The oligomeric state sets GABA(B) receptor signalling efficacy

Abstract

G protein-coupled receptors (GPCRs) have key roles in cell-cell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABA(B) receptor is one of the most abundant, being distributed in most brain regions, on either pre- or post-synaptic elements. Here, using specific antibodies labelled with time-resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABA(B) receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA(B1) subunits. Destabilizing the oligomers using a competitor or a GABA(B1) mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABA(B) receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA(B) heterodimers.

Figure 1

GB1–GB1 interaction in heterologous system. (A) GB1–GB1 interaction detected by cell-surface co-immunoprecipitation. Anti-HA (left) and anti-Flag (right) immunoblots performed on anti-Flag immunoprecipitate (top) and the corresponding lysate (bottom) obtained from COS-7 cells transfected with the indicated constructs. Lane 4 corresponds to mock-transfected cells. The gels are representative of three independent experiments. The bands at high molecular weight (over 250 kDa) represent the multimeric forms of the receptors. (B) GB1 cell-surface expression assessed by binding of the GB1 selective non-permeant antagonist [³H]-CGP54626A either on COS-7 cells expressing GB1 and GB2 or on mouse cortical neurons (E15.5)—15 DIV expressed as pmol of bound ligand per mg of total amount of proteins. No [³H]-CGP54626A binding could be detected on non-transfected COS-7 cells. Data are mean±s.e.m. of three individual experiments each performed in triplicate. P>0.05 in a paired t-test. (C) FRET intensity measured on COS-7 cells between the indicated Snap-tag-labelled subunits. Data are mean±s.e.m. of four individual experiments each performed in triplicate.

Figure 2

Detection of GABA_B tetramers on brain membrane. (A) FRET intensity measured using Lumi4-Tb (K) and d2-conjugated monoclonal anti-sushi antibodies on COS-7 cells expressing increasing amounts of GB1a and GB2 (black squares) or GB1b and GB2 (open squares). The background signal was determined in the absence of d2-conjugated antibodies. Cell-surface expression was measured by binding assay. (B) Specific K anti-sushi antibody labelling on brain membrane prepared from wild-type, and GB1^−/−, GB1a^−/− or GB1b^−/− mice determined in the presence of d2 anti-sushi antibodies with (white bars) or without (black bars) an excess of non-conjugated anti-sushi antibodies. The specific antibody labelling for each condition tested is given by the difference between the black and the white bars. (C) FRET intensity between K and d2-conjugated anti-sushi antibodies measured on brain membrane prepared from wild-type, GB1b^−/−, GB1a^−/− or GB1b^−/− mice or on synaptosomes prepared from wild-type mice. The background signal was determined on samples labelled with K and an optimized amount of non-conjugated antibodies to obtain the same donor emission as in the assay (data not shown). Data in (A–C) are mean±s.e.m. of three individual experiments each performed in triplicates. ^*, ^** and ^*** represent P<0.05, P<0.01 and P<0.001, respectively, in a paired t-test; in (C) samples were compared with GB1^−/−.

Figure 3

Determination of the GB1 domains involved in the interaction between GABA_B heterodimers. (A) FRET intensity using the fluorescent BG substrates measured on COS-7 cells expressing wild-type or chimeric GABA_B subunits bearing an ST or an HA tag. FRET signals for the same amount of HA construct at the cell surface (see Supplementary Figure S4) are represented over the BG-K emission. GB1 domains are illustrated in blue and GB2 in red. Wild-type GABA_B receptor was used as a positive control. (B) FRET intensity over the HA cell-surface expression measured after fluorescent anti-HA antibodies labelling on the same transfected COS-7 cells than that used for the experiment depicted in (A). For (A, B), data are representative of three independent experiments, each performed in triplicates. ^** and ^*** represent P<0.01 and P<0.001, respectively, in a t-test compared with Flag-ST-GB1+HA-GB2. (C) Schematic representation of the different favoured associations for each subunit combination used as suggested by the results of FRET experiments.

Figure 4

Functional implication of the GABA_B higher-ordered oligomers formation assessed using a competitor of the GB1–GB1 interaction. (A) Dissociation of the GABA_B tetramers using as a competitor HA-GB1-ΔB-ΔCT: a GB1 unable to bind GABA and deleted of its C-terminal tail as illustrated in the top scheme. Only the combinations reaching the cell surface are represented. ST-GB1 is represented in blue and Flag-GB2-KKXX in red. FRET intensity over the wild-type GABA_B cell-surface expression (ELISA) measured on cells expressing either ST-GB1 and Myc-GB2-KKXX (control) (black bars) or control with GB1-ΔB-ΔCT (white bars), (a) between ST-GB1 subunits labelled with BG-K and BG-d2, (b) between ST-GB1 and HA-GB1-ΔB-ΔCT labelled with BG-d2 and K anti-HA antibody and (c) between ST-GB1 and Myc-GB2-KKXX labelled with BG-d2 and K anti-Flag antibody. ^** and ^*** represent P<0.01 and P<0.001, respectively, in a t-test. (B) Calcium signal measured upon stimulation of the chimeric G protein Gqi9 by increasing concentration of GABA in HEK-293 cells transfected with GB1 and GB2 (control) (black-filled circles), or co-expressed with CD4 (negative control) (black triangles) or with GB1-ΔB-ΔCT (grey open squares). (C) Calcium release kinetics on cells expressing the GABA_B receptor recorded for a period of 300 s, including the addition of 10⁻⁷ M AVP and 10⁻⁴ M GABA at 20 and 240 s, respectively (see also Supplementary Figure S6). (D) Measurement of GABA_B signalling through native Gi/o coupling upon application of increasing concentration of GABA in a calcium assay after pre-stimulation of an endogenous Gq-coupled receptor. Experiments were conducted as in (B). (E) Schematic representation of the BRET assay with Go fused to Rluc and Gγ₂ to Venus leading to a BRET signal that is decreased upon activation of the G protein. (F) Variation of BRET signal between Go–Rluc and Venus-Gγ₂ measured on HEK-293 cells also expressing GB1 and GB2-KKXX (black bar) or GB1, GB2-KKXX and the competitor GB1-ΔB-ΔCT (white bar). The results are shown as the difference between the ΔBRET ratio recorded with PBS (basal) minus the ΔBRET ratio recorded with 1 mM GABA (stimulated). ^**P<0.01 in a paired t-test. (G) BRET variation kinetics determined on HEK-293 cells co-transfected with the same constructs as described in (F). GABA (1 mM) was added at 150 s of reading. The kinetics recorded for the control is in black and the kinetics measured on cells overexpressing the competitor GB1-ΔB-ΔCT is in grey. For (A, D, G), data are representative of three to six independent experiments. For (B, F), data are mean±s.e.m. of three to four independent experiments each performed in triplicates.

Figure 5

Mutation at the GB1–GB1 contact area destabilizes the tetramer and increase G protein coupling efficacy. (A) Crystal structure (left, pdb code 3KG2) and schematic representation (centre) of GluR2 N-terminal domain tetramer, illustrating the assembly of the four VFTs into a loose dimer of tight dimers. Putative organization of GABA_B tetramer (right). (B) Illustration of contact area of GluR2 N-terminal domain (left), 3D model of the corresponding GB1-VFT (right). The identified contact area in GluR2 is highlighted in orange and the position 380 in GB1 in red. (C) FRET intensity measured on HEK-293 cells between the indicated ST-labelled subunits. ^** represents P<0.01 in a t-test. (D) Calcium signal measured upon stimulation of the chimeric G protein Gqi9 by increasing concentration of GABA in HEK-293 cells transfected with wild-type (black-filled circles) or N380 (open squares) GB1 together with GB2. For (C, D) data are representative of four independent experiments.

Figure 6

One binding site per tetramer is sufficient to induce the maximal agonist response. (A) Schematic representation of the oligomeric GABA_B receptor in the absence (left) or in the presence of a GB1 unable to bind the GABA (GB1-ΔB), which can substitute one wild-type GB1 to form tetramers with a single functional heterodimer per tetramer (right). FRET intensity measured on cells expressing either ST-GB1 and Myc-GB2-KKXX (control) (black bars) or control with an optimized amount of GB1-ΔB (white bars), (a) between ST-GB1 subunits labelled with BG-K and BG-d2, (b) between ST-GB1 and HA-GB1-ΔB labelled with BG-d2 and K anti-HA antibody, (c) between ST-GB1 and Myc-GB2-KKXX labelled with BG-d2 and K anti-Myc antibody and (d) between HA-GB1-ΔB and Myc-GB2-KKXX labelled with K anti-HA and d2 anti-Myc antibodies. Data are mean±s.e.m. of three independent experiments each performed in triplicate. ^** and ^*** represent P<0.01 and P<0.001, respectively, in a paired t-test. (B) Intracellular calcium response curve mediated by GABA on cells expressing GB1 and GB2-KKXX (control) (black-filled circles), GB1-ΔB and GB2-KKXX (negative control) (black triangles) and control and GB1-ΔB (grey open squares) in the presence of a chimeric Gqi9. Data are representative of four independent experiments each performed in triplicate. (C) Displacement of [³H]-CGP54626A binding by GABA on wild-type GB1 measured on cells expressing GB1 and GB2-KKXX (control) (black-filled circles), and control with GB1-ΔB (grey open squares). Data are representative of three independent experiments, each performed in triplicates.

Figure 7

Regulation of G protein coupling depends on the dimerization of the GABA_B heterodimers. (A) Schematic representation of the oligomeric GABA_B receptor in the absence (left) or in the presence (right) of a GB2 subunit unable to activate the G protein and carrying a KKXX retention signal motif (GB2(L686P)-KKXX), which can substitute one wild-type GB2 to form tetramers with a single functional heterodimer per tetramer (right). FRET intensity measured on COS-7 cells expressing the indicated constructs: control (black bars) and control with GB2(L686P)-KKXX (white bars) (a) between the Myc-GB1 subunits after labelling with a mixture of K and d2 anti-Myc antibodies, (b) between Myc-GB1 and HA-GB2-KKXX after labelling with K anti-HA and d2 anti-Myc antibodies and finally (c) between Flag-GB2(L686P)-KKXX and Myc-GB1 labelled with K anti-Flag and d2 anti-Myc antibodies. Data are mean±s.e.m. of three independent experiments each performed in triplicates. ^** and ^*** represent P<0.01 and P<0.001, respectively, in a t-test. (B) Calcium dose-response recorded after stimulation by the GABA on cells expressing GB1 and GB2-KKXX (control) (black-filled circles), GB1 and GB2(L686P)-KKXX (negative control) (black triangles) and control with GB2(L686P)-KKXX (grey open squares) in the presence of a Gqi9 chimeric protein. (C) GABA-induced response of the native Gi/o protein by measurement of the calcium formation after pre-stimulation of an endogenous Gq-coupled receptor. For (B, C), representative of three to four independent experiments, each performed in triplicates.