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

Sophie Mary, Jesus GomezaDagger , Laurent Prézeau§, Joël Bockaert, and Jean-Philippe Pin

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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Although they possess 7 transmembrane domains, the G-protein-coupled metabotropic glutamate receptors (mGluRs)1 (1-3), Ca2+-sensing (4), and gamma -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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 mGluR1Delta 1139 and mGluR1Delta 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 mGluR1Delta 1139) and 5'-CTC CTT CTA GAA GGT GCT CAG GTG CAG GGG-3' (for mGluR1Delta 1093). The resulting PCR products were cut by SphI and XbaI and subcloned into pmGluR1a cut with the same enzymes. For the construction of mGluR1Delta 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.

The truncated mGluR5Delta (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 (5'-TG ACT TGG GCC CAG TAG GAT CCG AGT ACC CGG-3') and an antisense primer located downstream of the second ApaI site (position 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 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 mGluR1aDelta beta and mGluR1cDelta beta Receptors-- mGluR1aDelta beta 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 mGluR1cDelta beta 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.

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, chimeric, 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 (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) beta -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).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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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 µM Glu on oocytes expressing mGluR1a (left) or mGluR1c (right) and voltage-clamped at -70 mV. Scale bars: vertical, 200 nA; horizontal, 20 s. In the bottom graph, the time-to-peak values of individual responses are plotted against the maximal current amplitude measured upon Glu (300 µM) application (Imax). 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 mM Glu was 100 ± 2, 1078 ± 120, and 1075 ± 110 in mock-transfected cells and in cells expressing mGluR1a or mGluR1c, respectively (percent of the basal IP formation in mock-transfected cells, means ± S.E. of 26, 21, and 17 triplicate determinations). Values correspond to the [3H]IP produced divided by the amount of radioactivity in the membranes and are means ± S.E. of n independent 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 (open circle ) or mGluR1c (bullet ). Values are expressed as percentage of the maximal effect of Glu over basal activity and are means ± S.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 ± 9% (n = 4) that of mGluR1c.


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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 mGluR1aDelta beta and mGluR1cDelta beta are indicated by a thin white line. In the second column are the means ± S.E. of the time-to-peak values in seconds for responses smaller than 1000 nA obtained upon stimulation with 300 µM Glu 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.

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 mGluR1Delta 1139, 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 mGluR1Delta 1093, an additional segment rich in acidic residues was removed. Finally, an additional truncated receptor mGluR1Delta 879 with a carboxyl-terminal intracellular tail shorter than that of mGluR1c was also constructed (Fig. 3).


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Fig. 3.   Schematic representation of the carboxyl-terminal tail of mGluR1a and mGluR1c mutants. The positions where stop codons were introduced to generate the truncated mutants mGluR1Delta 1139, mGluR1Delta 1093, and mGluR1Delta 879 are indicated by vertical bars. Serine and threonine residues in the last 60 carboxyl-terminal sequence are represented by filled diamonds (black-diamond ). Glutamate and aspartate residues between position 1093 and 1139 are represented by thick circles (), and proline residues between position 879 and 1093 are represented by plus signs (+). Putative phosphorylation sites are indicated by the filled circles (bullet ) attached to the sequence. Specific mGluR1c residues are in black.

The coupling to PLC of these truncated receptors was first analyzed after expression in Xenopus oocytes. In oocytes expressing mGluR1Delta 1139, mGluR1Delta 1093 as well as in oocytes expressing mGluR1Delta 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 mGluR1Delta 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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Fig. 4.   The truncated receptor mGluR1Delta 879 with a carboxyl-terminal tail shorter than that of mGluR1c has functional properties different from those of mGluR1c, but similar to those of mGluR1a. a, mGluR1Delta 879 induces fast responses in Xenopus oocytes. Schematic representation of the mutant and typical current trace obtained upon application of 300 µM Glu on oocytes expressing mGluR1Delta 879 and voltage-clamped at -70 mV. Scale bars: vertical, 200 nA; horizontal, 20 s (left). On the right, the time-to-peak values of individual responses are plotted against the maximal current amplitude measured upon Glu (300 µM) application (Imax). b, truncated mGluR1Delta 879 mutant stimulates IP production in the absence of agonist. Basal IP formation occurs in mock-transfected cells and in cells expressing mGluR1c and mGluR1Delta 879. In these cells, the maximal IP formation induced by 1 mM Glu was 100 ± 2, 1075 ± 110, and 1326 ± 483 in mock-transfected cells, in cells expressing mGluR1c, and in cells expressing mGluR1Delta 879, respectively. Values correspond to the [3H]IP produced divided by the amount of radioactivity in the membranes and are means ± S.E. of 6-17 independent experiments performed in triplicate. c, truncated mGluR1Delta 879 mutant is expressed at a level similar to mGluR1c. Membranes prepared from mock-transfected cells or cells transfected with 0.5 µg of plasmid containing the mGluR1c or mGluR1Delta 879 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).

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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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 mGluR1Delta 879 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 mGluR5Delta is also indicated.


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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 ± S.E. of 4 independent experiments performed in triplicate. In c, mGluR5/1c displays significantly reduced IP production in the absence of agonist as compared with mGluR5/1a. The maximal IP formation induced by 1 mM Glu was 100 ± 2, 991 ± 160, and 880 ± 232 in mock-transfected cells, in cells expressing mGluR5/1a, and in cells expressing mGluR5/1c, respectively. Values are means ± S.E. of 4 independent experiments performed in triplicate.

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 (mGluR1Delta 939) 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 mGluR1aDelta beta and mGluR1cDelta beta , 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, mGluR1cDelta beta 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 mGluR1cDelta beta , 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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Fig. 7.   Mutation of 3 basic residues in the carboxyl-terminal tail of mGluR1c generates mGluR1cDelta beta with PLC-coupling properties of mGluR1a. In a, mGluR1cDelta beta induces faster responses in Xenopus oocytes than mGluR1c. In b, mGluR1cDelta beta but not mGluR1c stimulates IP production in the absence of agonist in HEK 293 cells. The maximal IP formation induced by 1 mM Glu was 100 ± 7, 1061 ± 243, and 927 ± 198 in mock-transfected cells, in cells expressing mGluR1c, and in cells expressing mGluR1cDelta beta , respectively (means ± S.E. of 11 experiments performed in triplicate). In c, mGluR1cDelta beta exhibits a higher affinity for the agonist glutamate than does mGluR1c. Values are means ± S.E. of 5 independent experiments performed in triplicate. d, relative levels of expression of mGluR1c and mGluR1cDelta beta 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 mGluR1cDelta beta cDNAs and the mGluR proteins (top panel) and actin (bottom panel) were detected using selective antibodies. The determination of the ratio intensity of the mGluR band over that of actin (using Molecular Imager quantification) indicates that the intensity of the mGluR1cDelta beta band was 48 ± 14% (n = 3) that of mGluR1c.

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, mGluR1Delta 879, or mGluR1cDelta beta .


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Fig. 8.   All wild-type and mutant mGluR1 receptors are targeted to the plasma membrane. HEK 293 cells transiently transfected with plasmid DNA encoding mGluR1a (a), mGluR1c (b), mGluR1Delta 879 (c), or mGluR1cDelta beta (d) receptors were fixed with 4% paraformaldehyde. The mGluR1 receptor proteins were detected using a polyclonal antibody directed against the amino-terminal domain and a secondary antibody coupled to fluorescein.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Fig. 9.   Schematic representation of the action of the cluster of basic residues in the carboxyl-terminal domain of mGluR1c and its inhibition by the proline-rich region of the mGluR1a carboxyl-terminal domain.

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 mGluR1cDelta beta , 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 beta -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 beta -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, mGluR1Delta 879, or in cells expressing the mutated mGluR1cDelta beta , 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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