Functioning of the dimeric GABAB receptor extracellular domain revealed by glycan wedge scanning
2008; Springer Nature; Volume: 27; Issue: 9 Linguagem: Inglês
10.1038/emboj.2008.64
ISSN1460-2075
AutoresPhilippe Rondard, Siluo Huang, Carine Monnier, Haijun Tu, Bertrand Blanchard, Nadia Oueslati, Fanny Malhaire, Ying Li, Eric Trinquet, Gilles Labesse, Jean‐Philippe Pin, Jianfeng Liu,
Tópico(s)Neuropeptides and Animal Physiology
ResumoArticle3 April 2008free access Functioning of the dimeric GABAB receptor extracellular domain revealed by glycan wedge scanning Philippe Rondard Philippe Rondard CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France Search for more papers by this author Siluo Huang Siluo Huang Sino-France Laboratory for Drug Screening, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, Hubei, China Search for more papers by this author Carine Monnier Carine Monnier CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France Search for more papers by this author Haijun Tu Haijun Tu Sino-France Laboratory for Drug Screening, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, Hubei, China Search for more papers by this author Bertrand Blanchard Bertrand Blanchard CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France Search for more papers by this author Nadia Oueslati Nadia Oueslati CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France Search for more papers by this author Fanny Malhaire Fanny Malhaire CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France Search for more papers by this author Ying Li Ying Li Sino-France Laboratory for Drug Screening, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, Hubei, China Search for more papers by this author Eric Trinquet Eric Trinquet CisBio International, Parc technologique Marcel Boiteux, Bagnols/Cèze, France Search for more papers by this author Gilles Labesse Gilles Labesse Centre de Biochimie Structurale, CNRS, UMR5048, Université Montpellier 1, Montpellier, France INSERM U414, Montpellier, France Search for more papers by this author Jean-Philippe Pin Corresponding Author Jean-Philippe Pin CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France Search for more papers by this author Jianfeng Liu Corresponding Author Jianfeng Liu Sino-France Laboratory for Drug Screening, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, Hubei, China Search for more papers by this author Philippe Rondard Philippe Rondard CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France Search for more papers by this author Siluo Huang Siluo Huang Sino-France Laboratory for Drug Screening, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, Hubei, China Search for more papers by this author Carine Monnier Carine Monnier CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France Search for more papers by this author Haijun Tu Haijun Tu Sino-France Laboratory for Drug Screening, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, Hubei, China Search for more papers by this author Bertrand Blanchard Bertrand Blanchard CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France Search for more papers by this author Nadia Oueslati Nadia Oueslati CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France Search for more papers by this author Fanny Malhaire Fanny Malhaire CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France Search for more papers by this author Ying Li Ying Li Sino-France Laboratory for Drug Screening, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, Hubei, China Search for more papers by this author Eric Trinquet Eric Trinquet CisBio International, Parc technologique Marcel Boiteux, Bagnols/Cèze, France Search for more papers by this author Gilles Labesse Gilles Labesse Centre de Biochimie Structurale, CNRS, UMR5048, Université Montpellier 1, Montpellier, France INSERM U414, Montpellier, France Search for more papers by this author Jean-Philippe Pin Corresponding Author Jean-Philippe Pin CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France Search for more papers by this author Jianfeng Liu Corresponding Author Jianfeng Liu Sino-France Laboratory for Drug Screening, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, Hubei, China Search for more papers by this author Author Information Philippe Rondard1,‡, Siluo Huang2,‡, Carine Monnier1, Haijun Tu2, Bertrand Blanchard1, Nadia Oueslati1, Fanny Malhaire1, Ying Li2, Eric Trinquet3, Gilles Labesse4,5, Jean-Philippe Pin 1 and Jianfeng Liu 2 1CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France and INSERM, U661, Montpellier, France and Université Montpellier 1, 2, Montpellier, France 2Sino-France Laboratory for Drug Screening, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, Hubei, China 3CisBio International, Parc technologique Marcel Boiteux, Bagnols/Cèze, France 4Centre de Biochimie Structurale, CNRS, UMR5048, Université Montpellier 1, Montpellier, France 5INSERM U414, Montpellier, France ‡These authors contributed equally to this work *Corresponding authors: CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France, and INSERM, U661, Montpellier, France, and Université Montpellier 1,2, Montpellier 34000, France. Tel.: +33 467 14 2988; Fax: +33 467 54 2432; E-mail: [email protected] Sino-France Laboratory for Drug Screening, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, Hubei, China. Tel.: +86 278 779 2031; Fax: +86 278 779 2024; E-mail: [email protected] The EMBO Journal (2008)27:1321-1332https://doi.org/10.1038/emboj.2008.64 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABAB1 and GABAB2. GABAB1 binds agonists, whereas GABAB2 is required for trafficking GABAB1 to the cell surface, increasing agonist affinity to GABAB1, and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABAB1 VFT leads to GABAB2 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABAB VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABAB2, including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation. Introduction GABA is the main inhibitory neurotransmitter in the central nervous system, regulating many physiological and psychological processes. It mediates fast synaptic inhibition through ionotropic GABAA receptors, as well as slow and prolonged synaptic inhibition through both pre- and post-synaptic metabotropic GABAB receptors (Couve et al, 2004; Bettler and Tiao, 2006). GABAB receptors represent promising drug targets for the treatment of epilepsy, pain, drug addiction, anxiety and depression (Cryan and Kaupmann, 2005; Bowery, 2006). GABAB agonists have demonstrated beneficial effects in humans, as illustrated by the anti-spastic activity of baclofen (Lioresal®). Recently, GABAB-positive allosteric modulators (PAM) have been identified as potentially better alternatives to agonists, as they can limit the development of tolerance and avoid the adverse effects observed with agonists (Pin and Prézeau, 2007). At the structural level, the GABAB receptor is composed of two homologous subunits, GABAB1 and GABAB2 (Jones et al, 1998; Kaupmann et al, 1998; White et al, 1998; Kuner et al, 1999), also called GB1 and GB2 (Figure 1A). Heterodimerization of GB1 and GB2 is required for the formation of functional receptors, both in recombinant systems and in native tissues. Cell-surface trafficking of the GABAB receptor is controlled by an endoplasmic reticulum retention signal (RSR motif) located in the intracellular C-terminal region of GB1. This signal can be masked through a coiled-coil (CC) interaction with the intracellular tail of GB2, such that GB1 reaches the cell surface only when associated with GB2 (Margeta-Mitrovic et al, 2000; Calver et al, 2001). The GABAB receptor is an allosteric complex similar to other class C G-protein-coupled receptors (GPCRs). Each subunit is composed of an extracellular domain, called Venus flytrap domain (VFT), which is linked to the N-terminus of a prototypical heptahelical transmembrane domain (7TM) (Pin et al, 2004). GABAB agonists and competitive antagonists (the orthosteric ligands) bind to the GB1 VFT, whereas the GB2 VFT is not bound by GABA nor, most likely, by any other ligand (Kniazeff et al, 2002). In contrast, the 7TM of GB2 is responsible for G-protein activation (Galvez et al, 2001) and contains the site of action of PAMs (Binet et al, 2004). Figure 1.Models and bioinformatic analysis of the GABAB VFTs. (A) Structural model of the dimeric GABAB receptor in the resting state. Corey–Pauling–Koltun representation of the GABAB VFT dimer model generated according to the resting state of the dimeric mGlu receptors (Protein Data Bank (PDB) accession number 1EWT), and apposition of two heptahelical domains (7TM) according to the rhodopsin dimer structure (PDB accession number 1n3m). GB1 (yellow) and GB2 (blue) are in the front and the back, respectively. The C-terminal regions of the two subunits are associated through a CC interaction that masks the RSR intracellular retention signal of GB1. (B) The phylogenetic tree was constructed using the sequences of the VFTs of the mGlu1 receptor, the amide-binding protein (AmiC) from the amidase operon, the NR2A subunit of the rat N-methyl-D-aspartate (NMDA) receptor, the leucine–isoleucine–valine-binding protein (LIVBP), the natriuretic peptide receptor types A and C (NPRA and NPRC, respectively), RTK1 from Schistosoma mansoni and the rat GB1 and GB2 subunits. Only branches with bootstrap values >600 are shown. (C, D) Evolutionary conservation of residues (upper panels) and electrostatic surfaces (lower panels) of the GB1 and GB2 VFTs visualized on both faces of the VFTs (Face 1 and Face 2). Conservation scores are indicated according to a colour scale, from variable (blue) to conserved (purple) residues. No conservation scores were calculated for the residues in grey. Electrostatic surface representations are provided (negative, red; neutral, white; positive, blue) for the VFT faces, in which the green ribbons correspond to the helices of the associated subunit in the inactive state, illustrating the possible dimerization interface. Download figure Download PowerPoint To better understand both the molecular functioning of the GABAB receptor and the mechanism of action of orthosteric and allosteric ligands, it is important to know how the GABAB VFTs dimerize and control 7TM activity. We recently demonstrated that the VFTs of the two subunits interact with each other, and also that the GB2 VFT controls agonist affinity for GB1 (Liu et al, 2004); this was recently confirmed using purified VFTs (Nomura et al, 2008). However, it is unknown whether the GABAB VFT dimer functions similarly to the VFT of mGlu receptors (Kunishima et al, 2000; Muto et al, 2007), of ANP receptors (He et al, 2001; van den Akker, 2001) or of tyrosine kinase receptors in Schistosoma (Vicogne et al, 2003), with which it shares equal evolutionary distance (Figure 1B). Here, we have identified the VFT dimerization interface and used a glycan wedge scanning approach to analyse its functional relevance. Our data demonstrate that a direct interaction between the VFTs of the GABAB subunits is required for cell-surface targeting and agonist activation of the receptor. We also provide direct evidence that a change in the dimerization interface takes place during agonist activation of the receptor. Results Bioinformatic prediction of the dimerization interface of GABAB VFTs Previous results have shown that GABAB VFTs form heterodimers at the surface of cells, even in the absence of the receptors' 7TM and C-terminal domains (Liu et al, 2004). To localize the dimerization interfaces of the GABAB VFTs, we first analysed the conservation of residues at their surfaces using a set of 20 GB1 VFT sequences and 22 GB2 VFT sequences, from Dictyostelium to mammals (see Supplementary Figure 1); the sequences were selected based on our previously established 3D models of the GABAB VFTs (Kniazeff et al, 2002). This analysis revealed that one face of the subunits is more conserved than the others (Face 1 in Figure 1C and D), suggesting that it might correspond to the dimerization interface, as observed with mGlu receptor dimers (Rondard et al, 2006). This possibility was also supported by an analysis of the charge distribution at the surface of the VFTs. Indeed, the conserved Face 1 is composed of a large patch of hydrophobic residues in both subunits (Figure 1C and D), and corresponds to the hydrophobic dimerization interface of the mGlu VFT dimer. In view of this correspondence, the 3D structure of the mGlu VFT dimer was used to build a model of the GB1–GB2 VFT heterodimer (Figure 2A). Interestingly, none of the putative N-glycosylation sites (consensus sequence Asn-X-Ser/Thr or NXS/T, where X can be any natural amino acid except proline) found in the GB1 and GB2 sequences from different species, from nematodes to mammals, were located within the proposed dimerization interface (Figure 2A). In contrast, most other faces contained at least one putative glycosylation site in at least one of the species examined. This further supported our model of GB1 and GB2 VFT interaction. Taken together, these observations were consistent with the VFT dimer interface in the GABAB receptor being similar to that in mGlu receptors, involving the same two helices of lobe 1. Figure 2.Native and engineered N-glycan sites in the heterodimeric GABAB VFTs. (A) Ribbon views of the heterodimeric VFTs are shown, with the putative N-glycosylation sites (Cα of Asn residue) in mammalian VFTs in cyan and orange for GB1 and GB2, respectively. Additional putative N-glycosylation sites in other species are in dark blue and magenta for GB1 and GB2, respectively. (B) The GABAB VFT interface is mainly composed of two helices (green) in lobe 1 of GB1 and GB2 that interact together. The positions of Cα of Asn residues modified by an N-glycan and resulting in a nonfunctional or functional receptor are depicted in red and blue, respectively. Download figure Download PowerPoint Introduction of N-glycans at the VFT interface abolishes receptor activity To examine the functional importance of the interaction between the VFTs, we tried to block the interaction by introducing N-glycans at the possible dimer interface in GB1 or GB2 (Figure 2B), creating a steric wedge. Experimentally, we introduced the consensus sequence NXS/T (where X can be any natural amino acid except proline), which typically results in the attachment of a bulky N-glycan moiety to the side chain of the Asn residue. These N-glycosylation sites were introduced at different positions within GB1 (225, 229, 232, 251, 255 and 258) and GB2 (110, 114, 118, 137, 141 and 145) (Figure 2B and see Supplementary Table 1). To ensure the correct trafficking of GB1 to the cell surface when expressed alone, these mutations were first introduced into a GB1 subunit that had a mutated ER retention signal (ASA instead of RSR) (Pagano et al, 2001; Brock et al, 2005). Western blot experiments revealed that all mutated subunits were expressed at their expected molecular weights (Figures 3A and 4A). Owing to the large size of the full-length subunits (130 kDa), it was impossible to detect any shifts in their apparent molecular weights due to the presence of additional N-glycan moieties (Figures 3A and 4A). However, such shifts were clearly seen with most mutants of GB1 and GB2 VFTs attached to the plasma membrane with a single transmembrane domain (Liu et al, 2004) (Figures 3B and 4B; see Supplementary Table 1). Moreover, treatment with glycosidase PNGase F restored gel mobility similar to that of wild-type VFT (Figures 3B and 4B), demonstrating that the mutants were indeed glycosylated. Figure 3.Analysis of the N-glycosylated GB1 mutants. (A) Western blot analysis of the full-length HA–GB1 mutants from membrane fractions of cells coexpressing HA–GB1 mutants and Flag–GB2-WT. Cartoons depict the GABAB subunits wild-type (white) and mutant (grey), and engineered N-glycan is indicated by a white star. (B) Western blot analysis of the truncated GB1 subunits deleted of both HD and C-terminal regions, with or without treatment with PNGase F, and comparison to the wild-type construct. (C) The amount of HA-tagged GB1 mutants coexpressed with Flag-tagged GB2-WT at the cell surface as measured by ELISA. (D) Inositol phosphate (IP) production for the HA-tagged GB1ASA mutants coexpressed with GB2-WT. Data are means±s.e. of at least three independent determinations. Download figure Download PowerPoint Figure 4.Analysis of the N-glycosylated GB2 mutants. (A) Western blot analysis of the full-length Flag–GB2 mutants from membrane fractions of cells coexpressing Flag–GB2 mutants and HA–GB1ASA. As indicated in Figure 3, cartoons depict the GABAB subunits wild-type (white) and mutant (grey), and engineered N-glycan is indicated by a white star. (B) Western blot analysis of the truncated GB2 subunits deleted of both HD and C-terminal regions, with or without treatment with PNGase F, and comparison to the wild-type construct. (C) The amount of Flag–GB2-WT mutants coexpressed with HA–GB1ASA at the cell surface as measured by ELISA. (D) IP production for the Flag-tagged GB2 mutants coexpressed with GB1ASA. Data are means±s.e. of at least three independent determinations. Download figure Download PowerPoint The surface expression of all the full-length glycosylation mutants was also examined using haemagglutinin (HA) or Flag epitopes inserted into the extracellular N-terminal end (after the signal peptide) of GB1ASA and GB2, respectively. ELISA assays performed on intact cells revealed that all mutants reached the cell surface. Although a lower level was observed with some of them at the cell surface (Figures 3C and 4C), their total expression level as measured on western blots was similar to that of the wild-type proteins, suggesting that some mutants had difficulties passing the quality control system. This was confirmed by quantifying both the surface and total expression levels of these subunits using ELISA performed on intact and permeabilized cells, respectively (Supplementary Figure 3A and B). The functional consequences of these additional glycosylation sites were then analysed by coexpressing either the mutated GB1 subunits together with wild-type GB2 (Figure 3D), or the mutated GB2 forms with GB1ASA (Figure 4D). In most cases, GABA was unable to generate a response in cells coexpressing the subunits, despite a sufficient expression level of both proteins at the cell surface. Among the different mutants tested, only two GB1 mutants (HA–GB1ASA-N225 and -N232) and one GB2 mutant (Flag–GB2-N137) were able to form a functional receptor when coexpressed with the wild-type partner. Notably, for those positions whose mutation generated nonfunctional receptors, it was sterically impossible to add an N-acetyl glucosamine group to the Asn residue in the 3D model of the GB1–GB2 VFT dimer, in contrast to those residues whose mutation did not interfere with the formation of a functional heterodimer (Supplementary Figure 2A and B). To address whether the negative effect of the NXS/T mutations was indeed due to the introduction of an additional glycan on the VFT, we first tested the effect of the N-glycosylation inhibitor tunicamycin (Luo et al, 2003). However, this molecule was toxic to our electroporated cells, preventing us from examining its effect on receptor function. Indeed, even the response mediated by the wild-type receptor could no longer be measured. Therefore, we compared the properties of certain N-glycosylation site mutants (HA–GB1ASA-N229 or -N251 and Flag–GB2-N114 or -N141) with those of analogous mutants in which the Asn residue was replaced by a Gln residue, which cannot be glycosylated. The Gln-containing mutants (HA–GB1ASA-Q229 and -Q251; and Flag–GB2-Q114 and -Q141) generated similar responses upon activation with GABA to those obtained with the wild-type receptor (Figure 5A and B). These data demonstrated that the lack of activity of the HA–GB1ASA-N229 or -N251 and Flag–GB2-N114 or -N141 mutants was likely due to the presence of the N-glycan on the VFT and not mutation per se. Figure 5.Loss of function is due to the N-glycan at the interface between the VFTs. (A) Comparison of the IP production stimulated by the GB1ASA NXS/T and QXS/T mutants. (B) Similar comparison of IP stimulation for the GB2 mutants. Data are means±s.e. of at least three independent measurements. (C) Effect of CGP7930 on Ca2+ signals in cells expressing the indicated GB2 mutants. Data are means±s.e. of triplicates from a typical experiment. Download figure Download PowerPoint Binding and G-protein coupling properties of GB1 and GB2 mutants Among the mutations introduced into GB1, those that led to nonfunctional receptors were unable to bind the radiolabelled antagonists 3H-CGP54626 and 125I-CGP64213, suggesting that the mutant proteins were not folded correctly (Supplementary Figure 4). It has recently been reported that the correct association of soluble GB1 and GB2 VFTs is required for the GB1 VFT to be able to bind ligands (Nomura et al, 2008). Then, the absence of antagonist binding on GB1-N229 and -N251 could well be the consequence of the lack of possible association with GB2 rather than a misfolding due to the N-glycan. We therefore introduced nine additional glycosylation sites into the GB1 VFT in a region devoid of any natural sites in the various GB1 subunits identified in different species (Figure 2). All these mutations generated GB1 VFTs with an additional N-glycan, as shown by a decrease in mobility in acrylamide gels (Supplementary Figure 5A). Moreover, all were expressed correctly to the cell surface, and eight led to a functional GABAB receptor upon coexpression with GB2, with GABA EC50 values in the same range as that of the wild-type receptor (Supplementary Figure 5B and C). Further information on the nonfunctional N315 mutant is provided at the end of the Results section (see Figure 11). Regarding the GB2 mutants, the absence of activation by GABA of the GB2-N114 and -N141 mutants was not due to the uncoupling of these subunits from G proteins. We previously reported that the PAM of the GABAB receptor, CGP7930, has agonist activity and can activate both GB2 expressed alone and a truncated version of GB2 that is deleted for the VFT (GB2 7TM) (Binet et al, 2004). Interestingly, the GB2 subunits carrying an additional N-glycan could still be activated by CGP7930 (Figure 5C), demonstrating that they all retained their ability to activate G proteins. These data revealed that perturbation of the putative dimer interface in both the GB1 and GB2 VFTs has important consequences for the activity of the receptor, but does not prevent the 7TM of GB2 from reaching an active state. N-glycan in GB2 lobe 1 VFT interface prevents receptor heterodimerization To further analyse the molecular mechanism by which the N-glycan modification of GB2 abolishes receptor activity, we focused our study on three mutants that were well expressed (N114, N137 and N141), but of which only one, GB2-N137, forms a functional GABAB receptor when coexpressed with GB1ASA. Using FRET measurements, we found that GB2-N114 and -N141 do not interact with GB1 at the cell surface in contrast to GB2-N137. These experiments were conducted on intact cells, using anti-HA antibodies conjugated with the energy donor fluorophore (europium cryptate PBP) to label the HA-tagged GB1 and anti-Flag antibodies linked to the fluorophore acceptor (d2) to label the Flag-tagged GB2 proteins (Figure 6A). Such an approach enables the detection of receptor dimers at the cell surface only, as previously shown with GB1 and GB2 subunits coexpressed in the same cells (Maurel et al, 2004). As shown in Figure 6B, a very low FRET signal was measured between Flag–GB2-N114 or -N141 and HA–GB1ASA and was not significantly different from a negative control (e.g. FRET between Flag–GB2 and HA–CD4; data not shown). In contrast, large FRET signals were obtained between HA–GB1ASA and either Flag–GB2-N137, -Q114, -Q141 or wild-type Flag–GB2 (Figure 6B). These experiments were conducted with similar amounts of GB1ASA and GB2 subunits at the cell surface for all the constructs tested, to ensure that differences in FRET signals did not reflect differences in the level of expression of one of the subunits (Figure 6C and D). These data strongly suggest that the glycosylation site prevents direct interaction of GB1 with GB2. Co-immunoprecipitation experiments confirmed that, among the GB2 mutants, only GB2-N137 interacts with GB1 (Supplementary Figure 6). Figure 6.N-glycan at the GB2 VFT interface prevents dimerization with GB1. (A) The schemes depict the experimental approach used to monitor receptor dimers at the cell surface using time-resolved FRET. The FRET signal is measured between an anti-HA antibody linked to a donor molecule (D) and an anti-Flag antibody linked to an acceptor molecule (A). (B) FRET signal between HA-tagged GB1ASA and Flag-tagged GB2 subunits in cells coexpressing the indicated constructs. (C, D) Amount of HA- and Flag-tagged subunits expressed at the cell surface, respectively, as measured by ELISA. Data are means±s.e. of triplicates from a typical experiment. Download figure Download PowerPoint One well-established consequence of GB1–GB2 interaction is an increase in agonist affinity for GB1. Indeed, GABA affinity, as measured by the displacement of 125I-CGP64213 on GB1ASA expressed alone, was 5- to 10-fold lower than the affinity measured in the presence of GB2 (Liu et al, 2004) (Figure 7). Coexpression of GB1ASA with GB2-N114 or -N141 did not increase agonist affinity for GB1ASA (Figure 7), whereas GB2-N137, -Q114 and -Q141 had the same effect as wild-type GB2. These data indicate that the presence of a glycosylation site at the GB2 VFT dimer interface suppresses the allosteric control of agonist affinity in GB1, consistent with a lack of interaction between GB2-N114 and -N141 and GB1 (Figure 6B). Figure 7.N-glycan at the GB2 VFT interface prevents allosteric interaction with GB1. Displacement of non-permeant antagonist [125I]CGP64213 by GABA on HA-tagged GB1ASA, expressed alone or in combination with the indicated Flag-tagged GB2 subunits. Download figure Download PowerPoint Presence of N-glycan at the GB2 lobe 1 VFT interface abolishes cell-surface targeting of the heterodimer Wild-type GB1 is retained in the endoplasmic reticulum in the absence of GB2 (Margeta-Mitrovic et al, 2000; Calver et al, 2001; Pagano et al, 2001). Till this point, all of the experiments had been conducted with a mutated version of GB1 in which the ER retention signal was changed to ASA. Therefore, we next examined if the glycosylated GB2 mutants could still target wild-type GB1 to the cell surface. Indeed, the inactive N-glycan-modified GB2-N114, -N118 and -N141 receptors were unable to target wild-type GB1 to the cell surface, as indicated by both ELISA and 125I-CGP64213 binding experiments on intact cells (Figure 8). In contrast, GB2-Q141 was able to target GB1 to the cell surface. These results indicate that the masking of the ER retention signal cannot occur if VFT interaction is prevented by the presence of an N-glycan, even at an early s
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