J Neurophysiol 104: 439 – 448, 2010.
First published May 12, 2010; doi:10.1152/jn.00799.2009.
Venus Fly Trap Domain of mGluR1 Functions as a Dominant Negative
Against Group I mGluR Signaling
Donald Beqollari and Paul J. Kammermeier
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York
Submitted 27 August 2009; accepted in final form 11 May 2010
INTRODUCTION
Metabotropic glutamate receptors (mGluRs) are class C G
protein coupled receptors (GPCRs) with widespread distribution in the CNS where they regulate physiological processes
such as synaptic plasticity (Bolshakov and Siegelbaum 1994;
Neyman and Manahan-Vaughan 2008; Rouach and Nicoll
2003; Volk et al. 2007) and electrical excitability (Nistri et al.
2006) and play important roles in a variety of pathologies (Kew
2004; Olive 2009; Varney and Gereau 2002). The structure of
class C GPCRs is unique in that they contain a large, modular,
N-terminal extracellular ligand binding domain, called a venus
fly trap domain (VFT) in which the closed state (which leads to
activation) is favored when ligand is bound in the VFT pocket
(Jingami et al. 2003; Kunishima et al. 2000; Tsuchiya et al.
2002). Class C GPCRs also contain a heptahelical domain that
anchors them to the plasma membrane, mediates G protein
association and activation (Francesconi and Duvoisin 1998),
Address for reprint requests and other correspondence: P. J. Kammermeier,
Dept. of Pharmacology and Physiology, University of Rochester Medical Center,
Box 711, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: paul_kammermeier
@urmc.rochester.edu).
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and interacts with some small molecule allosteric modulators
(Goudet et al. 2004; Knoflach et al. 2001). These domains are
linked by a short extracellular cysteine-rich domain believed to
play an important role in translating the conformational changes in
the VFT that result from ligand binding into G protein activation
in the heptahelical region (Rondard et al. 2006).
Like many GPCRs, mGluRs can dimerize (Robbins et al.
1999). In contrast to the class A GPCRs, however, the dimerization state of mGluRs is well established (Jingami et al.
2003; Kubo and Tateyama 2005), and the functional consequences of their dimerization are beginning to be elucidated
(Brock et al. 2007; Goudet et al. 2005; Kammermeier and Yun
2005; Kniazeff et al. 2004). Unlike the closely related GABAB
receptors, which form obligate heterodimers between GBR1
and GBR2 (Kaupmann et al. 1998; White et al. 1998), mGluRs
form stable, covalently linked homodimers (Jingami et al.
2003; Kunishima et al. 2000; Tsuchiya et al. 2002). This
dimerization appears to be essential, as the dimer molecules
function as a receptor unit. For example, group I mGluRs
(mGluR1 and 5) do not exhibit full activity unless bound by
ligand in both dimer subunits (Kammermeier and Yun 2005;
Kniazeff et al. 2004), and G protein activation seems to be
mediated by only one subunit at a time (Goudet et al. 2005). In
short, the currently accepted model seems to point to the
dimeric mGluR as the indivisible basic receptor unit.
While there is no evidence for heterodimerization across the
different members of the mGluR family (but see Goudet et al.
2005), less is known about the ability of different splice
variants to dimerize with each other. For example, there is
controversy about whether mGluR1a and mGluR1b can dimerize (Kumpost et al. 2008; Remelli et al. 2008; Robbins et al.
1999), but splice variants of mGluR5 do appear to cross
dimerize (Remelli et al. 2008). Further, truncated splice variants of several mGluR subtypes including mGluR1, mGluR6,
and mGluR8 (Malherbe et al. 1999; Valerio et al. 2001; Zhu et
al. 1999), that possess only the extracellular N-termini have
been reported. To date, the function of these splice variants is
not known, but it is likely that at least some can form dimeric
receptors with full-length mGluRs because similar recombinant
constructs of mGluR1, mGluR3, and mGluR5 can clearly dimerize with their full-length counterparts (Bates et al. 2002; Robbins
et al. 1999; Selkirk et al. 2002; Yao et al. 2004) and when
expressed alone are translated and secreted as covalently linked
dimers (Robbins et al. 1999; Selkirk et al. 2002; Yao et al. 2004).
Therefore the functional effects of the N-terminal domain of
mGluR1 were examined when co-expressed with full-length
mGluR1 and mGluR5, and with a point mutation of mGluR1
that expresses and functions like the wild type but does not
form covalently linked dimers. It was determined that the
0022-3077/10 Copyright © 2010 The American Physiological Society
439
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Beqollari D, Kammermeier PJ. Venus fly trap domain of
mGluR1 functions as a dominant negative against group I mGluR
signaling. J Neurophysiol 104: 439 – 448, 2010. First published
May 12, 2010; doi:10.1152/jn.00799.2009. Metabotropic glutamate receptors (mGluRs) form covalently linked homodimers and
contain large, N-terminal extracellular ligand binding, “venus fly trap”
(VFT) domains. These domains, when expressed separately, are secreted as disulfide linked dimers and can dimerize with full-length
receptors. mGluR splice variants have been described that contain
only this domain, but the consequences of their interaction on receptor
signaling have not been explored. Here it is shown that an mGluR1
mutant containing only the VFT is retained on the cell surface when
a full-length receptor is co-expressed. Further, when expressed in rat
superior cervical ganglion (SCG) neurons and modulation of native
calcium currents is used as an assay for receptor activity, the VFT acts
as a dominant negative with respect to mGluR1 signaling. Although
full-length mGluR1 and mGluR5 are not known to heterodimerize, the
mGluR5 VFT partially occludes mGluR1 signaling and the mGluR1
VFT potently occludes mGluR5 signaling in SCG neurons. In addition, an mGluR1 point mutant, mGluR1 C140G, which cannot covalently dimerize, functions like the wild-type receptor when expressed alone. The C140G mutant is inhibited by the mGluR1 VFT
construct but does not retain the mGluR1 VFT on the cell surface,
suggesting that the loss of C140 renders the interaction reversible.
Finally, a peptide designed to disrupt mGluR1 dimerization reduced
signaling through the C140G mutant receptor, but only when applied
intracellularly for several hours, indicating that loss of signaling
requires disruption of dimerization prior to plasma membrane
insertion.
440
D. BEQOLLARI AND P. J. KAMMERMEIER
truncated receptor could occlude signaling of both mGluR1
and mGluR5 as a dominant negative subunit. Further, the
mechanism of this occlusion of signaling was investigated to
determine whether it acted by preventing the formation of
full-length receptor dimers.
METHODS
Cell isolation, DNA injection, and plasmids
Electrophysiology and data analysis
Patch pipettes were made with a Sutter P-97 horizontal puller from
8250 glass (Garner Glass, Claremont, CA) and had resistances of 1–3
M⍀. Series resistances were 2–5 M⍀ prior to electronic compensation
of 80%. Whole cell patch-clamp recordings were made with an
Axopatch 200B patch clamp amplifier (Axon Instruments, Foster
City, CA) or EPC-7 patch clamp amplifier (Heka Elektronik, Germany). Voltage protocol generation and data acquisition were performed using custom software (courtesy Stephen R. Ikeda, NIAAA,
Rockville, MD) on a Macintosh G3 or G4 computer (Apple Computer, Cupertino, CA) with an InstruTech (Port Washington, NY; now
Heka Elektronik) ITC-18 or ITC-16 data acquisition board. Currents
were low-pass filtered at 3–5 kHz using the 4-pole Bessel filter in the
patch clamp amplifiers, digitized at 2–5 kHz and stored on the
computer for later analysis. Experiments were performed at 21–24°C
(room temperature). Patch-clamp data analysis was performed using
Igor Pro software (Wavemetrics, Lake Oswego, OR).
The external (bath) solution for patch-clamp recordings contained
(in mM): 155 tris hydroxymethyl aminomethane, 20 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 glucose, 10
CaCl2, and 0.0003 tetrodotoxin (TTX), pH 7.4. The internal (pipette)
solution contained (in mM) 120 N-methyl-D-glucamine (NMG) methJ Neurophysiol • VOL
Immunofluorescence and TIRF imaging
To detect surface expression of the myc-mG1-NT construct, a
Cy3-conjugated, mouse anti-myc, monoclonal antibody (Sigma) was
applied to live cells at room temperature for 25 min at a 1:150
dilution. Cells were subsequently washed five times in phosphate
buffered saline (PBS), and images were acquired from live cells.
Fluorescence images and figures were constructed using Adobe Photoshop (Adobe Systems, San Jose, CA) and Canvas software (ACD
Systems of America, British Columbia, Canada). Quantitative image
analysis was performed using Igor Pro.
TIRF and mGluR1-GFP epifluorescence images were obtained on an
Olympus IX71 inverted microscope using the total internal reflection
fluorescence (TIRF) ⫻60, 1.45 NA objective. Images were acquired with
a Cooke Sensicam QE digital cooled CCD camera (Cooke, Romulus, MI)
using TillVision software (Till Photonics GmbH). All epifluorescence
images described in Fig. 2C and images analyzed in Fig. 3C, as well as
all TIRF images, were acquired using identical parameters to permit
comparisons across cells. TIRF images were acquired with longer exposure times than epifluorescence (200 vs. 50 ms) to account for the lower
light levels using the TIRF method. Fluorescence levels reported are
average pixel intensities from a large rectangular region of interest within the
cell, taken from 12 bit tiff images. Background levels were indistinguishable
across images, so background subtraction was not reported, although subtraction did not alter the findings nor the interpretation (not shown).
RESULTS
Full-length mGluR1 retains a myc-tagged, N-terminal
mGluR1 construct on the plasma membrane
A deletion mutant consisting of the first 590 amino acids of
(rat) mGluR1 and incorporating a myc-epitope tag after amino
acid 33 was constructed. Similar mGluR1 and mGluR5 deletion constructs are translated and secreted as dimers in various
expression systems (Robbins et al. 1999; Selkirk et al. 2002).
To determine whether our construct, termed myc-mG1-NT,
could be detected on the plasma membrane in the presence of
full-length mGluR1, a Cy3-conjugated primary anti-myc antibody was used to detect cell surface myc expression in live rat
sympathetic neurons from the superior cervical ganglion
(SCG) under a variety of expression conditions (Fig. 1). When
myc-mGluR1 (the full-length wild-type construct) was expressed alone (with GFP), substantial Cy3 labeling was detectable (Fig. 1A, top). As expected, expression of myc-mG1-NT
or the untagged, full-length mGluR1 alone did not result in
detectable Cy3 labeling (Fig. 1A, center). However, when
myc-mG1-NT was co-expressed with the full-length mGluR1,
Cy3 labeling was clearly evident (Fig. 1A, bottom), suggesting
that the myc-tagged construct is retained on the plasma membrane due to its interaction with mGluR1.
Quantification of Cy3 fluorescence is shown in Fig. 1B.
Signal was quantified by averaging Cy3 fluorescence intensity
at the membrane measured from a line scan of each epifluorescence image. Peak GFP fluorescence is shown as a control
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A detailed description of the cell isolation and cDNA injection
protocol can be found elsewhere (Ikeda 1997). Animal protocols were
approved by the university committee on animal resources (UCAR).
Briefly, both SCGs were removed from adult male Wistar rats (175–
225 g) following CO2 euthanasia and decapitation and incubated in
Earle’s balanced salt solution (InVitrogen, Life Technologies Carlsbad, CA) containing 0.6 mg/ml trypsin (Worthington Biochemicals,
Freehold, NJ) and 0.8 mg/ml collagenase D (Boehringer Mannheim
Biochemicals, Indianapolis, IN) for 1 h at 35°C. Cells were transferred
to minimum essential medium (InVitrogen/Gibco), plated on poly-llysine (Sigma Chemical, St. Louis, MO) coated 35 mm tissue culture
dishes and incubated (95% air and 5% CO2; 100% humidity) at 37°C
for 2– 4 h before cDNA injection. Following injection, cells were
incubated overnight at 37°C, and patch clamp or immunofluorescence
experiments were performed the following day.
Injection of cDNA was performed with an Eppendorf 5247 microinjector and InjectMan (Madison, WI) NI 2 micromanipulator 4 – 6 h
following cell isolation. Injection electrodes were made with a Sutter
(Novato, CA) P-97 horizontal electrode puller using thin-walled,
borosilicate glass (World Precision Instruments, Sarasota, FL). Plasmids were stored at –20°C as a 1 ␮g/␮l stock solution in TE buffer (10
mM Tris, 1 mM EDTA, pH 8). All mGluR1 and mGluR5 constructs
were injected at 100 –130 ng/␮l (pCDNA3.1⫹; InVitrogen). The
C140G mutant was produced using the QuikChange Site-Directed
Mutagenesis strategy (Stratagene). All neurons were co-injected with
“enhanced” green fluorescent protein cDNA (0.02 ␮g/␮l; pEGFPN1;
BD Biosciences-Clontech, Palo Alto, CA) for identification of successfully injected cells.
All constructs were sequence confirmed. PCR products were purified with Qiagen (Valencia, CA) silica membrane spin columns prior
to restriction digestion and ligation. Plasmids were propagated in the
Top10 Escherichia coli strain (InVitrogen) and midipreps were prepared using Qiagen anion exchange columns.
anesulfonate, 20 TEA, 11 EGTA, 10 HEPES, 10 sucrose, 1 CaCl2, 4
MgATP, 0.3 Na2GTP, and 14 tris creatine phosphate, pH 7.2. lGlutamate (Sigma) was used as the agonist for mGluRs. All drugs and
control solutions were applied to cells using a custom gravity-driven
perfusion system positioned ⬃100 ␮m from the cell that allowed
rapid solution exchange (ⱕ250 ms). The degree of mGluR-mediated
calcium current inhibition was calculated as the maximal inhibition of
the current in the presence of drug compared with the last current
measurement prior to application of the drug.
mGluR1 N-TERMINUS FUNCTIONS AS A DOMINANT NEGATIVE
A
Bright field
GFP
441
cy3
myc-mGluR1
mGluR1
mGluR1
+
myc-mG1-NT
B
cy3 membrane fluorescence (arbitrary units)
0
5
10
15
20
myc-mG1-NT
+ mGluR1
25
(9)
ns
*
myc-mG1-NT
alone
0
20
(13)
40
60
80
100
GFP Fluorescence (arbitrary units)
for expression efficiency. While GFP fluorescence was similar
in both groups, a strong Cy3 signal was detected in cells
co-expressing myc-mGluR1-NT and WT mGluR1 but was
undetectable in cells expressing myc-mG1-NT alone. SCG
neurons co-expressing mGluR1 with myc-mG1-NT and those
expressing myc-mG1-NT alone had Cy3 fluorescence intensities (in arbitrary fluorescence units) of 19 ⫾ 4 (n ⫽ 9 cells) and
4 ⫾ 0.4 (means ⫾SE; n ⫽ 13), respectively. GFP fluorescence
from the same cells measured 51 ⫾ 9 and 77 ⫾ 17, respectively. These data are consistent with the conclusion that
myc-mGluR1-NT expressed alone is secreted and that fulllength mGluR1 retains myc-mGluR1-NT on the plasma membrane, presumably via dimerization (Robbins et al. 1999;
Selkirk et al. 2002).
Myc-mG1-NT functions as a dominant negative against
mGluR1 signaling
Although several reports have demonstrated that the Nterminal VFT domains of mGluR1 and mGluR5 can dimerize
J Neurophysiol • VOL
with their respective full-length receptors (Bates et al. 2002;
Robbins et al. 1999; Selkirk et al. 2002), the effect of this
interaction on receptor signaling has not been tested (but see
Tiao et al. 2008). To address this question, full-length mGluR1
was expressed alone and with myc-mG1-NT in SCG neurons,
and mGluR-mediated calcium current modulation was measured at several glutamate concentrations (Fig. 2). Consistent
with previous reports (Kammermeier and Yun 2005), when
mGluR1 was expressed alone, glutamate inhibited SCG calcium currents with an EC50 of 1–2 ␮M (Fig. 2, A and B) and
a maximal inhibition (at 30 ␮M) of 48 ⫾ 5% (n ⫽ 10).
Surprisingly, when myc-mG1-NT was co-expressed, the maximal inhibition at 30 ␮M glutamate was reduced to only 13 ⫾
3% (n ⫽ 11). This residual effect could be interpreted as
signaling through the small number of mGluR1 homodimers
because if mGluR1 and myc-mG1-NT are expressed at similar
levels and form “heterodimers” as well as homodimers, ⬃25%
of expressed receptors are expected to be full-length mGluR1
homodimers.
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myc-mG1-NT
FIG. 1. Coexpression of mGluR1 retains myc-mG1-NT on
the cell surface. A: bright field (left), GFP (center, positive
control for expression), and cy3 (right) epi-fluorescence. Top:
“myc-mGluR1” illustrates cy3 labeling of the myc-tagged,
full-length mGluR1, a positive control for antibody labeling.
Also note the absence of background labeling indicated by the
uninjected cell (no GFP fluorescence) in the left side of the
frame. Second row: “mGluR1,” illustrates the lack of cy3
labeling in a cell expressing only the full-length mGluR1.
Third row: “myc-mG1-NT”, illustrates the lack of cy3 labeling
in a cell expressing only myc-mG1-NT. Fourth row: “mGluR1 ⫹
myc-mG1-NT”, illustrates strong cy3 labeling in a cell expressing both the full-length mGluR1 and myc-mG1-NT. Exposure
times for cy3 images in the 2nd and 3rd rows were 2–3 times
those in rows 1 and 4. Longer exposures indicated that cy3
labeling in those experiments (rows 2–3) was not appreciably
above background (not shown). B: average (⫾SE) GFP fluorescence (green bars) and cy3 membrane fluorescence (red
bars) for cells expressing mGluR1 and myc-mG1-NT together
and those expressing myc-mG1-NT alone, as indicated. Statistical significance (P ⱕ 0.05; indicating a difference from
myc-mG1-NT ⫹ mGluR1 group) indicated by asterisk; ns, no
significant difference. Number of cells in each group shown in
parentheses.
442
D. BEQOLLARI AND P. J. KAMMERMEIER
B
A
mGluR1 alone
mGluR1 + myc-mG1 -NT
mGluR1 + myc-mG5-NT
mGluR1
3 0 µM Glu
0 .1 nA
Glu
% Ica Inhibit ion
5 0 sec
60
Con
3 µM Glu
Glu
0 .3 3 0
3
100
[ Glu] ( µM)
50
(10)
40
*
30
20
+10
+80 mV
-80
(12)
*
10
Con
(11)
0
+10
1
-80
10
100
mGluR1 -GFP alone
C
dSRed-nuc Epi
GFP Epi
GFP TIRF
mGluR1 -GFP + myc-mG1 -NT
GFP TIRF f luorescence ( arbit rary unit s)
mGluR1 -GFP +
myc-mG1 -NT
(12)
(12)
(16)
(16)
0
0
TIRF
As a negative control, mGluR1 was co-expressed with the
analogous N-terminal venus fly trap domain of (rat) mGluR5
(amino acids 1–574), myc-mG5-NT. Because mGluR1 and
mGluR5 are not known to heterodimerize, this construct was
expected to be without effect on mGluR1 signaling. Surprisingly, a significant reduction of the maximal calcium current
inhibition at 30 ␮M glutamate to 32 ⫾ 5% (n ⫽ 12) was
observed in co-expressing cells, suggesting that some interaction between mGluR1 and the VFT domain of mGluR5 can
occur. These results would suggest that the 7 transmembrane
domain of mGluRs can impose some negative influence on
mGluR dimerization, perhaps imparting selectivity to dimerization within the mGluR family.
An alternate interpretation of these data are that the truncated mutants reduce mGluR1 expression to varying degrees.
To rule out this possibility, the expression levels of a Cterminally GFP-tagged mGluR1 (mGluR1-GFP) were examined when expressed alone or with myc-mG1-NT (Fig. 2C).
Cells were also injected with cDNA for a nuclear targeted
dSRed (dSRed-nuc) to independently identify successfully
injected neurons. Total mGluR1-GFP expression and plasma
membrane expression were examined using epifluorescence
and total internal reflection fluorescence microscopy (TIRF),
respectively. TIRF is used to illuminate only those fluorescent
molecules within ⬃100 Å of the glass cover slip (Toomre and
Manstein 2001). Total mGluR1-GFP expression measured by
J Neurophysiol • VOL
GFP epif luorescence ( arbit rary unit s)
mGluR1 -GFP
*
EPI
epifluorescence was not detectably altered when myc-mG1-NT
was co-expressed. Average fluorescence intensity measured
from epifluorescence images was 735 ⫾ 115 (arbitrary fluorescence units ⫾SE; n ⫽ 16) and 836 ⫾ 135 (n ⫽ 12) in SCG
neurons expressing mGluR1-GFP alone and mGluR1-GFP
plus myc-mG1-NT, respectively. Surprisingly, plasma membrane expression of mGluR1-GFP, measured from TIRF images, appeared to be slightly enhanced when myc-mG1-NT
was co-expressed. Average fluorescence intensity from TIRF
images was 209 ⫾ 27 (n ⫽ 16) in control and 337 ⫾ 60 (n ⫽
12) in the myc-mG1-NT co-expressing cells (Fig. 2C; the same
cells were used for epifluorescence and TIRF imaging). Finally, to verify that the fluorescently tagged construct was
inhibited by myc-mG1-NT in a manner similar to the wild-type
mGluR1, mGluR1-GFP was expressed alone or with mycmG1-NT in SCG neurons and the magnitude of calcium
current inhibition in response to 100 ␮M glutamate application
was monitored. Consistent with the results using the untagged
mGluR1, mGluR1-GFP produced 27 ⫾ 6% (n ⫽ 7) inhibition
of the native calcium current when expressed alone but only
1 ⫾ 3% (n ⫽ 6) when myc-mG1-NT was co-expressed using
identical cDNA concentrations for intranuclear injection as in
the fluorescence experiments described above (Fig. 2C).
While the apparent increase in plasma membrane receptor in
the presence of myc-mG1-NT was unexpected and difficult to
interpret, the data provide strong evidence that the dominant
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[ Glu] ( µM)
FIG. 2. Dominant negative effect of mycmG1-NT on mGluR1 signaling. A: time course
of calcium current inhibition by a range of
glutamate concentrations in a representative
superior cervical ganglion (SCG) neuron expressing mGluR1 alone. Inset: sample control
and inhibited currents before and during application of 3 ␮M (bottom) and 30 ␮M (top)
glutamate. , the time of current measurement
(10 ms after the start of the ⫹10 mV test pulse)
for the plot in A. B: glutamate dose-response
curve showing average calcium current inhibition (⫾SE) at a range of concentrations from
SCG neurons expressing mGluR1 alone (),
mGluR1 ⫹ myc-mG1-NT (Œ), and mGluR1 ⫹
myc-mG5-NT (□). Number of cells in each
group shown in parentheses. C, left: epifluorescence images illustrating nuclear dSRed,
mGluR1-GFP and TIRF images showing
plasma membrane mGluR1-GFP expression in
representative SCG neurons expressing either
mGluR1-GFP alone or with myc-mG1-NT, as
indicated. Right: bar graph showing average
mGluR1-GFP fluorescence taken from epifluorescence and TIRF images from the same sets
of cells. *, statistical significance (P ⱕ 0.05)
compared with mGluR1-GFP alone. Number
of cells indicated in parentheses.
mGluR1 N-TERMINUS FUNCTIONS AS A DOMINANT NEGATIVE
negative actions of myc-mG1-NT do not result from loss of
full-length receptor expression. Together, these data indicate
that the N-terminal domain of mGluR1 can act as a strong
dominant negative against mGluR1 signaling.
Myc-mGluR1-NT also functions as a dominant negative
against mGluR5 signaling
B
mGluR5b alone
+myc-mG5-NT
+myc-mG1-NT
50
(13)
40
% inhibition Ica
Control
+myc-mG1-NT
60
50
% inhibition Ica
A
calcium current modulation via mGluR5b to 7 ⫾ 2% (n ⫽ 8)
at 100 ␮M glutamate. These results suggest that the N-terminal
region of mGluR1 can function as a potent dominant negative
against signaling of both mGluR1 and mGluR5, while the
mGluR5 N-terminus only weakly inhibits signaling in both
receptors. To test whether the differential effects of mycmG1-NT and myc-mG5-NT resulted from differences in their
expression levels, live SCG neurons expressing mGluR1 plus
either myc-mG1-NT or myc-mG5-NT were labeled with Cy3anti-myc antibody, as in Fig. 1. As controls, neurons expressing only mGluR1 or myc-mGluR1 were also labeled (Fig. 3C).
While no Cy3 labeling was evident in mGluR1 cells (0.75 ⫾
0.5, n ⫽ 4), no difference was detectable between the positive
controls (myc-mGluR1 alone: 6.6 ⫾ 1.7, n ⫽ 5) and cells with
mGluR1 plus either myc-mG1-NT (9.5 ⫾ 2.5, n ⫽ 5) or
myc-mG5-NT (11.8 ⫾ 2.4, n ⫽ 9). Thus the weaker effect of
myc-mG5-NT on mGluR1 and mGluR5 signaling is not likely
to be the result of weaker expression. Therefore regions in the
heptahelical domains of group I mGluRs must participate in
selectivity of dimer formation among group I mGluRs. By
contrast, co-expression of myc-mGluR1-NT did not alter calcium current modulation via 100 ␮M glutamate in mGluR2expressing SCG neurons, nor via native ␣2-adrenergic recep-
30
(7)
20
40
30
20
(8)
10
10
*
(7)
0
2
4
0.1
6 8
2
4
1
6 8
2
4
0
6 8
10
100
[Glu] (µM)
(7)
(7)
mGluR2
(100 µM Glu)
(7)
α2 AdrR
(10 µM NE)
Cy3 Epi-fluorescence (arb. Units)
C
0
mGluR1
myc-mGluR1
mGluR1+
myc-mG1NT
mGluR1+
myc-mG5NT
2
4
6
8
10
12
14
(4)
(5)
(5)
(9)
J Neurophysiol • VOL
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FIG. 3. myc-mG1-NT, but not mycmG5-NT functions as a strong dominant negative against mGluR5 signaling. A: glutamate
dose-response curves obtained in SCG neurons
expressing mGluR5 alone (), mGluR5 ⫹
myc-mG1-NT (□), and mGluR5 ⫹ mycmG5-NT (Œ). Number of cells in each group
shown in parentheses. B: bar graphs showing
SCG calcium current inhibition in the absence
(□) and presence () of myc-mG1-NT expression using as an agonist 100 ␮M glutamate in
cells expressing mGluR2, and 10 ␮M norepinephrine, which activates a native adrenergic
receptor, as indicated. C: average (⫾SE) Cy3
epifluorescence from cells labeled as in Fig. 1
with the indicated expression. Number of cells
in each group is shown in parentheses.
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 15, 2017
To gain a better understanding of the mechanism of the
dominant negative actions of the N-terminal VFT domain of
mGluRs, the effects of the these domains when co-expressed
with mGluR5 were examined. A glutamate dose-response
curve was generated in SCG neurons expressing mGluR5b
(Fig. 3). The EC50 for mGluR5b was similar to that for
mGluR1 (⬃1 ␮M), although the maximal calcium current
inhibition was slightly smaller. Surprisingly, co-expression of
the mGluR5 VFT domain, myc-mG5-NT, had only a moderate
effect on the mGluR5b dose-response in SCG neurons, decreasing the calcium current inhibition at 100 ␮M glutamate by
less than half from 38 ⫾ 5 (n ⫽ 13) in cells expressing
mGluR5b alone to 24 ⫾ 4% (n ⫽ 7) when co-expressed with
myc-mG5-NT. Further, co-expression of the mGluR1 VFT,
myc-mG1-NT, appeared to have as strong a dominant negative
effect on mGluR5 signaling as on mGluR1 (Fig. 3), reducing
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D. BEQOLLARI AND P. J. KAMMERMEIER
tors following application of 10 ␮M norepinephrine (Fig. 3B).
These data demonstrate that the dominant negative effect of the
mGluR1 VFT exhibits selectivity in that only group I mGluRs
appear to be affected.
Noncovalently dimerizing mGluR1 mutant is inhibited by
myc-mG1-NT but does not retain it on the cell surface
Homodimerization of group I mGluRs is mediated in part by
a covalent link between the C140 residues (C129 in mGluR5)
in each dimer subunit (Ray and Hauschild 2000; Romano et al.
2001). However, mutation of this residue does not abolish
dimerization because noncovalent dimers can form (Ray and
Hauschild 2000; Romano et al. 1996, 2001). To determine
whether the dominant negative actions of myc-mG1-NT require this covalent linkage, the truncated construct was coexpressed with the point mutant mGluR1 C140G in SCG
neurons, and receptor-mediated modulation of calcium currents was examined. When expressed alone, mGluR1 C140G
functioned like the wild-type receptor, with a similar maximal inhibition of 37 ⫾ 8% (n ⫽ 9; not significantly
different from the wild-type mGluR1; P ⫽ 0.23) at 30 ␮M
glutamate, and an EC50 of about 3 ␮M (Fig. 4, A and B).
Co-expression of myc-mG1-NT with mGluR1 C140G revealed that the deletion construct had as strong a dominant
negative effect on the mutant receptor as on the wild-type.
In cells co-expressing mGluR1 C140G and myc-mG1-NT,
the maximal calcium current inhibition at 100 ␮M glutamate
was only 8 ⫾ 2% (n ⫽ 7).
A
B
mGluR1 C140G
50 sec
40
% inhibition Ica
0.3
3
+10
30
(9)
30
20
100 µM Glu
+80 mV
-80
10
(7)
+10
-80
Glu
Con
C
A plausible explanation of the data in the preceding text may
be that the myc-mG1-NT construct occludes mGluR function
by preventing dimerization of full-length receptors. This possibility was further probed by testing whether a 20 amino acid
mGluR1 C140G
C140G+myc-mG1-NT
50
0.1 nA
Selective disruption of mGluR1 C140G signaling with a
dimer interface peptide
0
0.1
Glu (30 µM)
Con
1
10
100
[Glu] (µM)
cy3 Fluorescence
0.2 nA, 20 msec
0
10
20
30
40
(5) *
mGluR1 + myc-mG1NT
mGluR1 C140G + myc-mG1NT
(10)
ns
Uninjected
(7)
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FIG. 4. Dominant negative effect of mycmG1-NT on mGluR1 C140G signaling. A:
time course of calcium current inhibition by a
range of glutamate concentrations in a representative SCG neuron expressing mGluR1
C140G alone. Inset: sample control and inhibited currents before and during application of
30 ␮M glutamate as well as voltage protocol.
Circle indicates time of current measurement
for time course plot. B: glutamate dose-response curve showing average calcium current
inhibition (⫾SE) at a range of concentrations
from SCG neurons expressing mGluR1
C140G alone () and mGluR1 C140G ⫹
myc-mG1-NT (□). Number of cells in each
group shown in parentheses. C: average membrane cy3 immuno-fluorescence in SCG neurons expressing wild-type mGluR1 ⫹ mycmG1-NT as a positive control, mGluR1 C140G
⫹ myc-mG1-NT, and uninjected cells as a
negative control, as indicated. *, a statistically significant difference from the uninjected group. Number of cells in each group
shown in parentheses.
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 15, 2017
The strong inhibitory effect of myc-mG1-NT led to the
expectation that like the wild-type mGluR1, the C140G mutant
would likewise cause retention of myc-mG1-NT on the cell
surface. However, no myc-mG1-NT plasma membrane retention was detectable when the construct was co-expressed with
mGluR1 C140G (Fig. 4C). Using methodology identical to that
described for Fig. 1, the presence of the myc epitope on the
plasma membrane was probed with a Cy3 anti-myc antibody in
live cells. While SCG neurons co-expressing mGluR1 and
myc-mG1-NT yielded significantly greater plasma membrane
Cy3 fluorescence than uninjected cells, those expressing
mGluR1 C140G and myc-mG1-NT did not. Together these
data suggest that the dominant negative effects of mycmG1-NT may arise from the prevention of dimerization of
mGluRs with the caveat that dimers must form before insertion
into the plasma membrane. The lack of myc-mG1-NT on the
cell surface also suggests that the interaction between mGluR1
C140G and myc-mG1-NT is reversible, but the lack of signaling under these conditions suggests that mGluR1 C140G
dimerization is not initiated on the plasma membrane or occurs
there very slowly.
mGluR1 N-TERMINUS FUNCTIONS AS A DOMINANT NEGATIVE
min. Surprisingly, the magnitude of calcium current inhibition
by neither mGluR1 nor mGluR1 C140G was detectably altered
by acute peptide application in this way. Calcium currents were
inhibited before and after DI-pep application in mGluR1 expressing cells by 55 ⫾ 10 and 52 ⫾ 11% (n ⫽ 3), respectively.
Calcium currents in cells expressing mGluR1 C140G were
inhibited 55 ⫾ 7 and 51 ⫾ 9% (n ⫽ 5) before and after acute
DI-pep application, respectively. In addition, 1–2 h bath application of 10 ␮M DI-pep prior to recording was likewise
ineffective at reducing mGluR1 C140G activity (Fig. 5C).
Peptide-treated mGluR1 C140G-expressing cells showed calcium current inhibition of 42 ⫾ 6% (n ⫽ 9) in paired mGluR1
C140G-expressing, untreated control cells, 37 ⫾ 7% (n ⫽ 8) in
cells pretreated for 1 h, and 39 ⫾ 5% (n ⫽ 9) in those
pretreated for 2 h. Figure 5C shows the combined data for all
the bath applied, C140G experiments, pooling the control cells
from all of these experiments. Note that while there appears to
be a small reduction in inhibition after 1–2 h in the peptide, this
difference was not statistically significant. Further, when these
data are compared with the control cells recorded on the same
days (as reported in the preceding text), no reduction is evident.
Together these data strengthen the conclusion that mGluR
function is occluded when full-length dimerization is disrupted, but that dimerization is fairly stable once the receptors
are inserted in the plasma membrane, even for the C140G
mutant receptor. However, if dimerization can be prevented
before plasma membrane insertion, perhaps at the time of
translation, such as when myc-mG1-NT is co-expressed or
A
Dimer interface peptide:
N -WHSSVALEQSIEFIRDSLIS- COOH
B
Inj ec t ed p ep t id e
Control
+Peptide
70
60
(3)
(4)
(5)
C
70
Bat h- ap p lied p ep t id e
mGluR1 C140G
(5)
60
50
40
(9)
(8)
50
*
% inhibition Ica
40
(5)
30
30
20
20
10
10
s
hr
2
1
in
J Neurophysiol • VOL
hr
0
m
mGluR1 C140G
n
mGluR1 WT
1
0
co
% inhibition Ica
(14)
FIG. 5. Intracellular, but not bath-application of the dimer
interface peptide, DI-pep reduces signaling of mGluR1 C140G.
A, left: graphical representation of the structure of the Nterminal, VFD domain of mGluR1. Separate dimer subunits are
shown in blue and pink. The yellow highlighted residues illustrate the residues of the DI-pep, showing its close proximity to
the opposing dimer subunit. Right: sequence of DI-pep and
graphical representation in isolation. B: average (⫾SE) calcium
current modulation by 100 ␮M glutamate in SCG neurons
expressing mGluR1 or mGluR1 C140G, as indicated, untreated
(□) or preinjected cytoplasmically with 10 ␮M DI-pep (). *,
a statistically significant difference from control (P ⱕ 0.05).
C: average (⫾SE) calcium current modulation by 100 ␮M
glutamate in SCG neurons expressing mGluR1 C140G, untreated or following 2 h of bath applied, 10 ␮M DI-pep.
Number of cells in each group shown in parentheses.
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peptide corresponding to the helical mGluR1 dimer interface
(“DI-pep”; Fig. 5A) could disrupt mGluR function (Sato et al.
2003), possibly by preventing dimerization.
SCG neurons were initially isolated and double injected:
intranuclearly and cytoplasmically, using a micropipette containing 100 ng/␮l cDNA (encoding mGluR1) and 10 ␮M
DI-pep, or containing cDNA alone as a negative control. For
these mGluR1-expressing SCG neurons, no difference in
mGluR1-mediated calcium current modulation was detectable.
Average inhibition in control and peptide injected, mGluR1expressing cells was 55 ⫾ 10% (n ⫽ 3) and 58 ⫾ 1% (n ⫽ 4),
respectively (Fig. 5B). By contrast, when identical experiments
were performed with mGluR1 C140G expression, the magnitude of calcium current inhibition was significantly reduced
from 56 ⫾ 7% (n ⫽ 5) in control to 30 ⫾ 10% (n ⫽ 5) in
peptide injected cells (Fig. 5B). These data are consistent with
the interpretation that disruption of full-length mGluR dimerization can disrupt signaling but that the covalently linked
wild-type receptors are sufficiently stable that the DI-pep is
ineffective. It is possible that unlike the peptide, the mycmG1-NT has effective dominant negative activity against the
wild-type receptor because it contains the C140 residue, which
may permit covalent dimerization with the full-length
mGluR1.
To further characterize the activity of the DI-pep, glutamatemediated calcium current modulation was examined in SCG
neurons expressing either mGluR1 or mGluR1 C140G before
and after acute application of 10 ␮M DI-pep to the bath for 1
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D. BEQOLLARI AND P. J. KAMMERMEIER
when DI-pep is present intracellularly in the presence of the
C140G mutant receptor, full-length dimers may fail to form
and receptor signaling can be disrupted.
DISCUSSION
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The data in the preceding text demonstrate that an Nterminal mGluR1 construct, similar to a truncated splice variant (Zhu et al. 1999), is retained on the plasma membrane when
co-expressed with the full-length mGluR1 in rat sympathetic
neurons. Further, co-expression of the recombinant mycmG1-NT construct strongly inhibits signaling of mGluR1,
measured as the magnitude of mGluR-mediated calcium current modulation in rat SCG neurons. The remaining signal
observed is consistent with the expected small number of
full-length mGluR1 homodimers when both constructs are
expressed, perhaps indicating that the mGluR1/mG1-NT
dimers entirely lack the ability to signal. When the N-terminal
VFT region of mGluR5 was expressed with mGluR1 as a
negative control, some inhibition of mGluR1 signaling was
observed, but much less than with myc-mG1-NT, even though
myc-mGluR1-NT and myc-mG5-NT appeared to be expressed
at roughly equal levels (Fig. 3C). It is difficult to interpret how
equal levels of myc-mGluR1-NT and myc-mG5-NT on the cell
surface could produce different degrees of signal occlusion if
the mechanism relies on disruption of dimer formation. However, the immunofluorescence method used to measure the
surface myc-epitope is unlikely to be sensitive enough to tease
out subtle differences in expression levels. Thus it may be
prudent to interpret these data as something of a digital signal:
both of the NT constructs express reasonably well, and both
can interact with mGluR1 such that they are retained on the cell
surface.
Recent reports have suggested that mGluR activation involves a conformational change in which each dimer subunit
pivots with respect to the other on ligand binding and VFT
“closing” (Jingami et al. 2003; Sato et al. 2003). This change
may require plasma membrane anchoring of both subunits to
occur and to translate into G protein activation (Kunishima et
al. 2000; Rondard et al. 2006). The data presented here, in
which an unanchored mGluR1 VFT acts as a dominant negative subunit, support this model (Rondard et al. 2006; Sato et
al. 2003).
In addition to the effects of myc-mG1-NT on group I
mGluRs, the effect on mGluR1 C140G was tested. Mutation
of the C140 residue of mGluR1 and of the corresponding
cysteine in mGluR5 results in a point mutant that lacks
covalent dimerization (Ray and Hauschild 2000) but likely
retains the ability to form noncovalent dimers (Romano et
al. 2001) and appears to function normally. It remains
unclear, however, whether mGluR1 C140 mutants signal as
dimers, as monomers, or both. Further, it is not known how
stable mGluR1 C140 mutant dimers are, or whether they
occupy some equilibrium between monomeric and dimeric
states in live cells. Because the myc-mG1-NT construct
could occlude mGluR1 signaling presumably by disrupting
dimerization, the opportunity to address these questions was
presented. Indeed myc-mG1-NT was as potent a dominant
negative against mGluR1 C140G as against mGluR1, indicating that the C140 mutant does indeed derive its ability to
signal from a dimeric state. Otherwise, it’s likely that
signaling would be strong, or at least moderate, in the
presence of the myc-mG1-NT subunit.
Interestingly, although myc-mG1-NT acted to potently occlude mGluR1 C140G activity, retention of myc-mG1-NT on
the plasma membrane was not detectable when co-expressed
with mGluR1 C140G. These data suggested that while the VFT
of mGluR1 may prevent signaling of mGluR1 and mGluR1
C140G by an analogous mechanism, the basic interaction
between these subunits is different. These data are consistent
with the interpretation that like the wild-type mGluR1 (Robbins et al. 1999), mGluR1 C140G dimerizes prior to plasma
membrane insertion (e.g., in the ER or Golgi). Thus preventing
this dimerization causes loss of function. Unlike the myc-mG1-NT/
mGluR1 interaction, however, the myc-mG1-NT/mGluR1 C140G
interaction appears reversible, hence the lack of detectable mycmG1-NT retention. It may be inferred then that reformation of
mGluR1 C140G dimers on the plasma membrane does not occur or
occurs very slowly such that no signaling is detectable when mycmG1-NT is co-expressed. Thus these data argue against a model in
which mGluR1 C140 mutants exhibit dynamic equilibrium between
monomeric and dimeric states in living cells and therefore probably
exist, or at least signal, primarily as dimers.
The final set of experiments provide further support for these
conclusions by demonstrating that the DI-pep, which consists
of a 20 amino acid alpha helix that makes up a considerable
portion of the dimer interface (Jingami et al. 2003; Sato et al.
2003) fails to disrupt mGluR1 C140G signaling (and therefore
dimerization) when applied acutely to the bath, even for
prolonged periods of ⱕ2 h. This peptide can, however, disrupt
mGluR1 C140G signaling (and thus presumably dimerization)
when injected into cells several hours before the experiments
but failed to disrupt wild-type mGluR1 under any conditions
tested. While the inability of the DI-pep to disrupt wild-type
mGluR1 dimerization (signaling) was unsurprising due to the
covalent bond linking mGluR1 dimers, the inability to disrupt
mGluR1 C140G signaling provides further support for the
interpretation that this mutant primarily occupies and signals
from the dimeric state when expressed in live cells rather than
in an equilibrium between monomers and dimers. Otherwise,
the peptide would be expected to impart at least some inhibition of signaling when applied extracellularly at the relatively
high concentration of 10 ␮M, the same concentration that was
shown to disrupt the mutant receptor when injected intracellularly.
It should be noted that although the mechanism of action of
the DI-pep is assumed to be through prevention of dimerization
by occupying the dimer interface surface on a full-length
receptor, this assumption cannot be directly verified. The lack
of effect on wild-type mGluR1 provides a control against the
interpretation that the peptide works through some nonspecific
mechanism, such as inhibition of expression. But while it is
assumed that the differential effects observed with mGluR1
and mGluR1 C140G arise from the loss of a covalent interaction to link the dimers in the mutant, it is difficult to predict
the impact on the mGluR1 structure caused by the mutation
because the published structure (Jingami et al. 2003) leaves
a stretch of ⬃20 nearby amino acids unresolved, including
the C140 residue, which one assumes must come into close
proximity with the opposing dimer subunit at least for the
wild-type receptor. Thus a large change in the structure in
mGluR1 N-TERMINUS FUNCTIONS AS A DOMINANT NEGATIVE
ACKNOWLEDGMENTS
We thank A. V. Smrcka, S. Malik, D. Morrow, and and D. I. Yule
(University of Rochester) for use of equipment, reagents and for helpful
discussions.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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