Extreme C Terminus of G Protein α-Subunits Contains a Site That Discriminates between Gi-coupled Metabotropic Glutamate Receptors*

Metabotropic glutamate receptors (mGlu receptors), the Ca2+-sensing receptor, γ-aminobutyric acid type B receptors, and one group of pheromone receptors constitute a unique family (also called family 3) of heptahelical receptors. This original family shares no sequence similarity with any other G protein-coupled receptors. The identification and comparison of the molecular determinants of receptor/G protein coupling within the different receptor families may help identify general rules involved in this protein/protein interaction. In order to detect possible contact sites important for coupling selectivity between family 3 receptors and the G protein α-subunits, we examined the coupling of the cyclase-inhibiting mGlu2 and mGlu4 receptors to chimeric αq-subunits bearing the 5 extreme C-terminal amino acid residues of either Gαi, Gαo, or Gαz. Whereas mGlu4 receptor activated all three chimeric G proteins, mGlu2 receptor activated Gαqi and Gαqo but not Gαqz. The mutation of isoleucine −4 of Gαqz into cysteine was sufficient to recover coupling of the mutant G protein to mGlu2 receptor. Moreover, the mutation of cysteine −4 of Gαqointo isoleucine was sufficient to suppress the coupling to mGlu2 receptor. Mutations at positions −5 and −1 had an effect on coupling efficiency, but not selectivity. Our results emphasize the importance of the residue −4 of the α-subunits in their specific interaction to heptahelical receptors by extending this finding on the third family of G protein-coupled receptors.

Transduction of extracellular signals to intracellular responses via G protein-coupled receptors (GPCRs) 1 includes activation of the receptor and subsequent regulation of effectors via trimeric G proteins. A variety of heterotrimeric G proteins have different target proteins (1). Accordingly, the intracellular response depends on the G protein subtypes (among those located in the proximity of the receptor) that can be activated by the receptor (selectivity). Identification of the molecular basis of receptor/G protein coupling selectivity is therefore of interest since this determines the transduction mechanism of these receptors.
Three major families of GPCRs can be defined, and the members of each family share no sequence similarity with receptors from the other families. Receptors homologous to rhodopsin (receptors for catecholamines, acetylcholine, certain peptides, and glycoproteins) constitute the first family (family 1) which is to date the best characterized one. Family 2 receptors are those homologous to the vasoactive intestinal peptide and the glucagon receptors. Family 3 receptors comprise the metabotropic glutamate receptors (mGlu receptors) (2), the ␥-aminobutryric acid, type B receptors (3), the Ca 2ϩ -sensing receptor (4), and a recently discovered new family of putative pheromone receptors (5,6). The unique feature of family 3 GPCRs is a large extracellular N terminus that forms the ligand binding site (7,8). Common to all three families are some structural elements including seven transmembrane domains so that all GPCRs are also called heptahelical receptors.
Molecular determinants involved in the coupling between family 1 GPCRs and G proteins have been studied (9 -11). Specific motifs in the third intracellular loop (in most cases) and the second intracellular loop (in some cases) of these receptors have been shown to determine which G protein subtypes will be activated. Although the receptors probably contact both the ␣-subunit, and the ␤␥ dimer of the G protein (12)(13)(14), the direct interaction with the ␣ subunit is presumed to play a critical role in the coupling specificity. On the G protein ␣-subunit several elements have been shown to determine specificity of coupling to family 1 receptors (9,10,(15)(16)(17)(18)(19)(20). Among these, the extreme C terminus was shown to play a critical role by interacting with certain motifs within a cavity formed by the second and third intracellular loops of the receptor (9, 11, 20 -23).
Comparison of the molecular determinants of receptor/G protein coupling between members of the different GPCR families should help to establish some general principles for this interaction. Accordingly, we recently started to identify the structural determinants of G protein coupling in mGlu receptors that are used here as representatives of the family 3 GPCRs. Three groups of mGlu receptors have been defined based on their sequence similarity and G protein-coupling selectivity (2,24). In these receptors, the first and third intracellular loops (i1 and i3) are short with low variability. Major sequence differences are found in the second intracellular loop (i2). The highly variable intracellular C termini play a role in G protein interaction but probably not in a discriminatory manner (25)(26)(27)(28).
It was shown that the C terminus of the G protein ␣-subunit plays a critical role in the recognition of the G o /G i -coupled groups II and III mGlu receptors (29,30). The aim of the present study was to identify the important residues in the C-terminal end of the G protein ␣-subunit controlling the selectivity.

EXPERIMENTAL PROCEDURES
Materials-Chemicals including glutamate were obtained from Sigma (L'Isle d'Abeau, France) unless otherwise indicated. Serum, culture media, and other solutions used for cell culture were from Life Technologies, Inc. (Cergy Pontoise, France). The plasmids expressing mGlu2 and mGlu4 receptors were as described previously (29). The hemagglutinin epitope-tagged G␣ q and chimeric G proteins (21) were kindly provided by Dr. Bruce Conklin (The Gladstone Institute, San Francisco, CA).
Construction of Epitope-tagged Receptors-The mGlu2 and mGlu4 receptors were tagged with MRGS-His 6 (MRGS: methionine, arginine, glycine, serine) epitope at the C termini. A pRK5 plasmid containing the sequence encoding the epitope MRGS-His 6 flanked by a unique NheI site at its 5Ј end and an in-frame TAA stop codon at its 3Ј end was first constructed. To generate this vector, the following oligonucleotides were annealed and introduced into the HindIII unique restriction site of the pRK5 plasmid: 5Ј-agc tag cat gag agg atc gca tca cca tca cca tca cta aca t-3Ј; 5Ј-tcg tac tct cct agc gta gtg gta gtg gta gtg att gtc ga-3Ј. The mGlu2 and mGlu4 receptor-coding sequences were then introduced by a polymerase chain reaction using the unique NheI restriction site so that the stop codon was replaced by alanine followed by serine and the MRGS-His 6 epitope. The sequence of the epitope-tagged receptors was verified using 17-25 mer primers and Sequenase with the dideoxynucleotide method (U. S. Biochemical Corp.). The resulting fusion protein is detectable using specific antibodies recognizing this epitope (Qiagen, Paris, France). Functional studies after co-expression with various chimeric G proteins, and determination of the EC 50 of at least three distinct agonists revealed no differences between the wildtype and the epitope-tagged receptors in our system (data not shown).
G Protein Mutagenesis-Point mutations were introduced by a polymerase chain reaction into the plasmids encoding hemagglutinintagged G␣ qo or G␣ qz using specific oligonucleotides and Vent DNA polymerase (New England Biolabs, Ozyme, Montigny-le-Bretonneux, France). Polymerase chain reaction products were subcloned into the original plasmids using XhoI and HindIII sites. The sequences of the mutated G protein ␣-subunits were verified by sequencing. For the nomenclature of the chimeras and mutants, see Fig. 3.
Determination of Inositol Phosphate (IP) Accumulation-The procedure used for the determination of IP accumulation in transfected cells was adapted from previously published methods (31,32). Cells were washed 2 to 3 h after electroporation and incubated for 14 h in Dulbecco's modified Eagle's medium-Glutamax-I (Life Technologies, Inc., Paris, France) containing 1 Ci/ml myo-[ 3 H]inositol (23.4 Ci/mol) (NEN, Paris, France). Cells were then washed three times and incubated for 1-2 h at 37°C in 1 ml of Hepes saline buffer (NaCl 146 mM, KCl 4.2 mM, MgCl 2 0.5 mM, glucose 0.1%, Hepes 20 mM, pH 7.4) supplemented with 1 unit/ml glutamate pyruvate transaminase (Boehringer Mannheim, Meylan, France) and 2 mM pyruvate (Sigma, Lisle d'Abeau, France). Cells were then washed again twice with the same buffer, and LiCl was added to a final concentration of 10 mM. The agonist was applied 5 min later and left for 30 min. The reaction was stopped by replacing the incubation medium with 0.5 ml of perchloric acid (5%), on ice (32). Supernatants were recovered and the IPs were purified on Dowex columns (31). Total radioactivity remaining in the membrane fraction was counted after treatment with 10% Triton X-100, 0.1 N NaOH for 30 min and used as a standard. Results are expressed as the amount of IP produced over the radioactivity present in the membranes. The doseresponse curves were fitted according to the equation y ϭ ((y max Ϫ y min )/1 ϩ (x/EC 50 ) n ) ϩ y min using the kaleidagraph program (Abelbeck Software, Reading, PA).
Immunoblotting Analysis-The cells were transfected and treated as described above. Cells cultured in one well were harvested for immunodetection, and the rest of the cells were used for IP assay. Protein samples (10 g/lane) were separated using SDS-polyacrylamide gel electrophoresis (33) and blotted onto a nitrocellulose membrane. Prior to the immunoblotting, total protein on the membranes was visualized with Ponceau S to confirm that the same amounts of protein were loaded on the gel. The receptors and G proteins were detected using primary monoclonal antibodies against MRGS-His 6 epitope (Qiagen, Paris, France) or hemagglutinin epitope (generous gift of Dr. B. Mouillac, Montpellier, France), respectively. ECL chemiluminescence system was used to stain the secondary antibodies (Amersham Life Sciences, Paris, France).

Differential Coupling of Groups II and III mGlu Receptors to
Phospholipase C via Chimeric G␣ Proteins-Group II (mGlu2 and mGlu3 receptors) and group III mGlu receptors (mGlu4, 6, 7, and 8 receptors) inhibit adenylyl cyclase activity in transfected cells via pertussis toxin-sensitive G proteins and are not capable of stimulating IP formation (2,29,30). Their coupling to chimeric G protein ␣-subunits, that correspond to G␣ q with the 5 C-terminal residues of G␣ i2 (G␣ qi ), G␣ o (G␣ qo ), or G␣ z (G␣ qz ) (34), can therefore be easily determined by measuring their capability of stimulating IP formation upon activation with glutamate (29). We previously reported that both group II and group III mGlu receptors coupled to G␣ qi and G␣ qo , but that G␣ qz was activated by group III receptors only (29,30). By using hemagglutinin-tagged G protein ␣-subunits (21,35) and receptors tagged with the MRGS-His 6 sequence at their C terminus, similar data were obtained (Fig. 1, a and b, Table I). The group II mGlu2 receptor stimulated phospholipase C when co-expressed with G␣ qi or G␣ qo but not when expressed alone or with G␣ q or G␣ qz , even though these G proteins were expressed at very similar levels (Fig. 2). Like mGlu2 receptor, the group III mGlu4 receptor activated G␣ qi and G␣ qo , but not G␣ q (Fig.  1, c and d). Only mGlu4, but not mGlu2, activated G␣ qz (Fig. 1,  c and d), even though these two receptors were expressed at comparable levels (Fig. 2). These data indicate that the differential coupling of mGlu2 and mGlu4 receptors to G␣ qz is not due to a difference in the level of expression of either the receptor or the G protein. This raises the question of the sequence element and amino acid residues responsible for this coupling selectivity.
Isoleucine Ϫ4 in the C Terminus of G␣ qz Prevents Coupling to the mGlu2 Receptor-As shown in Fig. 3, the G␣ chimeras used in our study vary in a span of 5 extreme C-terminal amino acid residues, among which three differ between G␣ qo and G␣ qz (at positions Ϫ1, Ϫ4, and Ϫ5). All the mutant G proteins were expressed at a similar level in transfected HEK 293 cells (Fig.  2).
Reciprocal exchange of the residue at position Ϫ5 or Ϫ1 between G␣ qo and G␣ qz did not change their coupling specificity toward mGlu2 and mGlu4 receptors (Fig. 4, a and c). In contrast, swapping the residue at position Ϫ4 between G␣ qo and G␣ qz (mutants G␣ qo CI and G␣ qz IC where isoleucine replaces cysteine and vice versa, respectively) resulted in the exchange of coupling properties of the G proteins toward the mGlu2 receptor (Fig. 4, a and b). The mGlu2 receptor activated G␣ qz IC upon activation with glutamate in a concentration-dependent manner, but not G␣ qo CI (Fig. 4b). As expected, both are stimulated by mGlu4 receptor (Fig. 4, c and d). The potency of glutamate in stimulating IP formation was determined in cells expressing mGlu2 or mGlu4 receptors with any of the mutated G proteins. As shown in Table I, when a glutamate-induced IP formation was detected, a similar potency was observed, whatever G protein was co-expressed with the receptor.
Tyrosine Ϫ5 in the C Terminus of G␣ Proteins Enhances Coupling to mGlu2 Receptor-Chimeric and mutated G proteins that are activated by mGlu2 and mGlu4 receptors show differences in the maximal effect of glutamate (Fig. 4, a and c). Mutation of residue Ϫ1 or Ϫ5 in G␣ qo or G␣ qz resulted in small changes in the maximal effect of glutamate, indicating that these positions influence the coupling efficacy to these receptors. Among the various chimeric and mutant G protein ␣-subunits tested, those which possess a tyrosine at position Ϫ5 and a cysteine at position Ϫ4 (G␣ qo GY and G␣ qz IC) allow a better coupling of mGlu2 receptor to phospholipase C (Fig. 4a). These same mutated G proteins also allow a very good coupling to phospholipase C of mGlu4 receptor (Fig. 4c) as well as other group II and III mGlu receptors (data not shown). This indicates that a tyrosine residue at position Ϫ5 is often better accepted by mGlu receptors than is a glycine residue. Changing the Ϫ1 residue alone does not dramatically modify the coupling efficacy of the G protein to the mGlu2 receptor. Tyrosine at position Ϫ1 is often less accepted by the mGlu4 receptor than is cysteine. For example, the cysteine Ϫ1 to tyrosine mutation of G␣ qz , or G␣ qz YG generates G␣ qz CY and G␣ qo CI, respectively, that are less efficient in coupling mGlu4 receptor to phospholipase C (Fig. 4c). Taken together, residues at the other positions Ϫ1 and Ϫ5 weaken or enhance coupling efficiency to mGlu2 and mGlu4 receptors.  1. Differential coupling of mGlu2 (a and b) and mGlu4 (c and d) receptors to chimeric G␣ q proteins bearing various Cterminal ends. Basal (^) and 1 mM glutamate-induced (3) IP formations were determined in HEK 293 cells co-expressing the epitopetagged mGlu2 (a) or mGlu4 (c) receptors with the hemagglutinin-tagged G protein ␣ q -subunit. The G␣ q protein and its chimeras with the 5 C-terminal amino acids from G␣ q G␣q), G␣ i2 (G␣qi), G␣ o (G␣qo), or G␣ z (G␣qz) were used. In b, cells co-expressing the mGlu2 receptor with G␣ qo (E) or G␣qz (q) were stimulated with increasing concentrations of glutamate. d, same as in b, with the mGlu4 receptor. In a and c, values correspond to the radioactivity in the IP fraction divided by the total radioactivity in the membranes. In b, values correspond to the glutamate-induced IP formation expressed as the percentage of the maximum response measured in cells expressing mGlu2 receptor and G␣ qo .

FIG. 2. Western blot analysis of expression levels of the receptors tagged with the MRGS-His 6 epitope and the G␣ q proteins
and their chimeras tagged with the hemagglutinin epitope in HEK 293 cells. 10 g of total membrane proteins from cells transfected with pRK5 vector (mock) or vectors encoding the indicated proteins were separated on SDS-polyacrylamide gel electrophoresis. a, bands that migrated on SDS-polyacrylamide gel electrophoresis in molecular mass range corresponding to that of mGlu2 and mGlu4 receptors (around 100 kDa) were detected with the MRGS-His 6 -specific antibodies. b, expression levels of the G␣ q and its chimeras and c, the point mutants of G␣ qo and G␣ qz as detected with the hemagglutinin epitopespecific antibodies.
FIG. 3. Sequences of the 5 extreme C-terminal amino acid residues of G␣ proteins used in this study. Activation of the indicated G protein ␣-subunit by mGlu2 or mGlu4 receptors is indicated on the right. Boxed residues are those which are distinct between G␣ qo and G␣ qz and that were mutated. The C-terminal end of G␣ 15 and its activation by mGlu2 and mGlu4 receptors is indicated for information (29). ϩϩ, more than 3-fold stimulation of IP formation by glutamate in cells expressing the indicated mGlu receptor and the G protein ␣-subunit; ϩ, 2-3-fold stimulation of IP formation by glutamate; 0, no significant increase in IP formation by glutamate.

DISCUSSION
The present data extend our knowledge on the importance of the last few C-terminal amino acid residues of the G protein ␣-subunit in the specific interaction with heptahelical receptors, including the metabotropic glutamate receptors from family 3 GPCRs. Usage of chimeric G protein ␣-subunits that do not differ in other portions of the proteins than the extreme C terminus allowed us to study specifically the relevance of this region. We show that a single residue (at position Ϫ4) on the extreme C terminus is crucial for chimeras of G␣ q proteins to distinguish group II from group III mGlu receptors. Residues at position Ϫ5 and at position Ϫ1 do not control specificity, but still play a role in coupling efficiency to mGlu receptors.
Several studies using different approaches indicated that the extreme C-terminal part of the G protein ␣-subunit has a critical role in specifying interactions with family 1 GPCRs (for reviews, see Refs. 9 -11). These include the coupling of chimeric G␣-proteins and receptors (15,21,34,36,37), alanine scanning mutagenesis (20), functional competition with the ␣-subunit C-terminal-derived peptides (23,38), direct interaction of such C-terminal peptides with receptors (16,39), or functional inhibition with antibodies directed against the C terminus of the ␣-subunit (for reviews, see Refs. 1 and 10). The present study extends our knowledge of the role of this portion of the ␣-subunit in the specific interaction with family 3 GPCRs. Moreover, our data indicate that a single amino acid residue within this sequence is sufficient to cause discrimination between group II and group III mGlu receptors. Cysteine at position Ϫ4 is compatible with a coupling of G␣ qi and G␣ qo to either mGlu2 or mGlu4 receptors, whereas in G␣ qz isoleucine at position Ϫ4 is compatible with a coupling to mGlu4 receptor, but prevents coupling to the mGlu2 receptor. In agreement with our obser-vation, post-translational modifications of residue Ϫ4 has been shown to play an important role in the interaction with family 1 GPCRs. In G i and G o , this position corresponds to the cysteine residue that is ADP-ribosylated by pertussis toxin, which prevents coupling of these G proteins to their receptors (for reviews, see Refs. 1 and 10). In G q and G 11 a tyrosine Ϫ4 has to be phosphorylated to allow coupling to family 1 as well as to the mGlu1a receptor from family 3 GPCRs (40). Residues at positions Ϫ4 and Ϫ3 in G i have been reported to play an important role in discriminating between G i -and G q -coupled receptors (15). Moreover, residues at positions Ϫ3 and Ϫ5 in G␣ qs chimeras play a key role in coupling to muscarinic and vasopressin receptors and that a residue at position Ϫ5 discriminates between these two receptors from family 1 GPCRs (37). The three-dimensional structure of a peptide corresponding to the C-terminal end of transducin bound to rhodopsin was determined by nuclear magnetic resonance (16). This study revealed the glycine residue at position Ϫ3 which is conserved in G i , G o , G z , and G t ␣-subunits is involved in a ␤-turn, a structure that likely favors the contact between the receptor and the side chain of the Ϫ4 residue.
It is interesting to note that in G␣ 15 , which is activated by both group II and group III mGlu receptors (29,30), there is an isoleucine at position Ϫ4, as in G␣ qz . However, the residue at position Ϫ3 in G␣ 15 is not a glycine, but an asparagine. This possibly changes the flexibility in this area and thus neutralizes the decisive discriminatory effect of the (Ϫ4) residue. Taken together, these observations are consistent with the same sequence and/or structural element of the G protein ␣-subunit C-terminal end for the specific interaction with either family 1 or family 3 receptors.
Such similarity in the molecular determinants of the G protein ␣-subunit involved in the specific recognition of family 1 and family 3 GPCRs was not expected. The C terminus of the G protein ␣-subunit is likely to fit into a cavity formed by the second and the third intracellular loops of family 1 GPCRs (9,11). The third intracellular loop of these family 1 receptors is the longest and often plays a discriminatory role toward the G␣ protein, while the second contains a highly conserved sequence (DRY) important for G protein activation (41). In contrast, the third intracellular loop of mGlu receptors is very short, highly conserved (10 conserved residues out of 13), and involved in G protein activation (42), whereas the second is longer (24 -26 residues), more variable (only 5 are conserved in all mGlu receptors), and involved in G protein recognition (26,28,42). It would therefore be of interest to examine how family 1 and 3 GPCRs could recognize the same features of the C terminus of the G protein ␣-subunits with such different intracellular loops.
The C-terminal end of the ␣-subunit is only one of several areas that is likely to contact the receptor directly, these also include the N-terminal end and the ␣43␤6 loop (9,10,(15)(16)(17)(18)(19)(20). Accordingly, G␣ 15 and G␣ 16 which share identical C-terminal ends, but mostly differ in their ␣43␤6 loop, coupled differently to group II and group III mGlu receptors (29,30). One may therefore propose that all portions of heterotrimeric G proteins that are in contact with the receptor influence the receptor interaction with (and activation of) G␣, and therefore the coupling has to be seen as an outcome of network coordination. Moreover the ␤␥ dimer interaction with various ␣-subunits might play a role in the coupling efficiency as well (12)(13)(14). When interactions at certain contact points are weak, absent, or negative, coupling between the receptor and G protein can still be possible if this is overcome by a strong interaction at some other points, or when such regions that weaken coupling are removed either from the receptor or from the G protein (see, for example Refs. 27, 43, and 44). FIG. 4. Isoleucine ؊4 in G␣ qz prevents coupling to mGlu2 receptor. Basal (^) and 1 mM glutamate-induced (3) IP formations were determined in HEK 293 cells co-expressing the mGlu2 (a) or mGlu4 (c) receptors with the G protein ␣ q -subunit chimeras G␣ qo , G␣ qz , and their mutants. In b, cells co-expressing mGlu2 receptors with reciprocal mutants at position Ϫ4 G␣qoCI (E) or G␣qzIC (q) were stimulated with increasing concentrations of glutamate. d, same as in b, with the mGlu4 receptor. In a and c, values correspond to the radioactivity in the IP fraction divided by the total radioactivity in the membranes. In b, values correspond to the glutamate-induced IP formation expressed as the percentage of the maximum response measured in cells expressing mGlu2 receptor and G␣ qz IC. In d, values correspond to the glutamateinduced IP formation expressed as the percentage of the maximum. Values are means Ϯ S.E. of four to eight (a and b) and four (c and d) independent experiments performed in triplicate.