The Heptahelical Domain of GABA_B2 Is Activated Directly by CGP7930, a Positive Allosteric Modulator of the GABA_B Receptor*

The γ-aminobutyric acid, type B (GABA_B)¹ receptor is a G-protein-coupled receptor (GPCR) activated by the most abundant inhibitory neurotransmitter of the central nervous system, GABA. This receptor is involved in numerous physiological processes via the regulation of both GABAergic and glutamatergic synapses at either the pre- or post-synaptic level (1). Accordingly, GABA_B receptors are involved in various types of epilepsy, nociception and drug addiction, and spasticity associated with multiple sclerosis (2). Although it has been described pharmacologically for 20 years, only in 1998 was the first GABA_B receptor (GABA_B1) cloned (3). It belonged to the class-III of the GPCR superfamily together with the metabotropic glutamate (mGlu), the calcium-sensing, and some pheromone and taste receptors (4). In addition to the typical GPCR heptahelical domain (HD), GABA_B1 possesses a large extracellular domain (ECD), similar to most other class-III GPCRs (4). In contrast to the rhodopsin-like receptors (class-I GPCRs), the ligand binding site of class-III GPCRs is located within their large ECD in the so-called Venus flytrap module. Indeed, agonists bind within a cleft that separates the two lobes of the Venus flytrap module and stabilize a closed active conformation. This has been illustrated recently by the crystal structures of the mGlu1 ECD that have been solved both in the absence and presence of agonist (5) and confirmed in the case of GABA_B1 by multiple mutagenesis studies (6–8).

However, to form a receptor able to activate G-proteins efficiently, GABA_B1 needs to be associated with a homologous protein called GABA_B2 (9–13). GABA_B receptor then was the first described obligatory heterodimeric receptor. Several studies unraveled some specific roles dedicated to each subunit. First, GABA_B2 takes GABA_B1 to the cell surface, probably by masking a retention signal located in the GABA_B1 C-terminal tail (14–16). Second, GABA_B1 Venus flytrap module, but not that of GABA_B2, binds all of the known GABA_B agonists and antagonists, whereas GABA_B2 HD is critical for G-protein activation (17–20). Third, there are complex allosteric interactions between the ECD and the HD of both subunits (20–22), leading to optimal agonist affinity and coupling efficacy.

Although the co-expression of both GABA_B1 and GABA_B2 appears to be required for an efficient activation of G-proteins, some studies (9, 23) report a possible functioning of one GABA_B subunit independently of the other. In support of a functional role of GABA_B2 in the absence of GABA_B1, homodimeric GABA_B2 receptors have been observed at the surface of heterologous cells (24) and homodimeric GABA_B2 HDs are capable of activating G-proteins (20, 21). Moreover, localization studies revealed that some neurons in the brain express much more mRNA of one subunit than that of the other, consistent with a possible role of homomeric GABA_B receptors (25). Finally, GABA_B1 may be able to activate intracellular pathways independently of G-proteins (23, 26–28). The identification of selective compounds acting on GABA_B2 will probably help unravel this issue.

Within the last few years, allosteric modulators of class-III GPCRs have been identified for calcium-sensing and mGlu receptors (29–31). Such compounds act either as non-competitive antagonists (32–34) or as positive allosteric modulators (35–40). In each case, these compounds have been shown to bind within the HD of their targeted receptor (34, 41–43). They also are highly selective for one receptor subtype in contrast to most of the ligand acting at the orthosteric site. Urwyler et al. (44, 45) recently described the first GABA_B-specific positive allosteric modulators, CGP7930 and CGP13501, and described more recently the compound GS39783. Their site of action was not identified, but according to what was known for the mGlu-specific positive allosteric modulators and to the fact that GABA_B2 coupled to G-protein, we hypothesized that these compounds act in the GABA_B2 HD (46).

In this work, we not only demonstrate that CGP7930 indeed modulates the GABA_B receptor by directly acting in the GABA_B2 HD but that it also activates the homomeric GABA_B2 receptor, indicating that GABA_B2 could be functional by itself. Moreover, CGP7930 also activates a truncated version of GABA_B2 deleted of the ECD, demonstrating that this HD can behave as a rhodopsin-like receptor. These data bring much information on the mechanism of action of this GABA_B-positive modulator and reveal that GABA_B2-selective drugs can be identified. Such drugs will be useful to better dissect the specific role of GABA_B1 and GABA_B2 in the brain.

EXPERIMENTAL PROCEDURES

Materials—Aldehyde CGP13501 was synthetized according to the FR2237870 patent and was reduced subsequently with sodium borohydride to afford CGP7930 (melting point 82–84 °C, the melting point described in the U. S. patent 4333868 was 86 °C).

CGP54626 was purchased from Tocris (Fisher-Bioblock, Illkrich, France). Fetal bovine serum, culture medium, and other solutions used for cell culture were from Invitrogen. [³H]Myo-inositol (23.4 Ci/mol) was purchased from PerkinElmer Life Sciences. All of the other reagents used were of molecular or analytical grade where appropriate.

Plasmids and Site-directed Mutagenesis—The plasmids encoding the wild type and chimeric GABA_B1a and GABA_B2 subunits epitope-tagged at their N-terminal ends (pRK-GABA_B1a-HA, pRK-GABA_B1a/2-HA, pRK-GABA_B2/1-HA, and pRK-GABA_B2-HA or –c-Myc) under the control of a cytomegalovirus promoter were described previously (16, 20). pRK-HD2 was generated by the deletion of the sequence coding for the ECD in the pRK-GABA_B2-HA plasmid with the use of the MluI restriction site located just after the sequence coding for the HA tag and a MluI site created just after the proline residues at position 463 in GABA_B2.

Cell Culture and Transfection—Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and transfected by electroporation as described elsewhere (47, 48). Unless stated otherwise, 10 × 10⁶ cells were transfected with plasmid DNA containing the coding sequence of the receptor subunits and the amount of plasmid DNA was adjusted to 10 μg with the empty plasmid pRK₆. For determination of inositol phosphate accumulation, the cells also were transfected with the chimeric Gαqi9 G-protein, which allows the coupling of the recombinant heteromeric GABA_B receptor to phospholipase C (47).

Measurement of Inositol Phosphate Production—Determination of inositol phosphate (IP) accumulation in transfected cells was performed in 96-wells plates (0.2 × 10⁶ cells/well) after overnight labeling with [³H]myo-inositols (0.5 μCi/well) as already described (49). The stimulation was conducted for 30 min in a medium containing 10 mm LiCl and the indicated concentration of agonist or antagonist. The reaction was stopped with a 0.1 m formic acid solution. Supernatants were recovered, and IPs were purified by ion-exchange chromatography using DOWEX AG1-X8 resin (Bio-Rad) in 96-well filter plates (reference number MAHVN4550, Millipore, Bedford, MA). Total radioactivity remaining in the membrane fractions was counted after the treatment of cells with a solution containing 10% Triton X-100 and 0.1 n NaOH. Radioactivity was quantified using Wallac 1450 MicroBeta liquid scintillation counter. Data were expressed as IP/membrane (i.e. the amount of total IPs produced over the amount of radioactivity remaining in the membranes multiplied by 100). Unless stated otherwise, all of the data are means ± S.E. of at least three independent experiments. The dose-response curves were fitted using the Kaleidagraph program and the equation y = [(y_max – y_min)/(1 + (x/EC₅₀)^nH)) + y_min, where the EC₅₀ is the concentration of the compound necessary to obtain 50% of the maximal effect and n_H is the Hill coefficient.

Anti-HA ELISA for Quantification of Cell Surface Expression—24 h after transfection (10 × 10⁶ cells, HA-tagged GABA_B1 (2 μg) and c-Myc-tagged GABA_B2 (2 μg) subunits), cells were fixed with 4% paraformaldehyde and then blocked with PBS + 5% fetal bovine serum. After a 30-min reaction with primary antibody (monoclonal anti-HA clone 3F10 (Roche Applied Science) at 0.5 μg/ml) in the same buffer, the goat anti-rat antibody coupled to horseradish peroxidase (Jackson Immunoresearch, West Grove, PA) was applied for 30 min at 1 μg/ml. After intense washes with PBS, secondary antibody was detected and quantified instantaneously by chemiluminescence using Supersignal® ELISA femto maximum sensitivity substrate (Pierce) and a Wallac Victor² luminescence counter.

[γ-³⁵S]GTP Binding Measurements—Cells were transfected using PolyFect transfection reagent (Qiagen) under optimized conditions. The complex was formed using the total amount of 8 μg of plasmid DNA with 60 μl of Polyfect in 300 μl of serum-free antibiotic-free Dulbecco's modified Eagle's medium for 10 min and then added to cells at 40–60% confluence. According to the results of expression, the amount of DNA is 2 μg of GABA_B1, 1 μg of GABA_B2, 2 μ g of Gαo1c, and 3 μg of pRK₆ for wild type receptor. 48 h after transfection, cells were scraped in lysis buffer (15 mm Tris, 2 mm MgCl₂, 0.3 mm EDTA, pH 7.4) and centrifuged twice. The pellet was solubilized in Tris (50 mm), MgCl₂ (3 mm) buffer, pH 7.4, using a potter. The [γ-³⁵S]GTP binding was performed in 96-well filtration plates (reference number MAFCN0B50, Millipore) equilibrated with Tris (50 mm), and MgCl₂ (5 mm), pH 7.4. 5 μg of membrane preparation/wells were pre-incubated in 20 μl with antagonist (15 min) and afterward and/or with agonist (15 min). 60 μl of incubation buffer (50 mm Tris, 1 mm EDTA, 10 μm GDP, 5 mm MgCl₂, 0.01 mg/ml leupeptin, 100 mm NaCl) and 20 μl of H₂O/well were added, and then the plate was incubated for 1 h at 30 °C. After vacuum filtration and plate filter drying, the radioactivity was measured using a Wallac 1450 MicroBeta liquid scintillation counter. The dose-response curves were fitted using the Kaleidagraph program and the equation y = [(y_max – y_min)/(1 + (x/EC₅₀)^nH)) + y_min, where the EC₅₀ is the concentration of the compound necessary to obtain 50% of the maximal effect and n_H is the Hill coefficient.

RESULTS

CGP7930 Is a Positive Allosteric Modulator of the GABA_B Receptor—As previously described by Urwyler et al. (44), CGP7930 increased both the affinity and the maximal effect (∼40%) of GABA in stimulating [γ-³⁵S]GTP binding on Gαo-proteins (Fig. 1) (44). Dose-response curves indicate that the EC₅₀ for GABA was increased 3- and 10-fold in the presence of 0.1 and 1 mm CGP7930, respectively (42.0 ± 11.0, 30.0 ± 7.5, and 4.7 ± 1.04 μm in the absence or the presence of 0.1 and 1 mm CGP7930, respectively). A small increase in the binding of [γ-³⁵S]GTP binding also was observed in the absence of GABA, suggesting that CGP7930 also may be able to slightly activate the receptor. However, such an effect of CGP7930 could not be studied in details using this assay according to the high basal [γ-³⁵S]GTP binding, and to the low signal to noise ratio of this assay (2–3-fold increase observed with a saturating GABA concentration).

Therefore, the effect of CGP7930 was analyzed further using an IP production assay, which is supposed to give a higher signal to noise ratio. Indeed, the GABA_B receptor can activate efficiently phospholipase C when co-expressed with the chimeric G-protein Gqi9 (47). As noticed previously, the IP assay is much more sensitive than the [γ-³⁵S]GTP assay as indicated by the 100-fold lower EC₅₀ value measured for GABA compare with the [γ-³⁵S]GTP assay. This probably results from the large amplification of the signaling cascade between G-protein activation and IP formation.

As observed with the [γ-³⁵S]GTP assay, 100 μm CGP7930 increased GABA potency in stimulating IP formation 3-fold (EC₅₀ = 0.32 ± 0.10 μm and 0.11 ± 0.02 μm in the absence and presence of 100 μm CGP7930, respectively) as well as the maximal effect (Figs. 2 and 3A). The EC₅₀ for this effect of CGP7930 as determined by increasing concentrations of this compound in the presence of a fixed concentration of GABA was similar to that determined by Urwyler et al. (44) when using an IP-induced calcium signal assay (18.9 ± 7.7 and 10 ± 0.1 μm, respectively) (44). However, in this assay, CGP7930 also clearly stimulated IP production even in the absence of added GABA, further suggesting that the CGP7930 could be a GABA_B partial agonist (Figs. 2 and 3).

CGP7930 Is a Partial Agonist of the GABA_B Receptor—Additional experiments were performed to demonstrate that CGP7930 directly activates the GABA_B receptor. First, CGP7930 alone did not induce IP formation in pRK6- and Gqi9-transfected control cells or in cells co-expressing mGlu5, demonstrating that IP production did not result from a direct action on either the transfected G-protein or phospholipase C (data not shown). Second, the observed stimulation of IP production by CGP7930 (Fig. 3A) is dose-dependent with an EC₅₀ similar to that observed for the potentiating effect (32.5 ± 7.2 μm; Fig. 4).

Because we could not exclude that the observed effect of CGP7930 was attributed to overexpression of the receptors, we decided to examine the effect of CGP7930 at different receptor expression levels. To achieve this goal, the effect of saturating concentrations of GABA or CGP7930 (1 mm) was measured in cells transfected with increasing amounts of GABA_B1- and GABA_B2-expressing plasmids (Fig. 5). The expression levels of heteromeric GABA_B receptor were determined using ELISA performed on intact cells with an anti-HA antibody labeling the N-terminal HA-tagged GABA_B1 subunit. As shown in Fig. 5, the maximal agonist activity of CGP7930 was always lower than the maximal GABA activity and, of course, the maximal activity induced by CGP7930 together with GABA, indicating that CGP7930 was only a partial agonist.

The Heptahelical Domain of GB2 Is Required for the Action of CGP7930—To identify the site of action of CGP7930, we first examined whether the stimulatory effect was inhibited by the competitive antagonist CGP54626. As shown in Fig. 3B, a high concentration of 1 μm (about 250-fold its affinity) totally antagonized the effect of GABA, both compounds binding in the same site in the ECD of GABA_B1. However, CGP54626 did not inhibit completely (57.5 ± 0.5% of inhibition) the effect of CGP7930. This finding suggested that CGP7930 did not bind in the orthosteric site where GABA and competitive antagonists bind within the GABA_B1 ECD but did in another domain, an allosteric site. Thus, CGP54626 probably inhibited allosterically the CGP7930 effect.

GABA_B heterodimers can be considered as the association of four distinct domains (ECD1, ECD2, HD1, and HD2) that correspond to the ECD and HD of GABA_B1 and GABA_B2 subunits, respectively. To identify which of these domains is required for the effect of CGP7930, its action on various combinations of chimeric and mutated subunits was examined. The chimeric subunits used were GABA_B1/2 and GABA_B2/1 in which the entire ECD was swapped between GABA_B1 and GABA_B2 (20).

The GABA_B1/2 was expressed with GABA_B1 to form a receptor that does not contain the ECD2, and vice versa, GABA_B2/1 was expressed with GABA_B2 to form a receptor devoid of ECD1. Both combinations already have been shown to be expressed at the cell surface and to form heteromeric complexes (20). Although they are not sensitive to GABA, both activate Gqi9 as illustrated by the high constitutive IP formation measured in cells expressing these subunit combinations (20). As shown in Fig. 6, CGP7930 stimulated IP production in cells expressing either combination (Fig. 6, compare lanes 3 and 7). Accordingly, none of the ECD was required for the effect of CGP7930. Then, although we could not rule out that CGP7930 could act similarly on both ECD1 and ECD2 and that only one ECD would be enough for the effect of CGP7930, the more probable possibility was that CGP7930 acts in the HD of either GABA_B1 or GABA_B2.

To determine which HD could be the site of action of CGP7930, the effect of CGP7930 was analyzed on the combinations GABA_B1 + GABA_B2/1 and GABA_B2 + GABA_B1/2. The first combination possessed only HD1 and not HD2, whereas the second possessed HD2 only and not HD1 (Fig. 7). To allow the correct expression of both GABA_B1 and GABA_B2/1 at the cell surface, the ER retention signal RSR corresponding to the residues Arg-Ser-Arg located in the C-terminal tail of these subunits was mutated into ASA (20). Although the combination GABA_B1 + GABA_B2/1 was not activated by GABA, whereas the combination GABA_B2 + GABA_B1/2 was activated, both combinations were similarly expressed at the cell surface (for review see Ref. 20) (data not shown). As shown in Fig. 7, CGP7930 stimulated the combination containing only HD2 (a 4-fold increase of the IP production from 4.7- to 13.8-normalized cpm in the absence and presence of 100 μm CGP7930, respectively) but was devoid of activity on that possessing HD1 only. Taken together, these data illustrated the requirement of HD2 for the partial agonist activity of CGP7930 in the GABA_B heteromer. However, because GABA_B1 cannot activate the G-protein by itself, one cannot exclude that CGP7930 bound to GABA_B1 but failed at allowing it to stimulate G-proteins.

CGP7930 Activated GABA_B2 in the Absence of GABA_B1—As mentioned above, GABA_B2 possesses sufficient molecular determinants for G-protein activation (17–20). Even when transfected alone, GABA_B2 was expressed highly at the HEK293 cell surface (Fig. 8A), allowing us to examine whether CGP7930 could activate this subunit expressed alone. Indeed, whereas GABA is devoid of activity on GABA_B2, CGP7930 increased IP production 3-fold (Fig. 8B) with an EC₅₀ of 57.1 ± 3.8 μm (Fig. 9). A similar effect of CGP7930 was observed with the chimeric subunit GABA_B1/2, even though GABA was inactive (Fig. 8, A and B). This confirms that the HD of GABA_B2 is crucial for the CGP7930 effect. However, it is still possible that CGP7930 requires the presence of an ECD, either that of GABA_B1 or GABA_B2, to turn on the GABA_B2 HD.

CGP7930-activated GABA_B2 HD Expressed Alone—According to the data described above, it appeared that CGP7930 could activate GABA_B2 by a direct action in its HD. Thus, we looked for the action of CGP7930 on the HD of GABA_B2 alone. Indeed, we have shown recently that the HD of the mGlu5 receptor could be expressed alone and could be activated directly by a positive allosteric modulator of this receptor (49). Therefore, we generated a truncated version of GABA_B2 lacking the large ECD (Fig. 8). Thanks to the presence of a signal peptide and of a HA tag inserted at the N terminus, HD2 was found at the cell surface, although at a lower level than the wild type subunit (30% of the wild type in Fig. 8A). On cells expressing HD2, CGP7930 increased IP production more than 2-fold with an EC₅₀ of 64.8 ± 38.7 μm (Fig. 9). These data showed that CGP7930 directly stabilizes an active conformation of the GABA_B2 HD and can be considered as a first GABA_B2 ligand.

DISCUSSION

In this study, we explored the mechanism of action of CGP7930, one of the first described positive allosteric modulators of the GABA_B receptor (44). Using both [γ-³⁵S]GTP binding and IP production assays that reflect the activation of Gαo and Gαqi9, respectively, we confirmed the positive allosteric action of CGP7930. However, using the more sensitive IP assay, i.e. the activation of phospholipase C via Gαqi9, CGP7930 was found also to directly activate the GABA_B receptor but with a low efficacy. The action of CGP7930 on various combinations of wild type and chimeric subunits led us to propose that CGP7930 activated HD2 within the heteromer. This proposal then was demonstrated directly as we showed that CGP7930 acted as an agonist of HD2 expressed alone, demonstrating that this domain of the GABA_B receptor behaved as a rhodopsin-like receptor. It is noteworthy that, even if CGP7930 could bind to the HD of GABA_B1, it would not induced G-protein activation, because we never observed any activation of the recombinant G-proteins by GABA_B receptors lacking HD2 (17, 19, 20).

CGP7930, a Partial Agonist of the GABA_B Receptor—Not only could CGP7930 potentiate the effect of GABA as previously reported by others (44), it also could activate the wild type receptor directly. This effect occurred in a similar range of concentration as that observed for the potentiating effect, and various arguments excluded the possibility of a potentiation of the effect of a possible endogenous agonist present in the assay medium. Indeed, the effect of CGP7930 was not inhibited fully by a competitive antagonist. Moreover, the effect of CGP7930 still could be observed on various mutated GABA_B receptors not sensitive to GABA.

Because CGP7930 activated GABA_B2 in the absence of GABA_B1, the agonist effect observed could very well be the consequence of some GABA_B2 subunits not associated with GABA_B1. Although this possibility cannot be excluded firmly, we believe that it is unlikely because others and we (8, 50) observed that, in heterologous systems, GABA_B2 was less expressed than GABA_B1. Accordingly, it is very unlikely that there was enough isolated GABA_B2 subunits (either in a monomeric or homodimeric form) in cells transfected with both GABA_B1 and GABA_B2 to generate a CGP7930-induced response higher than that observed in cells expressing GABA_B2 only.

Of interest, CGP7930 activated not only the heteromeric GABA_B receptor and the GABA_B2 subunit expressed alone but also a GABA_B2 subunit deleted of its ECD. Moreover, all of these effects were observed in the same range of the concentration of CGP7930 (with very similar EC₅₀ values). This observation clearly indicates that neither GABA_B2 ECD nor the GABA_B1 is required for the agonist activity of CGP7930. Also, this further demonstrates that agonist binding is not required for CGP7930 interaction with HD2.

On a Possible Allosteric Control of CGP7930 Effect—The affinity of GABA on the ECD of GABA_B1 was regulated allosterically by the other domains of the heteromeric GABA_B receptor such as the ECD of GABA_B2. We recently hypothesized that this effect was probably due to the relief by the GABA_B2 ECD of an inhibitory action of the GABA_B1 HD on the GABA_B1 ECD (22). Accordingly, one would expect that the affinity of CGP7930 in the HD of GABA_B2 would also be under the allosteric control of the other domains of the heteromeric complex. The EC₅₀ values of CGP7930 on the wild type receptor, on GABA_B2, or on HD2 were quite similar whether the agonist effect or the potentiating effect of CGP7930 was measured. This finding suggests that CGP7930 affinity was not as dependent as GABA affinity on the specific state of the other domains. Alternatively, within this range of the concentration tested, CGP7930 may only bind and exert its effect on receptors in a specific state. The identification of radioactive compounds interacting at the same site than CGP7930 would be required to further study this point.

However, the antagonist CGP54626 that probably stabilizes the inactive open state of GABA_B1 ECD partly inhibited the agonist effect of CGP7930 on the wild type receptor. Such an effect was not observed on the GABA_B2 subunit expressed alone or on HD2. This effect then demonstrated that the action of CGP7930 was dependent on the specific state of the GABA_B1 ECD. To our actual knowledge of the mechanism of activation of the GABA_B receptor, GABA binding in the VFT of GABA_B1 leads to the closure of the VFT and to a possible re-orientation of the dimer of VFTs. As a consequence, this re-orientation stabilizes the active conformation of the dimer of HDs. The competitive antagonist such as CGP54626 is expected to prevent the closure of the GABA_B1 VFT and thus prevent the stabilization of the active state of the HD dimer by agonists. According to this model, any drug directly stabilizing the dimer of HDs also will stabilize the active conformation of the dimer of VFTs. In agreement with this proposal, CGP7930 increased agonist affinity. Because of allosteric coupling between the dimer of VFTs and the dimer of HD, locking the dimer of VFTs in the inactive state by a competitive antagonist is expected to make the change in conformation of the dimer of HDs required for its activation more difficult. This is expected to decrease the effect of CGP7930 as observed here.

CGP7930 as an Activator of the GABA_B2 HD—As reported recently (49) for mGlu5, the HD of GABA_B2 could be expressed as a membrane protein at the cell surface in HEK293 cells. Although the mGlu5-truncated receptor displayed constitutive activity, no such activity could be detected with GABA_B2. This was in agreement with the higher constitutive activity measured with mGlu5 compared with GABA_B receptor (51, 52). However, in both cases, these HDs were activated by positive allosteric regulators CGP7930 and DFB for GABA_B2 and mGlu5 HDs, respectively. This finding shows that, even though class-III and class-I rhodopsin-like GPCRs diverged early during evolution, their HDs still possess common structural properties and probably share a similar activation mechanism.

Whether GPCRs function as a monomer or as dimers is a matter of intense debate in the field (53–55). However, in the case of class-III GPCRs, it is well accepted that the dimeric nature of these receptors is crucial for the intramolecular transduction, i.e. transfer of information from the agonist binding domain to the heptahelical G-protein-activating domain (4). However, whether a dimeric nature of the HD of these receptors is necessary for the agonist effect of positive allosteric modulators is not known. Obviously, the demonstration that HDs of class-III GPCRs can function as class-I GPCRs will help unravel this important issue.

A Model for the Action of CGP7930 on the GABA_B Receptor—We recently proposed a model for the functioning of class-III GPCRs (56). This model integrated our common view of the functioning of Venus flytrap modules, i.e. the binding of the ligand in the cleft between the two lobes of the ECD, and the stabilization of a closed state by agonists or prevention of the closure of such a domain upon antagonist binding. Moreover, our model takes into account the putative mechanism of activation of the HD with the existence of at least two states, active and inactive, in which the equilibrium between these two states is under the control of the specific conformation of the ECD. As discussed in this paper, this model fits very nicely with a series of specific properties of class-III GPCRs. For example, this model provides a reasonable explanation for the lack of inverse agonist activity of competitive antagonists of mGluRs, whereas non-competitive antagonists interacting in the HD were found to be inverse agonists (56).

In this model, we proposed two options to explain the effect of positive allosteric modulators acting in the HD of class-III GPCRs. A first possibility is that such compounds act by stabilizing the active state of the HD and, as a consequence, stabilize the active state of the binding domain, thus increasing agonist affinity. According to this proposal, such positive allosteric modulators were expected to also activate with a low efficacy of the full-length receptor. This fits nicely with our observation that CGP7930 acted both as an activator and as a positive allosteric modulator of the full-length GABA_B receptor. The second possibility was that the positive modulators acted by increasing the allosteric coupling between the active ECD and the HD, rather than by directly activating the HD. According to the second possibility, the positive modulator directly activated neither the full-length receptor nor the HD. Our recent observation that the mGlu5-positive modulator, DFB (3,3′-difluorobenzaldazine), did not activate the full-length receptor but acted as a full agonist on the receptor deleted of its ECD did not fit with any of these two possibilities. Accordingly, a more complex model involving three states of the mGlu5 HD was proposed (49). This model was based on the recognized three states of rhodopsin (57, 58) Such an observation provides evidence for a different activation mechanism of GABA_B receptors and the other class-III GPCRs such as mGluRs. Indeed, although these two types of receptors share sequence similarities, the GABA_B receptor subunits lack the cysteine-rich domain that interconnects the ECD to the HD in the mGlu-like receptors. Further studies will be necessary to better clarify the specific properties of both types of class-III GPCRs.

CONCLUSION

The main observation of this study is that it is possible to identify compounds acting on the GABA_B2 subunit. As discussed above, such compounds will be useful to elucidate the activation mechanism of such a complex GABA_B heteromeric receptor. In addition, as pointed out in our introduction, it is well recognized that the vast majority of GABA_B1 and GABA_B2 subunits associate with each other to form a functional GABA_B receptor in the brain. However, some observations suggest that either GABA_B1 or GABA_B2 could be active on their own or in association with another type of subunit. Our data show that it should be possible to identify compounds acting on GABA_B2 specifically. Such compounds will help unravel the possible function of this subunit in the brain.