Amino Acid Mutagenesis of the Ligand Binding Site and the Dimer
Interface of the Metabotropic Glutamate Receptor 1
IDENTIFICATION OF CRUCIAL RESIDUES FOR SETTING THE ACTIVATED
STATE*
Toshihiro
Sato
,
Yoshimi
Shimada
,
Naoko
Nagasawa
,
Shigetada
Nakanishi§, and
Hisato
Jingami
¶
From the
Department of Molecular Biology,
Biomolecular Engineering Research Institute, 6-2-3 Furuedai,
Suita-City, Osaka 565-0874, Japan and the § Department of
Biological Sciences, Kyoto University Faculty of Medicine, Yoshida,
Sakyo-ku, Kyoto 606-8501, Japan
Received for publication, October 8, 2002, and in revised form, November 18, 2002
 |
ABSTRACT |
Previously, we determined the crystal structures
of the dimeric ligand binding region of the metabotropic glutamate
receptor subtype 1. Each protomer binds L-glutamate
within the crevice between the LB1 and LB2 domains. We proposed that
the two different conformations of the dimer interface between the two
LB1 domains define the activated and resting states of the receptor
protein. In this study, the residues in the ligand-binding site and the dimer interface were mutated, and the effects were analyzed in the
full-length and truncated soluble receptor forms. The variations in the
ligand binding activities of the purified truncated receptors are
comparable with those of the full-length form. The mutated full-length
receptors were also analyzed by inositol phosphate production and
Ca2+ response. The magnitude of the ligand binding
capacities and the amplitude of the intracellular signaling were almost
correlated. Alanine substitutions of four residues,
Thr188, Asp208, Tyr236, and
Asp318, which interact with the
-amino group of
glutamate in the crystal, abolished their responses both to glutamate
and quisqualate. The mutations of the Tyr74,
Arg78, and Gly293 residues, which interact with
the
-carboxyl group of glutamate, lost their responsiveness to
glutamate but not to quisqualate. Furthermore, a mutant receptor
containing alanine instead of isoleucine at position 120 located within
an
helix constituting the dimer interface showed no intracellular
response to ligand stimulation. The results demonstrate the crucial
role of the dimer interface in receptor activation.
 |
INTRODUCTION |
Glutamate is a major neurotransmitter in excitatory neurons in the
central nervous system. Glutamate released into the synaptic space is
recognized by two distinct receptors, glutamate-gated ion channels and
metabotropic glutamate receptors
(mGluRs)1 (1, 2). The mGluRs
consist of eight subtypes (mGluR1 to -8), which couple with a variety
of effector systems, including inositol phosphate pathway, adenylyl
cyclase, ion channels, etc. The mGluRs are considered to modulate
synaptic neurotransmission and thus to play roles in memory, learning,
and brain disorders such as epilepsy and neurodegenerative diseases.
The mGluR consists of three regions: a large extracellular region, a
seven-transmembrane-spanning region, and an intracellular region. Previously, we determined the crystal structures of the extracellular ligand-binding region (LBR) of mGluR1 (3). In combination
with biochemical studies (4, 5), the mGluR1-LBR (m1-LBR) was found to
be a homodimer consisting of two protomers. Each protomer consists of
an LB1 domain and an LB2 domain. The glutamate-binding structure is a
dimer composed of closed and open protomers, which differ in the
relative orientation of the LB1 and LB2 domains. Without glutamate, two
crystal forms of m1-LBR were obtained; one form exists as an open-open
dimer, and the other is an open-closed form. The two main functioning
sites were then elucidated: the ligand-recognition site and the LB1
dimer interface. In the ligand binding site, glutamate interacts mainly with 13 amino acid residues from the LB1 and LB2 domains of the protomer. We proposed that the ligand-binding domain of mGluR1 is in
dynamic equilibrium between the activated state and the resting state,
which are defined mainly by the different dimer interface conformations
of the three crystal forms. An antagonist binding crystal structure,
which was recently solved (6), maintained the resting dimer
conformation, supporting our proposal.
Recently, numerous dimer formation studies of G-protein-coupled
receptors have suggested a potential level of receptor complexity and
diversity (7). However, the precise structural basis of the dimer
formation has not been established. In this context, mGluR1, which is
considered to be a type C G-protein-coupled receptor (8), is unique in
that its extracellular dimer interface has been defined structurally,
as described above, and hence provides an opportunity to investigate
the functional role of dimerization.
Here, in order to elucidate the significance and the contribution of
the individual amino acid residues in ligand binding and the
correlation with intracellular events, such as inositol phosphate (IP)
formation and subsequent Ca2+ responses, we have analyzed
the functions of mutant receptors with amino acid mutations of the
ligand-binding residues. Furthermore, we also mutated the residues
within the helices forming the LB1 dimer interface and analyzed the
effects of the mutations. One of the dimer interface mutants lost the
Ca2+ response to ligand stimulus, indicating the
critical role of dimer interface relocation in receptor activation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
L-Quisqualic acid was purchased from
Tocris Cookson Ltd. (Langford Bristol, UK). L-Glutamic acid
monosodium salt was purchased from Nacalai Tesque (Kyoto, Japan).
LipofectAMINE 2000 and oligonucleotide primers were purchased from
Invitrogen. QF/C filters were purchased from Millipore Corp.
(Bedford, MA). The AG-1-X8 resin (100-200-mesh formate form) and the
Poly-Prep chromatography columns for anion exchange chromatography were
obtained from Bio-Rad. All other reagents used were of molecular or
analytical grade.
Construction of Expression Vectors and Oligonucleotide
Mutagenesis--
The pcDNAmGluR114His plasmid, which is an
expression vector for His6-tagged m1-LBR was made as
follows. A PCR was done with the primers TO1 and HJ113 (4), using
pVLmGluR113 (5) as a template. The PCR product was digested with
NcoI and XbaI and was cloned into the
NcoI/XbaI-digested fragment of pmGluR108 (4), resulting in pVLmGluR114His. The NotI/XbaI
fragment of pVLmGluR114His was subcloned into pcDNA3.1(+)
(Invitrogen). pcDNAmGluR1, an expression vector for the full-length
mGluR1, was made by subcloning the NotI/XbaI
fragment of pmGluR102 (4) into pcDNA3.1(+). For the construction of
the single amino acid Y74A, R78A, S164A, S165A, S166A, S186A, T188A,
L116A, I120A, L174A, L177A, and F178A mutants and the multiple amino
acid mutants, the first PCR was performed with mutagenic primers (Table
I) and TS35 (5'-TCCACTCTCGCCGGCATTCC-3'), using pcDNAmGluR114His as the template. The second PCR was
performed with the first PCR products and T7
(5'-TAATACGACTCACTATAGGG-3'), using pcDNAmGluR114His as the
template. The second PCR products were digested with NotI
and Eco81I and were subcloned in place of the corresponding
wild-type fragments of pcDNAmGluR114His and pcDNAmGluR1. For
the construction of the D208A, Y236A, E292A, G293A, R323A, and K409A
mutants, the first PCR was done with mutagenic primers and TS36
(5'-CCTGGCTCCGCCTCTGTGGC-3'), using pmGluR114His as the template, and
the second PCR was done with the first PCR products and the reverse
primer (5'-TAGAAGGCACAGTCGAGG-3'). The second PCR product was digested
with Eco81I and SfiI and was subcloned in place
of the corresponding wild-type fragments of pcDNAmGluR114His and pcDNAmGluR1. All of the PCR products mentioned above were confirmed by sequencing.
Cell Culture and Transfection into Human Embryonic Kidney (HEK)
293 Cells--
HEK 293 cells were cultured in a monolayer at 37 °C,
with a 5% CO2 atmosphere in Dulbecco's modified Eagle's
medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) lacking
L-glutamine, ribonucleosides, and deoxyribonucleosides and
supplemented with 2 mM GLUTAMAXTM-I
(Invitrogen), 10% dialyzed fetal bovine serum (JRH Biosciences), and
antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). Cells were grown to 90-95% confluence before the transient
transfection. The transfections were performed using the LipofectAMINE
2000 reagent according to the manufacturer's instructions. On day 3 after transfection, cells and culture medium were harvested to examine
expression and ligand binding.
Purification of the Wild-type and Mutant m1-LBRs--
The
conditioned medium from m1-LBR-producing HEK 293 cells was centrifuged
at 15,000 rpm for 20 min, filtered through a 0.22-µm filter, and
loaded on a 1-ml Ni2+-nitrilotriacetic acid-agarose column.
The column was washed with 10 ml of PBS, and the bound material was
eluted with PBS containing 200 mM imidazole. Aliquots of
the eluate were analyzed by a ligand binding assay.
Membrane Preparation--
Transfected cells (15-cm dishes) were
suspended in ice-cold homogenization buffer (20 mM HEPES
(pH 7.5) containing 320 mM sucrose, 1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml each of leupeptin,
aprotinin, benzamidine, and trypsin inhibitor) and were centrifuged at
10,000 × g for 10 min at 4 °C. The pellet was
homogenized with 20 strokes of a Potter-Elvehjem type homogenizer in 3 ml of ice-cold homogenization buffer and was centrifuged at 800 × g for 5 min at 4 °C. The supernatant was then centrifuged at 100,000 × g for 1 h at 4 °C to pellet the
membranes. The membranes were divided into aliquots and were stored at
80 °C until use.
Immunoblot Analysis--
The proteins within the culture medium
and the cell membranes were separated by SDS-PAGE and were
electroblotted onto a nitrocellulose membrane (Schleicher & Schuell).
The membrane was then blocked in TBST (10 mM Tris-HCl (pH
8.0), 150 mM NaCl, and 0.05% Tween 20) with 3% bovine
serum albumin and was incubated for 1 h at room temperature with
anti-His antibodies (Qiagen Inc., Valencia, CA), AB1551, polyclonal
antibodies against a synthetic carboxyl terminus peptide of mGluR1
(Chemicon International, Inc., Temecula, CA), or the monoclonal
antibody mG1Na-1 (4, 9), which was produced against the extracellular
region of mGluR1. The membrane was washed and then incubated with a
goat anti-mouse IgG conjugated with alkaline phosphatase. Color
development was done with a commercial detection kit (Promega).
Ligand Binding Assay--
For m1-LBRs, ligand binding was
performed with the polyethylene glycol precipitation method, as
described previously (10). Briefly, 20 nM
[3H]quisqualate (999 GBq/mmol) (Amersham Biosciences) and
m1-LBR samples were mixed in 150 µl of binding buffer (40 mM HEPES (pH 7.5), containing 2.5 mM
CaCl2) at 4 °C for 1 h. After the binding reaction,
6-kDa polyethylene glycol was added to the sample to a concentration of
15% with 3 mg/ml
-globulin. The precipitated material was washed
twice with 1 ml of 8% 6-kDa polyethylene glycol and was dissolved in 1 ml of water. After the addition of 14 ml of Scintisol EX-H (Wako Pure
Chemical Industries, Osaka, Japan), the radioactivity was counted in a
scintillation counter. For the mGluR1s, membrane fractions of
receptor-expressing HEK 293 cells were incubated with
[3H]quisqualate (20 nM) in a total volume of
150 µl of binding buffer for 1 h at room temperature. The
reaction mixture was aspirated onto GF/C filters. The filters were
washed with binding buffer, briefly dried, and counted by a
scintillation counter. Nonspecific binding was determined in the
presence of 1 mM L-glutamate. Binding data were
analyzed with the Prism 3 software (Graphpad Software, San Diego, CA).
Saturation binding curves were fitted to a one-site binding model, and
Kd and IC50 values were calculated.
Determination of IP Accumulation--
HEK 293 cells were grown
in Dulbecco's modified Eagle's medium supplemented with 10% dialyzed
fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin. Cells were plated at 2 × 105/well of a
poly-L-lysine-coated 24-well plate, and 1 µg of DNA was
transfected using 2 µl LipofectAMINE 2000 for 4 h. After
transfection, the medium was exchanged with inositol-free Dulbecco's
modified Eagle's medium (ICN Biomedicals Inc., Aurora, OH)
supplemented with 10% dialyzed fetal bovine serum, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 1 µCi/ml
[3H]myo-inositol (95.0 Ci/mmol) (Amersham
Biosciences), and the cultures were incubated at 37 °C for 16-18 h.
The cells were then washed with HEPES-buffered saline (146 mM NaCl, 4.2 mM KCl, 0.5 mM
MgCl2, 0.1% glucose, 20 mM HEPES (pH 7.4)) and
were incubated for 30 min with 2 mM pyruvate and 2 units/ml
glutamic-pyruvic transaminase (Sigma). After washes with HEPES-buffered
saline, the cells were incubated with HEPES-buffered saline containing the agonists and 10 mM LiCl at 37 °C for 1 h. The
reactions were stopped by adding an equal volume of ice-cold 40 mM formic acid, and then the mixture was incubated for 30 min at 4 °C. Cell extracts were loaded onto 1-ml packed volume
columns of AG-1-X8 anion exchange gel resin. After loading, the columns
were washed with 2 ml of 40 mM NH4OH (pH 9.0)
and 2 ml of 40 mM ammonium formate, and the bound material
was eluted with 3 ml of 2 M ammonium formate, 0.1 M formic acid. This procedure collects inositol mono-,
bis-, and trisphosphates (11). Results are expressed as the ratio of
the radioactivity collected in the IP fraction over the radioactivity recovered from the solubilized cellular membranes.
Expression of mGluRs in Oocytes and
Electrophysiology--
The 1330-bp NotI-SfiI
fragment of the mutant pcDNAmGluR1s and the 6.3-kb
NotI-SfiI fragment of pmGR1 (12) were ligated. The resulting plasmids (mutant pmGR1s) and pmGR1 were used as templates
for in vitro transcription to yield complementary RNA (cRNA). The plasmid DNA was linearized by NotI, and capped
cRNA was synthesized with the MEGAscript T7 kit (Ambion, Austin,
TX). Xenopus laevis oocytes were prepared,
injected with 10 ng of cRNAs, and incubated for 1-2 days. Current
measurements were conducted as previously described (5).
Measurement of [Ca2+]i--
HEK 293 cells were seeded on sterile polylysine-coated coverslips (Asahi Techno
Glass Corp., Tokyo, Japan) in a 12-well tissue culture plate (1.6 × 105/well), and were incubated overnight at 37 °C. The
cells were then transfected with 1.6 µg of either pcDNAmGluR1 or
mutant pcDNAmGluR1s and 0.4 µg of pDsRed2-N1
(Clontech) with 5 µl of LipofectAMINE 2000. After
24 h, the cells were loaded for 30-60 min with Fura-2/AM (5 µM) dissolved in balanced salt solution (BSS), containing
135 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl2, 0.9 mM MgCl2, and 10 mM HEPES (pH 7.4), postincubated for 30-60 min in BSS at
37 °C, and maintained in BSS until the assay. About 80% of the
cells in the field of view expressed mGluR1, as judged by
immunocytochemistry, and almost all of them expressed DsRed2 under
these conditions. No significant difference was observed in terms of
the [Ca2+]i response with and without pDsRed2
co-transfection (data not shown). Therefore, the fluorescence of DsRed2
through 499 nm was used to detect the transfectant-rich region. The
coverslips were mounted in a glass flow chamber with a flow rate of 2 ml/min at 22 °C. The cells were challenged with 100 µM
glutamate or 10 µM quisqualate in BSS for 1 min and then
were washed free of agonist. 100 µM of carbachol in BSS
was perfused for 1 min at the end of each assay in order to confirm the
integrity of the IP3/Ca2+ pathway. The chamber
was mounted on a Nikon inverted stage microscope. The
output from an intensified charge-coupled camera was digitized and
stored by a computerized imaging system, Argus 50 (Hamamatsu Photonics,
K.K., Hamamatsu, Japan). [Ca2+]i values were
calculated from the ratios of sequential 340/380-nm excitation image
pairs taken every 5 s by comparison with a standard curve
constructed with a Fura-2 calcium imaging calibration kit (Molecular
Probes, Inc., Eugene, OR). The results are expressed as
[Ca2+]i (nM) above the basal level,
subtracted from the peak [Ca2+]i. Data analysis
was performed using the Prism 3.0 software.
Immunocytochemistry--
HEK 293 cells seeded on sterile
polylysine-coated coverslips (IWAKI) were transfected with
pcDNAmGluR1 and the dimer interface mutants. After 24 h, the
cells were immunoreacted with the monoclonal antibody, mG1Na-1,
following the described procedures (13, 14). Briefly, the cells were
washed with PBS and directly immunoreacted with mG1Na-1 in PBS
containing 5% normal goat serum for 1 h at room temperature
without permeabilization. They were washed, fixed with 4%
paraformaldehyde in PBS for 5-10 min at room temperature, and
permeabilized with 0.1% Triton X-100 in PBS containing 5% normal goat
serum for 1 h. They were immunoreacted with the secondary antibody, fluorescein anti-mouse IgG (H + L) (Vector Laboratories, Burlingame, CA) and were visualized using a Carl Zeiss confocal microscope, model LSM510.
 |
RESULTS |
We constructed mutant receptors containing point mutations in the
ligand binding residues, as determined by our crystallographic study
(Fig. 1). We have designed two types of
receptors: one type is the soluble form of the receptor, including the
N terminus to Glu522, just before the start residue of the
cysteine-rich sequence ahead of the membrane-spanning domain, and the
other is the full-length membrane-bound form of the receptor.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 1.
A close-up view of the ligand binding site of
mGluR1. A, the ligand binding site residues of
m1-LBR contacting L-glutamate (yellow) are shown
as a ribbon diagram. L-Glutamate
contacts the LB1 interface (green) and the LB2 interface
(brown) of m1-LBR in the crystal structure (3). Red
balls are water molecules. Broken lines
indicate polar interactions. B, chemical structures of
L-glutamate and L-quisqualate. These compounds
are shown in the best orientation toward the binding site of
m1-LBR.
|
|
Fig. 2A shows the
immunoblot analysis of the mutant proteins expressed in the culture
medium of the HEK 293 cells transfected with plasmids encoding the
mutant soluble form of the receptors. The mutants sS164A, sS186A,
sG293A, and sK409A (where the small letter "s" represents the
soluble form) were expressed at levels similar to that of the
nonmutated soluble form of mGluR1 (sWT) (formerly designated
as mGluR114 (5) with a His tag). The sY74A, sS165A, sE292A, sD318A, and
sR323A mutants were expressed at lower levels. The sR78A, sT188A,
sD208A, and sY236A mutants were not detected in the culture medium.
Although Ser166 does not interact with the ligand in the
crystal, the sS166A mutant was made as a control and was expressed
well. These soluble forms of the receptors secreted into the culture
medium were partially purified on nickel resin, and their
ligand-binding capacities were examined, as shown in Fig.
2B. The partially purified soluble form receptor proteins,
sS164A, sS186A, sR323A, and sK409A, exhibited significant
[3H]quisqualate binding, as shown in Fig. 2B.
The sY74A and sG293A mutants lost their binding capacities. Since
sufficient amounts of purified protein were not obtained from the
mutants sR78A, sS165A, sT188A, sD208A, sY236A, sE292A, and sD318A, the
binding capacities of these mutants were not estimated. The
IC50 values of glutamate for the sS164A and sS186A mutants
were comparable with that of the wild type soluble form receptor, as
shown in Fig. 2C and Table II.
On the other hand, the sR323A and sK409A mutants showed 1-order of
magnitude higher values.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 2.
Biochemical and pharmacological
characterizations of m1-LBR mutants, which were expressed in the
culture medium. A, immunoblotting analysis of mutant
m1-LBRs. HEK 293 cells were transfected with vector alone
(mock), pcDNAmGluR114His (sWT), and
pcDNAmGluR114His mutants. Aliquots (20 µl) of culture medium from
cells transfected with each plasmid were loaded on a 4-20% gradient
SDS-polyacrylamide gel. Marker proteins are -galactosidase (122 kDa), bovine serum albumin (83.0 kDa), and ovalbumin (51.8 kDa).
Proteins were transferred onto a nitrocellulose membrane and probed
with anti-His antibodies. B, [3H]quisqualate
binding to m1-LBR mutants. The purified m1-LBR (1.8 µg) protein and
various mutant m1-LBRs (0.5-3.0 µg) were incubated in binding buffer
(40 mM HEPES, pH 7.5, containing 2.5 mM
CaCl2) with 20 nM [3H]quisqualate
on ice for 1 h. Nonspecific binding was measured in the presence
of 1 mM glutamate. Each binding was performed in triplicate
and is shown as the mean ± S.D. Representative data from two or
three experiments are shown. C, dose-response curves in inhibiting [3H]quisqualate
binding to the mutant m1-LBRs. The indicated concentrations of
glutamate and 20 nM [3H]quisqualate were
incubated with the purified m1-LBR and mutant m1-LBRs. Each
point shows the mean ± S.E. of a representative of
three experiments done in triplicate. The displacement curves were
obtained by sigmoidal fitting with the Prism 3 software.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Binding parameters of the ligand-binding site mutants
The inhibition of [3H]quisqualate binding to the mutant
m1-LBRs and mGluR1s by glutamate was examined. Mutant mGluR1s were also
tested in the saturation binding assay.
|
|
Next, the mutant full-length receptors, which possess an amino acid
point mutation, were constructed, and their expression and ligand
binding capacities were examined. An immunoblot analysis of membrane
preparations derived from the cells transfected with plasmids encoding
the mutant receptors and the wild type mGluR1 (WT) is shown
in Fig. 3A.
Although the R78A, T188A, and Y236A mutants were not detected in their
soluble forms, their full-length form mutants were detected in the
immunoblot. The D208A mutant was hardly detected. Although the ligand
binding values obtained with the membrane preparations in Fig.
3B were 2-3 orders of magnitude less than those of the
soluble forms in the medium (Fig. 2B) (see pmol
versus fmol in the units at the ordinate), the
variations in the expression levels among the mutants seem to parallel
those of the soluble forms of the mutants. Again, the S164A, S186A, R323A, and K409A mutants showed significant binding values.
Furthermore, the glutamate IC50 values for these mutants
are strikingly comparable with those for the soluble form mutants, as
shown in Fig. 3C and Table II. These data are compatible and
support our previous experiment involving the functional extraction of
the ligand binding region without the membrane region (4).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3.
Biochemical and pharmacological
characterizations of the wild-type and mutant mGluR1s.
A, immunoblotting analysis of mutant mGluR1s. Cells were
transfected with vector alone (mock), pcDNAmGluR1
(WT), and mutant pcDNAmGluR1s. Membrane fractions (40 µg) of HEK 293 cells transfected with each plasmid were loaded on a
4-20% gradient SDS-polyacrylamide gel. The marker proteins are myosin
heavy chain (212.0 kDa), -galactosidase (122.0 kDa), bovine serum
albumin (83.0 kDa), ovalbumin (51.8 kDa), carbonic anhydrase (36.2 kDa), and soybean trypsin inhibitor (29.9 kDa). Proteins were
transferred onto a nitrocellulose membrane and were probed with the
monoclonal antibody, mG1Na-1. B,
[3H]quisqualate binding to mutant mGluR1s. Membrane
fractions (100 µg) of HEK 293 cells transfected with plasmids
encoding the wild-type and mutant mGluR1s were incubated in binding
buffer (40 mM Hepes, pH 7.5, containing 2.5 mM CaCl2) with 20 nM
[3H]quisqualate on ice for 1 h. The reaction
mixtures were aspirated onto GF/C filters, and the material remaining
on the filters was counted with a scintillation counter. Nonspecific
binding was measured in the presence of 1 mM glutamate.
Each binding shows the mean ± S.D. of a representative of three
experiments done in triplicate. C, dose-response curve of
inhibiting [3H]quisqualate binding to the wild-type and
mutant mGluR1s. The indicated concentrations of glutamate and 20 nM [3H]quisqualate were incubated with
membrane fractions of HEK 293 cells transfected with plasmids for the
wild-type and mutant mGluR1s. Each point shows the mean ± S.E. of a representative of three experiments done in
triplicate.
|
|
In order to examine the effects of the amino acid mutations
in the ligand binding core on intracellular signaling, the enhancement of the IP concentration evoked by glutamate and quisqualate stimuli was
investigated in HEK 293 cells (Fig. 4). 1 mM glutamate stimulation of the cells expressing the S164A,
S186A, R323A, and K409A mutants evoked IP responses comparable with
that of the wild type mGluR1. The responses to the quisqualate
stimulation were almost parallel to those of the glutamate stimulation.
The S165A and E292A mutants showed an intermediate level response with
glutamate. Although a lower glutamate response level was observed with
the Y236A mutant, the response with quisqualate was almost lost.
Interestingly, in the Y74A and R78A mutants, the response to glutamate
was undetectable; however, a significant response to quisqualate was
observed. The amplitude of the quisqualate response in the G293A mutant
was very small but appears to be significant. No response to either ligand was detected with the T188A, D208A, and D318A mutants, which
hardly expressed the soluble form and exhibited weak ligand binding
capacities in the full-length forms. In the wild type mGluR1, the IP
level before ligand stimulation (white bar) was significantly higher than that in the cells transfected with the vector
plasmid alone (mock). Since the so-called basal level
of IP production by unliganded mGluR1 expression, also observed in Ref.
15, was lost in the mutants S165A and E292A, which showed reduced
ligand coupling, it seemed that the "basal level" required agonist-like conformational changes. In these mutants, movement of the
LB1-LB2 interdomain orientation angle might be restricted at the basal
state, and thus the dimer interface relocation, possibly related to
closing of at least one protomer, might be hampered.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 4.
Ligand-induced inositol phosphate production
in cells expressing the wild-type and mutant mGluR1s. Cells were
transfected with control vector alone, pcDNAmGluR1, and mutant
pcDNAmGluR1s. Transfected cells were labeled for 17 h with
[3H]myo-inositol and were stimulated with 1 mM glutamate or 0.1 mM quisqualate in the
presence of 10 mM LiCl. Inositol phosphates were extracted
and separated by anion exchange chromatography, as described under
"Experimental Procedures." Results are expressed as the ratio of
[3H]IPs accumulated over total radioactivity in the
membrane fraction and represent the means ± S.E. of at least
three independent experiments performed in duplicate or
triplicate.
|
|
Alterations of the intracellular Ca2+ concentrations, which
are a downstream effect of IP production, were examined, as shown in
Fig. 5A. Glutamate elicited an
enhancement of the intracellular Ca2+ concentration in
cells expressing the S164A, S165A, S186A, E292A, R323A, and K409A
mutants as well as the mGluR1 (WT) and the control mutant,
S166A. These data are comparable with the increment of the IP
concentration in these mutants. Thus, the intermediate level of IP
enhancement in the S165A and E292A mutants seems to be significant.
These data indicate the different sensitivities between the ligand
binding assay and the intracellular Ca2+ measurement. The
T188A, D208A, Y236A, and D318A mutants did not show any
Ca2+ response. In the Y74A, R78A, and G293A mutants, only
quisqualate showed a Ca2+ response. The Ca2+
responses in the Y74A and R78A mutants seemed to be comparable with
their IP responses.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 5.
Intracellular calcium responses in the ligand
binding site mutants. A, intracellular Ca2+
response to glutamate or quisqualate in cells transfected with plasmids
encoding ligand-binding site mutants as well as the wild type
(WT) were examined. Either 100 µM glutamate or
10 µM quisqualate in BSS was used in a 1-min perfusion.
The peak and basal intracellular Ca2+ concentrations were
determined, as described under "Experimental Procedures." Results
are expressed as Ca2+ concentrations above the basal
level. Data are means ± S.E. for greater than 14 individual cells
from at least two independent experiments. B,
electrophysiology of oocytes injected with RNAs for the wild-type and
mutant mGluR1s related to the ligand interaction. X. laevis
oocytes were injected with 10 ng of in vitro transcribed
cRNA as described under "Experimental Procedures." Between 24 and
48 h after the injection, the holding potential was set at 60 mV,
and the current was measured with the ligand stimulation, either
glutamate or quisqualate. Each point shows the mean ± S.E. of a representative of two experiments done in
quadruplicate.
|
|
We also examined the membrane currents in oocyte expression systems, as
shown in Fig. 5B. After the injection of cRNAs corresponding to the mGluR1 and its mutants, ligands were applied to the oocytes, and
their membrane potentials were measured. The characteristic variation
in the amplitude response was somewhat similar to that in the
intracellular Ca2+ response. The minor differences were
that both ligands elicited membrane currents in Y236A, and quisqualate
caused a small response in D318A.
In our previous structural studies, the LB1 dimer
interface, which mainly consists of helices B and C, is crucial for
setting and selecting the activation state in the ligand binding
region. Therefore, we mutated the five main amino acid residues in the interface and analyzed their properties. The Leu116 and
Ile120 residues are on the B helix, whereas
Leu174, Leu177, and Phe178 are on
the C helix. Single, double (Leu116/Ile120),
and triple (Leu174/Leu177/Phe178)
mutants were made and characterized. For these mutants as well as the
wild type receptor, an immunoreactive band around 144 kDa was detected
(Fig. 6A). The expression
levels of these dimer interface mutants appeared to be lower than that
of the wild type receptor, suggesting some difficulty in the protein
folding or stability. The ligand binding capacities of these mutants,
except for I120A, were not clearly detected, probably due to the low
expression levels and the low binding assay sensitivity (Fig.
6B). Next, functional assays were performed, as shown in
Figs. 7, A and B. The L116A, L174A, L177A, and F178A mutants showed IP enhancement at
magnitudes comparable with that of the wild type control. In contrast,
the IP response in the I120A mutant was obscure. Notably, the
Ca2+ response was completely lost in the I120A mutant (Fig.
7B). The membrane currents induced by the increased
intracellular Ca2+ concentration were measured by an oocyte
expression system. Again, the I120A mutant showed no membrane current
change, despite the maintenance of the current change in other mutants
(data not shown). The cell surface expression of these dimer interface
mutants was probed immunocytochemically, as shown in Fig.
8. The mG1Na-1 monoclonal antibody detected the mutants, including I120A, as well as the wild
type mGluR1 under the nonpermeabilized condition. Thus, the I120A
mutant appears to lose the signaling function, despite the maintenance of its ligand binding capability. We obtained an
~350 nM Kd value of
[3H]quisqualate for the I120A mutant, which
ruled out the possibility that the elimination of the
signaling capacity in the I120A mutant was due to a loss of
ligand affinity.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 6.
Immunoblotting analysis and ligand binding of
LB1 dimer interface mutants. A, cells were transfected
with vector alone (mock), pcDNAmGluR1 (WT),
and mutant pcDNAmGluR1s. Membrane fractions (40 µg) of HEK 293 cells transfected with each plasmid were subjected to SDS-PAGE.
Proteins were transferred onto the nitrocellulose membranes and were
probed with the AB1551 antibody as described under "Experimental
Procedures." B, membrane fractions (100 µg) of HEK 293 cells transfected with plasmids for the wild-type and mutant mGluR1s
were incubated in binding buffer (40 mM HEPES, pH 7.5, containing 2.5 mM CaCl2) with 20 nM
[3H]quisqualate on ice for 1 h. The reaction
mixtures were aspirated onto GF/C filters, and the labeled material
remaining on the filters was counted with a scintillation counter.
Nonspecific binding was measured in the presence of 1 mM
glutamate. Each binding shows the mean ± S.D. of a representative
of three experiments done in triplicate.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Glutamate-induced inositol phosphate
production and Ca2+ response in cells expressing the dimer
interface mutants. A, cells were transfected with
control vector (mock), pcDNAmGluR1 (WT), and
mutant receptors. Transfected cells were labeled for 17 h with
[3H]myo-inositol and were stimulated with 1 mM glutamate or 0.1 mM quisqualate in the
presence of 10 mM LiCl. Inositol phosphates were extracted
and separated by anion exchange chromatography, as described under
"Experimental Procedures." Results are expressed as the ratio of
[3H]IPs accumulated over total radioactivity in the
membrane fraction and represent the means ± S.E. of at least
three independent experiments performed in duplicate or triplicate.
B, cells were transfected with pcDNAmGluR1 and mutant
receptors. Glutamate (100 µM in BSS) was used for a 1-min
challenge. The peak and basal concentrations of intracellular
Ca2+ were determined, as described under "Experimental
Procedures." Results are expressed as values above the basal
concentration. Data are means ± S.E. for 10-17 cells of two
independent experiments.
|
|

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 8.
Cell surface expression of the dimer
interface mutants. HEK 293 cells were transfected with cDNAs
encoding the five single mutants and each of the double (positions 116 and 120) and triple (positions 174, 177, and 178) mutants in mGluR1.
After 24 h, the cells were directly stained with the mG1Na-1
monoclonal antibody under the nonpermeabilized condition.
|
|
 |
DISCUSSION |
Based on the crystal structure of the ligand-binding region of
mGluR1, we performed an amino acid mutagenesis of two groups of amino
acid residues, one series of residues interacting with the ligand,
glutamate, and the other series of residues residing in the dimer
interface. We prepared mutants of the full-length receptor and the
truncated receptor containing only the ligand-binding region. The
relationship between ligand-binding and intracellular signaling in the
mutant receptors was extensively examined along with the wild type receptor.
Among the 14 full-length mutants, the S164A, S186A, R323A, and K409A
mutants were expressed well and showed ligand binding capacities
comparable with that of the wild type. These mutants elicited normal IP
responses and intracellular calcium increments. The S165A and E292A
mutants did not show remarkable binding, but their intracellular
responses to both ligands were clear, indicating that the intracellular
signaling assay is more sensitive than the present ligand binding
assay. Interestingly, the Y74A, R78A, and G293A mutants lost their
responses to glutamate but maintained their responsiveness to
quisqualate. Jensen et al. (16) reported that the potency of
glutamate is much lower than that of quisqualate when Arg78
is mutated to leucine. The T188A, D208A, Y236A, and D318A mutants lost
their responses to both ligands. The side chains of these four
residues, which are conserved among all mGluR subtypes, interact with
the
-amino group of glutamate. The manner of the
-amino group
interaction with the hydroxyl group of Thr188 and the
carboxylate of Asp318 is a hydrogen bond, whereas the
interactions with Asp208 and Tyr236 are a salt
bridge and a cation-
interaction, respectively. The 4-fold increase
of the Kd value of quisqualate in the S186A mutant
(Table II) may be caused by an abrogation of the interaction between
the backbone carbonyl oxygen of Ser186 with the amino
group. As the expressions of the T188A, D208A, and Y236A mutants in the
soluble form were unsuccessful, these residues may also be important
for the folding of the receptor protein. Intriguingly, water molecules
are not involved in the recognition of the
-amino group, implying
the importance of the side chain orientations of the four residues.
This is in contrast to the adaptable recognition of the
-carboxyl
group through the water molecules. The binding mode and the environment
of the agonists, including glutamate, in the binding core of the AMPA
receptor (17) suggest that the interactions with the
-amino group
are conserved among the several agonists, although the interactions with the
-carboxyl group differ between the mGluR1 and AMPA
receptors. Thus, the so-called glutamate binding fold may have evolved
with the preservation of the binding property to the amino group of the ligand.
We used labeled quisqualate as a ligand in our binding assay. The
displacement experiment of labeled binding quisqualate by an excess of
unlabeled glutamate not quisqualate was performed to assess the
specific binding. Quisqualate differs from glutamate by the presence of
3,5-dioxo-1,2,4-oxadiazolidine, as shown in Fig. 1A. In the
crystal, the
-carboxyl group of glutamate interacts with
Tyr74, Arg78, Gly293,
Arg323, and Lys409. Although the crystal
structure complexed with quisqualate has not been solved, the
interactions of quisqualate with these residues may be different, due
to the presence of the ring. Quisqualate may utilize distinct binding
residues, which may cause its higher affinity as compared with that of
glutamate. This may reflect the loss of glutamate signaling and the
preservation of quisqualate signaling in the mutants Y74A, R78A, and
G293A. The backbone amide nitrogen of Gly293 interacts with
the
-carboxyl oxygen of the ligand glutamate through a water
molecule. According to the modeling analysis, the methyl moiety of
alanine, which replaces Gly293, collides with neighboring
residues, Arg323 and Gly319, distorting the
main chain and hindering the glutamate binding. The lower
Kd values of quisqualate in the R323A and K409A mutants (Table II) may also reflect the idea that the removal of their
side chains causes stronger quisqualate binding and thus produces
higher IC50 values of glutamate than those in the wild type.
Based on our crystal structure of mGluR1 and molecular modeling,
mutagenesis experiments have been performed on the Group II and III
mGluRs. For mGluR4, mutations of the amino acid residues possibly
corresponding to Arg78, Thr188,
Gly293, and Lys409 of mGluR1 abolished the
ligand binding (L-AP4) (18). For the mGluR2 mutants,
mutations of the residues corresponding to Tyr74,
Tyr236, and Asp318 abolished the ligand binding
(LY354740) (19). Those data are consistent with the present data. In
addition, alanine mutations of Ser159 in mGluR4 and
Ser145 in mGluR2, which correspond to Ser165 in
mGluR1, abolished the ligand binding and functional responses. The
S165A mutation did not completely eliminate the glutamate- and
quisqualate-induced signaling in the present study, which is consistent
with the report of O'Hara et al. (20) that the mutant
retained the functional activity, despite its reduced affinity. Thus,
Ser165 in mGluR1 does not appear to be as critical for
functional expression as the four residues mentioned above. However,
notably, the basal activity measured by the intracellular IP level was
lost in the S165A mutant. The inefficient expression of this mutant in
the truncated version, in addition to the reported key roles of the corresponding residues in related receptors such as the calcium-sensing receptor (21) and GABA-B receptor (22), suggests us that the residue is
important for the ligand binding domain fold. Since the main chain
nitrogen of Ser165 is hydrogen-bonded with the
-carboxyl
oxygen of glutamate, a similar main chain interaction may remain after
the alanine replacement. Thus, the corresponding residues in the other
subtypes may interact with glutamate in somewhat different manners.
In previous structural studies, we showed that mGluR1 is a homodimer
containing the dimer interface, which consists of the B and C helices
in each of the two LB1 domains. The construction of the dimer interface
defines the two states of the ligand binding domain, the activated
state and the resting state, which are supposed to be in dynamic
equilibrium. The recent structural analysis of the antagonist binding
crystal reinforces our proposal. Thus, to clarify the significance of
the LB1 interface in the signaling into the intracellular environment,
the dimer interface was analyzed. The ligand binding capacities of
these mutants were markedly decreased as compared with that of the wild
type. Out of the five single amino acid mutants in the hydrophobic
interface, four point mutants responded to the glutamate stimulus;
however, I120A lost the intracellular responses. Namely, the increments
of both the IP concentration and the intracellular Ca2+
were abolished. Although our ligand binding assay is not as sensitive as the signaling assay, the four other point mutants showed clear intracellular responses, despite their lower levels of ligand binding
than that of I120A mutant. Notably, the rotation axis of the dimer
interface passes in the close vicinity of Ile120. The
alanine replacement of Ile120 may hinder the ability of the
dimer interface to set the activated state, even upon ligand binding,
although the small reduction of the ligand affinity might contribute to
the signal loss to some extent. The structural determination of this
mutant is intriguing. These results demonstrate that the LB1 interface
performs the critical role of a gateway for determining the initial
activation status of the receptor molecule and also provides an
indispensable structural backbone for the dimer formation. Thus, the
dimer interface may provide a new target for the modification of signal
transmission in mGluRs. The dominant-negative character of the mutant
is under investigation.
In conclusion, we performed a systematic site-directed mutagenesis
study of mGluR1, focusing on the ligand binding site and the dimer
interface, which were revealed by the crystal structure. The mutant
analyses of the ligand binding site disclosed the critical residues
that directly bind to the
-amino group of glutamate. The alanine
replacement of isoleucine 120, located close to the relocation axis of
the dimer interface, abolished the Ca2+ response,
indicating the crucial role of the dimer interface relocation for the
initial activation of mGluR1.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Dai Watanabe and Dr. Jun Kitano
for assistance with Ca2+ response measurements and the
immunocytochemistry, respectively. We also thank Dr. Jun Otomo and Dr.
Tomoko Takeshita for the current measurements by oocyte expression. We
are grateful to Dr. Daisuke Tsuchiya and Dr. Kosuke Morikawa for Fig. 1
and critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by a research grant endorsed by the
New Energy Technology Development Organization.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.:
81-6-6872-8214; Fax: 81-6-6872-8219; E-mail: jingami@beri.or.jp.
Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M210278200
 |
ABBREVIATIONS |
The abbreviations used are:
mGluR, metabotropic
glutamate receptor;
m1-LBR, mGluR1-LBR;
LBR, ligand-binding region;
IP, inositol phosphate;
HEK, human embryonic kidney;
BSS, balanced salt
solution;
PBS, phosphate-buffered saline;
cRNA, complementary
RNA.
 |
REFERENCES |
1.
|
Nakanishi, S.,
and Masu, M.
(1994)
Annu. Rev. Biophys. Biomol. Struct.
23,
319-348[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Hollmann, M.,
and Heinemann, S.
(1994)
Annu. Rev. Neurosci.
17,
31-108[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
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]
|
4.
|
Okamoto, T.,
Sekiyama, N.,
Otsu, M.,
Shimada, Y.,
Sato, A.,
Nakanishi, S.,
and Jingami, H.
(1998)
J. Biol. Chem.
273,
13089-13096[Abstract/Free Full Text]
|
5.
|
Tsuji, Y.,
Shimada, Y.,
Takeshita, T.,
Kajimura, N.,
Nomura, S.,
Sekiyama, N.,
Otomo, J.,
Usukura, J.,
Nakanishi, S.,
and Jingami, H.
(2000)
J. Biol. Chem.
275,
28144-28151[Abstract/Free Full Text]
|
6.
|
Tsuchiya, D.,
Kunishima, N.,
Kamiya, N.,
Jingami, H.,
and Morikawa, K.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
2660-2665[Abstract/Free Full Text]
|
7.
|
Bouvier, M.
(2001)
Nat. Rev. Neurosci.
2,
274-286[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Böckaert, J.,
and Pin, J.-P.
(1999)
EMBO J.
18,
1723-1729[Abstract/Free Full Text]
|
9.
|
Neki, A.,
Ohishi, H.,
Kaneko, T.,
Shigemoto, R.,
Nakanishi, S.,
and Mizuno, N.
(1996)
Neurosci. Lett.
202,
197-200[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Miyazaki, J.,
Nakanishi, S.,
and Jingami, H.
(1999)
Biochem. J.
340,
687-692[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Paris, S.,
and Pouyssegur, J.
(1986)
EMBO J.
5,
55-60[Abstract]
|
12.
|
Masu, M.,
Tanabe, Y.,
Tsuchida, K.,
Shigemoto, R.,
and Nakanishi, S.
(1991)
Nature
349,
760-765[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Kitano, J.,
Kimura, K.,
Yamazaki, Y.,
Soda, T.,
Shigemoto, R.,
Nakajima, Y.,
and Nakanishi, S.
(2002)
J. Neurosci.
22,
1280-1289[Abstract/Free Full Text]
|
14.
|
Dev, K. K.,
Nishimune, A.,
Henley, J. M.,
and Nakanishi, S.
(1999)
Neuropharmacology
38,
635-644[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Prézeau, L.,
Gomeza, J.,
Ahern, S.,
Mary, S.,
Galvez, T.,
Böckaert, J.,
and Pin, J.-P.
(1996)
Mol. Pharmacol.
49,
422-429[Abstract]
|
16.
|
Jensen, A. A.,
Sheppard, P. O.,
O'Hara, P. J.,
Krogsgaard-Larsen, P.,
and Bräuner-Osborne, H.
(2000)
Eur. J. Pharmacol.
397,
247-253[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Armstrong, N.,
and Gouaux, E.
(2000)
Neuron
28,
165-181[Medline]
[Order article via Infotrieve]
|
18.
|
Rosemond, E.,
Peltekova, V.,
Naples, M.,
Thøgersen, H.,
and Hampson, D. R.
(2002)
J. Biol. Chem.
277,
7333-7340[Abstract/Free Full Text]
|
19.
|
Malherbe, P.,
Knoflach, F.,
Broger, C.,
Ohresser, S.,
Kraftzeisen, C.,
Adam, G.,
Stadler, H.,
Kemp, J. A.,
and Mutel, V.
(2001)
Mol. Pharmacol.
60,
944-954[Abstract/Free Full Text]
|
20.
|
O'Hara, P. J.,
Sheppard, P. O.,
Thøgersen, 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]
|
21.
|
Bräuner-Osborne, H.,
Jensen, A. A.,
Sheppard, P. O.,
O'Hara, P. J.,
and Krogsgaard-Larsen, P.
(1999)
J. Biol. Chem.
274,
18382-18386[Abstract/Free Full Text]
|
22.
|
Galvez, T.,
Parmentier, M.-L.,
Jolu, C.,
Malitschek, B.,
Kaupmann, K.,
Kuhn, R.,
Bittiger, H.,
Froestl, W.,
Bettler, B.,
and Pin, J.-P.
(1999)
J. Biol. Chem.
274,
13362-13369[Abstract/Free Full Text]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.