetitive antagonist effect (Fig.
3A). This proposal was
supported by a Schild analysis, where Glu concentration-response
experiments were performed in the presence of increasing concentrations
of Zn2+. Here, the maximal response of K260H was markedly
reduced with increasing concentrations of Zn2+ present,
whereas the EC50 values of Glu were relatively unaltered (Fig. 3B). Furthermore, much like the noncompetitive mGluR1
antagonist CPCCOEt and in contrast to the competitive antagonist
(S)-4CPG, Zn2+ did not influence
[3H]Quis binding to either WT or K260H mGluR1b (Fig.
4A). In conclusion, Zn2+ was clearly a noncompetitive antagonist at mutant
K260H. The metal ions Cu2+ and Co2+ were tested
on WT and K260H mGluR1b. However, CuCl2 and
CoCl2 were unable to antagonize the agonist-induced
response in both the WT and the mutant receptor in concentrations up to
100 µM (data not shown). In concentrations above 100 µM, CuCl2 had a marked toxic effect on the
tsA cells.
We wished to examine whether we had created a Zn2+ site in
K260H that actually involved the introduced histidine or whether it was
the removal of the positively charged lysine residue that had
eliminated a repulsive obstruction, making Zn2+ binding to
other amino acids in the region possible. Thus, we mutated
Lys260 to an alanine, and the K260A mutant was shown to
display a pharmacological profile similar to that of the WT receptor
upon exposure to Zn2+ (Table I). Hence, the histidine
introduced in K260H is in fact involved in and is essential for the
Zn2+ binding to and antagonism of the K260H mutant.
To identify the amino acid(s) forming the Zn2+ binding site
together with the introduced histidine in K260H, we mutated five amino
acids, which, according to our model of the open ATD of mGluR1,
are located in proximity to Lys260 (Fig.
5) (10). The five residues,
His231, Glu233, Glu238,
His257, and Asp259, are all potential
"Zn2+ ligands" (27-29). As can be seen from Table I,
single alanine mutations of these five residues in K260H did not reduce
the antagonistic potency of Zn2+. Mutants with the
Lys260 Substitution of the Participants in the Zinc Binding Site--
The
possibility cannot be excluded that the loss of Zn2+
antagonism of mutant K260H/H231A/E233A/E238A as compared with mutant K260H is the result of indirect structural changes within the region,
caused by the mutations. To study this, we substituted the three
"coligands" (His231, Glu233, and
Glu238) with other residues capable of coordinating the
metal ion, a principle previously used by Gether and co-workers (30) in
a study of an endogenous zinc site in the dopamine transporter. Interestingly, mutant K260H/H231E/E233H/E238H was not only still antagonized by zinc, the potency of the metal ion was in fact significantly increased as compared with K260H (Table I). The substitution of the three coligands appeared to have improved the
binding of Zn2+ to the receptor.
Agonist Pharmacology of WT and Mutant mGluR1bs--
In agreement
with a previous study of mGluR1b transiently expressed in tsA cells,
the WT receptor displayed a concentration-dependent 3-4-fold increase in IP accumulation when exposed to Glu (Fig. 6 and Table I) (22). The pharmacological
profiles of Glu on the majority of mutants tested were not
significantly different from that on the WT receptor (Table I).
However, mutants containing the Glu238
To investigate whether the functional profiles of the mutant and WT
receptors were mimicked in an agonist binding assay, we characterized [3H]Quis binding to WT, K260H, and
K260H/H231A/E233A/E238A mGluR1b and estimated the expression levels of
all mutants containing alanine substitutions of His231,
Glu233, or Glu238 (Table
II and Fig. 4). The Kd
values of the radioligand at mutants K260H and K260H/H231A/E233A/E238A
were not significantly different from that of the WT receptor (Fig.
4B and Table II). The expression levels of selected mutants
were estimated by measuring the specific binding at 250 nM
[3H]Quis. At this concentration, equilibrium is reached
for the binding to WT, K260H, and K260H/H231A/E233A/E238A mGluR1b. The expression levels of all of the mutants tested were comparable with
that of the WT receptor (Fig. 4C). Hence, the mutations in the ATD lobe did not appear to have affected agonist affinities or
expression densities of the receptor.
Dimerization of WT and K260H mGluR1a--
The mGluRs have been
demonstrated to exist as disulfide-linked homodimers (14, 15, 31). In a
recent preliminary study of mGluR5 by Romano and co-workers (32), the
cysteine residues Cys129 and Cys240 have been
reported to be crucial for the covalent and noncovalent dimerization of
the receptor, respectively. In agreement with studies of the functional
importance of Cys140 in mGluR1 and of Cys129
and Cys131 in CaR, mutation of Cys129 in mGluR5
did not impair the pharmacology of the receptor significantly (20, 32,
33). In contrast, mutation of Cys240 in mGluR5 resulted in
a nonfunctional receptor (32). Cys240 in mGluR5 is
conserved in all of the mGluRs, and since the corresponding cysteine
residue in mGluR1, Cys254, is located close to
Lys260, the effects of Zn2+ on the dimerization
process of WT and K260H mGluR1a were investigated by Western blotting,
using an antibody directed toward the
Glu1116-Leu1130 region in the C-terminal of
rat mGluR1a (34). As can be seen from Fig.
7, the expression level of K260H appeared
to be somewhat reduced compared with that of the WT receptor. This
contrasts with the findings from [3H]Quis binding
experiments, where the Bmax values obtained for WT and K260H mGluR1b were comparable in size (Fig. 4C and
Table II). Under nonreducing conditions, both WT and K260H mGluR1a
displayed two close bands at ~300 kDa. The smallest of the two bands
disappeared in the presence of 300 mM
In any case, it is quite clear that Zn2+ has no effect on
the oligomer/monomer ratio for either WT or K260H mGluR1a. In both cases, the oligomer bands were unaltered in their intensities, and no
band was observed at 150 kDa in the presence of the metal ion in
concentrations up to 10 mM (Fig. 7). In conclusion, zinc binding to the K260H mGluR1a mutant does not appear to interfere with
the receptor-receptor interactions necessary for dimerization.
Although key determinants for agonist binding to mGluR1 and the
other mGluRs have been proposed (11, 18, 22), very little is known
about the ATD contraction process following agonist binding to the
receptor or the transference of the activation signal from the ATD to
the G-protein coupling region. The agonist binding residues
Arg78 and Ser165 as well as Thr188
are located in one of the two ATD lobes in mGluR1 (11, 22). Hence, the
"closure" of the ATD may be a basic conformational change within
this lobe having no consequences or minute consequences for the
proximity of the two lobes. However, Galvez et al. (19) have
recently demonstrated that alanine mutations of the residues Ser247 and Gln312 located on the "lip" of
each ATD lobe in GABABR1a have a potentiating effect on
agonist affinities. Based on their model of the ATD of
GABABR1a, the authors suggested that these residues would
be located close to each other in the closed ATD conformation of the
receptor and that the mutations simply had improved the interaction between the lips of the two lobes (19). Furthermore, in a recent follow
up study, it was shown that the residues in both lobes of the ATD of
GABABR1a participate in the binding of the
GABAB agonist baclofen (38). Clearly, these observations
suggest a much more drastic structural change throughout the
entire ATD region of the family C receptor bringing the lips as well as
other parts of the two lobes in close proximity.
Creation of metal ion binding sites in GPCRs was introduced by Schwartz
and co-workers, and the technique has been used to gain structural
information about the spatial orientation of transmembrane regions
(TMs) in family A and B GPCRs and well as other transmembrane proteins
(30, 39-46). In the present study, we decided to explore the
structural flexibility within the ATD lobe opposite to the lobe
involved in agonist binding to mGluR1 and the implications of an
introduced structural constraint in this lobe for receptor functionality. Hence, we constructed a zinc site in the upper part of
this lobe by mutating Lys260 to a histidine. According to
our previously published model of the open form of the ATD of mGluR1
(11), the lysine residue is located in close proximity to several
histidine, glutamate, and aspartate residues (Fig. 5). Since it is well
established from protein crystal structures that histidines, cysteines,
glutamates, and aspartates are the prevailing ligands for
Zn2+ in proteins (27-29), some of these residues were
supposed to function as coligands for Zn2+ in the
constructed mutant. Zn2+ actually turned out to be a quite
potent noncompetitive antagonist of agonist-induced IP accumulation on
mutant K260H, whereas it had no measurable effects on the response of
the WT receptor or the mutant K260A at the Zn2+
concentrations used. The inability of Zn2+ to antagonize
the agonist response of K260A clearly demonstrates that the histidine
introduced in mutant K260H is an essential participant in the binding
of Zn2+. His231, Glu233, and
Glu238 were identified as other residues involved in the
binding of this ion. This is in excellent agreement with their location
and spatial orientation in our model of the open ATD of K260H mGluR1 (Fig. 5). His231, Glu233, and
Glu238 are located on the same strand in the ATD lobe,
whereas the histidine introduced instead of Lys260 is
located on another. Each of the three "coligands" are located ~6
Å from the introduced histidine. Considering that the distances in
metal ion-histidine and bidentate metal ion-carboxylate interactions in
proteins typically are 2-2.5 Å (27, 29, 47), Zn2+ binding
between the introduced histidine and these three residues seems feasible.
Removal of ligands from the tridentate zinc binding sites in enzymes
and transmembrane proteins and mutants of these have been demonstrated
to lead to decreased zinc affinities of the metal ion (30, 39, 48, 49).
Thus, it is surprising that all three endogenous Zn2+
ligands in K260H mGluR1b (His231, Glu233, and
Glu238) had to be mutated to alanines to eliminate
Zn2+ binding to the receptor. The typical coordination
arrangement of Zn2+ binding to proteins is tetrahedral or
distorted tetrahedral, where the protein most often contributes with
three or four ligands to the binding (27-29). However, zinc and other
metal ions are able to coordinate to proteins in a bidentate manner,
with solvent molecules acting as the remaining ligands (29, 50-52),
and several bidentate zinc sites have been introduced in the TMs of
GPCRs (39, 40, 42-46). Considering that the residues involved in zinc
binding to K260H mGluR1 are located in the top of the ATD lobe, the
region would be expected to be solvent-accessible. The idea that
Zn2+ is capable of binding to the introduced histidine and
three different coligands in K260H mGluR1 with roughly the same
affinity is still quite remarkable. However, a histidine introduced
instead of Glu193 in TM5 of the tachykinin NK-1 receptor
has been shown to be able to participate in two different bis-histidine
sites, which display comparable affinities of Zn2+, by
coordinating to introduced histidines in TM3 (Asn109 A second hypothesis for the observed elimination of Zn2+
antagonism in the K260H/H231A/E233A/E238A mutant is that the triple alanine mutation causes structural changes in the lip of the ATD lobe,
thereby disrupting a Zn2+ site between the introduced
histidine and ligands elsewhere in the receptor. However, a couple of
observations are not in line with this hypothesis. First, all of the
mutants where single or double alanine mutations have been introduced
in K260H display pharmacological profiles of Zn2+ not
significantly different from that of K260H (Table I). It seems unlikely
that this indirect disruption of the Zn2+ antagonism by the
triple alanine mutation would not be seen in any of the mutants
containing double alanine mutations. Second, the antagonistic effect of
zinc is retained when the three histidine/glutamate coligands are
mutated to glutamates and histidines, respectively (mutant
K260H/H231E/E233H/E238H). In many ways, this combination of
glutamate/histidine mutations represents a much more drastic electronic
and structural alteration of the receptor protein than the triple
alanine mutation in K260H/H231A/E233A/E238A. Furthermore, the increased
antagonistic potency of Zn2+ on mutant
K260H/H231E/E233H/E238H compared with K260H strongly supports the
proposal of the involvement of one or more of the three coligands in
zinc binding.
While only one of the three endogenous coligands had to be present in
the mGluR1 ATD for Zn2+ to exert its effect, the histidine
introduced instead of Lys260 appeared to be crucial for
Zn2+ binding. No antagonistic effect of Zn2+
was observed on the WT receptor or mutant K260A, most likely because
the metal ion does not bind to these receptors. Alternatively, zinc may
bind "silently" to the three endogenous "coligands" in WT and
K260A. In both scenarios, Zn2+ binding clearly has to
involve residues on two strands in the ATD lip to have any implications
for the signal transduction through the receptor. Analogously, both
antagonistic and agonistic zinc sites created by mutations in one TM of
family A and B GPCRs have been significantly "improved" by the
introduction of coordinating zinc ligands in another TM (39, 42-44,
46).
The fact that mGluR1 activation can be inhibited by Zn2+
binding to two strands in the ATD lip not involved in agonist binding is very interesting. Binding of the metal ion to K260H mGluR1 does not
appear to disrupt the dimerization of the receptor (Fig. 7). The
possibility that binding of the metal ion could cause a "twist" in
the orientation between the two receptors in the homodimer interface,
hereby affecting receptor functionality, cannot be completely excluded.
However, we favor an explanation where the effects of Zn2+
arise solely from intramolecular structural changes in the receptor protein. We propose that Zn2+ binding pins the two strands
in the lip of the ATD lobe together and introduces a conformational
"lock" on the region. The Zn2+-bound lobe is incapable
of participating in the contraction of the ATD, which becomes trapped
in a rigid, inactivated conformation. Alternately,
Zn2+ binding to the ATD lip of K260H could also be
speculated to interfere with the intramolecular communication between
the ATD and the seven-transmembrane moiety (7TM) of the family C
receptor. We and others have suggested a mechanism underlying the
signal transduction through the receptor, where the signal is
transferred to the G-protein coupling area by an intramolecular
interaction between the ATD and the 7TM of the receptor (1, 9, 53).
Hence, the antagonistic effect of Zn2+ binding could be
thought to arise from a disruption of a structural motif in the ATD
lobe necessary for the signal transfer process.
In contrast to the antagonistic effect of Zn2+ on K260H
mGluR1, Cu2+ and Co2+ displayed no measurable
inhibition of the agonist-induced response in the nontoxic
concentration range of the metal ions. This indicates that the site
constructed in K260H is relatively specific for Zn2+. The
effect of Cu2+ (as the free ion or complexed with
phenanthroline) on metal ion sites constructed in TMs of family A GPCRs
have varied from being just as potent to being significantly weaker
than that of that of Zn2+ (41, 42, 44). Whether
Cu2+ and Co2+ do not bind to K260H or whether
they bind but, unlike Zn2+, are unable to induce a
conformational lock on the region is impossible to determine.
It is quite interesting that introduction of a structural constraint in
the ATD lobe opposite to the "agonist-binding" lobe of mGluR1 has
such pronounced consequences for receptor functionality. The local
flexibility in the ATD lip, which may enable Zn2+ to
coordinate to the introduced histidine and three different endogenous
coligands is contrasted by the apparent small degree of overall
structural flexibility of the ATD region, resulting in its sensitivity
toward "locally applied rigidity." Hence, the zinc site
created in mGluR1 is quite interesting from a medicinal chemistry point
of view. From a therapeutic perspective the interest in the field of
mGluR ligands has classically been focused on antagonists for the group
I mGluRs and agonists for the group II and III mGluRs (1, 9, 10). The
vast majority of antagonists for mGluRs reported so far have been
competitive antagonists constructed from the amino acid structure of
the endogenous agonist (1, 10). However, in recent years a new
generation of noncompetitive antagonists has been introduced (54-58),
of which CPCCOEt and 2-methyl-6-(phenylethynyl)pyridine have been
demonstrated to bind to the 7TM of mGluR1 and mGluR5, respectively (23,
53, 59). The present study is the first report of noncompetitive
antagonism originating from ligand binding to a region in the ATD. The
"functional antagonism" exerted by Zn2+ on the ATD of
K260H mGluR1 in this study resembles the actions of CPCCOEt on mGluR1
in some respects. CPCCOEt binds to the top of TM7 in mGluR1, and it has
been speculated that it in this way interferes with the signaling
between the ATD and the 7TM (53). Analogously, Zn2+ also
blocks the signal transduction through the receptor, either through a
similar interference in the ATD/7TM communication or by blocking ATD
contraction a step prior to this "intramolecular cross-talk." We
propose that the lips of the two globular domains of the ATD could be
very interesting targets in the design of new antagonists for mGluR1
and the other mGluRs. Furthermore, certain compounds interacting with
the lips of the two ATD lobes could possibly also function as
allosteric modulators of mGluR activation. In that respect, the
increased basal response achieved by a Glu238 In conclusion, this study represents the first zinc site constructed in
a family C GPCR. Furthermore, it is the first time a metal ion site has
been created in a region outside of the seven transmembrane regions of
a GPCR and the loops connecting these. As it was to be expected from
the existence of numerous endogenous metal ion binding sites in
proteins (27-29, 48), the use of zinc as a structural probe in GPCRs
can clearly be extended to regions outside of the "confinement" of
the membrane. While not all ligands for Zn2+ in K260H
mGluR1 may have been identified, the findings in this study offer
valuable insight into the mechanism of ATD closure and family C
receptor activation, identify regions in the ATD as interesting targets
from a medicinal chemistry point of view, and strongly support our
previously published model of the ATD of mGluR1 (11).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid
receptors type B (GABABR1-2) (2), a calcium-sensing
receptor (CaR) (3), a family of putative pheromone receptors (4), and
three recently cloned orphan receptors (5-7). Molecular cloning has
revealed the existence of eight different mGluR subtypes, subdivided
into three subgroups based on their amino acid sequence similarities,
agonist pharmacology, and signal transduction pathways (1, 8-10).
Group I is constituted by mGluR1 and mGluR5, group II by mGluR2 and
mGluR3, and group III by the remaining four subtypes. Some of the
subtypes exist as multiple splice variants, which only differ in their
carboxyl termini (10). In the case of mGluR1, the splice variant
mGluR1a possesses a C-terminal of 359 amino acid residues, whereas the
termini of the other splice variants cloned so far only consist of
50-60 residues.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
His mutation from mutant K260H mGluR1b-pSI into
mGluR1a-pSI using the unique restriction enzymes XhoI and
ApaI. All amplified receptor DNAs were sequenced on an ABI
310 sequencer using the Big Dye Terminator Cycle Sequencing kit
(PerkinElmer Life Sciences).
-mercaptoethanol, 100 mM dithiothreitol, or 10 mM ZnCl2. The samples were heated for 5 min at
65 °C before electrophoresis, where the samples (5-15 µg of
membrane protein/well) were separated on 7.5% polyacrylamide gels. The
proteins were transferred to nitrocellulose membranes by transblotting
at 200 mA for 1 h. The blots were incubated in phosphate-buffered
saline solution (PBS) containing 0.05% Tween 20 and 2% powdered skim
milk overnight. The blots were washed three times for 30 min with PBS
containing 0.05% Tween 20 and incubated with rabbit anti-mGluR1a
polyclonal antibody (diluted 1:2000 in PBS containing 0.05% Tween 20 and 2% powdered skim milk) for 1 h. This was followed by three
30-min washes in PBS containing 0.05% Tween 20 and incubation in
anti-Rabbit IgG conjugated to horseradish peroxidase (diluted 1:1500 in
PBS containing 0.05% Tween 20 and 2% powdered skim milk) for 1 h, after which the membranes were developed in a solution of
3,3-diaminobenzidine (Kem-En-Tec A/S, Copenhagen, Denmark). Three
independent Western blot experiments were performed, and similar
results were obtained in all of them.
(Rmax/(1 + (IC50/[A]n))),
where [A] is the concentration of antagonist, n is the
Hill coefficient, and R is the response. Curves were
generated by nonweighted least-squares fits using the program
KaleidaGraph 3.08 (Synergy Software, Reading, PA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The effects of Zn2+ on
agonist-induced IP accumulation in tsA cells transiently transfected
with mGluR1b-pSI, CaR-pSI, or m5-pCD. The IP assays were performed
as described under "Experimental Procedures." The cells were
prelabeled overnight with 1 µCi/ml
myo-[2-3H]inositol, washed with HBSS, and
incubated for 10 min with HBSS supplemented with 0.9 mM
CaCl2, 1.05 mM MgCl2, 10 mM LiCl, and various concentrations of ZnCl2.
The buffer was removed, and the cells were incubated for 40 min in the
same buffer containing agonist (1 mM Glu, 3 mM
CaCl2, or 50 nM acetylcholine, respectively)
and various concentrations of ZnCl2. The buffer was
aspirated, and the reactions were stopped by the addition of ice-cold
20 mM formic acid. Total IP formation was determined by an
ion exchange assay. Data are given as percentage of the IP accumulation
in the absence of ZnCl2.
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Fig. 2.
Inhibition by ZnCl2 of
agonist-induced IP accumulation in tSA cells transiently transfected
with WT and mutant mGluR1b receptors. The IP assays were performed
as described under "Experimental Procedures." The cells were
prelabeled overnight with 1 µCi/ml
myo-[2-3H]inositol, washed with HBSS, and
incubated for 10 min with HBSS supplemented with 0.9 mM
CaCl2, 1.05 mM MgCl2, 10 mM LiCl, and various concentrations of ZnCl2 or
100 µM CPCCOEt. The buffer was removed, and the cells
were incubated for 40 min in the same buffer containing 1 mM Glu and various concentrations of ZnCl2 or
100 µM CPCCOEt. The buffer was aspirated, and the
reactions were stopped by the addition of ice-cold 20 mM
formic acid. Total IP formation was determined by an ion exchange
assay. Data (mean ± S.D.) are given as percentage of the
difference of the response in absence of antagonist and the response in
the presence of 100 µM CPCCOEt.
Pharmacological characterization of WT and mutant mGlu1b
receptors
View larger version (19K):
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Fig. 3.
Zn2+ is a noncompetitive
antagonist on K260H mGluR1b. The IP assays were performed as
described under "Experimental Procedures." A, inhibition
by ZnCl2 of IP accumulation induced by 3 µM,
50 µM, and 1000 µM Glu in tsA cells
transiently transfected with K260H mGluR1b-pSI. The cells were
prelabeled overnight with 1 µCi/ml
myo-[2-3H]inositol, washed with HBSS, and
incubated for 10 min with HBSS supplemented with 0.9 mM
CaCl2, 1.05 mM MgCl2, 10 mM LiCl, and various concentrations of ZnCl2.
The buffer was removed, and the cells were incubated for 40 min in the
same buffer containing three different concentrations of Glu (3, 50, or
1000 µM) and various concentrations of ZnCl2.
The buffer was aspirated, and the reactions were stopped by the
addition of ice-cold 20 mM formic acid. Total IP formation
was determined by an ion-exchange assay. Data (mean ± S.D.) are
given as percentage of the difference of the response in the absence
and presence of 10 mM ZnCl2. B,
Schild analysis of the inhibition by ZnCl2 of IP
accumulation induced by Glu in tsA cells transiently transfected with
K260H mGluR1b-pSI. The cells were prelabeled overnight with 1 µCi/ml
myo-[2-3H]inositol, washed with HBSS, and
incubated for 10 min with HBSS supplemented with 0.9 mM
CaCl2, 1.05 mM MgCl2, 10 mM LiCl, and four different concentrations of
ZnCl2 (0, 1, 4, or 16 µM). The buffer was
removed, and the cells were incubated for 40 min in the same buffer
containing the four different concentrations of ZnCl2 and
various concentrations of Glu. The buffer was aspirated, and the
reactions were stopped by the addition of ice-cold 20 mM
formic acid. Total IP formation was determined by an ion exchange
assay. Data are given as disintegrations/min (DPM) per well
(mean ± S.D).
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Fig. 4.
[3H]Quis binding to WT and
mutant mGlu1b receptors. Displacement of
[3H]Quis binding to WT and K260H mGluR1b (A)
and saturation binding to WT, K260H, and K260H/H231A/E233A/E238A
mGluR1b are shown. A, 50 µg of membrane fractions
of tsA cells transfected with WT (black bars) or
K260H (gray bars) mGluR1b were prepared as
described under "Experimental Procedures" and incubated at room
temperature for 1 h with 30 nM [3H]Quis
alone (Total) or in the presence of 1 mM Glu,
100 µM (S)-4CPG, 100 µM CPCCOEt,
or 100 µM ZnCl2. The assay mixture was
aspirated onto a GF/C filter, washed with 3 × 5 ml of ice-cold
assay buffer, after which the filters were dried and counted in a
scintillation counter. Data are given as disintegrations/min
(DPM) per well (mean ± S.D.). B, 5-15 µg
of membrane fractions of WT, K260H, or K260H/H231A/E233A/E238A mGluR1b
transfected cells prepared as described under "Experimental
Procedures" were incubated at room temperature for 1 h with
various concentrations of [3H]Quis alone or in the
presence of 1 mM Glu. [3H]Quis was diluted
with unlabeled (S)-quisqualic acid at concentrations above
300 nM. The assay mixture was aspirated onto a GF/C filter,
washed with 3 × 5 ml of ice-cold assay buffer, after which the
filters were dried and counted in a scintillation counter.
C, binding of 250 nM [3H]Quis to
5-7 µg of membrane fractions of WT and mutant mGluR1b-transfected
cells were performed as described for B and under
"Experimental Procedures" in the absence or presence of 1 mM Glu.
His mutation and different combinations of two
of these mutations were constructed as well, some of which are shown in
Table I. However, none of these combinations of mutations impaired the ability of Zn2+ to antagonize the receptor significantly.
In contrast, simultaneous mutation of the three residues
His231, Glu233 and Glu238 (mutant
K260H/H231A/E233A/E238A) resulted in a receptor displaying a
pharmacological profile of Zn2+ not significantly different
from that on the WT receptor, indicating that the binding of this ion
had been eliminated (Fig. 2).
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Fig. 5.
Detail from the molecular model of the open
form of the ATD of mGluR1. Shown is a depiction of the upper part
of the mGluR1 ATD lobe not involved in agonist binding.
Lys260 and the regions potential Zn2+ coligands
are shown. The construction of the model of the open form of the mGluR1
ATD has been published previously (11).
Ala and, to a
smaller extent, the Glu233
Ala mutation displayed
slightly elevated basal responses as compared with WT. Furthermore,
mutation of Glu238 to histidine resulted in a further
increased basal response (Fig. 6). The elevated basal activity could be
reduced with both the competitive mGluR antagonist, (S)-4CPG, and the
noncompetitive mGluR1 antagonist, CPCCOEt (data not shown). This is
quite interesting considering the reported increased agonist affinities
caused by an alanine mutation of Gln312 in the
corresponding region of GABABR1a (19). The functional importance of Glu238 in mGluR1 is currently under
investigation and will not be discussed further in this report.
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Fig. 6.
Glu-induced IP accumulation in tsA cells
transiently transfected with WT, K260H, or K260H/E238H mGluR1b.
The IP assays were performed as described under "Experimental
Procedures." The cells were prelabeled overnight with 1 µCi/ml
myo-[2-3H]inositol, washed with HBSS, and
incubated for 10 min with HBSS supplemented with 0.9 mM
CaCl2, 1.05 mM MgCl2, and 10 mM LiCl. The buffer was removed, and the cells were
incubated for 40 min in the same buffer containing various
concentrations of Glu. The buffer was aspirated, and the reactions were
stopped by the addition of ice-cold 20 mM formic acid.
Total IP formation was determined by an ion exchange assay. Data are
given as disintegrations/min (DPM) per well (mean ± S.D.).
[3H]-Quis binding to WT and mutant mGluRlb receptors
-mercaptoethanol
or 100 mM dithiothreitol, whereas the intensity of the
larger band was not altered significantly. Under these reducing
conditions, a band at 150 kDa corresponding to the monomeric receptor
appeared. The size of the monomeric receptor is in excellent agreement
with the predicted size of the recombinant receptor and with the
findings of recent studies (35, 36). None of the oligomeric bands or
the monomeric band were observed in membranes prepared from tsA cells
transfected with the pSI vector (data not shown). An identical
oligomeric/monomeric pattern and similar sizes of the oligomeric and
monomeric bands were observed, when membranes prepared from the
mGluR1a-CHO cell line were used (data not shown). To investigate
whether the upper band could be ascribed to nonspecific receptor
aggregation due to overexpression of the receptors on the tsA cell
surface, the tsA cells were transfected with 1.5 µg of WT or K260H
mGluR1a-pSI per 10-cm tissue culture plate (~30% of the cDNA
used in Fig. 7). However, a Western blot of membranes prepared from
these cells displayed the same two oligomeric bands under nonreducing
conditions, and only the lower band was converted into a monomer band
at 150 kDa in the presence of 300 mM
-mercaptoethanol
(data not shown). The only difference between this experiment and the
experiment depicted in Fig. 7 was that the expression levels of both WT
and K260H mGluR1a were markedly reduced. At present, a fully
satisfactory explanation of the presence of two dimer/oligomer bands is
not obvious. However, the oligomeric/monomeric pattern, where only one
of two oligomeric bands was converted to a monomeric band upon exposure
to a reducing agent, closely resembles that observed by two other
groups in studies of the dimerization of mGluR1 and another family C
receptor, CaR (36, 37).
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Fig. 7.
Imunoblotting analyses of WT and K260H
mGluR1a-transfected tsA cells. 7 µg of membrane protein prepared
from WT and K260H mGluR1a-pSI-transfected tsA cells were preincubated
in the absence (lane A) or in the presence of 100 mM dithiothreitol (lane B), 300 mM -mercaptoethanol (lane C), or
10 mM ZnCl2 (lane D). The
samples were separated on 7.5% SDS-polyacrylamide gels, transferred to
nitrocellulose membranes, and detected with a rabbit anti-mGluR1a
polyclonal antibody as described under "Experimental Procedures."
The left lane is a ladder, and the 150- and
250-kDa bands are given.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
His) or TM6 (Tyr272
His) as coligands (39, 40).
Clearly, some degree of flexibility in the orientation of the
introduced histidine in TM5 has to exist to achieve binding to both
TMs. Since the strands in the ATD lip of mGluR1 in contrast to the
transmembrane
-helices are not constricted by the surrounding plasma
membrane, they probably are more flexible in their intramolecular
orientation. Hence, the idea of bidentate Zn2+ sites
between the introduced histidine in K260H and each of the three
coligands must be considered a distinct possibility.
His
mutation in mGluR1b is quite interesting (Fig. 6). Allosteric activators of another family C GPCR, CaR, have been reported, but the
binding sites of these compounds have not been disclosed (60, 61).
Ligands directed toward the ATD lips of the family C GPCRs could be
very different structurally from that of agonists and competitive
antagonists, which again could be a way of overcoming traditional drug
delivery obstacles such as metabolism and low blood-brain barrier
penetration and, in the case of mGluR and GABAB
ligands, reduce the risk of the compound being taken up by
glutamate and GABA transporters.
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ACKNOWLEDGEMENTS |
---|
We thank Professor Shigetada Nakanishi, Professor Solomon H. Snyder, and Dr. Mark R. Brann for generous gifts of cDNAs encoding mGluR1a, CaR, and m5, respectively. Dr. Penelope S. V. Jones and Professor Nakanishi are also thanked for generous gifts of the tsA and mGluR1a-CHO cell lines, respectively. The kind guidance of Dr. Carl Romano with respect to the design of the Western blot experiments is greatly appreciated.
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Note Added in Proof |
---|
The recently published crystal structures of the mGluR1 ATD homodimer (Kunishima et al. (2000) Nature 407, 971-977) have shedded light on the created zinc site in mGluR1b. According to the crystal structures, the introduced histidine in K260H mGluR1b and the three proposed coligands for Zn2+, His231, Glu233, and Glu238 are located in close proximity in both of the ATDs in the dimer. Whereas the two Zn2+ sites in the two ATDs are located far apart in the "resting," inactive dimer conformation, they are facing each other across the cleft between the ATDs in the "active"dimer conformation. Hence, we propose that Zn2+ binding to one or both of the zinc sites prevents the conformational transition of the ATD dimer from the "resting" to the "active" state due to steric interactions.
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FOOTNOTES |
---|
* This work was supported by grants from the Danish Medical Research Council and the Novo Nordisk Foundation.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.
To whom correspondence should be addressed: Dept. of Medicinal
Chemistry, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark. Tel.: 45-3530-6518; Fax: 45-3530-6040; E-mail: hbo@dfh.dk.
Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M007220200
2 L. Prézeau and J.-P. Pin, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
Glu, (S)-glutamic acid;
mGluR, metabotropic glutamate receptor;
GPCR, G-protein-coupled receptor;
GABA, -aminobutyric acid;
GABABR,
-aminobutyric acid receptor type B;
CaR, calcium-sensing receptor;
ATD, amino-terminal domain;
PBP, periplasmic
binding protein;
[3H]Quis, [3H]quisqualic
acid;
(S)-4CPG, (S)-4-Carboxyphenylglycine;
CPCCOEt, 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylic acid
ethyl ester;
IP, inositol phosphate;
HBSS, Hanks' balanced saline
solution;
WT, wild type;
PBS, phosphate-buffered saline solution;
CHO, Chinese hamster ovary;
TM, transmembrane region;
7TM, seven-transmembrane moiety.
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