From the Pharma Division, Discovery Research CNS and
¶ Chemistry, F. Hoffmann-La Roche Ltd.,
CH-4070 Basel, Switzerland
Received for publication, November 19, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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A model of the rmGlu1 seven-transmembrane
domain complexed with a negative allosteric modulator,
1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)- 1,6-dihydro-pyrimidine-5-carbonitrile
(EM-TBPC) was constructed. Although the mGlu receptors belong to the
family 3 G-protein-coupled receptors with a low primary sequence
similarity to rhodopsin-like receptors, the high resolution crystal
structure of rhodopsin was successfully applied as a template in this
model and used to select residues for site-directed mutagenesis. Three
mutations, F8016.51A, Y8056.55A, and
T8157.39M caused complete loss of the
[3H]EM-TBPC binding and blocked the EM-TBPC-mediated
inhibition of glutamate-evoked G-protein-coupled inwardly
rectifying K+ channel current and
[Ca2+]i response. The mutation
W7986.48F increased the binding affinity of antagonist by
10-fold and also resulted in a marked decrease in the IC50
value (4 versus 128 nM) compared with wild
type. The V7575.47L mutation led to a dramatic reduction in
binding affinity by 13-fold and a large increase in the
IC50 value (1160 versus 128 nM).
Two mutations, N74745.51A and N75045.54A,
increased the efficacy of the EM-TBPC block of the glutamate-evoked [Ca2+]i response. We observed a striking
conservation in the position of critical residues. The residues
Val-7575.47, Trp-7986.48,
Phe-8016.51, Tyr-8056.55, and
Thr-8157.39 are critical determinants of the
EM-TBPC-binding pocket of the mGlu1 receptor, validating the rhodopsin
crystal structure as a template for the family 3 G-protein-coupled
receptors. In our model, the aromatic ring of EM-TBPC might
interact with the cluster of aromatic residues formed from
Trp-7986.48, Phe-8016.51, and
Tyr-8056.55, thereby blocking the movement of the TM6
helix, which is crucial for receptor activation.
The mGlu1 receptor
family currently comprises eight receptors that are divided into
three classes on the basis of their sequence similarities, signal
transduction, and agonist rank order of potency. Group I (mGlu1 and -5)
receptors are coupled to the stimulation of phosphoinositide
hydrolysis; group II (mGlu2 and -3) and group III receptors (mGlu4, -6, -7, and -8) are negatively coupled to cAMP production (1-3). Many
studies have demonstrated the involvement of mGlu receptors in the
modulation of synaptic transmission, ion channel activity, and synaptic
plasticity (4, 5), and dysfunction of these receptors has been
implicated in psychiatric and neurological diseases (6). The mGlu
receptors belong to the family 3 of G-protein-coupled receptors
(GPCRs). Other members of this family include the GABAB,
Ca2+-sensing, vomeronasal, pheromone, and putative taste
receptors (7). The family 3 GPCRs shares a low sequence similarity with the other families. In contrast to family 1, the family 3 receptors are
characterized by two distinctly separated topological domains: an
exceptionally long extracellular amino-terminal domain (500-600 amino
acids), which forms the agonist-binding pocket (8-10), and the 7TM
helical segments involved in receptor activation and G-protein coupling
(11).
Compounds acting at group I mGlu receptors can be grouped into two
categories. Category one comprises competitive agonists and
antagonists. These compounds are phenylglycine derivatives or
rigidified analogs of glutamate (12), which logically bind to the
glutamate-binding domain. Competitive group I ligands have achieved
only limited subtype selectivity and potency, perhaps due to the high
sequence homology of the mGlu receptor family agonist-binding site
supported by the three-dimensional structure of mGlu1 amino-terminal
domain (10). However, recent development of more sensitive technologies
for functional screening of GPCRs has resulted in the discovery of a
second category of compounds. These novel compounds, which interact
within the 7TMD of group I mGlu receptor, act as positive or negative
allosteric modulators (13). CPCCOEt was the first non-amino acid
derivative, subtype-selective antagonist of the mGlu1 receptor
(IC50 = 6.5 µM at hmGlu1b) to be described
(14). Litschig et al. (15) elucidated the site of action of
CPCCOEt, which binds within the 7TMD of mGlu1, in close contact with
the residues Thr-815 and Ala-818 of TM7. Similarly, methyl-6-(phenylethynyl)pyridine (MPEP), which was the first
noncompetitive, highly potent, mGlu5-selective antagonist
(IC50 = 36 nM at hmGlu5a) to be described (16),
was suggested to make close contact with the amino acid residues
Ala-810 in TM7 and Pro-655 and Ser-658 in TM3 of the mGlu5 receptor.
Moreover, it has been demonstrated that the CPCCOEt and MPEP interact
with residues that appear to form overlapping binding pockets in
homologous regions of the 7TMD of the mGlu1 and -5 receptors,
respectively (17). Recently, BAY36-7620, another highly potent
mGlu1-selective antagonist (IC50 = 160 nM at
rmGlu1a) that interacts within the 7TMD, has been reported (18).
Knoflach et al. (19) have described a novel class of ligands
RO 67-7476, RO 01-6128, and RO 67-4853 acting as positive allosteric
modulators of the mGlu1 receptor. Interestingly, their binding pocket
appears to be also located within the 7TMD of mGlu1. Furthermore, the
mutational analysis revealed (19) that RO 67-7476 binding site in the
TM3 region of mGlu1 appears to overlap with that of the MPEP binding
site in the homologous region of the mGlu5 receptor. Urwyler et
al. (20) concomitantly reported on the identification of CGP7930
and its aldehyde analog CGP13501, as positive modulators of
GABAB receptor function.
In the present study, we have probed the allosteric antagonist-binding
site of mGlu1 using molecular modeling, site-directed mutagenesis,
[3H]1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) binding, Ca2+ imaging, and G-protein-coupled
inwardly rectifying K+ channel (GIRK) current activation.
[3H]EM-TBPC is a highly potent, subtype-selective,
noncompetitive antagonist of rmGlu1 receptor (21, 22). Amino acid
residues in the TM3, -5, -6, and -7 and extracellular loop 2 (EC2)
regions, initially identified from an alignment of the 7TMD of rmGlu1
with bovine rhodopsin, were demonstrated by mutational analysis to be
important determinants of the noncompetitive antagonist binding pocket
of the mGlu1 receptor. A homology model constructed based on the x-ray
crystal of bovine rhodopsin (23) visualizes these findings and suggests
a possible binding mode of EM-TBPC.
Materials
EM-TBPC and
N-[2-[(2-chloro-6-fluorobenzyl)thio]ethyl]2-thiophenecarboxamide
(CFBTE-TPC) were synthesized at Hoffmann-La Roche Ltd. (21).
[3H]EM-TBPC (specific activity 33.4 Ci/mmol) was
synthesized by Dr. P. Huguenin at the Roche chemical and isotope
laboratories (21). [3H]Quisqualate (specific activity,
32-35 Ci/mmol; TRK 1070) was synthesized at Amersham Biosciences.
2-MPEP was obtained from Sigma.
Residue Numbering Scheme
The position of each amino acid residue in the 7TMD of mGlu
receptor is identified both by its sequence number (including the
signal peptide) and by the generic numbering system proposed by
Ballesteros and Weinstein (24) which is shown as superscript. In this
numbering system, amino acid residues in the 7TMD are given two
numbers; the first refers to the TM number, and the second indicates
its position relative to a highly conserved residue of family 1 GPCRs
in that TM which is arbitrarily assigned 50. The amino acids in
the extracellular loop EC2 are labeled 45 to indicate their location
between the helix 4 and 5. The highly conserved cysteine, thought to be
disulfide-bonded, is given the index number 45.50 (SWISS-PROT:
opsd_bovin C187), and the residues within the EC2 are then indexed
relative to the "50" position.
Molecular Modeling
Alignment--
An alignment of the seven-transmembrane helices
of rmGlu1 toward the transmembrane helices of bovine rhodopsin (Protein
Data Bank code 1f88) were obtained with help of our in-house program Xsae,2 using a modified
version of ClustalV (25). Sequences were obtained from SWISS-PROT:
rmGlu1, P23385. The sequence of bovine rhodopsin was read directly from
the rhodopsin structure (Protein Data Bank code 1f88).
Model Building--
All modeling calculations were made on a
Silicon Graphics Octane with a single R12000 processor using our
in-house modeling package Moloc (26, 27) (available on the World Wide
Web at www.Moloc.ch). An initial C-
In a following optimization step, only C- Plasmids, Cell Culture, and Membrane Preparation
cDNAs encoding the rmGlu1a and rmGlu5a receptors in
pBlueScript II were obtained from Prof. S. Nakanishi (Kyoto, Japan).
hmGlu1a receptor cDNA was amplified from a human fetal brain
cDNA library in pCMV.SPORT2 (Invitrogen) using primers derived from
the hmGlu1a sequence (AC:U31216). All point mutants were constructed
using the QuikChange site-directed mutagenesis kit (Stratagene). The entire coding regions of all point mutants were sequenced from both
strands using an automated cycle sequencer (Applied Biosystems).
HEK-293 cells were transfected as previously described (29). 48 h
posttransfection, the cells were harvested and washed three times with
cold PBS and frozen at [3H]EM-TBPC and [3H]Quisqualate
Bindings
After thawing, the membrane homogenates were centrifuged at
48,000 × g for 10 min at 4 °C, the pellets were
resuspended in the 20 mM HEPES-NaOH (pH 7.4) binding buffer
to a final assay concentration of 20 µg of protein/ml. Saturation
isotherms were determined by the addition of various
[3H]EM-TBPC concentrations (0.3, 1, 3, 10, 30, 100, and
300 nM) to these membranes for 1 h at room temperature
(equilibrium binding conditions determined in kinetic experiments). At
the end of the incubation, membranes were filtered onto Filtermate
(unitfilter Packard: 96-well white microplate with bonded GF/B filter
preincubated 1 h in 0.1% polyethyleneimine) and washed three
times with cold binding buffer. Nonspecific binding was measured in the
presence of 100 µM CFBTE-TPC, a noncompetitive,
subtype-selective antagonist of mGlu1 with Ki = 390 nM, which is a compound from the same class as EM-TBPC,
Fig. 1 (21). The radioactivity on the filter was counted on a Packard
Top-count microplate scintillation counter with quenching correction
after the addition of 40 µl of microscint 40 (Canberra Packard S.A.).
Saturation experiments were analyzed by Prism 3.0 (GraphPad Software,
San Diego, CA) using the rectangular hyperbolic equation derived from
the equation of a bimolecular reaction and the law of mass action,
B = (Bmax *
[F])/(KD + [F]), where B is the
amount of ligand bound at equilibrium, Bmax is
the maximum number of binding sites, [F] is the concentration of free
ligand, and KD is the ligand dissociation constant.
The experiments were performed three times in triplicate, and the
mean ± S.D. of the individual KD values were
calculated and are reported in Table I. [3H]Quisqualate
binding was performed as previously described (30).
Single Cell Calcium Imaging
HEK-293 cells were plated at 5 × 104 cells on
glass coverslips (diameter 15 mm) coated with 100 µg/ml
poly-D-lysine. After 24 h, the cells were
co-transfected with a 2:1 (w/w) mixture of mGlu/enhanced green
fluorescent protein plasmids using LipofectAMINE 2000 (Invitrogen).
48 h later, the transfected cells were incubated with 20 µM fura-2 acetoxymethyl ester plus 0.5% Pluronic F-127 (Molecular Probes, Inc., Eugene, OR) for 40 min at room temperature with 20-min postincubation in balanced salt solution. Cells were stimulated at room temperature in artificial cerebrospinal fluid with drug as indicated. Glutamate applications (drug + 30 µM glutamate, 30-s exposure) were separated by 10-min
intervals (3-min wash, 7-min antagonist incubation). Imaging
measurements were made on an inverted microscope with a long distance
×40 objective (Axiovert 405M; Zeiss). A cooled CCD camera (CH-250;
Photometrics) was used to acquire image pairs at 340- and 380-nm
excitation wavelengths (with dark correction) to a computer. Exposure
times were 400 ms. The intrinsic fluorescence in cells not dye-loaded
was less than 5% and did not contribute a significant error to the
measurements. Fluorescence ratio values were calculated as previously
described (31). Inhibition curves were fitted according to the Hill
equation: y = 100/(1 + (x/IC50)nH), where
nH represents slope factor.
Electrophysiology
A Chinese hamster ovary (CHO) cell line stably expressing human
GIRK1-GIRK2c dimer was co-transfected with a 1:1 (w/w) mixture of
mGlu/enhanced green fluorescent protein plasmids using LipofectAMINE 2000. GIRK channel currents were recorded 24-96 h after transfection using the whole-cell configuration of the patch clamp technique as
described previously (19). Briefly, for GIRK current recordings, the
pipette solution contained 130 mM KCl, 1 mM
MgCl2, 10 mM HEPES, 5 mM
K4BAPTA, 3 mM Na2ATP, 0.3 mM Na2GTP, adjusted to pH 7.2 with KOH, and
osmolarity was adjusted to 310 mosM with sucrose. The drug
was applied locally to the cell by fast perfusion from a double-barreled pipette assembly. The rate of solution exchange was
around 20 ms. The CHO cells were held at Molecular Modeling of 7TM Domain of the rmGlu1
Receptor--
[3H]EM-TBPC,
1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile
(Fig. 1) is a highly potent,
subtype-selective, noncompetitive antagonist that binds only to rat
mGlu1 (KD = 6.6 ± 0.5 nM,
Ki = 11 ± 2 nM). It has a low
affinity for human mGlu1 and none for the rat mGlu5 (22). Using a
series of chimeric rmGlu1/5a and rmGlu5/1 Generation of Point Mutations and the [3H]EM-TBPC
Binding--
20 mutations (19 in rmGlu1
Finally, the conversion of the tryptophan 798 to a phenylalanine
(W798F) or a tyrosine (W798Y) significantly increased the binding
affinity of the radioligand by 10- and 5-fold (KD = 0.7 ± 0.2 nM and KD = 1.3 ± 0.2 nM), respectively.
In order to examine effects of the mutations on the glutamate binding
pocket and to check for receptor expression, saturation analyses were
performed on membranes from selected mutated receptors using
[3H]quisqualate. The KD values and
maximum number of binding sites (Bmax) derived
from the saturation isotherms are given in Table I. As seen, the
mutations have no significant effect on quisqualate affinity, and the
level of receptor expression was comparable even with mutants
that completely lost the antagonist binding (Table I).
Effect of Mutations on Inhibition of Glutamate-induced
[Ca2+]i Response by EM-TBPC as Measured by
Single-cell Ca2+ Imaging--
In HEK-293 cells transiently
transfected with rmGlu1
As expected from the binding studies, the antagonist inhibition of
glutamate-induced [Ca2+]i response was not
affected by the mutation A818S. In cells expressing the mutants Y805A
and T815M, which did not bind [3H]EM-TBPC, EM-TBPC was
not able to efficiently inhibit glutamate-evoked [Ca2+]i response and thus resulted in the large
increases in IC50 values (>10,000 nM for Y805A
and T815M). In agreement with the binding experiments, the
glutamate-induced functional response with the mutant V757L was
inhibited by EM-TBPC with an increase in the IC50 value
(1160 versus 128 nM) as compared with WT.
The mutant W798F, which showed increased radioligand binding affinity,
exhibited a marked decrease in the IC50 value (4 versus 128 nM) relative to WT. Surprisingly, two
mutations, N747A and N750A, which had no effect on the binding affinity
of [3H]EM-TBPC, resulted in a decrease in the antagonist
IC50 values (30 and 10 versus 128 nM
WT, respectively) and with Hill coefficient values somewhat lower than
found with WT receptors.
Effect of Point Mutation on the Inhibition of the GIRK Channel
Current by EM-TBPC--
As we have shown previously (19), the
electrophysiological assay based on the activation of GIRKs by group I
mGlu receptors can be employed to assess the pharmacology of the
compounds. Therefore, we transiently expressed the wild type and
selected critical mutated rmGlu1 Although the mGlu receptor and rhodopsin are distant in sequence
identity, we have used the 7TM domains of rhodopsin as a general model
for a GPCR receptor to gain further insight into the mechanism of
action of a negative allosteric modulator, EM-TBPC. cis-Retinal, the inverse agonist of rhodopsin, served as
template for the location of EM-TBPC within the TM domains. Amino acids of the TM regions, in close proximity to cis-retinal and
very often identified as critical residues in family 1 GPCR mutation studies (33), were selected as candidates for mutation studies. To
visualize the mutation data, we constructed a three-dimensional model
of the 7TMD of the mGlu1 receptor using the atomic coordinates of
bovine rhodopsin (Protein Data Bank code 1f88). Fig.
5 shows the amino acids in the TM region,
found to affect the binding affinity of EM-TBPC, and suggests a
possible binding mode for this allosteric modulator.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
model of rat mGlu1 was built by fitting the aligned rmGlu1 sequence lacking the extracellular domain on
the bovine rhodopsin template C-
structure. Loops were optimized
with the Moloc C-
force field. In a next step, a full atom model was
generated.
and
angles were obtained for aligned amino acids
from the rhodopsin template.
angles were also adopted from the
bovine rhodopsin structure where possible or in case of nonidentical
amino acids generated by using the most probable value applying the
Ponder-Richards method (28). An energy calculation of the initial full
peptide structure revealed regions with bad van der Waals contacts of
amino acid side chains that were subsequently improved by manually
adjusting the relevant
angles. Repulsive van der Waals interactions
were removed manually where necessary. Refinement of the model was done
by keeping all backbone atoms in fixed positions and allowing only the
side chains to move.
atoms were kept in a fixed
position while all other atoms were allowed to move. In a third round
of optimization, no atoms were kept stationary, but constraints were
applied to C-
atoms. The quality of the model was then checked with
Moloc internal programs. EM-TBPC was manually docked into the 7TMD
region taking cis-retinal as template for location. Where
necessary, nonconserved amino acid side chains in the rmGlu1 model were
rotated such that no van der Waals conflicts with the antagonist
occurred. All amino acid side chains reaching within a 6-Å distance of
the antagonist were subsequently included in a round of optimization.
80 °C. The pellet was suspended in cold 20 mM HEPES-NaOH buffer containing 10 mM EDTA (pH
7.4) and homogenized with a Polytron homogenizer (Kinematica, AG) for
10 s at 10,000 rpm. After centrifugation at 48,000 × g for 30 min at 4 °C, the pellet was resuspended in cold
20 mM HEPES-NaOH buffer containing 0.1 mM EDTA
(pH 7.4), homogenized, and respun as above. The pellet was resuspended
in a smaller volume of a cold 20 mM HEPES-NaOH buffer
containing 0.1 mM EDTA (pH 7.4). After homogenization for
10 s at 10,000 rpm, the protein content was measured using the BCA
method (Pierce) with bovine serum albumin as the standard. The membrane
homogenate was frozen at
80 °C before use.
70 mV, and the recordings were made under conditions in which K+ currents would be
inward ([K+]i = 150 mM,
[K+]o = 30 mM).
Concentration-response curves were obtained by applying 20-s pulses of
varying concentrations of the compound every 90 s to the cells.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptors (19), the
binding pocket of EM-TBPC has been localized to the 7TMD of mGlu1 (data
not shown). To elucidate the binding mode of EM-TBPC further, an
alignment of the seven-transmembrane helices of the whole rmGlu family
toward the transmembrane helices of bovine rhodopsin (Protein Data Bank
code 1f88) was made. The inverse agonist of rhodopsin,
cis-retinal, was employed as a template for the location of
EM-TBPC. Amino acids in mGlu1 located within 6.0 Å away from retinal
in the x-ray crystal structure of rhodopsin (23, 32) were considered as
likely candidates to affect binding of EM-TBPC. In Fig.
2, the alignment of these amino acids of
the rmGlu1 and the rmGlu family with rhodopsin is shown. From this
preselection, amino acids in the TM3, -4, -5, -6, and -7 and EC2
regions (Fig. 2, boldface type) were chosen for
the mutational studies.
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Fig. 1.
Structures of [3H]EM-TBPC,
CFBTE-TPC, ( )-CPCCOEt, BAY36-7620, RO 67-7476, and MPEP.
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Fig. 2.
Alignment of amino acid residues of 7TMD and
the EC2 loop in the rmGlu sequences relative to bovine rhodopsin.
The amino acids shown were found to be in close proximity to the
inverse agonist cis-retinal in the x-ray crystal structure
of the bovine rhodopsin (6 Å, Protein Data Bank code 1f88) and were
considered as guidance for mutational studies. The Ballesteros
Weinstein numbering scheme of the amino acids is given to facilitate
the comparison with other GPCRs (see "Experimental Procedures").
The conserved residue in each TM of rhodopsin that is assigned as 50 is
shown in the bottom row. The asterisks
indicate identical residues among mGlu and rhodopsin. Colons
indicate identical residues among mGlus. The residues that have been
point-mutated in rmGlu1 are shown in boldface
type.
and 1 in hmGlu1
) were
accordingly introduced in the 7TMD region by site-directed mutagenesis. Saturation binding analyses were performed on membranes isolated from
the HEK-293 transfected with the WT and mutated receptors using
0.3-300 nM of [3H]EM-TBPC. The dissociation
constants (KD) derived from the saturation isotherms
are given in Table I. Eleven mutations, A699V, Y672F, T723, N747A, S749T, S749A, N750A, N750Q, V802M, A818S,
and A818I, did not significantly affect the [3H]EM-TBPC
affinity compared with the WT rmGlu1
receptor (Table I). Three
mutations, F801A, Y805A, and T815M, completely abolished [3H]EM-TBPC binding, and the mutation Y672V led to a
5-fold decrease in affinity (KD = 32 ± 5.4 nM). [3H]EM-TBPC does not bind to hmGlu1
under our experimental conditions (Table I). Interestingly, only one
amino acid differs between the rat and human receptor in the TM domain;
it is a valine at position 757 in the rat and a leucine in the human
receptor. As expected, the replacement of the leucine residue at
position 757 of the hmGlu1 receptor by a valine (L757V) gave to the
mutant receptor a binding affinity for [3H]EM-TBPC
comparable with that of WT rmGlu1
(KD = 9.9 ± 2.9 nM for the hmGlu1
L757V versus
6.6 ± 0.5 nM rmGlu1
). Conversely, the replacement
of the valine 757 with a leucine (V757L) or an alanine (V757A) led to a
dramatic, although not complete, reduction in [3H]EM-TBPC
affinity by 13-fold (KD = 84 ± 39 nM and KD = 85 ± 19 nM), respectively, in both mutants.
Binding properties of WT mGlu1 and mutants
receptors, glutamate elicited a
concentration-dependent increase in intracellular free
calcium [Ca2+]i, as assayed by single-cell fura-2
imaging (EC50 = 6.8 µM). All mutated
receptors elicit an increase in [Ca2+]i response
upon application of 30 µM glutamate, indicating the
presence of functional receptors. However, co-application of EM-TBPC at
various concentrations with 30 µM glutamate in the cells
expressing WT rmGlu1
resulted in a
concentration-dependent inhibition of glutamate-evoked
[Ca2+]i response with IC50 = 128 nM (Fig. 3). The
concentration-dependent inhibition of glutamate-evoked
increases in [Ca2+]i by EM-TBPC in the cells
expressing various mutated receptors is shown in Fig. 3, A
and B, and their derived IC50 and
nH values are shown in Table
II.
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Fig. 3.
The effect of mutations on inhibition of
glutamate-stimulated increases in [Ca2+]i by
EM-TBPC. Concentration-dependent inhibition of 30 µM glutamate-stimulated increases in
[Ca2+]i by EM-TBPC as assayed by single cell
fura-2 imaging of HEK-293 transiently transfected with the rmGlu1 WT
and mutated receptors. In A, the mutations that shifted the
dose-response curve to the left, and in B, the
mutations that shifted the dose-response curve to the right
or have no effect are shown. Data are mean ± S.E. of four
separate experiments with 15-20 cells/experiment. Responses are
normalized to the first control response.
Effect of mutations on inhibition of glutamate-induced
[Ca2+]i response by EM-TBPC
WT and mutated receptors. Data are
means ± S.E. of four separate experiments with 15-20
cells/experiment.
receptors in a CHO line stably
expressing concatenated human Kir3.1 and Kir3.2c GIRK subunits. First,
we examined the subtype selectivity of EM-TBPC for rmGlu1
receptor.
Rapid application of glutamate (10 µM) induced an inward
current in GIRK-CHO cells expressing the rmGlu1
or rmGlu5a
receptors. Co-application of EM-TBPC at various concentrations with 10 µM glutamate in the GIRK-CHO cell expressing
rmGlu1
resulted in a dose-dependent inhibition of
glutamate-evoked GIRK current with IC50 = 15.3 nM (pIC50 = 7.81 ± 0.08, nH = 1.69 ± 0.17), whereas the
mGlu5a-selective antagonist MPEP had no effect (Fig.
4A). Similarly, MPEP inhibited glutamate-evoked GIRK current in the GIRK-CHO cells expressing rmGlu5a
with a dose-dependent manner (pIC50 = 8.1 ± 0.1, IC50 = 8 nM, and
nH = 0.92 ± 0.13), whereas EM-TBPC had no
effect on GIRK current (Fig. 4B). The effect of 1 µM EM-TBPC on the 10 µM glutamate-evoked
GIRK current in the critical point-mutated mGlu1
receptors is shown
in Fig. 4C. EM-TBPC lost its ability to block the
glutamate-evoked GIRK currents from rmGlu1
with the point-mutations of F801A, Y805A, or T815M (<45%, <35%, or <10% inhibition by 1 µM EM-TBPC, respectively), which all led to complete loss
of binding affinity for [3H]EM-TBPC. The
concentration-response inhibition curve by EM-TBPC of glutamate-evoked
current in GIRK-CHO cell expressing the mutant W798F is shown in Fig.
4D. In good agreement with the binding and Ca2+
imaging studies, the inhibition curve is shifted to the left, indicating a marked decrease in IC50 value for this
mutation (pIC50 = 8.69 ± 0.08, IC50 = 2 nM, and nH = 1.87 ± 0.41).
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Fig. 4.
Effect of allosteric antagonists on
glutamate-evoked GIRK current in WT and mutated receptors.
Concentration-response curves for the effect of the mGlu1 receptor
antagonist EM-TBPC and the mGlu5 receptor antagonist MPEP on 10 µM glutamate-evoked GIRK current in GIRK-CHO cells
transiently transfected with either rmGlu1 (A) or rmGlu5a
(B) receptor cDNA. C, block of the 10 µM glutamate-evoked GIRK current by 1 µM
EM-TBPC in GIRK-CHO expressing WT and mutated rmGlu1
receptors.
D, concentration-dependent inhibition of 10 µM glutamate-induced GIRK current by EM-TBPC in GIRK-CHO
cells transiently transfected with the WT and W798F mutated rmGlu1
receptors. The values are normalized to the control responses obtained
with glutamate alone (10 µM, 100%). Each point
represents the mean ± S.E. of five cells. The sigmoidal curves
were generated with the mean IC50 and Hill
coefficient.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
Molecular modeling of the rmGlu1 complexed
with EM-TBPC. Shown is a homology model of rmGlu1 based on the
high resolution structure of bovine rhodopsin. A three-dimensional side
view of the 7TM binding pocket of the rmGlu1 receptor is shown with a
suggested binding mode of EM-TBPC. C- traces are represented as
ribbons: TM1 (cyan), TM2 (yellow), TM3
(red), TM4 (blue), TM5 (orange), TM6
(gray), and TM7 (magenta). The important residues
that were mutated in the current work are labeled in one-letter
code. Color coding of Tyr-672, Asn-747,
Asn-750, Val-757, Trp-798, Phe-801, Tyr-805, and Thr-815
(blue) and EM-TBPC (magenta) atoms is as follows:
red, oxygen; blue, nitrogen; white,
carbon.
Among the 19 point mutations that are located in TM3, -4, -5, -6, and
-7 and EC2 of rmGlu1, we observed that the F8016.51A,
Y8056.55A, and T8157.39M mutations resulted in
complete loss of the [3H]EM-TBPC binding affinity and
also blocked the inhibition by EM-TBPC of glutamate-evoked GIRK current
or [Ca2+]i response. In our model, the aliphatic
hydroxyl group of Thr-8157.39 is positioned at sufficient
proximity to EM-TBPC to form a hydrogen bond. In an earlier study,
Litschig et al. (15), probing the CPCCOEt binding pocket of
hmGlu1
, demonstrated that the two nonconserved residues
Thr-8157.39 and Ala-8187.42 are important
molecular determinants for the binding of the antagonist CPCCOEt. We
have also found that the mutation Thr8157.39M had a
detrimental effect on [3H]EM-TBPC affinity; however,
conversion of Ala-8187.42 to Ser or Ile did not
affect EM-TBPC binding affinity or functional IC50.
With the exception of the mutation Y6723.40V, which led to a decrease of 5-fold in [3H]EM-TBPC binding affinity, the other mutated residues in TM3 and TM4 had no significant effect on [3H]EM-TBPC affinity. In the three-dimensional structure of rhodopsin, cis-retinal is in close contact to the residues in EC2, which connects TM4 and TM5. There is also evidence that the EC2 loop has an important functional role in the entire GPCR family 1 (33). Interestingly, we identified two mutations in the EC2 loop, N74745.51A and N75045.54A, which had no significant effect on the binding affinity but increased the efficacy of the EM-TBPC block of glutamate-evoked [Ca2+]i response (IC50 of 30 and 10 nM, respectively, versus IC50 of 128 nM for WT). Thus, it seems that although these residues are not in direct contact with the antagonist, they might nevertheless form an important linker to transduce the glutamate binding event in the extracellular domain into the TM region in the mGlu family.
[3H]EM-TBPC had very low affinity for human mGlu1 and none for mGlu5. The sequence comparison of the 7TMD showed that, with the exception of rat mGlu1, which carries a valine at position 757, all mGlu receptors have leucine at this position. This is the only amino acid difference found in the 7TMD region between rat and human mGlu1. Therefore, Val-7575.47 was identified as the critical residue for the binding selectivity of [3H]EM-TBPC at the rat versus human mGlu1 receptor. The mutation L7575.47V in hmGlu1 converted the affinity of this receptor for [3H]EM-TBPC to a value comparable with that of WT rmGlu1 (KD values 6.6 and 9.9 nM for rmGlu1 and hmGlu1 L757V, respectively). Moreover, mutation of rmGlu1 Val-7575.47 to leucine or alanine both led to a similar decrease in [3H]EM-TBPC binding affinity by 13-fold, indicating the importance of valine for selectivity for rat mGlu1. As we have shown previously (19), Val-7575.47 is also a critical residue for the enhancing effect of the positive allosteric modulator of mGlu1, RO 67-7476.
It is interesting to compare the critical residues involved in the
EM-TBPC-binding site of rmGlu1 with those of the
cis-retinal-binding pocket of rhodopsin or in reported
ligand recognition sites of other family 1 GPCRs (Table
III). We observed a striking conservation in the position of critical residues. We found that the five
residues Val-7575.47, Trp-7986.48,
Phe-8016.51, Tyr-8056.55, and
Thr-8157.39 are crucial in the EM-TBPC-binding pocket
of rmGlu1, thus validating the application of rhodopsin
three-dimensional structure (23, 32, 34) as a template for the family 3 GPCRs. Furthermore, conservation in the position of these critical
residues was also observed in the previously reported ligand
recognition sites for h2 adrenergic, hA3
adenosine, or h5HT4 receptors (33, 35-37) (Table III).
Therefore, the allosteric binding site crevice of mGlu1 shares a
similar structural feature as rhodopsin-like family 1 GPCRs.
|
The residue Trp-7986.48 of mGlu1 is of special
interest, because it is highly conserved in all mGlus and in family 1 GPCRs. In the case of rhodopsin, spin labeling and Cys cross-linking
studies (38, 39) demonstrated the requirement for a rigid body movement of the cytoplasmic end of TM6 away from TM3 upon rhodopsin activation and that the Trp-2656.48 serves to transmit the chromophore
motion to TM6 helix. The cross-linking experiment (40) also showed that
in the inactivated form (in the dark) of rhodopsin, the -ionone ring
of cis-retinal is linked to Trp-2656.48.
Similarly, in the three-dimensional structure of rhodopsin, the indole
side chain of Trp-2656.48 comes within about 3.8 Å of the
cis-retinal C20 (23, 32). Taken together, all of these
studies are consistent with a pivotal role for the Trp6.48,
acting as a switch for the transition of the receptor between different
allosteric states. In our model, the crucial residues Phe-8016.51 and Tyr-8056.55 are located in
proximity to Trp-7986.48 in the TM6 helix. Thus, we propose
that the aromatic cluster formed from Trp-7986.48,
Phe-8016.51, and Tyr-8056.55 interacts with the
aromatic ring of EM-TBPC, blocking the Trp-7986.48 movement
in TM6 helix, which is required for receptor activation. In good
agreement with this model, we observed that replacement of
Trp-7986.48 with a phenylalanine led to a large decrease of
EM-TBPC IC50 value for the inhibition of the
glutamate-mediated signal, due to the more preferable
-interaction
with Phe side chain. Joubert et al. (37) have recently
reported that three residues of h5HT4 receptor,
Asp-1003.32, Trp-2726.48, and
Phe-2756.51, are important molecular determinants for
agonist-independent activity, since mutation of these residues to
alanine completely blocked the effects of inverse agonists.
Interestingly, two of these residues are homologous to the critical
residues Trp-7986.48 and Phe-8016.51 that we
identified in the EM-TBPC-binding pocket of rmGlu1. Although the
relative inverse agonist activity of EM-TBPC is presently unknown, such
activity has been previously reported for BAY36-7620 and MPEP, the
selective noncompetitive mGlu1 (18) and mGlu5 (17) antagonists, respectively.
In conclusion, we have demonstrated for the first time that the x-ray
crystal structure of rhodpsin can be successfully applied as a template
for the family 3 GPCRs. The allosteric binding pocket on mGlu receptors
represents an attractive drug target. Hence, detailed information about
the contact sites of allosteric antagonist with the receptor should
further facilitate the rational design of drugs with improved subtype
selectivity and potency.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Birgit Molitor, Rachel Fimbel, Michael Weber, and Klaus Christensen for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* 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. Tel.: 41-61-688-6286; Fax: 41-61-688-1720; E-mail: parichehr.malherbe@roche.com.
Present address: Psychiatry Centre of Excellence for Drug
Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Ave., Harlow, Essex CM19 5AW, UK.
** Present address: Addex Pharmaceuticals SA, 12 Chemin Des Aulx, CH-1228 Plan Les Ouates, Switzerland.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M211759200
2 C. Broger, unpublished work.
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ABBREVIATIONS |
---|
The abbreviations used are:
mGlu, metabotropic
glutamate;
hmGlu, human mGlu;
TM, transmembrane;
7TM, seven-transmembrane;
7TMD, seven-transmembrane domain;
EM-TBPC, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile;
GPCR, G-protein-coupled receptor;
GIRK, G-protein-coupled inwardly
rectifying K+ channel;
WT, wild type;
GABAB, -aminobutyric acid, type B;
EC, extracellular
loop;
CPCCOEt, 7-(hydroxyimino)cyclopropan[b]chromen-1a-carboxylic
acid ethyl ester;
MPEP, 2-methyl-6-(phenylethynyl)pyridine;
BAY36-7620, [(3aS,6aS)-6a-naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on];
RO 67-7476, (S)-2-(4-fluorophenyl)-1-(toluene-4-sulfonyl)-pyrrolidine;
RO 01-6128, diphenylacetyl-carbamic acid ethyl ester;
RO 67-4853, (9H-xanthene-9-carbonyl)-carbamic acid butyl ester;
CGP7930, 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethylpropyl)-phenol;
CFBTE-TPC, N-[2-[(2-chloro-6-fluorobenzyl)thio]ethyl]2-thiophenecarboxamide;
CHO, Chinese hamster ovary;
5HT4, 5-hydroxytryptamine type 4.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Conn, P. J., and Pin, J. P. (1997) Annu. Rev. Pharmacol. Toxicol. 37, 205-237[CrossRef][Medline] [Order article via Infotrieve] |
2. | Pin, J. P., De Colle, C., Bessis, A. S., and Acher, F. (1999) Eur. J. Pharmacol. 375, 277-294[CrossRef][Medline] [Order article via Infotrieve] |
3. | De Blasi, A., Conn, P. J., Pin, J. P., and Nicoletti, F. (2001) Trends Pharmacol. Sci. 22, 114-120[CrossRef][Medline] [Order article via Infotrieve] |
4. | Holscher, C., Gigg, J., and O'Mara, S. M. (1999) Neurosci. Biobehav. Rev. 23, 399-410[CrossRef][Medline] [Order article via Infotrieve] |
5. | Nakanishi, S. (1994) Neuron 13, 1031-1037[Medline] [Order article via Infotrieve] |
6. | Bordi, F., and Ugolini, A. (1999) Prog. Neurobiol. 59, 55-79[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Bockaert, J.,
and Pin, J. P.
(1999)
EMBO J.
18,
1723-1729 |
8. | O'Hara, P. J., Sheppard, P. O., Thogersen, H., Venezia, D., Haldeman, B. A., McGrane, V., Houamed, K. M., Thomsen, C., Gilbert, T. L., and Mulvihill, E. R. (1993) Neuron 11, 41-52[Medline] [Order article via Infotrieve] |
9. |
Galvez, T.,
Prezeau, L.,
Milioti, G.,
Franek, M.,
Joly, C.,
Froestl, W.,
Bettler, B.,
Bertrand, H. O.,
Blahos, J.,
and Pin, J. P.
(2000)
J. Biol. Chem.
275,
41166-41174 |
10. | Kunishima, N., Shimada, Y., Tsuji, Y., Sato, T., Yamamoto, M., Kumasaka, T., Nakanishi, S., Jingami, H., and Morikawa, K. (2000) Nature 407, 971-977[CrossRef][Medline] [Order article via Infotrieve] |
11. | Parmentier, M. L., Prezeau, L., Bockaert, J., and Pin, J. P. (2002) Trends Pharmacol. Sci. 23, 268-274[CrossRef][Medline] [Order article via Infotrieve] |
12. | Schoepp, D. D., Jane, D. E., and Monn, J. A. (1999) Neuropharmacology 38, 1431-1476[CrossRef][Medline] [Order article via Infotrieve] |
13. | Gasparini, F., Kuhn, R., and Pin, J. P. (2002) Curr. Opin. Pharmacol. 2, 43-49[CrossRef][Medline] [Order article via Infotrieve] |
14. | Annoura, H., Fukunaga, A., Uesugi, M., Tatsuoka, T., and Horikawa, Y. (1996) Bioorg. Med. Chem. Lett. 6, 763-766[CrossRef] |
15. |
Litschig, S.,
Gasparini, F.,
Rueegg, D.,
Stoehr, N.,
Flor, P. J.,
Vranesic, I.,
Prezeau, L.,
Pin, J. P.,
Thomsen, C.,
and Kuhn, R.
(1999)
Mol. Pharmacol.
55,
453-461 |
16. | Gasparini, F., Lingenhohl, K., Stoehr, N., Flor, P. J., Heinrich, M., Vranesic, I., Biollaz, M., Allgeier, H., Heckendorn, R., Urwyler, S., Varney, M. A., Johnson, E. C., Hess, S. D., Rao, S. P., Sacaan, A. I., Santori, E. M., Velicelebi, G., and Kuhn, R. (1999) Neuropharmacology 38, 1493-1503[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Pagano, A.,
Ruegg, D.,
Litschig, S.,
Stoehr, N.,
Stierlin, C.,
Heinrich, M.,
Floersheim, P.,
Prezeau, L.,
Carroll, F.,
Pin, J. P.,
Cambria, A.,
Vranesic, I.,
Flor, P. J.,
Gasparini, F.,
and Kuhn, R.
(2000)
J. Biol. Chem.
275,
33750-33758 |
18. |
Carroll, F. Y.,
Stolle, A.,
Beart, P. M.,
Voerste, A.,
Brabet, I.,
Mauler, F.,
Joly, C.,
Antonicek, H.,
Bockaert, J.,
Muller, T.,
Pin, J. P.,
and Prezeau, L.
(2001)
Mol. Pharmacol.
59,
965-973 |
19. |
Knoflach, F.,
Mutel, V.,
Jolidon, S.,
Kew, J. N. C.,
Malherbe, P.,
Vieira, E.,
Wichmann, J.,
and Kemp, J. A.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
13402-13407 |
20. |
Urwyler, S.,
Mosbacher, J.,
Lingenhoehl, K.,
Heid, J.,
Hofstetter, K.,
Froestl, W.,
Bettler, B.,
and Kaupmann, K.
(2001)
Mol. Pharmacol.
60,
963-971 |
21. | Adam, G., Binggeli, A., Maerki, H. P., Mutel, V., Wilhelm, M., and Wostl, W. (2001) European Patent Application, pp. 1-85, Priority EP 99-1074549 A2 |
22. | Knoflach, F., Mutel, V., Kew, J. N. C., Waselle, L., Vieira, E., Jolidon, S., Wichmann, J., Malherbe, P., and Kemp, J. A. (2001) Soc. Neurosci. Abstr. 705, 13 |
23. |
Palczewski, K.,
Kumasaka, T.,
Hori, T.,
Behnke, C. A.,
Motoshima, H.,
Fox, B. A.,
Le Trong, I.,
Teller, D. C.,
Okada, T.,
Stenkamp, R. E.,
Yamamoto, M.,
and Miyano, M.
(2000)
Science
289,
739-745 |
24. | Ballesteros, J. A., and Weinstein, H. (1995) Methods Neurosci. 25, 366-428 |
25. | Higgins, D. C., Bleasby, A. J., and Fuch, R. (1992) Comput. Appl. Biosci. 8, 189-191[Abstract] |
26. | Gerber, P. R., and Muller, K. (1995) J. Comput. Aided Mol. Des. 9, 251-268[Medline] [Order article via Infotrieve] |
27. | Gerber, P. R. (1998) J. Comput. Aided Mol. Des. 12, 37-51[CrossRef][Medline] [Order article via Infotrieve] |
28. | Ponder, J. W., and Richards, F. M. (1987) J. Mol. Biol. 193, 775-791[Medline] [Order article via Infotrieve] |
29. |
Malherbe, P.,
Knoflach, F.,
Broger, C.,
Ohresser, S.,
Kratzeisen, C.,
Adam, G.,
Stadler, H.,
Kemp, J. A.,
and Mutel, V.
(2001)
Mol. Pharmacol.
60,
944-954 |
30. | Mutel, V., Ellis, G. J., Adam, G., Chaboz, S., Nilly, A., Messer, J., Bleuel, Z., Metzler, V., Malherbe, P., Schlaeger, E.-J., Roughley, B. S., Faull, R. L. M., and Richards, J. G. (2000) J. Neurochem. 75, 2590-2601[CrossRef][Medline] [Order article via Infotrieve] |
31. | Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450[Abstract] |
32. | Teller, D. C., Okada, T., Behnke, C. A., Palczewski, K., and Stenkamp, R. E. (2001) Biochemistry 40, 7761-7772[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Ballesteros, J. A.,
Shi, L.,
and Javitch, J. A.
(2001)
Mol. Pharmacol.
60,
1-19 |
34. | Sakmar, T. P., Menon, S. T., Marin, E. P., and Awad, E. S. (2002) Annu. Rev. Biophys. Biomol. Struct. 31, 443-484[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Gao, Z. G.,
Chen, A.,
Barak, D.,
Kim, S. K.,
Muller, C. E.,
and Jacobson, K. A.
(2002)
J. Biol. Chem.
277,
19056-19063 |
36. | Lopez-Rodriguez, M. L., Murcia, M., Benhamu, B., Olivella, M., Campillo, M., and Pardo, L. (2001) J. Comput. Aided Mol. Des. 15, 1025-1033[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Joubert, L.,
Claeysen, S.,
Sebben, M.,
Bessis, A. S.,
Clark, R. D.,
Martin, R. S.,
Bockaert, J.,
and Dumuis, A.
(2002)
J. Biol. Chem.
277,
25502-25511 |
38. |
Farrens, D. L.,
Altenbach, C.,
Yang, K.,
Hubbell, W. L.,
and Khorana, H. G.
(1996)
Science
274,
768-770 |
39. | Sheikh, S. P., Zvyaga, T. A., Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Borhan, B.,
Souto, M. L.,
Imai, H.,
Shichida, Y.,
and Nakanishi, K.
(2000)
Science
288,
2209-2212 |