A Cluster of Basic Residues in the Carboxyl-terminal Tail of the Short Metabotropic Glutamate Receptor 1 Variants Impairs Their Coupling to Phospholipase C

Abstract

Among phospholipase C-coupled metabotropic glutamate receptors (mGluRs), some have a surprisingly long carboxyl-terminal intracellular domain (mGluR1a, -5a, and -5b), and others have a short one (mGluR1b, -1c, and -1d). All mGluR1 sequences are identical up to 46 residues following the 7th transmembrane domain, followed by 313, 20, 11, and 26 specific residues in mGluR1a, mGluR1b, mGluR1c, and mGluR1d, respectively. Several functional differences have been described between the long isoforms (mGluR1a, -5a, and -5b) and the short ones (mGluR1b, -1c, and -1d). Compared with the long receptors, the short ones induce slower increases in intracellular Ca2+, are activated by higher concentration of agonists, and do not exhibit constitutive, agonist-independent activity. To identify the residues responsible for these functional properties, a series of truncated, chimeric, and mutated receptors were constructed. We found that the deletion of the last 19 carboxyl-terminal residues in mGluR1c changed its properties into those of mGluR1a. Moreover, the exchange of the long carboxyl-terminal domain of mGluR5a with that of mGluR1c generated a chimeric receptor that possessed functional properties similar to those of mGluR1c. Mutagenesis of specific residues within the 19 carboxyl-terminal residues of mGluR1c revealed the importance of a cluster of 4 basic residues in defining the specific properties of this receptor. Since this cluster is part of the sequence common to all mGluR1 variants, we conclude that the long carboxyl-terminal domain of mGluR1a suppresses the inhibitory action of this sequence element.

Full text

A Cluster of Basic Residues in the Carboxyl-terminal Tail of the
Short Metabotropic Glutamate Receptor 1 Variants Impairs
Their Coupling to Phospholipase C*
(Received for publication, July 8, 1997, and in revised form, September 26, 1997)
Sophie Mary, Jesus Gomeza‡, Laurent Pre´ zeau§, Joe¨ l Bockaert, and Jean-Philippe Pin¶
From the Me´canismes Mole´culaires des Communications Cellulaires, Unite´ Propre de Recherche 9023-CNRS, Centre
CNRS Inserm de Pharmacologie Endocrinologie, 141 rue de la Cardonille, 34094 Montpellier Cedex 05, France
Among phospholipase C-coupled metabotropic gluta-
mate receptors (mGluRs), some have a surprisingly long
carboxyl-terminal intracellular domain (mGluR1a, -5a,
and -5b), and others have a short one (mGluR1b, -1c, and
-1d). All mGluR1 sequences are identical up to 46 resi-
dues following the 7th transmembrane domain, followed
by 313, 20, 11, and 26 specific residues in mGluR1a,
mGluR1b, mGluR1c, and mGluR1d, respectively. Sev-
eral functional differences have been described between
the long isoforms (mGluR1a, -5a, and -5b) and the short
ones (mGluR1b, -1c, and -1d). Compared with the long
receptors, the short ones induce slower increases in
intracellular Ca
21
, are activated by higher concentra-
tion of agonists, and do not exhibit constitutive, agonist-
independent activity. To identify the residues responsi-
ble for these functional properties, a series of truncated,
chimeric, and mutated receptors were constructed. We
found that the deletion of the last 19 carboxyl-terminal
residues in mGluR1c changed its properties into those
of mGluR1a. Moreover, the exchange of the long carbox-
yl-terminal domain of mGluR5a with that of mGluR1c
generated a chimeric receptor that possessed functional
properties similar to those of mGluR1c. Mutagenesis of
specific residues within the 19 carboxyl-terminal resi-
dues of mGluR1c revealed the importance of a cluster of
4 basic residues in defining the specific properties of
this receptor. Since this cluster is part of the sequence
common to all mGluR1 variants, we conclude that the
long carboxyl-terminal domain of mGluR1a suppresses
the inhibitory action of this sequence element.
Although they possess 7 transmembrane domains, the G-
protein-coupled metabotropic glutamate receptors (mGluRs)
1
(1–3), Ca
21
-sensing (4), and
g
-aminobutyric acid, type B
(GABA
B
)receptors (5) share no sequence homology with any
other G-protein-coupled receptors (GPCRs) and constitute
therefore a distinct family of receptor proteins. Whereas the
agonist binding site is located within a hydrophobic cleft
formed by the 7 transmembrane domains in most GPCRs, it is
located within the large extracellular domain homologous to
bacterial periplasmic binding proteins in this receptor family
(6, 7). Moreover, the second intracellular loop of mGluRs likely
plays a role equivalent to that of the third intracellular loop of
the other GPCRs for G-protein coupling and activation (8–10).
Among the mGluR subtypes cloned so far, three that are
coupled to phospholipase C (PLC), mGluR1a and the two splice
variants mGluR5a and mGluR5b, possess a surprisingly long
(350 residues) carboxyl-terminal intracellular domain (11–15).
The role of this domain is not yet fully characterized, but it may
be involved in specific regulation of the receptor function, pos-
sibly by interacting with specific proteins (16, 17). Several
splice variants have been isolated for mGluR1 that differ in the
length of their intracellular tail (18–21). In the rat mGluR1b,
mGluR1c, and mGluR1d, the 313 carboxyl-terminal residues of
mGluR1a are replaced by 20, 11, and 26 residues, respectively.
All these splice variants do couple to PLC indicating that the
long carboxyl-terminal domain of mGluR1a is not critical for
this function of the protein. Differences in PLC coupling have
been reported between the long forms, mGluR1a, -5a, and -5b,
and the short forms, mGluR1b, mGluR1c, and mGluR1d. In
contrast to the long forms, which generate fast chloride current
responses upon agonist application when expressed in Xenopus
oocytes, mGluR1c induces slowly developing responses (14, 18),
and in baby hamster kidney (BHK) cells stably expressing
mGluR1b, glutamate (Glu) induced slower Ca
21
responses
than in cells expressing mGluR1a (22). Moreover, only the long
forms display a significant constitutive, agonist-independent
activity when expressed in human embryonic kidney (HEK)
293 cells (14, 23, 24), and mGluR1a possesses a higher appar-
ent affinity (EC
50
) for agonists than the short mGluR1 isoforms
(25, 26). Accordingly, it has been proposed that the long car-
boxyl-terminal domain enables better PLC coupling efficacy
(23, 27) .
To identify the sequence responsible for the specific func-
tional properties of mGluR1 splice variants, a series of trun-
cated and chimeric receptors were constructed and analyzed
both in Xenopus oocytes and HEK 293 cells. This approach
allowed us to identify a basic tetrapeptide in the carboxyl-
terminal end of these receptors that confers to the short vari-
ants mGluR1b, -c, and -d their specific PLC-coupling proper-
ties, i.e. slow responses in oocytes, lower potency of agonists,
and absence of constitutive activity. Since this sequence is
* This work was supported in part by grants from the CNRS,
Biomed2 Program Grant BMH4-CT96-0228 and Biotech2 Program
Grant BIO4-CT96-0049 from the European Community, ACC-SV5
Grant 9505077 from the French Ministry of Education, Research and
Professional Insertion, Direction des Recherches et Etudes Techniques
Grant DRET 91/161, and the Bayer Company (France and Germany).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
‡ Supported by the Spanish Ministry of Education. Present address:
National Institutes of Health, NIDDK, Laboratory of Bioorganic Chem-
istry, Bethesda, MD 20892.
§ Present address: Vanderbilt University School of Medicine, Dept. of
Pharmacology, Nashville, TN 37232-6600.
¶To whom correspondence should be addressed. Tel.: 334-67-14-29-
33; Fax: 334-67-54-24-32; E-mail: pin@ccipe.montp.inserm.fr.
1
The abbreviations used are: mGluRs, metabotropic glutamate re-
ceptors; GPCRs, G-protein coupled receptors; PLC, phospholipase C;
BHK, baby hamster kidney; Glu, glutamate; HEK, human embryonic kidney; PCR, polymerase chain reaction; IP, inositol phosphate; LLC-
PK1, porcine kidney epithelial; PBS, phosphate-buffered saline.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 1, Issue of January 2, pp. 425–432, 1998
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 425
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conserved in mGluR1a, we propose that the apparent inhibi-
tory action of this basic tetrapeptide is prevented by the pres-
ence of the long carboxyl-terminal tail of this receptor.
MATERIALS AND METHODS
Construction of Truncated Receptors—The obtention of mGluR
cDNAs and the construction of the eucaryotic expression plasmids have
been described previously (14, 18, 21). To generate mGluR1D1139 and
mGluR1D1093, polymerase chain reactions (PCR) were performed us-
ing mGluR1a cDNA as template (20 ng), dNTP (200
m
M), 2 units of Vent
DNA polymerase (New England Biolabs), and 100 pmol of sense and
antisense primers in a 100-
m
l reaction made with the 10 3buffer
supplied by the manufacturer. The PCR amplification was performed
for 30 cycles of 1 min at 95 °C, 1 min at 50 °C, and 2 min at 72 °C. The
sense primer was 59-CGG TGG CCC TGG GGT GC-39, and the anti-
sense primers containing the additional stop codon and an XbaI site
were 59-CTT GCT CTA GAT GGG CAG GTC CTC CTC CTC-39(for
mGluR1D1139) and 59-CTC CTT CTA GAA GGT GCT CAG GTG CAG
GGG-39(for mGluR1D1093). The resulting PCR products were cut by
SphI and XbaI and subcloned into pmGluR1a cut with the same en-
zymes. For the construction of mGluR1D879, a similar PCR reaction
was performed with 59-CTC AAC ATT TTC CGG AGA TAG AAG ACC
GGG-39and 59-GTA CCT CTA GAG AAG GTT TTT GAA TAA TTC-39as
sense and antisense primers, respectively. The resulting product was
subcloned into pmGluR1a after digestion with BspEI and XbaI.
The truncated mGluR5D(N887Stop) was constructed by PCR using
pmGluR5a as a template DNA, a sense primer containing the stop
codon just downstream of the ApaI site located at position 2651 (59-TG
ACT TGG GCC CAG TAG GAT CCG AGT ACC CGG-39) and an
antisense primer located downstream of the second ApaI site (posi-
tion 3151) in the mGluR5a sequence. The resulting product was
digested by ApaI and ligated into the pmGluR5a previously cut by ApaI
and dephosphorylated.
Construction of Chimeric mGluR5/1 Receptors—To generate pm-
GluR5/1a and pmGluR5/1b, the SphI-XbaI fragment from pmGluR1a
and pmGluR1b were subcloned into pmGluR5a cut with XbaI and
partially digested with SphI. To generate pmGluR5/1c, the 2544-bp
fragment obtained after digestion of pmGluR5a by EcoRI and partial
digestion by SphI was subcloned into pmGluR1c cut with EcoRI and
SphI.
Construction of mGluR1aD
b
and mGluR1cD
b
Receptors—
mGluR1aD
b
was constructed by PCR as described above using
pmGluR1a as a template DNA (100 ng), 59-ACA TTT TCC GGA TGG
CAG CTC CAG GGG CAG GGA ATG-39as sense primer containing the
mutations, 59-TTG GGA TTC CCT TGG TAA CTT TTA GTG AGG-39as
antisense primer and Vent DNA polymerase. The resulting product was
digested by BspEI-KasI and inserted into pmGluR1a previously di-
gested with the same enzymes. The mutant mGluR1cD
b
was con-
structed by PCR using pmGluR1c as a template DNA, the same sense
primer as described above, and T7 as antisense primer. The resulting
product was digested by BspEI-XhoI and inserted into pmGluR1c pre-
viously digested with the same enzymes.
For functional expression into mammalian cells, all constructed
cDNAs were inserted into the pRK5 expression vector, downstream of
the cytomegalovirus promoter (14). The sequence of truncated, chi-
meric, or mutant receptor DNA was verified by double strand DNA
sequencing on both strands with 17–25 mere primers using the
dideoxynucleotide method and Sequenase (U.S Biochemical Corp.).
Expression into Xenopus Oocytes—The preparation of oocytes and the
in vitro synthesis of RNA transcripts from the cloned cDNA were
performed as described previously (18). Recordings were performed in
Barth’s medium using the two-electrode voltage-clamp technique (Ax-
oclamp-2A) 3–4 days after injection. Data were analyzed using the
pclamp software (Axon Instrument, Foster City, CA).
Culture and Transfection of HEK 293 Cells—HEK 293 cells were
cultured in Dulbecco’s modified Eagle’s medium (Life Technologies,
Inc.) supplemented with 10% fetal calf serum and transfected by elec-
troporation as described previously (28). Electroporation was carried
out in a total volume of 300
m
l with 10
m
g of carrier DNA, 500 ng (unless
otherwise specified) of plasmid DNA containing the wild type or mu-
tated mGluR, and 10 million cells.
Determination of Inositol Phosphate (IP) Accumulation—Determina-
tion of IP accumulation in transfected cells was performed as described
previously after labeling the cells overnight with [
3
H]myoinositol (23.4
Ci/mol, NEN Life Science Products, France) (28). The stimulation was
conducted for 30 min in a medium containing 10 mMLiCl and 1 mMGlu.
The basal IP formation was determined after a 30-min incubation in the
presence of 10 mMLiCl. The Glu degrading enzyme glutamate pyruvate
transaminase (1 unit/ml) and 2 mMpyruvate were also added to avoid
the possible action of Glu released from the cells. Results are expressed
as the amount of IP produced over the radioactivity present in the
membranes. The dose-response curves were fitted according to the
equation y5((y
max
2y
min
)/1 1(x/EC
50
)n
H
)1y
min
) where EC
50
is the
concentration of agonist giving a response equal to 50% of the maxi-
mum, y
max
and y
min
correspond to the maximal and minimal values, and
n
H
is the Hill coefficient, using the kaleidagraph program.
SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting—
HEK 293 cell membranes were prepared as described previously (23).
Samples (40
m
g of protein) were solubilized in Laemmli sample buffer
(2.5% (w/v) SDS, 25 mMTris-HCl, pH 6.8, 5% (v/v)
b
-mercaptoethanol,
and 6.25% glycerol), resolved by SDS-polyacrylamide gel electrophore-
sis (7.5% acrylamide) and transferred by electroblotting onto a Hybond
C extra membrane (Amersham, France). Immunodetection of mGluR1
and actin proteins was performed as described previously (23). Chemi-
luminescent blots were quantitated using the GS-525 Molecular Imager
(Bio-Rad) for which volume analysis of the bands is calculated as pixel
density units. For normalization of the results we measured the ratio of
metabotropic Glu receptor to the actin signal for each sample.
Immunofluorescence of Transfected Cells—The generation and char-
acterization of the mGluR1 antibody (generous gift of Drs. V. Matarese
and F. Ferraguti, Glaxo, Verona, Italy) raised against a chimeric pro-
tein containing part of the extracellular domain has been described
previously (29). Eighteen hours after transfection, HEK 293 cells grown
on coverslips were washed three times with PBS, fixed for 20 min at
room temperature in 4% paraformaldehyde in PBS, and washed three
times in PBS. The cells were then incubated for1hatroom tempera-
ture in PBS containing 3% bovine serum albumin and rabbit anti-
mGluR1 (1:250). Cells were washed in PBS containing 3% bovine serum
albumin, and bound primary antibodies were detected with a fluores-
cein-labeled mouse anti-rabbit secondary antibody (1:50; Sigma, L’Isle
d’Abeau, France) for 45 min at room temperature. Cells were washed,
and the coverslips were mounted with Mowiol 4.88 and visualized with
a Zeiss (Axiophot) microscope.
Statistical Analysis—Statistical differences were examined using the
Stat-View Student program (Abacus Concept, Berkeley, CA) using ttest
or analysis of variance (Fisher’s PLSD test).
RESULTS
As shown in Fig. 1, several functional differences were ob-
served between the long mGluR1 isoform mGluR1a and the
short variant mGluR1c in agreement with our previous studies
(18, 23, 25). When expressed in Xenopus oocytes, mGluR1a
induced faster Chloride currents than mGluR1c upon activa-
tion with Glu (Fig. 1a). When the time needed to reach the
maximal amplitude of the current after the beginning of the
response was measured (time to peak), it was found to be5sin
oocytes expressing mGluR1a and 15 s in oocytes expressing
mGluR1c (p,0.001) (Fig. 2a), even though the amount of
cRNA injected was adjusted to obtain responses similar in
amplitudes (414 628 nA (n5111) for mGluR1a and 385 635
nA (n5140) for mGluR1c). In mGluR1a-expressing HEK 293
cells, a 2-fold higher basal Glu-independent PLC activity was
measured compared with mock-transfected cells or cells ex-
pressing mGluR1c (Figs. 1band 2a). Glu stimulated IP forma-
tion to similar extents in cells expressing mGluR1a or
mGluR1c, but the EC
50
value for Glu was smaller when deter-
mined in mGluR1a-expressing cells than in cells expressing
mGluR1c (1.08 60.12
m
M(n
H
51.03 60.14; n58) and 5.67 6
0.89 (n
H
51.13 60.20; n58) (p,0.001), respectively) (Fig.
1c). These functional differences did not result from a lower
level of expression of the short variant as shown by Western
blotting of membrane proteins prepared from both cell types
(Fig. 1d).
To identify the sequence element within the mGluR1 car-
boxyl-terminal tail responsible for the different functional
properties of the long versus the short variant, a series of
truncated receptors was constructed (Fig. 3). In mGluR1D1139,
a stop codon was introduced at position 1139 so that the last 60
amino acids including a large number of serine and threonine
PLC-coupling Inhibitory Sequence in mGluR1 Splice Variants426
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residues and a PDZ interacting sequence (17) were removed. In
mGluR1D1093, an additional segment rich in acidic residues
was removed. Finally, an additional truncated receptor
mGluR1D879 with a carboxyl-terminal intracellular tail
shorter than that of mGluR1c was also constructed (Fig. 3).
The coupling to PLC of these truncated receptors was first
analyzed after expression in Xenopus oocytes. In oocytes ex-
pressing mGluR1D1139, mGluR1D1093 as well as in oocytes
expressing mGluR1D879, Glu induced fast responses similar in
shape to those measured in oocytes expressing mGluR1a (Figs.
2band 4a). These truncated receptors were also expressed in
HEK 293 cells. In these cells all truncated and wild type re-
ceptors stimulated IP formation to a similar extent when acti-
vated with Glu (data not shown). All truncated receptors also
exhibited constitutive activity like mGluR1a (Figs. 2band 4b).
Taken together, these results indicate that the truncated re-
ceptor mGluR1D879, which possesses a carboxyl-terminal in-
tracellular domain shorter than that of mGluR1c, displays the
same functional properties as the long variant mGluR1a both
in Xenopus oocytes and HEK 293 cells (Fig. 4). This suggests
that sequence elements within the 19 carboxyl-terminal resi-
dues of mGluR1c are responsible for the specific functional
properties of this short mGluR1 variant.
To examine whether the carboxyl-terminal end of mGluR1c
was sufficient to explain its functional properties, we ex-
changed the carboxyl-terminal domain of the other PLC-cou-
pled mGluR (mGluR5a) with that of mGluR1a or mGluR1c
taking advantage of a conserved SphI site in the mGluR1 and
mGluR5 sequences (Figs. 2cand 5). Like the wild type
mGluR5a that also possesses a large carboxyl-terminal domain
(Fig. 2c), mGluR5/1a induced fast responses in oocytes (Figs. 2c
and 6a) and possessed high constitutive activity (Figs. 2cand
6b). In contrast, the chimeric mGluR5/1c receptor with the
carboxyl-terminal tail of mGluR1c induced slowly developing
responses in oocytes (Figs. 2cand 6a) and had reduced consti-
tutive activity (Figs. 2cand 6b). Moreover, when various con-
centrations of Glu were used to stimulate IP formation in cells
expressing these chimeric receptors, a lower EC
50
value was
measured with mGluR5/1a-expressing cells than with cells ex-
pressing mGluR5/1c (0.74 60.25
m
M(n
H
50.62 60.12; n54)
and 4.39 61.13 (n
H
51.05 60.20; n54) (p,0.02), respec-
tively) (Fig. 6c). Finally, a truncated mGluR5 receptor with a
carboxyl-terminal intracellular tail similar in length to that of
mGluR1c (see Fig. 5) has the same functional properties as the
wild-type mGluR5a (Fig. 2c). The presence of the carboxyl-
terminal half of the mGluR1c intracellular tail is therefore
sufficient to confer to mGluR5 the specific PLC coupling prop-
erties of mGluR1c.
The carboxyl-terminal sequences of mGluR1b, mGluR1c, and
mGluR1d display no sequence homology after the splice junc-
tion site (Fig. 5). They share however similar functional proper-
ties (18, 21, 25, 30) suggesting that their specific few carboxyl-
terminal residues may not play a critical role in these
properties. In agreement with this proposal, a truncated
mGluR1a receptor (mGluR1D939) with a carboxyl-terminal tail
slightly longer than that of mGluR1d shares functional prop-
erties with these short receptor variants: it induces slow re-
sponses in oocytes and displays no constitutive activity (data
not shown). Therefore, the short sequence located between
Arg-878 and the splice junction site (KKPGAGNA, see Fig. 5)
may be responsible for the specific functional properties of
these mGluR1 variants. Interestingly, residue Arg-878 is the
second of a cluster of 4 basic residues, RRKK (Fig. 5). To
examine if this cluster of basic residues could be responsible for
the specific properties of the short mGluR1 splice variants, we
constructed mutated mGluR1a and mGluR1c receptors in
FIG.1. mGluR1a and mGluR1c variants have different PLC-
coupling properties. a, mGluR1a induces faster current than does
mGluR1c. Schematic representation of mGluR1a and mGluR1c and
typical current traces obtained upon application of 300
m
MGlu on
oocytes expressing mGluR1a (left) or mGluR1c (right) and voltage-
clamped at 270 mV. Scale bars:vertical, 200 nA; horizontal,20s.Inthe
bottom graph, the time-to-peak values of individual responses are plot-
ted against the maximal current amplitude measured upon Glu (300
m
M) application (I
max
). b, mGluR1a but not mGluR1c stimulates IP
production in the absence of agonist. Basal IP formation in mock-
transfected HEK 293 cells and in cells expressing mGluR1a or
mGluR1c. The maximal IP formation induced by 1 mMGlu was 100 6
2, 1078 6120, and 1075 6110 in mock-transfected cells and in cells
expressing mGluR1a or mGluR1c, respectively (percent of the basal IP
formation in mock-transfected cells, means 6S.E. of 26, 21, and 17
triplicate determinations). Values correspond to the [
3
H]IP produced
divided by the amount of radioactivity in the membranes and are
means 6S.E. of nindependent experiments performed in triplicate. c,
mGluR1a exhibits a higher affinity for the agonist glutamate than does
mGluR1c. IP formation stimulated by various concentrations of Glu in
HEK 293 cells expressing mGluR1a (E) or mGluR1c (●). Values are
expressed as percentage of the maximal effect of Glu over basal activity
and are means 6S.E. of 5 experiments performed in triplicate. d,
relative levels of expression of mGluR1a and mGluR1c as revealed by
Western blot analysis. Membranes prepared from mock-transfected
HEK 293 cells or cells transfected with 500 ng of plasmid containing the
mGluR1a or mGluR1c cDNAs and the mGluR proteins (top panel) and
actin (bottom panel) were detected using selective antibodies. The basal
IP formation was also determined in parallel in these cells and were
found to be in total agreement with the data presented in panel b. The
upper band observed with the mGluR1 antibody likely corresponds to
mGluR dimer, as already described (23, 49). The determination of the
ratio intensity of the mGluR band over that of actin (using Molecular
Imager quantification) indicates that the intensity of the mGluR1a
band was 45 69% (n54) that of mGluR1c.
PLC-coupling Inhibitory Sequence in mGluR1 Splice Variants 427
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which Arg-878, Lys-879, and Lys-880 were replaced by Met,
Ala, and Ala, respectively. These mutated receptors were
named mGluR1aD
b
and mGluR1cD
b
, respectively. Mutation of
these 3 basic residues in mGluR1a did not modify its functional
properties when examined either in Xenopus oocytes or in HEK
293 cells (Fig. 2d). However, mGluR1cD
b
induced fast current
responses when expressed in Xenopus oocytes (Figs. 2dand 7a)
and displayed agonist-independent constitutive activity (Figs.
2dand 7b) and an increased potency of glutamate (6.39 61.76
m
M(n
H
51.03 60.21; n54) and 0.97 60.53 (n
H
50.96 60.21;
n54) (p,0.01) for mGluR1c and mGluR1cD
b
, respectively)
(Fig. 7c) even though it appeared to be expressed at a lower
level than the wild type mGluR1c (Fig. 7d).
To verify that all wild type and mutated mGluR1 receptors
were correctly targeted to the plasma membrane, immuno-
staining of HEK 293 cells expressing these receptors was per-
formed using an antibody directed against their conserved ex-
tracellular domain. As shown in Fig. 8, all receptors were found
at the plasma membrane level. Interestingly, the labeling was
found as large patches along the plasma membrane in many
cells expressing mGluR1c. Such large patches were never ob-
served in cells expressing mGluR1a, mGluR1D879, or
mGluR1cD
b
.
DISCUSSION
Our data indicate that a cluster of 4 basic residues located 36
amino acid residues after the 7th transmembrane domain is
responsible for the specific PLC coupling properties of the short
mGluR1 variants. Since this sequence is conserved in the long
isoform mGluR1a, the long extra carboxyl-terminal domain of
this receptor may simply prevent the action of this cluster of
basic residues (Fig. 9).
The functional properties due to this cluster of basic residues
in the short mGluR1 variants include a slow activation of the
Cl
2
current in Xenopus oocytes, a very low or no constitutive
activity, and a low potency of Glu in stimulating IP formation.
FIG.2.Summary table of the wild type and mutant receptors constructed and analyzed in this study. a, wild type receptors mGluR1a
and mGluR1c; b, truncated mGluR1a receptors; c, wild type, truncated, and chimeric mGluR5 receptors; d, mGluR1a and -1c mutants. In the first
column are the names of the receptors with the scheme of their carboxyl-terminal intracellular tail. The end of the 7th transmembrane domain is
indicated. The mGluR1a and mGluR5 sequences are in black and white, respectively. The specific sequence of mGluR1c is indicated with a hatched
rectangle. The position of the 3 mutated basic residues in mGluR1aD
b
and mGluR1cD
b
are indicated by a thin white line. In the second column
are the means 6S.E. of the time-to-peak values in seconds for responses smaller than 1000 nA obtained upon stimulation with 300
m
MGlu of
oocytes injected with 0.5–10 ng of cRNA. In a,b, and d,asterisks indicate that the values are statistically different (**, p,0.01) from that measured
in mGluR1a-expressing cells. In c,asterisks indicate that the values are statistically different (**, p,0.01; *, p,0.05) from that measured in
mGluR5a-expressing cells.
FIG.3.Schematic representation of
the carboxyl-terminal tail of mGluR1a
and mGluR1c mutants. The positions
where stop codons were introduced to gen-
erate the truncated mutants mGluR1D-
1139, mGluR1D1093, and mGluR1D879
are indicated by vertical bars. Serine and
threonine residues in the last 60 carboxyl-
terminal sequence are represented by filled
diamonds (l). Glutamate and aspartate
residues between position 1093 and 1139
are represented by thick circles (G), and
proline residues between position 879 and
1093 are represented by plus signs (1). Pu-
tative phosphorylation sites are indicated
by the filled circles (●) attached to the se-
quence. Specific mGluR1c residues are in
black.
PLC-coupling Inhibitory Sequence in mGluR1 Splice Variants428
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These properties may be explained if this sequence element
impairs the expression of the receptor in the plasma mem-
brane. This hypothesis cannot be tested directly using binding
experiments because of the absence of high affinity radioligand
for this receptor. However, our previous and present data in-
dicate that this is unlikely to be the case (18, 23). Slowly
developing currents are rarely observed in oocytes expressing
mGluR1a even when very low amounts of mRNA are injected
(18). By changing the amount of plasmid DNA transfected into
HEK 293 cells, we previously reported that the ratio of basal
over Glu-stimulated PLC activity was independent of the
amount of receptor protein and was higher with mGluR1a than
with mGluR1c (23). Finally, Western blots suggest a higher
level of expression of mGluR1c than mGluR1a or mGluR1cD
b
,
and immunostaining experiments revealed the presence of all
wild type and mutated receptors at the plasma membrane
level. Accordingly, it can be proposed that the cluster of basic
residues decreases the PLC coupling efficacy of the short
mGluR1 variants. In agreement with this hypothesis, several
authors reported slowly developing currents induced in oocytes
by receptors that have a low PLC coupling efficacy (8, 31–35).
Moreover, GPCR constitutive activity is often associated with a
higher G-protein coupling efficacy (for example, see Refs. 36
and 37) and higher potency of agonists (36, 38–40). A higher
PLC coupling efficacy of mGluR1a may be associated with a
higher Glu-induced IP formation in cells expressing this recep-
tor compared with cells expressing mGluR1c. However, under
our experimental conditions, Glu stimulated IP formation to a
similar extent in cells expressing any of the wild type or mu-
tated receptors. This may be explained if a high level of expres-
sion of these receptors is reached so that the PLC pathway is
saturated upon activation with Glu. In agreement with this
hypothesis, the maximal Glu-induced IP formation is lower in
mGluR1c-expressing cells than in cells expressing mGluR1a
when lower amounts of plasmid are transfected or when por-
cine kidney epithelial (LLC-PK1) cells, which expressed fewer
receptors
2
than the HEK 293 cells are used (18).
Finding that the carboxyl-terminal end of a GPCR decreases
G-protein coupling is not unique to mGluRs. Truncation of the
last 59 amino acid residues of the thyrotropin-releasing hor-
mone receptor causes constitutive activity (41). The last 12
residues of bovine rhodopsin have also been proposed to oper-
ate as a negative regulator of guanine nucleotide exchange
(42–44). In that case, the inhibitory action of the carboxyl
terminus is abolished when the receptor is depalmitoylated
(45). Similarly, removal of the extended carboxyl-terminal do-
main of the avian
b
-adrenergic receptor increases its activity
(46). Finally, removal of the carboxyl-terminal tail of the hu-
man parathyroid hormone receptor suppresses its G-protein
coupling selectivity, suggesting that this region inhibits cou-
pling to some G-proteins (47). However, there are no primary
sequence similarities between these domains.
Several hypotheses can be proposed to explain the lower PLC
coupling efficacy due to this basic tetrapeptide in the carboxyl
terminus of the short mGluR1 variants. One possibility is that
this basic tetrapeptide directly interacts with the G-protein
2
J. Gomeza, S. Mary, L. Pre´zeau, J. Bockaert, and J.-P. Pin, unpub-
lished results.
FIG.4.The truncated receptor mGluR1D879 with a carboxyl-
terminal tail shorter than that of mGluR1c has functional prop-
erties different from those of mGluR1c, but similar to those of
mGluR1a. a, mGluR1D879 induces fast responses in Xenopus oocytes.
Schematic representation of the mutant and typical current trace ob-
tained upon application of 300
m
MGlu on oocytes expressing
mGluR1D879 and voltage-clamped at 270 mV. Scale bars:vertical, 200
nA; horizontal,20s(left). On the right, the time-to-peak values of
individual responses are plotted against the maximal current ampli-
tude measured upon Glu (300
m
M) application (I
max
). b, truncated
mGluR1D879 mutant stimulates IP production in the absence of ago-
nist. Basal IP formation occurs in mock-transfected cells and in cells
expressing mGluR1c and mGluR1D879. In these cells, the maximal IP
formation induced by 1 mMGlu was 100 62, 1075 6110, and 1326 6
483 in mock-transfected cells, in cells expressing mGluR1c, and in cells
expressing mGluR1D879, respectively. Values correspond to the [
3
H]IP
produced divided by the amount of radioactivity in the membranes and
are means 6S.E. of 6–17 independent experiments performed in trip-
licate. c, truncated mGluR1D879 mutant is expressed at a level similar
to mGluR1c. Membranes prepared from mock-transfected cells or cells
transfected with 0.5
m
g of plasmid containing the mGluR1c or
mGluR1D879 cDNAs and the mGluR proteins (top panel) and actin
(bottom panel) were detected using selective antibodies after transfer on
membrane. The upper band observed with the mGluR1 antibody likely
corresponds to mGluR dimer, as already described (23, 49).
FIG.5.Alignment of the sequence of the carboxyl-terminal domain of mGluR1 splice variants and that of mGluR5a. The position of
the conserved SphI site used for the generation of the mGluR5/1 chimeric receptor is indicated. The position where the amino acid sequences of
mGluR1 splice variants diverge (splice site) and where the stop codon is inserted in mGluR1D879 are indicated. The specific sequence of the
different mGluR1 variants is in lowercase. The cluster of basic residues in mGluR1 is highlighted in black. The position where the stop codon is
inserted in mGluR5Dis also indicated.
PLC-coupling Inhibitory Sequence in mGluR1 Splice Variants 429
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and inhibits GDP/GTP exchange as observed with the carboxyl
terminus of bovine rhodopsin (44). Another possibility is that
the presence of this cluster of basic residues decreases the
affinity of the receptor for the G-protein. This could be due to
an interaction of the cluster of basic residues with one of the
intracellular loop of the receptor, masking the G-protein recog-
nition site as proposed for rhodopsin, or to the interaction of
these basic residues with the membrane phospholipids, pre-
venting the positive action of the amino-terminal part of the
intracellular tail on G-protein coupling (8, 10, 48). Alterna-
tively, this sequence element may stabilize the receptor in the
inactive state. Another possibility is that, like the avian
b
-ad-
renergic receptor (46), the carboxyl terminus of the short
mGluR1 variants reduces their accessibility to G-proteins pos-
sibly by decreasing their plasma membrane mobility. This
could result either from its interaction with a cytoskeletal
protein or from a clustering of the receptor. Interestingly, our
preliminary immunostaining experiments revealed that in
many cells expressing mGluR1c, large clusters of receptors can
be seen at the level of the plasma membrane. No such clusters
are seen with mGluR1a, mGluR1D879, or in cells expressing
the mutated mGluR1cD
b
, suggesting that the clustering of the
receptors and their low PLC coupling efficacy are related.
Although mGluR1a also contains this cluster of basic resi-
dues, it has a higher PLC coupling activity than the short
variants. The long carboxyl-terminal tail of mGluR1a may
therefore prevent the inhibitory action of this sequence (Fig. 9).
The functional analysis of our deletion mutants indicates that
the carboxyl-terminal acidic residue-rich region or serine/thre-
onine-rich domain do not impair PLC coupling indicating that
these sequences are not necessary to prevent the action of the
cluster of basic residues nor are the last few carboxyl-terminal
residues interacting with PDZ domains (17). This further sug-
gests that the long intracellular tail of mGluR1a has numerous
other regulatory roles such as the control of receptor desensi-
FIG.6. In contrast to the long carboxyl-terminal tail of
mGluR1a, the carboxyl-terminal end of mGluR1c impairs PLC
coupling of mGluR5. In a, mGluR5/1a induces a chloride current at
faster onset than does mGluR5/1c. In b, mGluR5/1a exhibits a higher
affinity for the agonist glutamate than does mGluR5/1c. Values are
means 6S.E. of 4 independent experiments performed in triplicate. In
c, mGluR5/1c displays significantly reduced IP production in the ab-
sence of agonist as compared with mGluR5/1a. The maximal IP forma-
tion induced by 1 mMGlu was 100 62, 991 6160, and 880 6232 in
mock-transfected cells, in cells expressing mGluR5/1a, and in cells
expressing mGluR5/1c, respectively. Values are means 6S.E. of 4
independent experiments performed in triplicate.
FIG.7.Mutation of 3 basic residues in the carboxyl-terminal
tail of mGluR1c generates mGluR1cD
b
with PLC-coupling prop-
erties of mGluR1a. In a, mGluR1cD
b
induces faster responses in
Xenopus oocytes than mGluR1c. In b, mGluR1cD
b
but not mGluR1c
stimulates IP production in the absence of agonist in HEK 293 cells.
The maximal IP formation induced by 1 mMGlu was 100 67, 1061 6
243, and 927 6198 in mock-transfected cells, in cells expressing
mGluR1c, and in cells expressing mGluR1cD
b
, respectively (means 6
S.E. of 11 experiments performed in triplicate). In c, mGluR1cD
b
ex-
hibits a higher affinity for the agonist glutamate than does mGluR1c.
Values are means 6S.E. of 5 independent experiments performed in
triplicate. d, relative levels of expression of mGluR1c and mGluR1cD
b
as revealed by Western blot analysis. Membranes prepared from mock-
transfected HEK 293 cells or cells transfected with 500 ng of plasmid
containing the mGluR1c or mGluR1cD
b
cDNAs and the mGluR proteins
(top panel) and actin (bottom panel) were detected using selective an-
tibodies. The determination of the ratio intensity of the mGluR band
over that of actin (using Molecular Imager quantification) indicates
that the intensity of the mGluR1cD
b
band was 48 614% (n53) that of
mGluR1c.
PLC-coupling Inhibitory Sequence in mGluR1 Splice Variants430
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tization and/or down-regulation, a possible role for the serine/
threonine-rich carboxyl-terminal end of mGluR1a, and interac-
tion with specific proteins like homer (17). However, a further
truncation of this carboxyl-terminal tail up to position 939 or
the natural truncation by alternative splicing generates recep-
tors with functional properties similar to those of mGluR1c.
The presence of this portion of the mGluR1a tail, downstream
of the splice site up to Phe-1092 is therefore necessary to
restore all mGluR1a functional properties: fast activation of
the chloride current in oocytes, high potency of agonists, and
high constitutive agonist-independent activity in HEK cells,
indicating that these additional residues are sufficient to pre-
vent the action of the inhibitory domain. Within this part of the
long intracellular domain of mGluR1a is the proline-rich do-
main. How this part of the carboxyl-terminal tail of mGluR1a
prevents the action of the inhibitory domain remains to be
determined. It is possible that the general conformation of the
carboxyl-terminal tail is such that the inhibitory sequence can
no longer interact with another domain of the receptor or with
another protein responsible for the impaired coupling.
In conclusion, the truncation of the long carboxyl-terminal
domain of mGluR1a by alternative splicing unmasks a short
sequence that decreases the ability of the receptor to activate
PLC. Such an inhibitory sequence is not found in mGluR5 for
which no short carboxyl-terminal tail splice variant has been
described yet. Additional experiments need to be performed to
see whether this inhibitory sequence also affects other signal
transduction pathways activated by mGluR1, such as Ca
21
-
and K
1
-channel modulation or phospholipase A
2
and adenylyl
cyclase activation which may be mediated by G-proteins differ-
ent from the G
q
type.
Acknowledgments—We thank Drs. Y. Grau and M. L. Parmentier for
constructive discussions all during this work and Drs. J. Blahos, M.
Bouvier, V. Homburger, L. Journot, and A. Varrault for critical reading
of the manuscript. We would like to acknowledge C. Joly for expert
technical assistance. We also gratefully acknowledge Drs. V. Matarese
and F. Ferraguti (Glaxo, Verona, Italy) for the generous gift of the
purified anti-mGluR1 antibody.
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Sophie Mary, Jesus Gomeza, Laurent Prézeau, Joël Bockaert and Jean-Philippe Pin
Glutamate Receptor 1 Variants Impairs Their Coupling to Phospholipase C
A Cluster of Basic Residues in the Carboxyl-terminal Tail of the Short Metabotropic
doi: 10.1074/jbc.273.1.425
1998, 273:425-432.J. Biol. Chem.
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