The second intracellular loop of metabotropic glutamate receptors recognizes C-termini of G protein -subunits

Heptahelical receptor coupling selectivity to G-proteins is controlled by a large contact area that involves several portions of the receptor and each subunit of the G-protein. In the G-protein alpha subunit, the C-terminal 5 residues, the N terminus, and the alpha N-beta 1 and alpha 4-alpha 5 loops play important roles. On the receptor side, both the second and third (i2 and i3) intracellular loops as well as the C-terminal tail probably contact these different regions of the G-protein. It is now accepted that the C terminus of the alpha subunit binds in a cavity formed by the i2 and i3 loops. Among the various G-protein-coupled receptors (GPCRs), class III receptors that include metabotropic glutamate (mGlu) receptors greatly differ from the rhodopsin-like GPCRs, but the contact zone between these receptors and the G-protein is less understood. The C terminus of the alpha subunit has been shown to play a pivotal role in the selective recognition of class III GPCRs. Indeed, the mGlu2 and mGlu4 and -8 receptors can discriminate between alpha subunits that differ at the level of their C-terminal end only (such as Gqo and Gqz). Here, we examine the role of the i2 loop of mGluRs in the selective recognition of this region of the alpha subunit. To that aim, we analyzed the coupling properties of mGlu2 and mGlu4 or -8 receptors and chimeras containing the i2 loop of the converse receptor to G-protein alpha subunits that only differ by their C termini (Gqo,Gqz, and their point mutants). Our data demonstrate that the central portion of the i2 loop is responsible for the selective recognition of the C-terminal end of the alpha subunit, especially the residue on position -4. These data are consistent with the proposal that the C-terminal end of the G-protein alpha subunit interacts with residues in a cavity formed by the i2 and i3 loops in class III GPCRs, as reported for class I GPCRs.


Introduction
G-Protein Coupled Receptors (GPCRs) modulate specific signaling pathways depending on the subset of G-proteins activated. This requires positive interaction of certain parts of the GPCR with portions of the heterotrimeric G-protein. Recent progress has been made in defining the structure of G-proteins and functional studies using site-directed mutagenesis of receptors and/or G-proteins. This progress has provided increasingly more specific and precise descriptions of the contact sites between these proteins (1). From the G-protein α, β and γ subunits, probably both the Gα and the βγ dimer contact the receptors. The Gα subunit has a decisive role in discriminating between different receptor subtypes (2)(3)(4) and also between different functional states of the receptor (5,6). Several regions along the sequence of the Gα-proteins are involved in the selective coupling to GPCRs (1,7,8). The best characterized region is the extreme C-terminus where residues at positions -3 and -4 are decisive for coupling Gα proteins to some receptors (1,(9)(10)(11)(12)(13). Residue -4 in the Gi family of G-proteins is the cysteine residue which is ADPribosylated by pertussis toxin, a covalent modification that prevents the G-protein to interact with the receptor. In the Gq family, the residue -4 must be a bulky aromatic residue (14) or alternatively, this Tyr residue may be phosphorylated (15) to efficiently couple to the PLCactivating receptors. Conformational changes in the C-terminal structure upon coupling to the receptor may play a role in the activation process. Other regions of the α subunit determining coupling selectivity are the extreme N-terminus (16) and the region between α4 and α5 helices, that includes the L9 loop and β6 sheet (17). This latter region is involved in the coupling selectivity, probably by interacting directly with either class I receptors (rhodopsin-like family) during activation (8,(18)(19)(20)(21), or class-III GPCRs.
On the receptor side, several intracellular portions are involved in the selective coupling to G-proteins. These have been extensively studied in class-I GPCRs and include both the i2 and i3 loops, as well as the amphipathic helix, called H8 (and previously referred to as the i4 loop) and the C-terminal tail of the receptor (3,22). For example, studies using the splice variants of prostanoid receptors pointed to the C-terminus to be one region that discriminates between Gproteins (23). A study that employed chimeras between muscarinic and beta adrenergic receptors pointed to the i2 and i3 loops to control coupling selectivity (24). Within the class-III GPCRs, although most of these regions have been shown to control coupling efficacy (25)(26)(27), only the i2 loop has been found to play a pivotal role in coupling selectivity (25,28,29).
Surprisingly, several regions of both the G-protein α subunit and class-III receptors have been identified as being involved in coupling selectivity. However, which part of the receptor recognizes a specific part of the G-protein α subunit, remains to be elucidated. The present study was aimed at identifying the specific region of the G-protein α subunit that is recognized by the by guest on March 23, 2020 http://www.jbc.org/ Downloaded from i2 loop of mGluRs. Our data revealed that this i2 loop, and more precisely the central part of it, discriminates between G-protein α subunits that differ at the level of their extreme C-terminus, especially at position -4. This is consistent with what has been proposed for class-I rhodopsinlike receptors and support the idea that receptors from these two classes couple to G-proteins in a similar way.

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 Gibco BRL (Cergy Pontoise, France). The plasmids expressing mGlu receptor subunits and their chimeras were described previously (12). The Gαq proteins were kindly provided by Dr. Bruce Conklin (The Gladstone Institute, San-Francisco, USA). The generation of the plasmids expressing the subunits Gqo, Gqz, GqoCI and GqzIC was described previously (30).

Determination of inositol phosphates (IP) accumulation.
The procedure used for the determination of IP accumulation in transfected cells was adapted from previously published methods (31). Cells were washed 2 to 3 hours after electroporation and incubated with 1 ml Hepes buffer saline containing 1U GPT/ml and 2 mM pyruvate for 1h. After washing ones with Hepes buffer saline, LiCl was added to a final concentration of 10 mM. The agonist was applied 5 min later and left for 30 min at 37°C. Replacing the incubation medium with 0.5 ml perchloric acid (5%) stopped the reaction and the clusters were kept on ice for 30 minutes.
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 standard. Results are expressed as the percentage of IP produced over the radioactivity present in the membranes. The dose-response curves were fitted according to the equation y = ((y max -y min ) / 1 + (x / EC 50 ) n ) + y min using the Kaleidagraph program (Abelbeck Software, USA).

Construction of chimeric mGlu receptors
In order to be able to analyze the expression and correct plasma membrane targeting of the wildtype and chimeric receptors studied, mGlu2, mGlu4 and mGlu8 receptors were first epitope tagged at their amino-terminal end. For the insertion of either a HA or c-myc epitope at the amino terminus, a Mlu-I restriction site was introduced just after the signal peptide using the QuickChange TM technology (Stratagene, France). The coding sequence after the Mlu-I site was then introduced in place of the equivalent mGlu5 coding sequence in the pRKG5-NHA or pRKG5-Nmyc described previously (32). Using this strategy, the mGlu4 and mGlu8 receptors were tagged with a HA epitope whereas both HA and c-myc mGlu2 receptors were generated. In the case of the mGlu4 receptor, a MRGSHis 6 sequence was also introduced just before the stop codon. For these three receptors, as previously reported for either mGlu5 (32) or mGlu7 (33) receptors, the presence of the tag did not modify the functional expression and pharmacological properties of the receptors.
The sequence encoding the i2 loop (as defined in Fig. 1) within the mGlu2 and mGlu4 receptor cDNA were swapped using a modification of the PCR overlap methodology as described (28). The resulting subunits were called mGlu2/4i2 and mGlu4/2i2. The central region of the i2 loop (as indicated by the horizontal bar in Fig. 1) or only part of it (see Fig.10 and 11) was swapped between mGlu2 and mGlu8 receptor cDNAs using the QuickChange TM technology (Stratagene, France). The resulting subunits were called mGlu2/8i2 (mGlu2 with the i2-loop sequence of mGlu8) and mGlu8/2i2 (mGlu8 with the i2-loop sequence of mGlu2), mGlu2-1st (mGlu2, with the N-terminal portion of the central zone of mGlu8 i2), mGlu8-1st (mGlu8 with the N-terminal portion of the central zone of mGlu2 i2), mGlu2-VTA and mGlu8-AQR for the mGlu2 and mGlu8 receptors in which the VTA/AQR sequences were swapped. The correct replacement of the loop was checked by double strand DNA sequencing.

Quantification of cell surface receptors using ELISA
Determination of expression level of surface receptors was performed using an ELISA assay adapted from (34). Twenty four hours after transfection (10.10 6 cells), cells were fixed with 4% paraformaldehyde and then blocked with PBS + 5% FBS. After 30 minutes reaction with primary antibody (monoclonal anti-HA clone 3F10 (Roche) at 0.5µg/ml) in the same buffer, the goat Anti-Rat antibody coupled to horseradish peroxidase (Jackson Immunoresearch) was applied for 30 minutes at 1µg/ml. After intense washes with PBS, secondary antibodies were detected and quantified instantaneously by chemiluminescence using Supersignal® ELISA femto maximum sensitivity substrate (Pierce) and a Wallac Victor 2 luminescence counter.

Immunofluorescence assay on non-permeabilized cells
The cells were cultured and transfected with wild-type and chimeric mGlu receptors as described previously. After transfection, cell suspension in DMEM was poured on cover-slips placed in 6well clusters (Falcon, Paris, France). The day after transfection cells immobilized on cover-slips were washed twice with PBS (phosphate buffer saline) 37°C. Then cover-slips were incubated at 37°C with primary anti-HA (anti-HA-Roche-12CA5, mouse) and anti-cMyc (anti-cMyc-Roche-9E10, mouse) antibodies diluted 1/300 in DMEM without serum. After 1.5 hours, cells were washed three times 10 min with gelatin in PBS (2g/l) and fixed with 4% paraformaldehyde in PBS supplemented with 0.15M glucose. After washing three times with PBS-glycin (0.1M), cover-slips were incubated with secondary antibody (Cy TM 3-conjugated AffiniPure Goat Anti-Mouse IgG, Jackson ImmunoResearch Laboratoires) diluted 1/1000 in PBS-gelatin. The incubation was performed in the dark for 1.5 hour. The preparations were then washed three times 10 min with PBS-gelatine and fixed. Pictures were taken using fluorescent microscope the next day.

Differential coupling of Gqo and Gqz and their -4 point mutants to mGlu4, mGlu8 and mGlu2 receptors.
Many Gi/o-coupled GPCRs have been shown to activate chimeric G-proteins corresponding to Gαq in which the C-terminal 5 residues are replaced by those of either Gαi2, Gαo or Gαz. Since these chimeric G-proteins activate PLCβ, the effective coupling between the receptor and the chimeric G-protein can easily be assessed by measuring IP accumulation after receptor activation. Group-II (mGlu2,3) and Group-III (mGlu4,6,7 and 8) mGlu receptors couple to pertussis toxin-sensitive Gαi/o proteins in vivo. However, whereas group-III receptors (mGlu4, 7 and 8) couple to both Gqo and Gqz, mGlu2 as well as the group-II Drosophila mGluA receptor couple to Gqo only (35,36) ( Fig. 1). This differential coupling is not due to a difference in the level of expression of these Gα subunits as shown by our group in previous studies using western blot analysis (12) as well as by others (37). Moreover, this is in agreement with the recent demonstration that group-II mGlu receptors do not activate Gz in transfected superior cervical ganglion neurons (38). Residue -4 which is an Ile in Gαz and the PTX-ADP ribosylated Cys in Gαi and Gαo, is responsible for the selective coupling of Gαz to mGlu2 (12). Indeed, replacing this Ile by a Cys in Gαqz is sufficient to allow coupling to mGlu2, whereas the converse mutation (Cys to Ile in Gαqo) suppresses coupling to this receptor (12). As a positive control, all these various G-protein α subunits can be efficiently activated by either mGlu4 (12) or mGlu8 receptors (Fig. 1).

Sequence differences of the intracellular loops of mGlu2, mGlu4 and mGlu8 receptors
Analysis of the intracellular portions of these receptors revealed highly conserved and short intracellular loops 1 and 3. Within the i1 loop, a single residue is different between group-II and group-III mGluRs, and in the i3 loop, only two positions are clearly different (see (35)). In contrast, the i2 loop is more variable (Fig.2a), and the C-terminal tail is the less conserved region.
We therefore decided to examine the role of the i2 loop in the specific recognition of the Cterminal tail of the α-subunit, and more specifically residue -4. To that aim, we constructed a series of chimeric receptors in which the i2 loop or a portion of it has been swapped between mGlu2 and either mGlu4 or mGlu8 receptors (Fig.2b) . These constructs also include a c-myc or HA epitope at the N-terminal end, just after the signal peptide. We previously reported that the presence of such an epitope at an equivalent position in either mGlu5 (32,39), mGlu7 (33,40) or GABA B (41,42) receptors affect neither their functional expression in HEK293 cells and in neurons, nor their pharmacology.

Sequence within the i2 loop recognizes the extreme C-terminus of G subunit
In order to examine the role of the i2 loop of mGlu receptors in the selective recognition of the C-terminal tail of the G-protein α subunit, the coupling of mGlu2/4i2 to Gqo and Gqz was first examined. As shown in Fig.3a, these receptors were expressed at a similar level at the cell surface, as revealed using an ELISA assay performed on intact cells (Fig.3a). The chimeric mGlu2/4i2 was found to activate both G-proteins, like mGlu4, but unlike mGlu2 that did not activate Gqz efficiently (Fig.3b). On these 3 receptors, when glutamate was active, its effect was dose-dependent and always displayed a similar EC50 value (Fig.4). This mGlu2/4i2 receptor retains the pharmacological properties of the mGlu2 receptor, since it is potently activated by the group-II selective agonists LY354740 and 2R,4R-APDC, but not by the group-III agonist DL-AP4 (Fig.5). These data highlight the i2 loop of mGlu4 as being the structure that enables coupling to Gqz. Unfortunately, we were unable to obtain large enough responses in cells expressing the converse chimera, mGlu4/2i2 with any of the tested G-proteins.
We decided to pursue our analysis using this receptor, because we always obtained robust and reproducible responses with the mGlu8 receptor that shares the same G-protein coupling selectivity with mGlu4 (36) (Fig.1), To this aim, the chimeric receptor subunits mGlu2/8i2 and mGlu8/2i2, in which only the central portion of the i2 loop has been swapped, were tested for their ability to activate Gqo and Gqz. As depicted in Fig.2, the central portion of the i2 loop contains most of the residues that differ between group-II and group-III mGluRs. When expressed in HEK293 cells, these constructs were correctly targeted to the cell surface (Fig.6), and found at a same density (Fig. 7a). As shown in Fig.7b, the mGlu2/8i2 couples well to Gqz, like the wild-type mGlu8 (Fig.1b), whereas the mGlu8/2i2 coupling to this G-protein is largely reduced compared to that obtained with the wild-type mGlu8. The low effective stimulation of the IP formation in cells expressing mGlu2/8i2 and Gqz challenged with 1 mM glutamate was not due to a large right shift of the glutamate dose-response curve. As shown in Table 1 and Fig. 8, the EC50 value measured in these cells was in the 10 µM range, only 3 times higher than that measured with Gqo.

The central portion of the i2 loop recognizes the -4 residue of the G subunit
The coupling of mGlu2/8i2 and mGlu8/2i2 to GqoCI and GqzIC was tested to analyze the role of the central portion of the i2 loop in the selective recognition of the -4 residue. . As shown in Fig. 7b and 8 The VSA/AQR segment plays an important role in the recognition of the C-terminus of the G subunit As illustrated in Fig.2, there are two tripeptides within the central portion of the i2 loop of group-II and group-III mGluRs that share no similarity. In order to identify the specific role of these two regions in the selective coupling to Gz C-terminus, these regions were swapped individually between mGlu2 and mGlu8 receptors. First we verified that under our experimental conditions, all these mutant receptors were expressed at a similar level at the cell surface (Fig. 9).
When co-expressed with the chimeric α-subunits, mGlu2-1st and mGlu8-1st were found to activate the same G-proteins as the wild-type mGlu2 and mGlu8 respectively. Interestingly, replacing the AQR motif of mGlu2 by VTA is sufficient to allow the resulting mutant mGlu2-VTA to activate Gqz (Fig. 10). The reverse chimera mGlu8-AQR still significantly activate Gqz, but to a lower extent than the wild-type mGlu8 (Fig. 10).

Discussion
Previous studies have identified that the 5 C-terminal residues of the G-protein α-subunit play important roles in determining the coupling not only to class-I (3,4) but also to class-III GPCRs (12,35). However, other regions of the G-protein α-subunit also play a role in the selective interaction to class-III GPCRs (30). On the receptor side, the second intracellular loop of the class-III GPCRs has been reported to be the main determinant controlling G-protein coupling selectivity (25)(26)(27)(28), although in this case also, other regions such as the C-terminal tail are involved. The present study demonstrates the pivotal role played by the i2 loop, and especially the central portion of it, in the selective recognition of the extreme C-terminal end of the α-subunit, and more precisely residue -4.
The crystal structure of both a 7TM receptor (rhodopsin) (43) and several states of different G-proteins have been solved (44). Several regions of both receptors and G-proteins have also been shown to be part of the contact area between these two proteins (3,4,45). However, the precise positioning of the G-protein on the receptor remains to be elucidated. Even though there is growing evidence indicating GPCRs may function as dimers, it is still not known whether both 7TM proteins contact a unique G-protein (45,46), or whether both 7TM proteins contact a single G-protein as recently suggested for rhodopsin (47), and shown for the LTB4 receptor (48). The crystal structure of a receptor-G-protein complex will be necessary to clarify this important issue. However, in the meantime, other approaches may help propose models of this interaction.
Using biochemical methods, as well as specific cross-linkers, the i3 loop of rhodopsin was shown to be in close proximity to both C-and N-terminal parts of transducin (49,50). Mutagenesis experiments aimed at identifying the regions involved in the selectivity of muscarinic receptor coupling to G-proteins also identified the i3 loop as well as the i2 loop as being involved in the recognition of the 5 C-terminal residues of the α-subunit (11,51). According to these and other data, the C-terminal peptide of the α-subunit has been proposed to interact in a cavity formed by the i2 and i3 loop of the class-I GPCRs (3,4).
Compared to the GPCRs from the other classes, class-III GPCRs are unique, not only because of their large N-terminal domain containing the agonist binding site, but also because of their very short (30 residues at most for i2) and highly conserved intracellular loops. Among these, i3 is the most conserved within all class-III GPCRs, and i2 the most variable, consistent with this loop playing a critical role in G-protein coupling selectivity. This is in contrast to the class-I GPCRs in which the i3 loop is often the longest and more variable loop, whereas i2 contains the highly conserved DRY motif. Interestingly, it has recently been reported that the i3 loop of rhodopsin can be replaced by the i2 loop of a class-III GPCR (mGlu6) without preventing G-protein coupling (29). In contrast, no coupling could be observed if the i3 loop is replaced by the i3 loop of mGlu6. This suggests that class-III and class-I GPCRs do not contact G-proteins the same way, although both classes of receptors recognize the same determinants of the G-protein α-subunit. However, our data indicate that the C-terminal end of the G-protein αsubunit is recognized by the i2 loop, consistent with the possibility that this part of the αsubunit also interacts in a cavity lined by the i2 and i3 loops in class-III GPCRs.
In class-I muscarinic receptors, the C-terminal end of the i3 loop that corresponds to the intracellular portion of transmembrane helix 6 (TM6) has been shown to play a major role in the selective recognition of the C-terminal end of the α-subunit (11,37). The equivalent region of the class-III GPCRs is also likely to be involved in an alpha helix that prolongs TM6 in the intracellular side of the receptor. However, the region corresponds to the NFNEAKxI motif conserved in all class-III GPCRs (52) and is therefore unlikely involved in G-protein coupling selectivity. In a 3D model of the heptahelical domain of the mGlu receptors (generated based on the crystal structure of rhodopsin, Dr. Gilles Labesse, Montpellier, France), the residues GGA (mGlu2) and EQG (mGlu4 or 8) (Fig.2) are just at the bottom of TM3, and in very close proximity to the bottom of TM6, facing a putative binding pocket formed by i2 and i3. However, they are not directly facing the NFNEAKxI motif but are located deeper in the cytoplasm, an observation that would not be consistent with the C-terminal end of the Gα-subunit being in close proximity to the NFNEAK motif. In agreement with this proposal, swapping this region between mGlu2 and mGlu8 did not change their specificity of interaction with the G-proteins tested (Fig.11). Indeed, in our 3D model, the residue of the i2 loop that is different between mGlu2 and mGlu8 and closer to this motif, is the A658 (mGlu2) or H674 (mGlu8) that is also not responsible for the recognition of the -4 residue of the α-subunit, according to our data. In agreement with this finding, this residue is not directed towards TM6, but rather towards the lipids. Another tripeptide segment that mostly differs between group-II and group-III mGlu receptors (AQR / V(S/T)(A/P)) aligns with a segment of rhodopsin, the structure of which has not been solved. Accordingly, it cannot be introduced with confidence in our 3D model. This is unfortunate because our data point to this short segment as playing an important role in the recognition of the C-terminus of the Gα subunit. Indeed, swapping this tripeptide between mGlu2 and mGlu8 allows the resulting mGlu2-VTA to activate Gqz, and decreases the efficacy of mGlu8-AQR to activate this G-protein (Fig.11). However, the AQR motif did not suppress coupling to Gz when in the mGlu8 i2 loop environment. This may result from a different position of one of the three side chains of this tripeptide when in the i2 loop of mGlu2 rather than in that of mGlu8. Although we did not analyze further the role of the individual residues, it is likely that the Arg residue with its positive charge does not tolerate the binding of a C-terminal tail with an Ile at position -4, whereas replacing this Arg by an Ala in mGlu8 is compatible with the Cterminal end of Gz. The side chain of Arg is long and its conformation largely depends on its environment. This can result in the long hydrophobic side chain being more exposed and ready to better accept the Ile-4 of the G-protein. As such, a role for this residue fits with our observation that the AQR sequence within the i2 loop of mGlu8 is still compatible with the activation of Gqz, though with a lower efficacy. More work using either modeling studies of structural analysis of the intracellular loops of mGluRs will be required to solve this issue.
Taken together, the present data are in agreement with the possible interaction of the Cterminal tail of the G-protein α-subunit in a cavity lined by i2 and i3 loops of class-III GPCRs.
However, other possibilities exist but more data will be necessary to demonstrate that these two classes of receptors couple similarly to G-proteins.   (12). N.D. not determined, n.e.: no effect.