From the Mécanismes Moléculaires des Communications Cellulaires, Unité Propre de Recherche 9023-CNRS, Centre CNRS Inserm de Pharmacologie Endocrinologie, 141 rue de la Cardonille, 34094 Montpellier Cedex 05, France
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ABSTRACT |
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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.
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INTRODUCTION |
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Although they possess 7 transmembrane domains, the
G-protein-coupled metabotropic glutamate receptors
(mGluRs)1 (1-3),
Ca2+-sensing (4), and -aminobutyric acid, type B
(GABAB)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, possibly 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 Ca2+ 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 apparent affinity (EC50) for agonists than the short mGluR1 isoforms (25, 26). Accordingly, it has been proposed that the long carboxyl-terminal domain enables better PLC coupling efficacy (23, 27) .
To identify the sequence responsible for the specific functional properties of mGluR1 splice variants, a series of truncated 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 variants mGluR1b, -c, and -d their specific PLC-coupling properties, i.e. slow responses in oocytes, lower potency of agonists, and absence of constitutive activity. Since this sequence is conserved in mGluR1a, we propose that the apparent inhibitory action of this basic tetrapeptide is prevented by the presence of the long carboxyl-terminal tail of this receptor.
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MATERIALS AND METHODS |
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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 mGluR11139
and mGluR1
1093, polymerase chain reactions (PCR) were performed
using mGluR1a cDNA as template (20 ng), dNTP (200 µM), 2 units of Vent DNA polymerase (New England Biolabs), and 100 pmol of sense and antisense primers in a 100-µl reaction made with the 10 × buffer 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 5
-CGG
TGG CCC TGG GGT GC-3
, and the antisense primers containing the
additional stop codon and an XbaI site were 5
-CTT GCT CTA GAT GGG CAG GTC CTC CTC CTC-3
(for mGluR1
1139) and 5
-CTC CTT CTA
GAA GGT GCT CAG GTG CAG GGG-3
(for mGluR1
1093). The resulting PCR
products were cut by SphI and XbaI and subcloned
into pmGluR1a cut with the same enzymes. For the construction of
mGluR1
879, a similar PCR reaction was performed with 5
-CTC AAC ATT
TTC CGG AGA TAG AAG ACC GGG-3
and 5
-GTA CCT CTA GAG AAG GTT TTT GAA TAA TTC-3
as sense and antisense primers, respectively. The resulting product was subcloned into pmGluR1a after digestion with
BspEI and XbaI.
Construction of Chimeric mGluR5/1 Receptors-- To generate pmGluR5/1a 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 mGluR1a and mGluR1c
Receptors--
mGluR1a
was constructed by PCR as described above
using pmGluR1a as a template DNA (100 ng), 5
-ACA TTT TCC GGA TGG CAG CTC CAG GGG CAG GGA ATG-3
as sense primer containing the mutations, 5
-TTG GGA TTC CCT TGG TAA CTT TTA GTG AGG-3
as antisense primer and
Vent DNA polymerase. The resulting product was digested by BspEI-KasI and inserted into pmGluR1a previously
digested with the same enzymes. The mutant mGluR1c
was
constructed 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 previously digested with the same enzymes.
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 (Axoclamp-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 electroporation as described previously (28). Electroporation was carried out in a total volume of 300 µl with 10 µg of carrier DNA, 500 ng (unless otherwise specified) of plasmid DNA containing the wild type or mutated mGluR, and 10 million cells.
Determination of Inositol Phosphate (IP)
Accumulation--
Determination of IP accumulation in transfected
cells was performed as described previously after labeling the cells
overnight with [3H]myoinositol (23.4 Ci/mol, NEN Life
Science Products, France) (28). The stimulation was conducted for 30 min in a medium containing 10 mM LiCl and 1 mM
Glu. The basal IP formation was determined after a 30-min incubation in
the presence of 10 mM LiCl. The Glu degrading enzyme
glutamate pyruvate transaminase (1 unit/ml) and 2 mM
pyruvate 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 y = ((ymax ymin)/1 + (x/EC50)nH) + ymin) where EC50 is the
concentration of agonist giving a response equal to 50% of the
maximum, ymax and ymin
correspond to the maximal and minimal values, and
nH 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 µg of protein) were solubilized in
Laemmli sample buffer (2.5% (w/v) SDS, 25 mM Tris-HCl, pH
6.8, 5% (v/v) -mercaptoethanol, and 6.25% glycerol), resolved by
SDS-polyacrylamide gel electrophoresis (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). Chemiluminescent 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 characterization of the mGluR1 antibody (generous gift of Drs. V. Matarese and F. Ferraguti, Glaxo, Verona, Italy) raised against a chimeric protein 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 for 1 h at room temperature 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 fluorescein-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 t test or analysis of variance (Fisher's PLSD test).
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RESULTS |
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As shown in Fig. 1, several functional differences were observed 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 activation 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 be 5 s in 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 ± 28 nA (n = 111) for mGluR1a and 385 ± 35 nA (n = 140) 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 expressing mGluR1c (Figs. 1b and 2a). Glu stimulated IP formation to similar extents in cells expressing mGluR1a or mGluR1c, but the EC50 value for Glu was smaller when determined in mGluR1a-expressing cells than in cells expressing mGluR1c (1.08 ± 0.12 µM (nH = 1.03 ± 0.14; n = 8) and 5.67 ± 0.89 (nH = 1.13 ± 0.20; n = 8) (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).
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To identify the sequence element within the mGluR1
carboxyl-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 mGluR11139, a stop codon was
introduced at position 1139 so that the last 60 amino acids including a
large number of serine and threonine residues and a PDZ interacting sequence (17) were removed. In mGluR1
1093, an additional segment rich in acidic residues was removed. Finally, an additional truncated receptor mGluR1
879 with a carboxyl-terminal intracellular tail shorter than that of mGluR1c was also constructed (Fig. 3).
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The coupling to PLC of these truncated receptors was first analyzed
after expression in Xenopus oocytes. In oocytes expressing mGluR11139, mGluR1
1093 as well as in oocytes expressing
mGluR1
879, Glu induced fast responses similar in shape to those
measured in oocytes expressing mGluR1a (Figs. 2b and
4a). These truncated receptors were also expressed in HEK
293 cells. In these cells all truncated and wild type receptors
stimulated IP formation to a similar extent when activated with Glu
(data not shown). All truncated receptors also exhibited constitutive
activity like mGluR1a (Figs. 2b and 4b). Taken
together, these results indicate that the truncated receptor
mGluR1
879, which possesses a carboxyl-terminal intracellular 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 residues of mGluR1c
are responsible for the specific functional properties of this short
mGluR1 variant.
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To examine whether the carboxyl-terminal end of mGluR1c was sufficient to explain its functional properties, we exchanged the carboxyl-terminal domain of the other PLC-coupled mGluR (mGluR5a) with that of mGluR1a or mGluR1c taking advantage of a conserved SphI site in the mGluR1 and mGluR5 sequences (Figs. 2c and 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. 2c and 6b). In contrast, the chimeric mGluR5/1c receptor with the carboxyl-terminal tail of mGluR1c induced slowly developing responses in oocytes (Figs. 2c and 6a) and had reduced constitutive activity (Figs. 2c and 6b). Moreover, when various concentrations of Glu were used to stimulate IP formation in cells expressing these chimeric receptors, a lower EC50 value was measured with mGluR5/1a-expressing cells than with cells expressing mGluR5/1c (0.74 ± 0.25 µM (nH = 0.62 ± 0.12; n = 4) and 4.39 ± 1.13 (nH = 1.05 ± 0.20; n = 4) (p < 0.02), respectively) (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 properties of mGluR1c.
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The carboxyl-terminal sequences of mGluR1b, mGluR1c, and mGluR1d
display no sequence homology after the splice junction site (Fig. 5).
They share however similar functional properties (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 (mGluR1939) with a
carboxyl-terminal tail slightly longer than that of mGluR1d shares
functional properties with these short receptor variants: it induces
slow responses 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 which Arg-878, Lys-879, and Lys-880 were replaced by Met, Ala, and
Ala, respectively. These mutated receptors were named mGluR1a
and
mGluR1c
, 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, mGluR1c
induced fast current responses when expressed in
Xenopus oocytes (Figs. 2d and
7a) and displayed
agonist-independent constitutive activity (Figs. 2d and
7b) and an increased potency of glutamate (6.39 ± 1.76 µM (nH = 1.03 ± 0.21;
n = 4) and 0.97 ± 0.53 (nH = 0.96 ± 0.21; n = 4) (p < 0.01)
for mGluR1c and mGluR1c
, respectively) (Fig. 7c) even
though it appeared to be expressed at a lower level than the wild type
mGluR1c (Fig. 7d).
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To verify that all wild type and mutated mGluR1 receptors were
correctly targeted to the plasma membrane, immunostaining of HEK 293 cells expressing these receptors was performed using an antibody
directed against their conserved extracellular 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 observed in cells expressing mGluR1a, mGluR1879,
or mGluR1c
.
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DISCUSSION |
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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).
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The functional properties due to this cluster of basic residues in the
short mGluR1 variants include a slow activation of the Cl
current in Xenopus oocytes, a very low or no constitutive
activity, and a low potency of Glu in stimulating IP formation. These
properties may be explained if this sequence element impairs the
expression of the receptor in the plasma membrane. 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 indicate 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 mGluR1c
, 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
receptor 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 mutated receptors.
This may be explained if a high level of expression 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 porcine
kidney epithelial (LLC-PK1) cells, which expressed fewer
receptors2 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 hormone receptor causes
constitutive activity (41). The last 12 residues of bovine rhodopsin
have also been proposed to operate 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 domain of
the avian -adrenergic receptor increases its activity (46). Finally,
removal of the carboxyl-terminal tail of the human parathyroid hormone
receptor suppresses its G-protein coupling selectivity, suggesting that
this region inhibits coupling 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 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 recognition
site as proposed for rhodopsin, or to the interaction of these basic
residues with the membrane phospholipids, preventing the positive
action of the amino-terminal part of the intracellular tail on
G-protein coupling (8, 10, 48). Alternatively, this sequence element may stabilize the receptor in the inactive state. Another possibility is that, like the avian -adrenergic receptor (46), the carboxyl terminus of the short mGluR1 variants reduces their accessibility to
G-proteins possibly 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, mGluR1
879,
or in cells expressing the mutated mGluR1c
, suggesting that the
clustering of the receptors and their low PLC coupling efficacy are
related.
Although mGluR1a also contains this cluster of basic residues, 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/threonine-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 suggests that the long intracellular tail of mGluR1a has numerous other regulatory roles such as the control of receptor desensitization and/or down-regulation, a possible role for the serine/threonine-rich carboxyl-terminal end of mGluR1a, and interaction 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 receptors 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 prevent the action of the inhibitory domain. Within this part of the long intracellular domain of mGluR1a is the proline-rich domain. 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 Ca2+- and K+-channel modulation or phospholipase A2 and adenylyl cyclase activation which may be mediated by G-proteins different from the Gq type.
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ACKNOWLEDGEMENTS |
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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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FOOTNOTES |
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* 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. The 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 Chemistry, 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{at}ccipe.montp.inserm.fr.
1 The abbreviations used are: mGluRs, metabotropic glutamate receptors; 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.
2 J. Gomeza, S. Mary, L. Prézeau, J. Bockaert, and J.-P. Pin, unpublished results.
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REFERENCES |
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