Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration
2009; Springer Nature; Volume: 28; Issue: 15 Linguagem: Inglês
10.1038/emboj.2009.177
ISSN1460-2075
AutoresM. Rives, Claire Vol, Yugo Fukazawa, Norbert Tinel, Eric Trinquet, Mohammed Akli Ayoub, Ryuichi Shigemoto, Jean‐Philippe Pin, Laurent Prézeau,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoArticle9 July 2009free access Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration Marie-Laure Rives Marie-Laure Rives Department of Molecular Pharmacology, CNRS, UMR 5203, Institut de Génomique fonctionnelle, Montpellier, France INSERM U661, Montpellier, France Université Montpellier, Montpellier, France Search for more papers by this author Claire Vol Claire Vol Department of Molecular Pharmacology, CNRS, UMR 5203, Institut de Génomique fonctionnelle, Montpellier, France INSERM U661, Montpellier, France Université Montpellier, Montpellier, France Search for more papers by this author Yugo Fukazawa Yugo Fukazawa Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki, Japan Search for more papers by this author Norbert Tinel Norbert Tinel CisBio International, Parc technologique Marcel Boiteux, Bagnols/Cèze Cedex, France Search for more papers by this author Eric Trinquet Eric Trinquet CisBio International, Parc technologique Marcel Boiteux, Bagnols/Cèze Cedex, France Search for more papers by this author Mohammed Akli Ayoub Mohammed Akli Ayoub Department of Molecular Pharmacology, CNRS, UMR 5203, Institut de Génomique fonctionnelle, Montpellier, France INSERM U661, Montpellier, France Université Montpellier, Montpellier, France Search for more papers by this author Ryuichi Shigemoto Ryuichi Shigemoto Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki, Japan Search for more papers by this author Jean-Philippe Pin Jean-Philippe Pin Department of Molecular Pharmacology, CNRS, UMR 5203, Institut de Génomique fonctionnelle, Montpellier, France INSERM U661, Montpellier, France Université Montpellier, Montpellier, France Search for more papers by this author Laurent Prézeau Corresponding Author Laurent Prézeau Department of Molecular Pharmacology, CNRS, UMR 5203, Institut de Génomique fonctionnelle, Montpellier, France INSERM U661, Montpellier, France Université Montpellier, Montpellier, France Search for more papers by this author Marie-Laure Rives Marie-Laure Rives Department of Molecular Pharmacology, CNRS, UMR 5203, Institut de Génomique fonctionnelle, Montpellier, France INSERM U661, Montpellier, France Université Montpellier, Montpellier, France Search for more papers by this author Claire Vol Claire Vol Department of Molecular Pharmacology, CNRS, UMR 5203, Institut de Génomique fonctionnelle, Montpellier, France INSERM U661, Montpellier, France Université Montpellier, Montpellier, France Search for more papers by this author Yugo Fukazawa Yugo Fukazawa Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki, Japan Search for more papers by this author Norbert Tinel Norbert Tinel CisBio International, Parc technologique Marcel Boiteux, Bagnols/Cèze Cedex, France Search for more papers by this author Eric Trinquet Eric Trinquet CisBio International, Parc technologique Marcel Boiteux, Bagnols/Cèze Cedex, France Search for more papers by this author Mohammed Akli Ayoub Mohammed Akli Ayoub Department of Molecular Pharmacology, CNRS, UMR 5203, Institut de Génomique fonctionnelle, Montpellier, France INSERM U661, Montpellier, France Université Montpellier, Montpellier, France Search for more papers by this author Ryuichi Shigemoto Ryuichi Shigemoto Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki, Japan Search for more papers by this author Jean-Philippe Pin Jean-Philippe Pin Department of Molecular Pharmacology, CNRS, UMR 5203, Institut de Génomique fonctionnelle, Montpellier, France INSERM U661, Montpellier, France Université Montpellier, Montpellier, France Search for more papers by this author Laurent Prézeau Corresponding Author Laurent Prézeau Department of Molecular Pharmacology, CNRS, UMR 5203, Institut de Génomique fonctionnelle, Montpellier, France INSERM U661, Montpellier, France Université Montpellier, Montpellier, France Search for more papers by this author Author Information Marie-Laure Rives1,2,3, Claire Vol1,2,3, Yugo Fukazawa4, Norbert Tinel5, Eric Trinquet5, Mohammed Akli Ayoub1,2,3, Ryuichi Shigemoto4, Jean-Philippe Pin1,2,3 and Laurent Prézeau 1,2,3 1Department of Molecular Pharmacology, CNRS, UMR 5203, Institut de Génomique fonctionnelle, Montpellier, France 2INSERM U661, Montpellier, France 3Université Montpellier, Montpellier, France 4Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki, Japan 5CisBio International, Parc technologique Marcel Boiteux, Bagnols/Cèze Cedex, France *Corresponding author. Department of Molecular Pharmacology, Institut de Génomique Fonctionnelle, 141 rue de la Cardonille, Montpellier, Cedex 5 34094, France. Tel.: +33 0467 1429 33; Fax: +33 0467 1429 96; E-mail: [email protected] The EMBO Journal (2009)28:2195-2208https://doi.org/10.1038/emboj.2009.177 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info G protein-coupled receptors (GPCRs) have critical functions in intercellular communication. Although a wide range of different receptors have been identified in the same cells, the mechanism by which signals are integrated remains elusive. The ability of GPCRs to form dimers or larger hetero-oligomers is thought to generate such signal integration. We examined the molecular mechanisms responsible for the GABAB receptor-mediated potentiation of the mGlu receptor signalling reported in Purkinje neurons. We showed that this effect does not require a physical interaction between both receptors. Instead, it is the result of a more general mechanism in which the βγ subunits produced by the Gi-coupled GABAB receptor enhance the mGlu-mediated Gq response. Most importantly, this mechanism could be generally applied to other pairs of Gi- and Gq-coupled receptors and the signal integration varied depending on the time delay between activation of each receptor. Such a mechanism helps explain specific properties of cells expressing two different Gi- and Gq-coupled receptors activated by a single transmitter, or properties of GPCRs naturally coupled to both types of the G protein. Introduction G protein-coupled receptors (GPCRs) are encoded by the largest mammalian gene family; there are about 900 receptors in humans. Owing to the critical functions they have in intercellular communication and their involvement in all major physiological functions, these receptors and their associated signalling complexes represent major targets for drug development (Overington et al, 2006). Each cell usually expresses several subtypes of these GPCRs, integrating their numerous signals both spatially and temporally to produce the cellular response (Selbie and Hill, 1998). However, the molecular mechanisms allowing such signal integrations are still not clearly understood and may occur at the level of receptor signaling pathways or even at the level of receptors themselves. A recent proposal suggests that such signal integration is facilitated through the ability of GPCRs to form dimers or larger oligomers. Indeed, two different GPCRs can physically associate together in complexes, either as heterodimers or hetero-oligomers, which could in turn show specific pharmacological and signaling behaviour, have an impact on cellular physiology, or be involved in pathologies (Bouvier, 2001; Angers et al, 2002; Milligan, 2004; Franco et al, 2007), making them interesting new targets for more selective drugs (Ferre et al, 2007; Pin et al, 2007; Gurevich and Gurevich, 2008). For example, the pharmacologically well-defined κ, μ and δ opioid receptor subtypes have been proposed to assemble into hetero-oligomers showing different ligand binding profiles and G protein-coupling properties (Jordan and Devi, 1999; George et al, 2000), as well as be involved in specific functional responses in animals (Waldhoer et al, 2005). Furthermore, in rat, the oligomerization of the metabotropic glutamate receptor type 2 (mGlu2) and the serotonin receptor 5-HT2A has recently been shown to control the action of hallucinogens in the prefrontal cortex (Gonzalez-Maeso et al, 2008). Another interesting example is the orphan receptor, GPR50, which interacts with the melatonin MT1 receptor to abolish both high-affinity agonist binding and G protein coupling, thus modifying the melatonin-induced cellular response (Levoye et al, 2006). In cerebellar Purkinje cells, activation of the GABAB receptor increases calcium responses generated by the mGlu1a receptor (Hirono et al, 2001; Tabata et al, 2004). Such a phenomenon would have an important function in generation of long-term depression at the parallel fibre—Purkinje cell synapses (Kamikubo et al, 2007). Both the receptors are indeed co-expressed in these neurons, and both were described in dendritic spines (Ige et al, 2000). Moreover, it was reported that both receptors could be co-immunoprecipitated (Tabata et al, 2004), suggesting that the observed crosstalk could result from the existence of possible mGlu1a–GABAB heteromers. Such a proposal is supported by the absence of potentiation of the mGlu1a response by other Gi-coupled receptors like the adenosine A1 receptors also expressed in Purkinje cells (Hirono et al, 2001). Consequently, it would be of interest to determine whether such a GABAB–mGlu1a oligomer could be selectively targeted pharmacologically, thus enabling a specific effect on this cross-regulation. However, it may well also result from a functional crosstalk, as previously reported for other Gq- and Gi-coupled receptor pairs (Carroll et al, 1995). The aim of this study was to analyse the molecular basis for such physiological crosstalk between GABAB and mGlu1a receptors. Our data show that the functional crosstalk can be observed with other pairs of Gi- and Gq-coupled receptors and reproduced in primary cells, again without a direct physical interaction occuring between these receptors in transfected cells. This general phenomenon may improve our understanding of the mechanisms involved in the spatial and temporal integration of GPCR signals in any given cell. Results mGlu1a and GABAB are co-localized in dendritic spines of Purkinje cells Previous data have suggested that the functional crosstalk between GABAB and mGlu1a receptors is the result of their physical association in the dendritic spines of Purkinje cells facing the parallel fibre terminals (Hirono et al, 2001; Tabata et al, 2004; Kamikubo et al, 2007). Such a proposal was primarily based on the co-immunoprecipitation of both receptors from cerebellar extracts. Using an SDS freeze-fracture replica labelling technique coupled to electronic microscopy, we visualized the precise co-localization of both receptors in Purkinje-cell dendritic spines on a nanometric scale (Figure 1). We observed mGlu1a labelling distributed in a circular pattern within the spine plasma membrane and surrounding the postsynaptic membrane specialization in individual spines. The distribution of GB1 immunoreactivity was similar but was also found in other areas, such as within the specialization (Figure 1). Specificity of the replica labelling with the primary antibodies against GB1 and mGlu1a was previously confirmed using respective knockout mice (Kulik et al, 2006; Kaufmann et al, 2009). Although not definitive, these data are consistent with a possible physical association between both receptors, leading us to examine whether the assembly of mGlu1a and GABAB receptors is necessary for their functional crosstalk. Figure 1.Co-localization of GB1 and mGlu1a receptors in Purkinje-cell spines as shown by SDS freeze-fracture replica labelling. Freeze-fracture replicas prepared from mouse cerebellum were labelled with 5-nm (small black dots pointed by arrows in panel B) and 10-nm (bold black dots in panels A and B) immunoparticles to detect GB1 and mGlu1a receptors, respectively. (A) Low magnification view of a Purkinje-cell dendrite from which two dendritic spines emerge. Post-synaptic membrane specialization in individual spines was indicated by a dotted line. (B) High magnification view of a detailed Purkinje-cell dendrite spine from A. Scale bars=500 nm in A; 100 nm in B. Download figure Download PowerPoint GABAB enhanced mGlu1a-mediated responses in cortical neurons and transfected cells We first examined whether it was possible to observe the GABAB–mGlu1a crosstalk in other neuronal cell types. Indeed, as reported in Purkinje cells, GABAB activation enhanced the mGlu1a-mediated increase in intracellular calcium level (Ca2+i), both in cultured cortical neurons (Figure 2A) and in HEK293 cells co-expressing both GABAB and mGlu1a receptors (Figure 2B–E). Indeed, in HEK293 cells, the GABAB-induced potentiation of the mGlu1a-induced calcium response also led to an increased efficacy and potency of glutamate (2.41-±0.96-fold increase in the EC50 by 50 μM GABA). Notably, activation of the Gi/o-coupled GABAB receptor did not lead to a significant change in intracellular Ca2+ concentration. Conversely, the maximal calcium response induced by 100 μM glutamate was increased in a dose-dependent manner by GABA with an EC50 of 0.37±0.10 μM, in agreement with the known potency of GABA at this receptor (0.31±0.07 μM; (Binet et al, 2007)). Surprisingly, the strength of the potentiation seemed to depend on the initial amplitude of the mGlu1a-induced calcium response; a low mGlu1a-induced calcium response was generally more potentiated by GABAB activation than a stronger response, suggesting the involvement of complex signal integration pathways (Figure 2C). These results were not because of a ceiling effect, as the experiments were carried out in conditions in which the mGlu1a calcium responses were not saturating (Supplementary data 1C). Figure 2.Functional crosstalk between GABAB and mGlu1a receptors in cortical neurons and in transfected HEK293 cells. (A) Activation of GABAB receptor alone by Baclofen 100 μM (filled squares) and mGlu1a by DHPG in the presence (open circles) or in the absence (filled circles) of baclofen (100 μM), in cortical neurons. (B, C) Potentiation of the glutamate response by co-activation of the GABAB receptors in HEK293 cells. In (B), Ca2+ responses mediated by various concentrations of glutamate (circles) and GABA (triangles) in cells expressing both mGlu1a and GABAB receptors. Glutamate responses were measured in the absence (filled circles) and in the presence (open circles) of 50 μM GABA. The GABA-mediated responses were measured in the absence (filled triangles) and in the presence (open triangles) of 100 μM glutamate. (C) Glutamate responses, as in (B), in cells expressing either a low (triangles) or a high (circles) density of mGlu1a. (D, E) The GABAB effect needs Gi/o protein activation. (D) Same as in (C) in cells co-expressing the WT (circles) or a G protein activation-deficient (L686P represented by triangles) GABAB receptor and mGlu1a receptor. (E) Same as in (C), in cells expressing GABAB and mGlu1a receptors, treated OVN with PTX (squares) or not (circles). The Gαo protein was co-expressed in all of these experiments. Data are means±s.e.m. of triplicate determinations from a representative experiment reproduced at least three times. Download figure Download PowerPoint When the G protein-activating GABAB receptor GB2 subunit bore the L686P mutation that suppresses coupling to G proteins (Duthey et al, 2002), potentiation was not observed, showing the necessity for functional coupling of the GABAB receptor to G proteins (Figure 2D). Similarly, inhibiting the Gi/o proteins with pertussis toxin (PTX) also prevented GABAB-mediated potentiation of the mGlu1a response (Figure 2E). Moreover, although this potentiation occurred without co-transfecting the G protein αo or i1 subunits (1.25-±0.05-fold increase of the maximal response, Supplementary data 1A), co-expression of the Gαo or i1 subunits further increased the GABAB potentiating effect, consistent with the necessity for Gi/o activation (Figure 2 and Supplementary data 1). Taken together, these observations led us to question the requirement for mGlu1a–GABAB receptor oligomerization for such crosstalk to occur, rather than a functional signal integration mechanism. GABAB and mGlu1a receptors did not form hetero-oligomers in transfected cells Using various approaches, we observed no oligomerization of GABAB and mGlu1a receptors in HEK293 cells (Figures 3, 4 and 5). First, we used antibody-based time-resolved (TR)-FRET technology, allowing the detection of energy transfer between anti-HA antibodies bearing the donor fluorophore europium-cryptate (HA–K) and anti-Flag antibodies bearing the acceptor fluorophore d2 (Flag–d2) (Maurel et al, 2004). Under these conditions, we observed a highly significant FRET signal in cells expressing HA-tagged GB1 (HA–GB1) and Flag-tagged GB2 (Flag–GB2) subunits of the heterodimeric GABAB receptor (Figure 3A and B). We obtained a similar FRET signal within the mGlu1a homodimer (between the HA-tagged mGlu1a and the Flag-tagged mGlu1a), the β2-adrenergic receptor dimer (between the HA-tagged β2AR and the Flag-tagged β2AR), as well as a significant FRET signal between two different receptors known to form hetero-oligomeric complexes, 5-HT2a and mGlu2 (Gonzalez-Maeso et al (2008); Figure 3A). In contrast, in a similar range of receptor expression levels (Supplementary data 2A), we detected no significant FRET signal between HA–mGlu1a and GABAB receptors, in which either GB1 or GB2 was tagged with the Flag epitope (Figure 3A and B). Similarly, we observed no significant TR-FRET signal in cells expressing either mGlu3 and GABAB (Figure 3A and B) or mGlu4 and GABAB (Figure 3A, data not shown). Figure 3.Absence of FRET between GABAB and mGlu1a receptors co-expressed in HEK293 cells. The FRET experiments were carried out using antibody- and SNAP-tag-based HTRF technologies. (A) Antibody-based HTRF experiments. The receptors were labelled with anti-HA and anti-Flag antibodies coupled to cryptate (HA–Ab–K) or D2 (Flag–Ab–D2) dyes. (B) FRET signal in function of the expression of the Flag-tagged receptors, in antibody-based HTRF experiments. Open squares: HA–GB1+Flag–GB2. Filled squares: HA–mGlu1a, GB1 and Flag–GB2. Open circles: HA–mGlu3+GB1+Flag–GB2. Cells were transfected with a constant amount (30 ng) of GB1, mGlu1a, mGlu3 or mGlu4 plasmids, and increasing amounts of the Flag–GB2 plasmid (0–30 ng per well) Expression of the receptors was determined with an ELISA assay against the Flag epitopes. (C) Combined antibody- and SNAP-tag-based TR-FRET experiment. The FRET experiment was carried out after labelling of the SNAP-tag with BG–K2 and then incubation of the anti-Flag antibodies coupled to D2. FRET signal is shown as a function of the cell-surface expression of the receptors, determined by an ELISA assay against the Flag epitope. Filled circles: ST–GB1 and Flag–GB2. Open circles: ST–5HT2a and Flag–mGlu2. Open squares: ST–GB1+GB2 and Flag–mGlu1a. Data are means±s.e.m. of triplicate determinations from a representative experiment reproduced at least three times. Download figure Download PowerPoint Figure 4.No significant BRET signal detected between mGlu1a and GABAB in HEK293 cells. (A, B) BRET signal was monitored in cells expressing combinations of receptors fused to YFP or RLuc proteins. (A) Cells were transfected with a constant amount (30 ng) of GB2-RLuc plasmid with increasing amounts of GB1–YFP plasmid (0–50 ng per well) (open circles), or with constant amount of GB2–RLuc and GB1 plasmids (30 ng each per well) and increasing amounts (0–50 ng per well) of PAR1–YFP plasmid (filled squares), or mGlu1a–YFP (open squares). (B) Cells were transfected with a constant amount (30 ng) of mGlu1a-RLuc plasmid and increasing amounts (0–50 ng per well) of mGlu1a–YFP (open squares), YFP(venus)–Homer3 (filled squares), GB1–YFP and GB2 (open circles), or GB2–YFP and GB1 (filled circles) plasmids. Data are means±s.e.m. of triplicate determinations from a representative experiment reproduced at least three times. Download figure Download PowerPoint Figure 5.No cell-surface co-immunoprecipitation between mGlu1a and GABAB in HEK293 cells. Upper panel: Cell-surface expression of the tagged receptors, determined by an ELISA assay. Lower panel: Cell-surface co-immunoprecipitation of the Flag-tagged receptors and western Blot carried out using anti-HA and anti-Flag antibodies from cells transfected with indicated plasmids. Data are representative of several experiments. Download figure Download PowerPoint To exclude the possibility that the absence of FRET was because of steric hindrance imposed by the large size of the antibodies, we replaced one epitope tag by a SNAP-tag, allowing specific labelling with either the europium cryptate or d2 fluorophores as previously reported (Maurel et al, 2008). The SNAP-tag is a 20 KDa modified alkyl guanine transferase that can be covalently labelled with fluorophore-coupled benzyl guanines (BG): europium cryptate-coupled BG (BG-K) or d2-coupled BG (BG-d2). A high FRET signal was detected in cells expressing SNAP-tagged GB1 (ST–GB1) and Flag–GB2, whereas a lower but significant FRET signal was also detected within the 5HT2a–mGlu2 receptor complex (Figure 3C). However, no (or a very weak) FRET signal was measured between Flag–mGlu1a receptor and the ST–GB1-containing GABAB receptor over a range of receptor expressions (Figure 3C). Moreover, a combination of ST–GB2-containing GABAB receptor and ST–mGlu1a (in which only one protomer was labelled) did not show any significant FRET signal (Supplementary data 2D). The overexpression of the Gαo protein, found to increase the functional crosstalk, did not modify the TR-FRET signal between GABAB and mGlu1a receptors (Supplementary data 2C). Consistent with our FRET results, a low linear BRET signal was observed in cells expressing mGlu1a–YFP and GB2–Rluc-containing GABAB receptors. This signal was in the same range as the one in negative control cells expressing PAR1–YFP and GB2–Rluc-containing GABAB receptors (Figure 4A). In contrast, BRET signals in positive control cells expressing GB1–YFP and GB2–Luc, fit a parabolic curve with a high maximum value (max=340.9 milliBRET). Similar results were obtained on inverting the fluorescent tags on the receptors (Figure 4B). A low linear BRET signal was observed in cells expressing mGlu1a–RLuc and either GB1–YFP- or GB2–YFP-containing GABAB receptors (Figure 4B). We validated the ability of mGlu1a constructs to generate BRET signals by measuring a saturating BRET signal in cells expressing either mGlu1a–RLuc and mGlu1a–YFP (Figure 4B); mGlu1a–RLuc and Homer3–YFP(Venus) or mGlu1a–YFP and Homer3–RLuc (Figure 4B and data not shown). GABAB and mGlu1a receptors did not co-immunoprecipitate from transfected HEK293 cell-surface extract Although GABAB and mGlu1a receptors have been found to co-immunoprecipitate from brain extract (Tabata et al, 2004), no co-immunoprecipitation of HA–GB1-containing GABAB and Flag–mGlu1a receptors was obtained from HEK293 cell-surface extracts (Figure 5). In contrast, HA–GB1 co-immunoprecipitated with Flag–GB2 in cells expressing both subunits, as well as with Flag–GB1 in cells expressing HA–GB1, Flag–GB1 and GB2, as GABAB receptors form tetramers through interaction between the GB1 subunits (Maurel et al, 2008). As expected, we also succeeded in detecting a high level of cell-surface interaction between the Flag-tagged mGlu2 and the HA-tagged 5HT2a receptors, but no co-immunoprecipitation between GABAB and mGlu4 receptors used as a negative control. Although the C-terminal tail of the mGlu1a receptor known to interact with intracellular partners (Fagni et al, 2004) has been reported to be involved in mGlu1a assembly with other receptors (Ciruela et al, 2001), functional crosstalk could still be observed between the GABAB receptor and the mGlu1 short splice variant mGlu1b receptor possessing a short C-terminal tail (Pin and Duvoisin (1995); data not shown). Taken together, the data above provide evidence against oligomerization between GABAB and mGlu1a receptors in HEK293 cells, however, we did observe a functional crosstalk between them, such as that reported in neurons. Mechanism of the functional interaction: involvement of the βγ subunits of the Gi/o protein Evidence for the involvement of the βγ subunits of Gi/o G proteins was obtained from different sets of results from cells expressing GABAB, mGlu1a and αo G protein subunit. First, the GABAB-induced potentiation of the mGlu1a-induced calcium response (Figure 6A) was decreased by trapping the endogenous βγ subunits, using the co-expressed C-terminus of the β-adrenergic receptor kinase (βARK-CT), which is known to interact with βγ subunits. Second, the co-expression of β1γ2 in the presence of αo increased the potentiation (Figure 6B). We then overexpressed β1γ2 alone (not αo), with the hope that, due to a limitation in the amount of α subunits, free β1γ2 will be constantly available (Supplementary data 3). As such, activating the GABAB receptor was not expected to have an effect, as it would have already been generated by the free β1γ2 overexpressed in the cells. Consistent with this idea, GABAB receptor activation did not enhance mGlu1a-mediated response in cells overexpressing β1γ2 (Figure 6C). Such an effect of β1γ2 probably results from their action on PLC activity (Quitterer and Lohse, 1999) that has been clearly shown for PLCβ type 3 (Park et al, 1993), which is expressed in the HEK293 cells (Supplementary data 6). Figure 6.The crosstalk was dependent on the βγ subunits. (A) Ca2+ responses induced by various concentrations of glutamate was monitored in HEK293 cells expressing both mGlu1a and GABAB (GB1+GB2) receptors and the αo G protein subunit, in the absence (circles) or in the presence (triangles) of βARK C-terminal domain (βARK-CT), a chelator of the βγ subunits. Glutamate responses were measured in the absence (filled shapes) and in the presence (open shapes) of 50 μM GABA. (B) Ca2+ responses mediated by various concentrations of glutamate in cells expressing both mGlu1a and GABAB (GB1+GB2) receptors and the αo G protein subunit, in the absence (circles) or in the presence (triangles) of co-expressed β1 and γ2 subunits. Glutamate responses were measured in the absence (filled shapes) and in the presence (open shapes) of 50 μM GABA. (C) Ca2+ responses mediated by various concentrations of glutamate in cells expressing both mGlu1a and GABAB (GB1+GB2) receptors and βγ subunits, but not the Gαo subunit. Glutamate responses were measured in the absence (filled circles) and in the presence (open circles) of 50 μM GABA. Data are means±s.e.m. of triplicate determinations from a representative experiment reproduced at least three times. Download figure Download PowerPoint Kinetics of the GABAB receptor-mediated potentiation If βγ production is responsible for the GABAB receptor-mediated potentiation of the mGlu1a-induced calcium response, one might expect that the activation of the GABAB receptor initiated before the activation of mGlu1a could still induce a potentiation of the mGlu1a Ca2+ signal. As shown in Figure 7D, this was clearly the case, as a stronger potentiation of the mGlu1a-induced calcium response was obtained when activating the GABAB receptor 100 s before the mGlu1a receptor. Such a kinetic profile probably reflects the kinetics of GABAB-induced activation of the G protein. Indeed, when Go-protein activation was recorded in living cells using a BRET approach (based on the use of αo—RLuc and β1 (Figure 7B and C) or γ2 (Supplementary data 7) fused to YFP (YFP–β1 and YFP–γ2, respectively)), G-protein activation was observed on GABAB receptor activation by GABA, as seen by the decrease in BRET signal (Figure 7B; Gales et al (2006)). Moreover, when the GABAB receptor was first activated and then antagonized with CGP54626, a full inhibition of G-protein activation was observed and correlated with an inhibition of the potentiation of the mGlu1a-mediated response (Figure 7A and B). These data highlight the importance of the temporal integration of receptor-induced signals. Figure 7.Correlation between Ca2+-response potentiation and kinetics of association of the αo–βγ G protein subunits. (A) Ca2+ responses mediated by various concentrations of glutamate in HEK293 cells expressing both mGlu1a and GABAB receptors and Gαo subunit. Glutamate responses were measured in the absence of GABA (filled squares), after incubation with 10 μM of GABA for 100 s (open squares), or after incubation with 10 μM of GABA for 40 s followed by a second incubation with 10 μM of GABA+50 μM of the GABAB antagonist CGP54626 for 60 s (open circles). (B, C) Kinetics of the BRET signal generated by Gαo–RLuc and β1–YFP on GABAB receptor activation, in cells transfected with mGlu1a and GABAB receptors. (B) BRET signal detected after activation of the GABAB receptor with GABA (10 μM), followed 40 s later by the application of the antagonist CGP54626 (50 μM). Data are collected every 0.5 s, and each point corresponds to the average of three consecutive measurements. (C) BRET signal detected after application of CGP54626 (50 μM) followed 40 s later by application of GABA (10 μM). (D) In cells transfected with mGlu1a and GABAB and Gαo subunit, the mGlu1a calcium responses (filled circles) were measured on simultaneous co-activation of the GABAB receptor (open circles) or incubation with GABA for 100 s before glutamate application (filled triangles). Data are means±s.e.m. of tripl
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