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INTRODUCTION |
Glutamate is the major excitatory neurotransmitter in the central
nervous system (1), and its function through ionotropic and
metabotropic (mGlu)1
glutamate receptors can be modulated by other
neurotransmitters/neuromodulators (2). Eight members of the mGlu
receptor family have been identified and categorized into three
subgroups on the basis of their sequence homology, agonist selectivity,
and signal transduction pathway. Group I contains mGlu1 and
mGlu5 subtypes, which are coupled to phospholipase C in
transfected cells, and have quisqualic acid as their most potent
agonist. Five splice variants of mGlu1 receptor have been described,
mGlu1
, mGlu1
, mGlu1c,
mGlu1d, and mGlu1e receptors (3, 4), all of
them differing in the length of their C-terminal tail. The functional
significance of the different splice variants has not yet been fully
explored. It has been suggested that the C-terminal tail, which is
intracellular, might play a role in the subcellular targeting of the
receptor (5). Recently, we have reported that the C terminus of
mGlu1
receptor interacts with tubulin (6) and that it
can regulate the cell surface expression of the receptor (7) and its
plasma membrane anchoring (8, 9).
Adenosine is an important neuromodulator implicated in a variety of
brain activities, particularly those related to sleep and
ischemic-hypoxic episodes (10). This ubiquitous nucleoside exerts its
actions via specific receptors, four of which (A1, A2A, A2B, and A3)
have been cloned (11). The A1R is functionally coupled to members of
the pertussis toxin-sensitive family of G proteins (Gi1,
Gi2, Gi3, and Go), and its
activation regulates several membrane and intracellular proteins such
as adenylate cyclase, Ca2+ channels, K+
channels, and phospholipase C (11). Of the multiple neurophysiological actions of adenosine, inhibition of glutamate neurotransmission has
been observed in several brain regions (12) and is probably a result of
the inhibition of presynaptic calcium influx (13). Apart from this
inhibitory effect, there is some evidence documenting functional
interactions between adenosine and glutamate receptors in the central
nervous system. Of particular interest are reports of group I mGlu
receptors signaling being enhanced by group II mGlu receptors in
hippocampal and cerebrocortical slices (14-16) and by adenosine A1
receptors in cultured hippocampal type 1 astrocytes (17). Very
recently, Toms and Roberts (18) have described that type 2 astrocytes
contain group I mGlu receptors coupled to [Ca2+]i
signaling and that co-activation of adenosine A1 receptors enhances
group I mGlu-evoked [Ca2+]i responses in these
cells via Gi/o G protein-mediated mechanism. Despite these
observations, no clear molecular mechanism of this interaction between
glutamate and adenosine receptors has been provided yet.
Here we report a molecular interaction between metabotropic glutamate
receptor type 1
and the adenosine A1 receptor, two members of
different GPCR families. This interaction suggests that both receptors
may form part of a signaling complex in vivo that could play
a critical role in fine-tuning neurotransmission at
glutamatergic synapses.
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EXPERIMENTAL PROCEDURES |
Cell Culture, Generation of mGlu1 Truncated Mutant,
and Transfection--
Human embryonic kidney cells, HEK-293, were
grown as described (9). Rat mGlu1 receptor was truncated
after amino acid position 885 (see Fig. 4). A stop codon was introduced
into the coding sequence of the FLAG epitope containing
mGlu1
receptor cDNA by polymerase chain
reaction (8). Forward primer was MGR1-F7 (5'-GGCCCTGGGGTGCATGTTTACTC-3', position 2833-2855 of the
mGlu1 receptor cDNA (GenBankTM accession
number X57569)), and reverse primer was MGR1-R22 (5'-CGTCTCGAGTTTAATTCCCTGCCCCGGGCTTCTTT-3',
position 3026 to 2992, but also encoded XhoI
restriction site (underlined) and a stop codon (shown in bold)). The
BspEI-XhoI fragment of the amplified cDNA
sequence, which contained a stop codon, was used to substitute
corresponding fragment of rat mGlu1
receptor cDNA in
pcDNA3 vector thus resulting in mGlu1-M7 construct (see Fig. 4). The truncation introduced was confirmed by DNA sequencing.
For the transient expression of mGlu1
,
mGlu1
, mGlu1-M7, and/or A1 receptors, cells
were transiently transfected with 10 µg of cDNA encoding the rat
metabotropic glutamate receptors and/or rat adenosine A1 receptor
(ratio 1:1; pcDNA containing LacZ reporter was used to equilibrate
the amount of total DNA) by calcium phosphate precipitation (19). The
cells were used for experimentation at either 24 or 48 h after
transfection. Cells were grown in glutamate-free medium (ICN,
Basingstoke, UK) in the absence of both glutamine and glutamic acid for
3 h before their use.
Primary Cultures and N-Methyl-D-Aspartate
(NMDA)-induced Neurotoxicity--
Cortical hemispheres from E16 rat
embryos were dissected, and primary cultures of rat cortical neurons
were prepared as described previously (9) and used after 14-21 days
in vitro. To determine the NMDA-mediated neurotoxicity, the
culture-conditioned medium was collected, and the cortical neurons were
washed once with serum-free B27-supplemented Neurobasal medium (Life
Technologies, Inc.) containing 50 µg/ml gentamicin (Sigma) (BNG
medium) and preincubated with or without 30 µM NMDA
(Tocris, Bristol, UK) for 10 min at 37 °C, as described previously
(20). Quisqualic acid (100 µM) (Tocris, Bristol, UK)
and/or (R)-phenylisopropyladenosine (R-PIA) (100 nM) (Sigma) were transiently applied for 1 min during the 5 min prior to the addition of NMDA (20) and then added together with the
NMDA. Neurons, after being washed with the BNG medium, were returned to
the culture-conditioned medium and further incubated at 37 °C for
24 h. After the NMDA pulse (24 h), neurons were stained with
propidium iodide (80 µg/ml) for 5 min and examined immediately with a
standard epi-illumination fluorescence microscope. Neuronal injury was
determined by the ability of propidium iodide (Sigma) to penetrate and
interact with DNA in damaged neurons, yielding red fluorescence.
Immunostaining--
For immunohistochemistry, rat cerebellum was
embedded in OCT and frozen in liquid nitrogen-cooled isopentane. Eight
micrometer sections were cut on a cryostat cooled to
18 °C.
Sections were collected onto SuperFrost Plus (BDH, Darmstadt, Germany)
slides, air-dried, and stored at
70 °C. Sections were blocked for
30 min in 10% donkey serum in Tris-buffered saline (TBS) (150 mM NaCl, 50 mM Tris-HCl, pH 7.5). Slides were
incubated with affinity-purified anti-mGlu1
receptor
(F2-Ab, 2-4 µg/ml) (21) and affinity-purified anti-A1R (PC21, 5-10
µg/ml) (22) for 1 h at room temperature and then washed twice
for 5 min in TBS. Fluorescein isothiocyanate-conjugated donkey
anti-rabbit IgG (Jackson ImmunoReserach Laboratories Inc., West Grove,
PA) was applied in TBS at a dilution of 1:100. Section were rinsed and
mounted with Vectashield immunofluorescence medium (Vector
Laboratories, Orton Southgate, UK).
For immunocytochemistry, HEK-293 cells transiently transfected (see
above) and primary cultures of rat cortical neurons were fixed in 4%
paraformaldehyde for 15 min and washed with phosphate-buffered saline
containing 20 mM glycine (buffer A) to quench the aldehyde groups. Where indicated, cells were permeabilized with buffer A
containing 0.2% Triton X-100 for 5 min. Cells were labeled for 1 h at room temperature with the indicated primary antibody, washed, and
stained with fluorescein-conjugated donkey anti-rabbit IgG antibody
(1/100) and Texas Red-conjugated donkey anti-mouse IgG antibody
(1/100). Coverslips were rinsed for 30 min in buffer B and mounted with
Vectashield immunofluorescence. Confocal microscope observations were
made with a Leica TCS NT (Leica Lasertechnik GmbH, Heidelberg, Germany)
confocal scanning laser microscope adapted to an inverted Leitz DMIRBE microscope.
Immunoprecipitation--
Rat cerebellum synaptosomes (6) or
transfected HEK cells were solubilized in ice-cold lysis buffer
(phosphate-buffered saline, pH 7.4, containing 1% (v/v) Nonidet P-40,
0.5% (w/v) sodium deoxycholic acid, and 0.1% (w/v) SDS) for 1 h
on ice. The solubilized preparation was then centrifuged at 80,000 × g for 90 min. The supernatant (1 mg of protein/ml) was
processed for immunoprecipitation as described before (6) using the
anti-A1R antibody (PC21-Ab) or anti-FLAG monoclonal antibody (Sigma,
Clone M2; 10 µg/ml). The immune complexes were dissociated in
SDS-PAGE sample buffer by heating to 100 °C for 5 min and resolved
by SDS-polyacrylamide gel electrophoresis in 7% gels (23). Proteins
were transferred to polyvinylidene difluoride membranes (Immobilon-P,
Millipore, Watford, UK) using a semi-dry transfer system and
immunoblotted using the primary antibodies indicated in the figure
legends and horseradish-peroxidase-conjugated swine anti-rabbit IgG
(Dako, Ely, UK) as a secondary antibody. The immunoreactive bands were developed with the enhanced chemiluminescence detection kit (Pierce), as described previously (6).
Ligand Binding Experiments--
Membrane suspensions from
transiently transfected HEK cells were obtained according to
Casadó et al. (24). For competition experiments to
determine the KD values, 30 nM
[3H]quisqualic acid (Amersham Pharmacia Biotech) or 20 nM [3H]R-PIA (Amersham Pharmacia Biotech)
binding to membrane suspensions of HEK cells transiently expressing
mGlu1
FLAG receptor or A1R (0.5 mg protein/ml) were
carried out at 25 °C in 50 mM Tris-HCl buffer, pH 7.4, in the absence or presence of increasing amounts of quisqualic acid or
R-PIA, as described previously (24). After 2 h of radioligand
incubation, free and membrane-bound radioligand were separated by rapid
filtration in a Brandel cell harvester through Whatman GF/C filters.
Filters were transferred to scintillation vials containing 10 ml of
Formula 989 (PerkinElmer Life Sciences). Radioactivity was counted
using a Packard 1600 TRI-CARB scintillation counter with 50% of
efficiency. Competition data were fitted using a non-linear regression
program as described previously (24, 25).
To determine the density of expressed receptors in HEK cells
transiently transfected with A1R, mGlu1
-FLAG or A1R plus mGlu1
FLAG, the binding of 70 nM
[3H]quisqualic and 15 nM
[3H]R-PIA was performed as described above, and the
Bmax was deduced taking into account the
KD values as described previously (24).
Calcium Determination--
Transiently transfected HEK cells
(106 cell/ml) were loaded with 5 µM Fura-2/AM
for 30 min at 37 °C. Cells were washed and subsequently incubated in
HBSS containing 0.2 units/ml adenosine deaminase. Calcium peak
induction was achieved by the addition of R-PIA or quisqualic acid.
Intracellular calcium was determined at 37 °C in a dual-wavelength
Shimadzu RF-5000 spectrofluorophotometer (Shimadzu Europe, Duisberg,
Germany) by using the excitation wavelength ratio of 334/366 nm with
emission cut-off at 500 nm. Free calcium concentration was calculated
as described previously (26).
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RESULTS |
Interaction of mGlu1
and A1 Receptors in Rat
Cerebellum--
Immunohistochemical studies showed that
mGlu1
receptors are present in cerebellum, and its expression
is mainly restricted to the cell body and the dendritic tree of
Purkinje cells and basket cells located in the molecular layer (Fig.
1a). This is in agreement with
the previously described location of these receptors in an annulus,
which surrounds the post-synaptic density (27). The A1R had a more
ubiquitous distribution in cerebellum being expressed in Purkinje cells
and basket cells, as mGlu1
receptor, and also in granule
cells (Fig. 1a). This fits with the localization of
adenosine A1 receptors both presynaptically and postsynaptically (28).
In addition, metabotropic glutamate receptor type 1
and adenosine A1
receptors showed a similar distribution in human cerebral cortex being
expressed in large pyramidal cells located at layers V and II/III (data
not shown).

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Fig. 1.
Interaction of
mGlu1 and A1 receptors in rat
cerebellum. a, 8-µm cryosections from rat cerebellum
were stained with anti-mGlu1 receptor antibody (F2-Ab; 4 µg/ml) and anti-A1R antibody (PC21-Ab; 10 µg/ml). F2-Ab
immunoreactivity is confined mainly to the molecular layer
(ML) and it stains Purkinje cells and basket cells. PC21-Ab
stains Purkinje cells and basket cells in the molecular layer as well
as granule cells in the granular layer (GL). b,
co-immunoprecipitation of mGlu1 and A1 receptors from
extracts of rat cerebellum synaptosomes. Extracts and
immunoprecipitates (IP) (see "Experimental Procedures")
were analyzed by SDS-PAGE and immunoblotted (IB) using anti-
mGlu1 receptor antibodies (F2-Ab; 5 µg/ml) or anti-A1R
antibodies (PC11-Ab; 10 µg/ml). Immunoreactive bands were detected
with swine anti-rabbit (1:5000) secondary antibody conjugated to
horseradish peroxidase followed by chemiluminescence detection. IgG
indicates the position of the immunoglobulins used in the
immunoprecipitation.
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The in vivo co-distribution of mGlu1
and A1
receptors in some cerebellar neurons (Fig. 1a) suggests a
potential interaction between both receptors at precise brain areas.
The existence of mGlu1
/A1 receptors' heteromers was
assayed by co-immunoprecipitation experiments using a soluble extract
from rat cerebellum synaptosomes that had been shown by Western
blotting to contain both adenosine A1 (Fig. 1b, lane 1) and
mGlu1
receptors (Fig. 1b, lane 2). When this
soluble extract was immunoblotted using a specific antibody against A1
receptor (PC21-Ab), two bands of around 39 and 74 kDa of molecular
size were shown (Fig. 1b, lane 3); these bands, which are glycosylated proteins (29), have been demonstrated previously to
correspond to the adenosine A1 receptor monomer and dimer, respectively
(22). On the other hand, a specific antibody against the
mGlu1
receptor (F2-Ab) immunoblotted in the cerebellum synaptosomes extract a band with apparent molecular size of 150 kDa
that corresponds to the mGlu1
receptor (Fig. 1b,
lane 4), which is a glycosylated protein as it has been described
previously (21, 30). From this extract, the antibody against A1R
(PC11-Ab) immunoprecipitated a band of 150 kDa that was detected by the F2-Ab (Fig. 1b). This band was also immunoprecipitated using
a different antibody against mGlu1 receptor (F1-Ab) but was
not present in immunoprecipitates generated with an irrelevant antibody (Fig. 1b, lane 5). It should be noted that the efficacy of
immunoprecipitation of mGlu1
receptor by the
anti-adenosine receptor antibody was much less than when an
anti-mGlu1 receptor antibody was used. Overall, these
results indicate that there are zones in which mGlu1
and
A1 receptors do not co-distribute and that there are zones in which the
two receptors co-distribute and form aggregates (heteromers).
Interaction of mGlu1
and A1 Receptors in Transiently
Transfected HEK-293 Cells--
The close association of
mGlu1
and A1 receptors was subsequently studied in
co-transfected HEK-293 cells by co-immunoprecipitation and double
immunolabeling experiments. By confocal microscopy analysis of HEK-293
cells transiently transfected with the cDNAs for
mGlu1
-FLAG and A1 receptors, a marked overlap in the distribution of the two proteins was found (Fig.
2). As deduced from horizontal optical
sections of permeabilized (+Triton) and nonpermeabilized
(
Triton) cells, co-localization was not restricted to the
plasma membrane but extended to intracellular compartments. Interestingly, when the double immunolabeling experiment was performed on HEK-293 cells transiently transfected with the cDNAs for
mGlu1
-FLAG, the C-terminal splice variant of
mGlu1 receptor (Fig. 4a), and A1 receptors, no
co-localization between both proteins was observed at the plasma
membrane level (Fig. 2), suggesting a specificity for the
co-localization between mGlu1
-FLAG and A1 receptors.

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Fig. 2.
Immunofluorescence localization of
mGlu1 ,
mGlu1 , and A1 receptors in HEK-293
cells. Cells were transiently transfected with cDNAs encoding
for mGlu1 FLAG or mGlu1 FLAG and A1R.
After 48 h cells were washed, fixed ( Triton), and/or
permeabilized (+Triton) and processed for immunostaining
with anti-FLAG monoclonal antibody (Sigma, Clone M2; 10 µg/ml) and
anti-A1R affinity-purified antibody (PC21-Ab, 5 µg/ml). The bound
primary antibodies were detected using either fluorescein-conjugated
donkey anti-mouse IgG antibody (1/50) or Texas Red-conjugated donkey
anti-rabbit (1/50). Cells were analyzed by double immunofluorescence
with a confocal microscopy. Superimposition of images reveals
mGlu1 (green) and A1 (red)
receptors co-localization in yellow. The images show a
single horizontal section of representative cells. Scale
bar, 10 µm.
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From co-transfected HEK cell extracts, the antibody against A1R
(PC11-Ab) co-immunoprecipitated a band of 150 kDa, which corresponds to
the mGlu1
-FLAG receptor (Fig.
3, lane 5). This band did not
appear in immunoprecipitates from cells transfected with the cDNA
for either A1 receptors (Fig. 3, lane 4) or
mGlu1
(Fig. 3, lane 6) or when an irrelevant
antibody was used (data not shown). Conversely, when we
immunoprecipitate with the FLAG antibody to pull down
mGlu1
-FLAG receptor in the transiently co-transfected HEK-293 cells, and subsequently the immunoprecipitates were analyzed by
SDS-PAGE and immunoblotted using anti-A1R antibodies, a band which
corresponds to the A1R was observed (Fig. 3, lane 11).
Interestingly, no immunoprecipitation of mGlu1
receptor,
the C-terminal splice variant of mGlu1 receptor (Fig.
4a), was obtained from co-transfected cells using the antibody against A1R (PC11-Ab) (Fig.
4b, lane 7). Additionally, the construct
mGlu1-M7, a deleted mutant close to the splice variant site
of the mGlu1 receptor (Fig. 4a), also did not
interact with A1R (Fig. 4b, lane 8). Overall, these results
suggest that the C-terminal tail of the mGlu1
receptor
(Fig. 4a) is implicated in the interaction of both
receptors.

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Fig. 3.
Interaction of
mGlu1 and A1 receptors in
transiently transfected HEK-293 cells. Cells transiently
expressing A1R alone (lanes 1 and 7), A1R plus
mGlu1 FLAG (lanes 2 and 8), or
mGlu1 -FLAG alone (lanes 3 and 9)
were washed and solubilized in ice-cold lysis buffer and processed for
immunoprecipitation using anti-A1R antibodies (PC11-Ab; 10 µg/ml;
lanes 4-6) and anti-FLAG monoclonal antibody (Sigma, Clone
M2; 10 µg/ml; lanes 10-12). Solubilized membranes
(Crude, lanes 1-3 and
7-9) and immunoprecipitates (IP, lanes
4-6 and 10-12) were analyzed by SDS-PAGE and
immunoblotted (IB) using anti-mGlu1 receptor
antibodies (F2-Ab; 5 µg/ml) or anti-A1R antibodies (PC21-Ab; 10 µg/ml). Immunoreactive bands were detected as before. IgG indicates
the position of the immunoglobulins used in the
immunoprecipitation.
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Fig. 4.
Specificity of the interaction between
mGlu1 and A1 receptors in
transiently transfected HEK-293 cells. a, schematic
representation of the primary structure of rat mGlu1 and
mGlu1 receptors. The gray arrowhead indicates
the position of the stop codon introduced to generate the deleted
mutant mGlu1-M7. b, HEK-293 cells transiently
expressing A1R alone (lane 1), A1R plus
mGlu1 FLAG (lane 2), A1R plus
mGlu1 -FLAG (lane 3), and A1R plus
mGlu1-M7-FLAG (lane 4) were processed for
immunoprecipitation using anti-A1R antibodies (PC11-Ab; 10 µg/ml).
Solubilized membranes (Crude, lanes 1-4) and
immunoprecipitates (IP, lanes 5-8) were analyzed
by SDS-PAGE and immunoblotted (IB) using
anti-mGlu1 antibodies (F1-Ab; 5 µg/ml). Immunoreactive
bands were detected as before. IgG indicates the position of the
immunoglobulins used in the immunoprecipitation.
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To test the functional significance of an mGlu1
/A1
receptors interaction, measurements of calcium mobilization in
co-transfected HEK-293 cells were performed. In HEK cells transiently
expressing A1R or A1R plus mGlu1
-FLAG R-PIA mobilized
intracellular calcium in a concentration-dependent manner,
with an EC50 value of 46.8 ± 4.4 nM for
the single expressing A1R cells or 45.7 ± 5.1 nM for
the doubly A1R plus mGlu1
-FLAG expressing cells. On the
other hand, in these cells quisqualic acid also mobilized intracellular
calcium in a concentration-dependent manner, with an
EC50 value of 4.1 ± 1.1 µM for the
single expressing mGlu1
-FLAG cells or 5.6 ± 1.1 µM for the doubly A1R plus mGlu1
-FLAG expressing cells. The densities of the transiently expressed receptors were controlled by means of ligand binding experiments performed in
these cells (see "Experimental Procedures"). Cells transfected with
the cDNA for A1R alone express 3.4 ± 0.4 pmol of A1R/mg
protein as detected by [3H]R-PIA binding with a
KD of 27 ± 3 nM. In these cells the A1R agonist R-PIA leads to a calcium peak (Fig.
5). On the other hand, cells transfected
only with the cDNA for mGlu1
receptor express
3.3 ± 0.9 pmol of mGlu1
receptor/mg protein as
detected by [3H]quisqualic binding with a
KD of 177 ± 46 nM, and a weak
calcium peak was detected when treated with the agonist for the
metabotropic glutamate receptor, quisqualic acid (Fig. 5). Interestingly, in HEK-293 cells co-transfected with both receptors (3.3 ± 0.2 pmol of A1R/mg of protein and 2.8 ± 1.5 pmol of
mGlu1
/mg of protein), preincubation with quisqualic acid
markedly potentiated the calcium peak obtained in response to A1R
activation (140 ± 10%; n = 3). Conversely,
preincubation of co-transfected cells with the agonist for A1R led to a
marked enhancement of the signal provided by quisqualic acid (180 ± 20%; n = 3) (Fig. 5). Quisqualic acid or R-PIA
failed to bind or to provide any signal in nontransfected HEK-293
cells. These results clearly show a heterologous sensitization or
synergistic effect upon mGlu1
and A1 receptors
activation.

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Fig. 5.
Ca2+ mobilization in HEK-293
transiently expressing mGlu1 and
A1 receptors. Intracellular Ca2+ concentrations were
measured in suspended cells loaded with FURA-2/AM after stimulation
with the mGlu1 receptor agonist quisqualic acid (100 µM) (Quis) or A1R agonist
N6-(R)-phenylisopropyladenosine (500 nM) (R-PIA).
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Interaction of mGlu1
and A1 Receptors in Primary Rat
Cortical Neurons--
To assess the physiological relevance of the
mGlu1
/A1 receptors interaction, we analyzed the
distribution of both receptors in primary rat cortical neurons. Both
receptors showed a similar punctate distribution throughout the
proximal and distal dendrites, and the degree of co-localization at
these locations was very high (Fig. 6,
a-c). In fact, some of the mGlu1
receptor
or A1R-containing puncta co-distributed with the synaptic marker protein synaptophysin (Fig. 6, d-i), suggesting that they
could be localized to synapses. Thus, synapses are one of the specific cellular sites where mGlu1
and A1 receptors
interact.

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Fig. 6.
Co-localization of
mGluR1 and A1R in primary cultures of rat
cortical neurons. Neurons (days in vitro 14-21) were
fixed, permeabilized, and processed for immunostaining using
rhodamine-conjugated anti-A1R antibody (PC21-Ab; 15 µg/ml) and
fluorescein-conjugated anti-mGlu1 receptor antibody
(F2-Ab; 15 µg/ml) (a-c), anti-A1R antibody (PC21-Ab, 5 µg/ml) and anti-synaptophysin monoclonal antibody (1/20)
(d-f), or anti-mGlu1 receptor antibody
(F2-Ab; 5 µg/ml) and anti-synaptophysin monoclonal antibody (1/20)
(g-i). Primary antibodies in d-i were detected
using fluorescein-conjugated donkey anti-mouse IgG antibody (1/50) or
Texas Red-conjugated donkey anti-rabbit (1/50)(d-i). Cells
were analyzed by double immunofluorescence with a confocal microscopy.
Images show A1R receptor (a) in red
mGlu1 receptor (b) in green, A1R
(d) and mGlu1 (g) receptors in
red, and synaptophysin (h) in green.
Superimposition of images reveals co-localization in yellow
(c, f, and i). Scale bar, 10 µm.
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Glutamate/adenosine receptors interaction may be important for
modulating the role of mGlu1
receptor in
neurodegeneration/neuroprotection, an issue that is still
controversial. When examining this role a number of factors, including
the heteromeric composition of NMDA receptors, the time of exposure to
drugs or to ambient glutamate, and the function of astrocytes clearing
extracellular glutamate and producing neurotoxic or neuroprotective
factors must be taken into account (20). On the other hand, glutamate
could also modulate the well known function of adenosine as
neuroprotective factor (12). It is thus likely that the interaction of
mGlu1
/A1 receptors could be beneficial in situations of
enhanced neuronal activity, in which potentiation of postsynaptic
adenosine A1 receptor limits evoked depolarization and results in
decreased activation of voltage-dependent Ca2+
channels and NMDA receptor ion channels, through which Ca2+
enters cell bodies (30). We have therefore examined the effect of
activating of both mGlu1 and A1 adenosine receptors on
NMDA-mediated neurotoxicity in primary neuronal cultures. In agreement
with previously described data (20), submaximal concentrations of NMDA
induced neuronal death, which was enhanced by the presence of
quisqualic acid during the NMDA treatment (Fig.
7). In contrast, when the adenosine A1
receptor agonist, R-PIA, was present during the NMDA treatment, the
induced neurotoxicity was reduced by nearly 50%. When added
simultaneously, R-PIA reduced the enhancement of the neurotoxicity
induced by quisqualic acid (Fig. 7). On the other hand, pre-exposure of
neurones to R-PIA or to quisqualic acid also reduced the NMDA-induced
neurotoxicity. This reduction was more marked if both quisqualic acid
and R-PIA were present during the pre-exposure, showing that the
simultaneous activation of both receptors appears to increase the
protection of the neurones against the NMDA treatment compared with the
effect evoked by either quisqualate or R-PIA applied separately. These
results show the relevance of the interaction of
mGlu1
/A1 receptors and support the concept of
specificity and complexity of this interaction, being the
spatiotemporal segregation profile of adenosine/glutamate during
synaptic activity of special importance to achieve a neuroprotective or
a neurotoxic effect.

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Fig. 7.
Excitotoxicity in rat cortical neurons.
Cortical neurons were exposed for 10 min to 30 µM of
NMDA. Quisqualic acid (Quis, 100 µM) and/or
R-PIA (100 nM) were transiently applied (for 1 min) 5 min
prior to the addition of NMDA (Pre-exposure) and/or added
together with NMDA for 10 min (Treatment). Quisqualic acid
treatments were always made in the presence of 50 µM
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, Sigma) to block
non-NMDA receptors. Death neurons were computed by assessing the
propidium iodide staining in photomicrographs of 10 representative
fields from each monolayer of treated neurons and expressed as percent
of NMDA toxicity. Asterisks denote differences from the
control (*, p < 0.05; **, p < 0.01, two-tail t test).
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DISCUSSION |
Here we describe a novel interaction between two unrelated G
protein-coupled receptors (GPCR), namely the metabotropic glutamate type 1
and the adenosine A1 receptors. Although the cooperativity of
the agonist binding to GPCR suggested the possibility of
oligomerization of G proteins and their associated receptors (31), the
existence and the precise function of GPCR homo- or heteromerization
has not been fully elucidated.
Metabotropic glutamate type 1
and adenosine A1 receptors show a high
degree of co-localization in co-transfected cells and in more
physiological systems as neurons from rat cerebellum and from rat
cortex. Both receptors co-immunoprecipitate from co-transfected cells
and from rat cerebellum synaptosomes, suggesting that
mGlu1
and A1 receptors interact and that this
interaction is physiologically relevant. From a functional point of
view, heteromerization often results in facilitatory responses or
synergistic effects. Thus, GABABR1 and GABABR2 receptors need to be
co-expressed and assembled into heteromers to reach the cell surface
(32-35). On the other hand, two functional opioid receptors,
and
, can heteromerize, which changes the pharmacology of the individual
receptors and potentiates signal transduction (36). Also, it has been
described for heterodimerizations for the CC chemokine receptor 2 (CCR2) and the CX chemokine receptor 4 (CXCR4) and/or CC chemokine
receptor 5 (CCR5) (37). Recently, it has been demonstrated that
and
opioid receptors can form heteromers with
2-adrenergic receptors affecting their trafficking
properties without any significant alteration in the ligand binding or
coupling properties of the receptors (38). There are also some papers
reporting similar interactions between GPCR and non-GPCR proteins, for
example the receptor activity-modifying protein, a small protein
containing only one putative transmembrane domain and a short cytosolic
tail, acting as a chaperone protein that facilitates the cell surface targeting and modulator function of the calcitonin-receptor-like receptor (39). Interestingly, recent data (40) indicated that a GPCR,
the D5-dopamine receptor, physically interacts with the
-aminobutyric acid A (GABAa) receptor, a GABA-operated
Cl
channel. This physical interaction was dependent on
the presence of agonist for both receptors and necessary for the
functional cross-talk between the D5 and GABAa receptors (40). In the
case of cells co-expressing mGlu1
and A1 receptors, a
glutamate/adenosine synergism was found at the level of calcium
mobilization (Fig. 5). Furthermore, in experiments of neuroprotection
performed in neuronal cultures, preincubation with quisqualic acid plus
adenosine was much more effective than pretreatment with any of the
compounds. These results suggest that activation of
mGlu1
and A1 receptors in the same neuron results in
synergism. Heteromerization, however, does not always lead to
facilitation or synergistic events. Thus, in basal ganglia, there is an
adenosine-mediated antagonism of dopaminergic neurotransmission. This
antagonism is in part due to cross-talk at the level of second
messengers but is also mediated by formation of adenosine/dopamine
receptor heteromers (41). Although it is difficult to ascertain to what
extent the antagonism is mediated by heteromerization or to
interference in signaling, there is evidence indicating that both
events operate and are closely interrelated. Thus, in cells where A1R
and D1-dopamine receptors are present, as in nigrostriatal GABAergic
neurons, adenosine leads to both the disappearance of the high affinity site of D1 receptors, probably via conformational changes in A1R/D1R heteromers, and a reduction in dopamine-induced cAMP increases, an
effect due to cross-talk at the adenylate cyclase level (41).
There is experimental and molecular modeling evidence that
intramembrane domains are involved in the formation of homodimers of G
protein-coupled receptors. Gouldson et al. (42) have
hypothesized that domain swapping with involvement of transmembrane
regions 5 and 6 is responsible for homo- and heteromerization of G
protein-coupled receptors. In contrast, heterodimerization of GABAR1
and GABABR2 receptors is mediated by the coiled-coil interaction of the
C-terminal cytoplasmic tails (35). In the case of mGlu1a/A1
receptors' heteromers the interaction depends on the C terminus of
mGlu1
receptor as its splice variant,
mGlu1
receptor, which has a short and different C
terminus, does not interact with A1R. Also a deleted mutant of
mGlu1 receptor, close to the splice variant site and
missing nearly all the C terminus of the receptor, does not interact
with A1R. The co-immunoprecipitation of mGlu1
and A1
receptors might be due to a physical association between them, but it
also could be the case that both receptors are recruited into a
specific signaling complex at specific synapses via common interactions
with other proteins.
One possible mechanism for this mGlu1
/A1 receptor
coupling is that it is directed by interactions of the cytoplasmic C
terminus with specific targeting proteins. This type of targeting mechanism appears to operate for the synaptic localization of the
ionotropic glutamate receptors and a number of different proteins, containing PDZ domains, which interact with specific C-terminal sequences of these receptors (43-47). Also, the EVH1-like domain (ENA/VASP homology domain
1)-containing protein, which binds specifically to the
C-terminal residues of mGlu1
receptor, has been described (48). This
protein, termed Homer-1A, was isolated as a synaptic plasticity-regulated gene from rat hippocampus (48, 49). Additional proteins related to Homer-1A have also been described, namely Homer-1B,
Homer-1C, Homer-2A, Homer 2B, Homer-2C, Homer-2D, Homer-3A, Homer-3B,
Homer-3C, and Homer-3D (50-53). The ability of Homer to link
mGlu1
receptor to Shank, a scaffolding multimeric signaling protein, may contribute to anchoring the mGlu1
receptor to specific sites at the plasma membrane (8, 9). Our efforts
are directed to find a network of protein interactions that are shared
by both receptors and that likely play a key role in the signaling
mechanisms of both mGlu1
and A1 receptors. The nature of
the signaling complexes formed would depend on the type of receptors
present in a given neuron, and the effect will be determined by the
balance between concentrations of agonists in the synaptic cleft and
timing of receptor activation. It has been proposed that the mechanism
of the biphasic effects of quisqualic acid to increase NMDA toxicity if
added together with NMDA or to reduce it if pretreated before NMDA is
in part due to the functional switch described for the
mGlu1
receptor (54, 55, 20). In mixed cortical or pure
hippocampal neuronal cultures a first application of
3,5-dihydroxyphenylglycine, an agonist of the group I mGlu receptors,
potentiated toxicity induced by submaximal concentrations of NMDA,
whereas the same drug applied shortly after a brief pre-exposure, protected against neuronal death (20). Interestingly, the switch in the
regulation of excitotoxic neuronal death was sensitive to protein
kinase C inhibitors (20). This mechanism may explain the opposite
results obtained with group I mGlu receptors, assuming that the
influence of group I mGlu receptors on excitotoxic neuronal death will
depend on the "functional status" of group I mGlu receptors (naive
versus experienced or unphosphorylated versus
phosphorylated receptors) (20). In Fig. 7 we show that the timing in
mGlu1
and A1 receptors activation is very important to
achieve a maximum effect in adenosine- and glutamate-mediated
neuroprotection/neurodegeneration. In fact, although adenosine added
together with glutamate was protective for cultured neurons, this
protection was nearly total by preincubation with the metabotropic
glutamate receptor agonist.
Our data provide biochemical and functional evidence for
mGlu1
and A1 receptor-receptor interaction. The way A1R
is involved in interactions with receptors for other neurotransmitters,
using different receptor system to synchronize synaptic transmission, opens new perspectives to understand the actual role of this autacoid. Since mGlu1
receptors seem to be involved in the
pathophysiology of neuropsychiatric diseases such as Alzheimer's and
related disorders, this molecular interaction offers a new basis for
the design of novel strategies to study the genesis and evolution of
these diseases and of novel agents to treat them.