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Allosteric interactions between GB1 andGB2 subunits are required for optimalGABAB receptor function

2001; Springer Nature; Volume: 20; Issue: 9 Linguagem: Inglês

10.1093/emboj/20.9.2152

1460-2075

Thierry Galvez, Béatrice Duthey, Julie Kniazeff, Jaroslav Blahoš, Giorgio Rovelli, Bernhard Bettler, Laurent Prézeau, Jean‐Philippe Pin,

Neuroscience and Neuropharmacology Research

Article1 May 2001free access Allosteric interactions between GB1 and GB2 subunits are required for optimal GABAB receptor function Thierry Galvez Thierry Galvez Mécanismes Moléculaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, 141 Rue de la Cardonille, F-34094 Montpellier, France Search for more papers by this author Béatrice Duthey Béatrice Duthey Mécanismes Moléculaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, 141 Rue de la Cardonille, F-34094 Montpellier, France Search for more papers by this author Julie Kniazeff Julie Kniazeff Mécanismes Moléculaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, 141 Rue de la Cardonille, F-34094 Montpellier, France Search for more papers by this author Jaroslav Blahos Jaroslav Blahos Present address: Laboratory of Molecular Physiology, Department of Physiology, Charles University 3rd Faculty of Medicine and Institute of Physiology, Czech Academy of Science, Ke Karlovu 4, Prague 2, Czech Republic Search for more papers by this author Giorgio Rovelli Giorgio Rovelli TA Nervous System, Novartis Pharma AG, CH-4002 Basel, Switzerland Search for more papers by this author Bernhard Bettler Bernhard Bettler TA Nervous System, Novartis Pharma AG, CH-4002 Basel, Switzerland Search for more papers by this author Laurent Prézeau Laurent Prézeau Mécanismes Moléculaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, 141 Rue de la Cardonille, F-34094 Montpellier, France Search for more papers by this author Jean-Philippe Pin Corresponding Author Jean-Philippe Pin Mécanismes Moléculaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, 141 Rue de la Cardonille, F-34094 Montpellier, France Search for more papers by this author Thierry Galvez Thierry Galvez Mécanismes Moléculaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, 141 Rue de la Cardonille, F-34094 Montpellier, France Search for more papers by this author Béatrice Duthey Béatrice Duthey Mécanismes Moléculaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, 141 Rue de la Cardonille, F-34094 Montpellier, France Search for more papers by this author Julie Kniazeff Julie Kniazeff Mécanismes Moléculaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, 141 Rue de la Cardonille, F-34094 Montpellier, France Search for more papers by this author Jaroslav Blahos Jaroslav Blahos Present address: Laboratory of Molecular Physiology, Department of Physiology, Charles University 3rd Faculty of Medicine and Institute of Physiology, Czech Academy of Science, Ke Karlovu 4, Prague 2, Czech Republic Search for more papers by this author Giorgio Rovelli Giorgio Rovelli TA Nervous System, Novartis Pharma AG, CH-4002 Basel, Switzerland Search for more papers by this author Bernhard Bettler Bernhard Bettler TA Nervous System, Novartis Pharma AG, CH-4002 Basel, Switzerland Search for more papers by this author Laurent Prézeau Laurent Prézeau Mécanismes Moléculaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, 141 Rue de la Cardonille, F-34094 Montpellier, France Search for more papers by this author Jean-Philippe Pin Corresponding Author Jean-Philippe Pin Mécanismes Moléculaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, 141 Rue de la Cardonille, F-34094 Montpellier, France Search for more papers by this author Author Information Thierry Galvez1, Béatrice Duthey1, Julie Kniazeff1, Jaroslav Blahos2, Giorgio Rovelli3, Bernhard Bettler3, Laurent Prézeau1 and Jean-Philippe Pin 1 1Mécanismes Moléculaires des Communications Cellulaires, CNRS-UPR9023, CCIPE, 141 Rue de la Cardonille, F-34094 Montpellier, France 2Present address: Laboratory of Molecular Physiology, Department of Physiology, Charles University 3rd Faculty of Medicine and Institute of Physiology, Czech Academy of Science, Ke Karlovu 4, Prague 2, Czech Republic 3TA Nervous System, Novartis Pharma AG, CH-4002 Basel, Switzerland ‡T.Galvez and B.Duthey contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:2152-2159https://doi.org/10.1093/emboj/20.9.2152 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Recent studies on G-protein-coupled receptors revealed that they can dimerize. However, the role of each subunit in the activation process remains unclear. The γ-amino-n-butyric acid type B (GABAB) receptor is comprised of two subunits: GB1 and GB2. Both consist of an extracellular domain (ECD) and a heptahelical domain composed of seven transmembrane α-helices, loops and the C-terminus (HD). Whereas GB1 ECD plays a critical role in ligand binding, GB2 is required not only to target GB1 subunit to the cell surface but also for receptor activation. Here, by analysing chimeric GB subunits, we show that only GB2 HD contains the determinants required for G-protein signalling. However, the HD of GB1 improves coupling efficacy. Conversely, although GB1 ECD is sufficient to bind GABAB ligands, the ECD of GB2 increases the agonist affinity on GB1, and is necessary for agonist activation of the receptor. These data indicate that multiple allosteric interactions between the two subunits are required for wild-type functioning of the GABAB receptor and highlight further the importance of the dimerization process in GPCR activation. Introduction G-protein-coupled receptors (GPCRs) recognize various stimuli, including light, odours, pheromones, tastes, ions and a large variety of hormones and neurotransmitters (Bockaert and Pin, 1999). These proteins, which are encoded by >1% of the mammalian genes, are therefore involved in a large variety of physiological regulations and functions. Several subfamilies of GPCRs have been defined based on their proposed general structure and sequence similarity (Kolakowski, 1994; Bockaert and Pin, 1999). All possess seven α-helices, which contain extra- and intracellular loops connecting them, as well as the C-terminal tail. The intracellular loops and C-terminus are involved in the recognition and activation of the heterotrimeric G-proteins (Bourne, 1997). Helices, loops and C-terminus are referred to herein as the 'heptahelical domain' (HD). For a long time, it has been assumed that one molecule of GPCR interacts with one molecule of G-protein. However, recent studies have revealed that these receptors can form dimers, either homodimers or heterodimers (Salahpour et al., 2000). Therefore, in light of this emergent concept, the role of each subunit in the recognition and activation of the G-protein needs to be identified. γ-amino-n-butyric acid (GABA) is a major neurotransmitter in the mammalian brain that controls neuronal excitability by activating ionotropic GABAA and GABAC receptors and G-protein-coupled GABAB receptors. The latter inhibits adenylyl cyclase and modulates the activity of a variety of ion channels. Two GABAB receptor types have been identified: GABAB1 (GB1) (Kaupmann et al., 1997b) and GABAB2 (GB2) (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998; Kuner et al., 1999). These proteins share 35% sequence identity and are related to the family 3 GPCRs, which also includes the metabotropic glutamate (mGlu) and calcium-sensing (CaS) receptors. These GPCRs have a large extracellular domain (ECD), which contains the agonist binding site and shares the same protein folding as bacterial periplasmic binding proteins (O'Hara et al., 1993; Galvez et al., 1999; Kunishima et al., 2000). Another specific feature of these family 3 receptors is that they are likely to function as dimers (Romano et al., 1996; Bai et al., 1999; Kunishima et al., 2000). In contrast to the mGlu and CaS receptors, which form functional homodimers, the GABAB receptor is a heteromer constituted of both GB1 and GB2 proteins (Kaupmann et al., 1998). Indeed, the coexpression of both subunits is required for a fully functional GABAB receptor (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998; Kuner et al., 1999). Why are two subunits required for the formation of a functional GABAB receptor? The GB1 subunit binds all known GABAB ligands (Kaupmann et al., 1997b) and is responsible for the ligand recognition by the heteromeric GABAB receptor (Galvez et al., 2000a,b), whereas the GB2 subunit is necessary for the correct trafficking of GB1 to the cell surface (Couve et al., 1998; White et al., 1998). Indeed, the C-terminal tail of the GB2 subunit, by interacting with that of GB1, masks a signal responsible for the retention of GB1 in the endoplasmic reticulum (ER) (Margeta-Mitrovic et al., 2000; Calver et al., 2001; Pagano et al., 2001). However, even when this ER retention signal is mutated such that the GB1 subunit reaches the cell surface alone, no functional GABA response can be measured despite the correct binding of GABAB ligands (Margeta-Mitrovic et al., 2000; Calver et al., 2001; Pagano et al., 2001). This indicates that the GB2 subunit is necessary not only for the correct targeting of the GB1 subunit to the cell surface, but also for the correct functioning of the receptor. The present study revealed new roles for the heterodimerization of the GABAB receptor and explained why the two subunits are required for the formation of a functional GABAB receptor. Here we show that although the ECD of GB1 is sufficient to bind GABAB ligands (Galvez et al., 1999; Malitschek et al., 1999), the ECD of GB2 is required for agonist-dependent activation of the receptor. Conversely, although the HD of GB2 contains the molecular determinants required for the recognition and activation of the same heterologous G-proteins as the wild-type heteromer, the HD of GB1 increases the coupling efficacy. These data reveal a modular organization of the GABAB receptor in which four functional and specialized domains cooperate. Results Individual chimeric GB1/2 or GB2/1 constructs are not functional When the ER retention signal RSRR in the GB1 C-terminal tail is mutated into ASAR, the mutant receptor GB1ASA can reach the cell surface alone, bind GABAB ligands, but is still not functional (Margeta-Mitrovic et al., 2000; Calver et al., 2001; Pagano et al., 2001). We therefore speculated that the HD of GB2 was responsible for G-protein coupling, and so generated a chimeric GABAB receptor (called GB1/2) in which the HD of GB1 was replaced by that of GB2 (Figure 1A). The converse chimeric receptor (GB2/1 as well as its ER retention mutant GB2/1ASA) was also constructed. For all the constructs described, the GB1a variant (Kaupmann et al., 1997b) has been used. As shown in Figure 1A, all constructs were expressed and displayed the expected molecular weight. In addition, due to the presence of either a c-myc or an HA epitope added at their extracellular N-terminus, GB1ASA, GB2, GB1/2 and GB2/1ASA were shown to reach the cell surface (Pagano et al., 2001; data not shown). Among these, only GB1ASA and GB1/2 were able to bind the GB1 selective antagonist [125I]CGP64213, allowing us to show that both subunits bind GABA (Table I). The functionality of these constructs was then tested after their coexpression with the chimeric G-protein Gqi9 (Gαq in which the last nine C-terminal residues are replaced by those of Gαi2). This latter G-protein α-subunit has been shown to couple a wide variety of Gi-coupled receptors to PLC (Conklin et al., 1993), including the wild-type GABAB heteromeric receptor (Franek et al., 1999). When expressed alone, none of these constructs, not even the GB1/2 chimera, activated PLC upon application of GABA (Figure 1B) or baclofen (data not shown). Only a slight increase in the basal level of IP formation could be detected in cells expressing GB1/2, an activity that was inhibited by GABA. These data show that GB1/2, comprising the GABA-binding domain of GB1 and the GB2 HD, does not form a functional receptor that can be activated by GABA. Therefore, we examined whether the coexpression of any of these wild-type or chimeric subunits could form a functional receptor. Figure 1.Expression of the wild-type and chimeric GABAB receptor subunits. (A) Schematic representation and western blot analysis of the constructs used in this study. The GB1 and GB2 subunits are represented in white and black, respectively; the ECD is represented as a circle, whereas the HD is a rectangle with a tail showing the C-terminal tail. The horizontal bar means that the ER retention signal RSRR has been mutated into ASAR. (B) Basal (open bars) and 1 mM GABA-induced (grey bars) IP formation were measured in cells expressing the indicated constructs and Gqi9. Values correspond to the percentage of the GABA response measured in cells expressing the wild-type receptor and are the means ± SEM of at least three experiments performed in triplicate. Download figure Download PowerPoint Table 1. Affinity values of CGP64213 and GABAa Subunit combination [125I]CGP64213 binding CGP642313 Ki (nM) Hill coefficient GABA Ki (μM) Hill coefficient GB1 1.4 ± 0.2b 1.0 ± 1.1b 22.3 ± 1.9b 0.9 ± 0.1b GB1ASA 3.5 ± 0.8 1.4 ± 0.1 25.7 ± 3.9 0.7 ± 0.1 GB1 + GB2 2.9 ± 0.2 1.4 ± 0.1 3.3 ± 0.6 1.0 ± 0.1 GB1ASA + GB2/1ASA 3.6 ± 0.6 1.4 ± 0.1 1.2 ± 0.4 0.8 ± 0.1 GB1/2 4.0 ± 1.2 0.9 ± 0.2 8.2 ± 0.9 1.5 ± 0.1 GB1/2 + GB2/1ASA 2.8 ± 0.1 1.0 ± 0.1 2.5 ± 0.6 1.0 ± 0.2 GB1/2 + GB1 3.9 ± 1.2 1.0 ± 0.2 6.0 ± 1.5 0.9 ± 0.2 GB1/2 + GB2 3.3 ± 0.9 1.2 ± 0.1 3.3 ± 0.9 1.1 ± 0.3 a Affinity values (Ki, determined as described in Materials and methods) of CGP64213 and GABA as determined from displacement of [I]CGP64213 binding on intact cells expressing the indicated subunit combinations. b Binding experiments were performed on crude membranes [data from Galvez et al. (2000a)]. Values are means ± SEM of at least three independent determinations. Coexpression of GB1/2 and GB2/1 leads to a functional GABAB receptor with wild-type properties The possible formation of a functional GABAB receptor after coexpression of GB1/2 and GB2/1 was tested first, since all four modules (GB1 and GB2 ECDs and HDs) of the GABAB heteromer were conserved in that combination. As observed in cells expressing GB1 + GB2, a large GABA-induced IP formation was measured in cells expressing both GB1/2 and GB2/1 (Figure 2A). Indeed GABA, baclofen (Figure 2C) and APPA (data not shown), three GABAB agonists, stimulated IP formation to similar extents and with similar EC50 values to cells coexpressing the wild-type subunits. Moreover, the agonist-induced response could be inhibited by the GABAB antagonist CGP64213 with a similar potency to wild-type receptor (Figure 2C, Table I). Similar data were obtained when GB2/1ASA was used instead of GB2/1 (data not shown). These data revealed that both GB1/2 and GB2/1 are correctly folded. Moreover, they show that the modules can be swapped between the two subunits without affecting the function of the heteromer. Figure 2.Functional analysis of GABAB receptor combinations in which the heteromeric nature of the HDs is conserved. (A) Basal (open bars) and 1 mM GABA-induced (grey bars) IP formation were measured in cells expressing GB1 + GB2, GB1/2 + GB2/1, GB1 + GB1/2 or GB2/1 + GB2 and the chimeric G-protein Gqi9. The dotted line indicates the basal IP formation measured in mock-transfected cells. Values correspond to the percentage of the GABA response measured in cells expressing the wild-type receptor. (B) Total (open bars) and non-specific (grey bars; 1 mM GABA) [125I]CGP64213 binding measured in intact cells expressing GB1 + GB2, GB1/2 + GB2/1, GB1 + GB1/2 or GB2/1 + GB2. Values correspond to the amount of bound radioactivity per well expressed as a percentage of the total binding measured in cells expressing the wild-type receptor. (C) Dose–effect curves of GABA, baclofen and CGP64213 on cells expressing GB1 + GB2 (open circles) or GB1/2 + GB2/1 (filled circles). The effect of CGP64213 was analysed in the presence of 0.1 μM GABA. Values correspond to the percentage of the maximal agonist-induced IP formation measured in cells expressing the wild-type receptor. Values are means ± SEM of at least three experiments performed in triplicate. Download figure Download PowerPoint Heteromeric nature of ECD is required for agonist activation, but not for G-protein coupling We then examined the possible formation of a functional receptor after coexpression of two subunits such that the wild-type heteromeric nature of the HDs was conserved, but not that of the ECDs (Figure 2A). In cells expressing GB2/1 + GB2, GABA did not stimulate IP formation, but a large increase in the basal IP formation was detectable (Figure 2A). Surprisingly, the same high basal activity and no agonist-induced response was also observed in cells expressing GB1 and GB1/2, even though both subunits expressed alone or in combination clearly bind GABAB ligands (Figure 2B and Table I) and form heterodimers. Indeed, the GB1 subunit could be immunoprecipitated from cells expressing both GB1 and GB1/2 using a GB2 C-terminal antibody (data not shown). The basal activity observed with the combination GB1 + GB1/2 could not be inhibited by the competitive antagonists CGP64213 (data not shown). However, a significant inhibition of basal IP formation was induced by 1 mM GABA (Figure 2A), an effect antagonized by CGP64213 (data not shown). Taken together, these data revealed that either of these two combinations of GABAB subunits can activate Gqi9, but that the heteromeric nature of the ECDs is required for the activation by GABA. Coexpression of GB2 and GB1/2 leads to a functional GABA receptor that couples to the same heterologous G-proteins as the wild-type receptor In order to determine whether the heteromeric nature of HDs is important or not for coupling, combinations in which the heteromeric nature of the ECDs, but not that of the HDs, is maintained were tested (Figure 3). Figure 3.Functional and biochemical analysis of GABAB receptor combinations in which the heteromeric nature of the ECDs is conserved. (A) Basal (open bars) and 1 mM GABA-induced (grey bars) IP formation were measured in cells expressing GB1 + GB2, GB1/2 + GB2, GB1 + GB2/1 or GB1ASA + GB2/1ASA and the chimeric G-protein Gqi9. Values correspond to the percentage of the GABA response measured in cells expressing the wild-type receptor. (B) Total (open bars) and non-specific (grey bars; 1 mM GABA) [125I]CGP64213 binding measured in intact cells expressing GB1, GB1ASA, GB1 + GB2, GB1ASA + GB2/1ASA or GB1/2 + GB2. Values correspond to the amount of bound radioactivity per well expressed as a percentage of the total binding measured in cells expressing the wild-type receptor. (C) Specific [125I]CGP71872 labelling immunoprecipitated with the HA antibody in cells expressing GB1 + HA-GB2, GB1ASA + HA-GB2/1ASA, GB1/2 + HA-GB2, GB1/2 + HA-GB2/1 or GB1 + HA-mGlu5. Non-specific labelling was determined in the presence of 1 mM CGP54624A. Specific 125I-labelled material immunoprecipitated is plotted as a percentage of that obtained in cells expressing GB1 + HA-GB2. Values are means ± SEM of at least three experiments performed in triplicate. Download figure Download PowerPoint Coexpression of GB2 and GB1/2 leads to a functional GABAB receptor that possesses GB2 HD only (Figure 3A). The wild-type GABAB receptor has been reported to couple not only to Gqi9, but also to Gqi5 and Gqo5, two chimeric Gαq proteins in which the five C-terminal residues have been replaced by those of Gαi and Gαo, respectively (Franek et al., 1999). However, it does not efficiently couple to the promiscuous G-proteins G15 and G16 (Figure 4), although these two G-proteins were activated by mGlu8 receptors (data not shown), as previously described (Blahos et al., 2001). Like the wild-type and the heteromeric GB1/2 + GB2/1 receptors, the coexpression of GB2 and GB1/2 leads to a receptor that can efficiently activate Gqi9, Gqi5 and Gqo5, but not G15 and G16 (Figure 4). Figure 4.Coupling of various combinations of GABAB subunits to different G-proteins. Basal (empty bars) or 1 mM GABA-induced (grey bars) IP formation were measured in cells expressing the indicated combinations of subunits and the indicated G-proteins. Values are expressed as a percentage of the GABA-induced response measured in cells expressing GB1, GB2 and Gqi9. Values are the means ± SEM of three experiments performed in triplicate. Download figure Download PowerPoint In contrast, the coexpression of the subunits GB1 and GB2/1 did not lead to a functional receptor (Figure 3A) even when the ER retention signal of both subunits was mutated (GB1ASA + GB2/1ASA). Such an absence of coupling was observed whether GABA, APPA or baclofen was used as agonist, and with all the G-proteins tested (Figure 4). This absence of coupling is not due to a low level of expression of the receptor at the cell surface. Indeed, a similar amount of specific binding of the membrane GB1-specific impermeant radioligand [125I]CGP64213 was detected on intact cells expressing GB1 + GB2 or GB1ASA + GB2/1ASA (Figure 3B). Moreover, the affinities of CGP64213 determined on cells expressing GB1 + GB2 or GB1ASA + GB2/1ASA were similar (Table I). Taken together, these data indicate that within the heterodimers, the GB2 HD is necessary to allow coupling to G-proteins, and that it possesses the molecular determinants for G-protein recognition and signalling. GB1ASA and GB2/1ASA form a heteromeric complex unable to activate G-proteins The absence of a GABA-induced response in cells coexpressing GB1ASA and GB2/1ASA is not due to the inability of these two subunits to form a heteromeric complex. Indeed, we found that material specifically labelled with the GB1 photo-affinity ligand [125I]CGP71872 (Kaupmann et al., 1997b, 1998; Malitschek et al., 1999) was co-immunoprecipitated with the HA-tagged GB2/1ASA in cells coexpressing this subunit and GB1ASA (Figure 3C). After separation on acrylamide gels, this immunoprecipitated 125I-labelled material appears as a single band with the expected molecular weight of the GB1ASA protein (data not shown). As a control experiment, the coexpression of GB1 and HA-tagged mGlu5 receptor did not lead to the co-immunoprecipitation of specific [125I]CGP71872 labelling with the HA antibody. Moreover, we found that the amount of [125I]CGP71872 specific labelling that could be co-immunoprecipitated in cells coexpressing GB1ASA and HA-GB2/1ASA represented ∼70% of that obtained in cells expressing GB1 + HA-GB2 or GB1/2 + HA-GB2/1 (Figure 3C). Therefore, GB1ASA and GB2/1ASA form heteromers like GB1 and GB2, and like GB1/2 and GB2/1. Although they can interact with each other, can GB1 and GB2/1 cross-talk with each other? It has been reported that the affinity of GABAB agonists on GB1 was increased in the presence of GB2 (Kaupmann et al., 1998). Here we show that the affinity of GABA measured in intact cells expressing GB1ASA was increased 5- to 8-fold in the presence of either GB2 or GB2/1ASA (Table I). This indicates that the cross-talk between GB1ASA and GB2 can also be observed between GB1ASA and GB2/1ASA. Then, GB1ASA and GB2/1ASA cannot activate the heterologous G-proteins even though they are targeted to the cell surface, interact with and 'talk' to each other. Heteromeric receptors with the HD of GB2 only are not as efficient as the wild-type receptor in coupling to Gqi9 In order to identify any possible role of the HD of GB1 in the G-protein coupling of the wild-type receptor, the functional coupling of the GB1/2 + GB2 combination was compared further with that of the wild-type combination. The maximal effects obtained with saturating concentration of agonists on cells expressing GB1/2 + GB2 were found to be about half of those obtained with cells expressing the wild-type or GB1/2 + GB2/1 combinations (Figures 2, 4 and 5). Moreover, the EC50 values of agonists were always higher than those measured in cells expressing the wild-type receptor (Figure 5). These differences are not due to a different level of GB1/2 + GB2 at the cell surface. Indeed, a similar amount of [125I]CGP64213 binding was measured in intact cells expressing either GB1 + GB2 or GB1/2 + GB2 (Figure 3B). Moreover the amount of [125I]CGP71872-labelled GB1/2 co-immunoprecipitated with GB2 represented 70% of that immunoprecipitated in cells expressing GB1 + GB2 (Figure 3C), indicating that the amount of heteromers in GB1/2 + GB2-expressing cells is not drastically different from that in GB1 + GB2-expressing cells. Finally, the GB1/2 subunit is in a high agonist affinity state in the presence of GB2 (Table I). Taken together, these data reveal that the heteromeric GB1/2 + GB2 receptor is less efficient in activating Gqi9 than the wild-type GABAB receptor, suggesting that the presence of the HD of GB1 is necessary for full coupling to G-proteins of the heterodimer. Figure 5.Effects of various concentrations of agonists and antagonists on IP formation in cells expressing GB1 + GB2 (open circles) or GB1/2 + GB2 (filled diamonds) together with Gqi9. Values are the percentage of the maximal GABA-induced formation of IP measured in GB1 + GB2-expressing cells. The effect of CGP64213 was tested in the presence of 0.1 μM GABA (GB1 + GB2) or 15 μM GABA (GB1/2 + GB2). Values are the means ± SEM of at least three experiments performed in triplicate. Download figure Download PowerPoint Discussion Among all GPCRs, the GABAB receptor is the only one known so far that requires the presence of two subunits, GB1 and GB2, for efficient coupling to G-proteins. We and others have recently shown that GB1 binds GABAB ligands, and that GB2 is necessary for GB1 insertion in the plasma membrane. The present study revealed new roles of both subunits for the formation of a fully functional receptor. We show that the HD of GB2 contains the molecular determinants for the recognition and activation of G-proteins. However, for an efficient coupling to G-proteins, the GB1 HD is necessary. In addition, we show that both GB1 and GB2 ECDs are required for GABA-induced activation of the receptor. Functional specialization of GABAB subunits We recently reported that the GABA binding site located within the ECD of GB1 plays a critical role in the recognition of GABAB ligands not only by the GB1 subunit expressed alone, but also by the heteromeric GABAB receptor (Galvez et al., 2000a,b). Here we showed that the HD of GB2 can activate the same G-proteins as those activated by the wild-type receptor. In contrast, such coupling could not be measured in combinations of subunits that have the HD of GB1 only, even though the subunits could reach the cell surface (since the ER retention signal was mutated). It seems likely, therefore, that the GB1 HD does not couple to G-proteins, or at least does not activate the G-proteins tested here. In order to ascertain further the critical role played by the HD of GB2 in the heteromeric wild-type receptor in G-protein activation, it would be of interest to prevent GB2 coupling to G-proteins and determine whether the heteromer is functional or not. We therefore introduced point mutations in the i3 loop of GB2 (and also in GB1 as a control), similar to those that prevent G-protein coupling in mGlu (Francesconi and Duvoisin, 1998) and CaS (Pollak et al., 1993) receptors. However, all mutant subunits (R679W or R679D in GB2 and K791W or I798S in GB1) still form functional receptors when expressed either with the complementary wild-type subunit or in combination (B.Duthey, J.-P.Pin and L.Prézeau, unpublished data), showing that these mutations do not prevent the G-protein coupling of the GABAB receptor subunits. Within the family 3 GPCRs, the second and third intracellular loops play a critical role in G-protein recognition (Pin et al., 1994; Gomeza et al., 1996) and activation (Pollak et al., 1993; Francesconi and Duvoisin, 1998; Chang et al., 2000), respectively. Indeed, the second intracellular loops of GB1 and GB2 share no sequence similarity, in agreement with the difference noticed here for the G-protein coupling properties of GB1 and GB2. This does not mean that the HD of GB1 does not play a role in the signalling of the heteromeric receptor. Indeed, the GB1 subunit can associate with the transcription factors ATF4 and ATFx (Nehring et al., 2000; White et al., 2000), suggesting that this subunit may be involved in other transduction cascades not necessarily involving G-proteins. Cooperative interactions between different GABAB functional domains Although the ECD of GB1 clearly binds GABAB ligands, and although the HD of GB2 can couple and activate G-proteins, a chimeric receptor comprising these two domains (GB1/2) did not lead to a functional receptor that can be activated by GABA when expressed alone. However, it did so when coexpressed with either GB2 or the converse chimera GB2/1. This observation highlights further the importance of cooperative interactions between different GABAB functional domains for a correct activation of the HD of GB2 by the agonist-occupied ECD of GB1. In agreement with this proposal, the heterodimer in which the two ECDs have been swapped between the two subunits (GB1/2 + GB2/1) behaves exactly like the wild-type receptor in all aspects studied here, i.e. coupling efficacy, G-protein selectivity, agonist and antagonist potencies and cross-talk between the two subunits. Indeed, our data indicate that cooperativity occurred at the level of both the ECDs and the HDs between the two subunits. Concerning the ECD, we found that among all wild-type and chimeric GABAB subunits exp