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The Journal of Neuroscience, October 15, 2001, 21(20):8043-8052

GABA_B2 Is Essential for G-Protein Coupling of the GABA_B Receptor Heterodimer

Melanie J. Robbins², Andrew R. Calver¹, Alexander K. Filippov⁴, Warren D. Hirst¹, Robert B. Russell³, Martyn D. Wood², Shabina Nasir¹, Andrés Couve^{4, 5}, David A. Brown⁴, Stephen J. Moss^{4, 5}, and Menelas N. Pangalos¹

Departments of ¹ Neurology Centre of Excellence for Drug Discovery (CEDD) and ² Psychiatry CEDD and ³ Bioinformatics Research Group, GlaxoSmithKline Pharmaceuticals, Harlow, Essex CM19 5AW, United Kingdom, and ⁴ Department of Pharmacology and ⁵ Medical Research Council Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

GABA_B receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA_B1 and GABA_B2, for functional expression. Whereas GABA_B1 is capable of binding receptor agonists and antagonists, the role of each GABA_B subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA_B2 that are critical for the coupling of GABA_B receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA_B2 subunit in G-protein coupling of the GABA_B receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.

Key words: GABA_B; GPCR; dimerization; signaling; G-protein coupling; receptor subunits

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

GABA_B receptors are G-protein-coupled receptors (GPCRs) that mediate slow synaptic inhibition in the brain and spinal cord (for review, see Bowery, 1993; Kerr and Ong, 1995; Couve et al., 2000). They are unique among type C GPCRs in that they are heterodimers of GABA_B1 (Kaupmann et al., 1997) and GABA_B2 subunits (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998; Kuner et al., 1999; Ng et al., 1999), and it is now generally accepted that each subunit is unable to form a functional receptor when expressed in isolation (Kaupmann et al., 1997; Jones et al., 1998; White et al., 1998; Ng et al., 1999). With respect to GABA_B1, this is attributable to its retention within the endoplasmic reticulum on homomeric expression (Couve et al., 1998; Calver et al., 2000; Filippov et al., 2000; Margeta-Mitrovic et al., 2000). In this context, GABA_B2 has been shown to play a key role in trafficking the GABA_B1 subunit to the cell surface (Couve et al., 1998; Filippov et al., 2000). This exposes the N-terminal ligand binding domain of GABA_B1, which is not present in GABA_B2 (Galvez et al., 2000a,b), enabling it to bind extracellular agonist and subsequently activate downstream signaling pathways. Interestingly, it has been shown recently that C-terminal GABA_B1 mutants, which are expressed on the cell surface in the absence of GABA_B2, are completely nonfunctional, despite their ability to bind GABA (Calver et al., 2000; Margeta-Mitrovic et al., 2000). GABA_B receptor function is restored, however, when these mutants are coexpressed with GABA_B2, demonstrating that the GABA_B2 subunit may not only be important for correct trafficking of GABA_B1 but also for the mediation of agonist-induced G-protein coupling. This hypothesis has been given additional strength by a recent report by Galvez et al. (2001), demonstrating that the extracellular domain of GABA_B2 is essential for agonist activation of the heterodimeric receptor.

Mechanisms for G-protein coupling have been investigated widely for members of the rhodopsin--adrenergic GPCR family. Growing evidence suggests that the second and third intracellular loops are critical interaction sites important for both G-protein coupling and selectivity. In addition, these intracellular domains have been demonstrated to be important for G-protein coupling of type C GPCRs, such as metabotropic glutamate receptors (mGluRs) (Gomeza et al., 1996; Francesconi and Duvoisin, 1998). At present, the role of the two GABA_B subunits in G-protein coupling and downstream signal transduction is unknown. Here, using a site-directed mutagenesis approach, we investigated the importance of residues in the second intracellular loop (il2) of GABA_B1 and GABA_B2 subunits. We demonstrated that mutations within il2 of GABA_B2 can dramatically decrease responses of the heterodimer complex to agonist, to the extent that receptor signaling can be effectively abolished by the introduction of three negatively charged residues into this region. In contrast, reciprocal mutations made in il2 of the GABA_B1 subunit have no apparent effect on heterodimer signaling. These results are consistent with a model in which agonists bind to the GABA_B1 subunit, resulting in signal transduction via G-protein coupling through the GABA_B2 subunit. This would therefore suggest the existence of a novel "sequential and sideways" signaling cascade that may be of relevance to the growing number of reported GPCR heterodimers (Milligan and Rees, 2000).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Site-directed mutagenesis. The GABA_B1b and GABA_B2 subunits were tagged with either an N-terminal c-myc (wtGABA_B1) or hemaglutinin (HA) (wtGABA_B2) epitope tag (Calver et al., 2001). PCR primers were designed to include single, double, triple, or quintuple amino acid changes in both the wtGABA_B1 and wtGABA_B2, resulting in 12 mutant constructs: GABA_B1^E579K, GABA_B1^E583K, GABA_B1^E579K/E580K, GABA_B1^{E579K/E580K/E583K} (referred to as GABA_B1^tripleK), GABA_B2^K586E, GABA_B2^K590E, GABA_B2^K586E/M587E, GABA_B2^{K586E/M587E/K590E} (referred to as GABA_B2^tripleE), GABA_B1^K577/578A, GABA_B1^K581/582A, GABA_B1^{K577/578/581/582/586A} (referred to as GABA_B1^KQA), and GABA_B1^{K577/578/581/582/586E} (referred to as GABA_B1^KQE). Mutagenesis experiments were performed using the Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the instructions of the manufacturer, and all amino acid changes were confirmed by full double-stranded sequencing.

C-terminal truncates (from amino acid 747 of GABA_B1) of the GABA_B1 mutant constructs (glutamate to lysine only) were made by PCR amplification using forward primer 5'-CCCGAATTCATGGG GCCCGGGGCC-3' and reverse primer 5'-CCCAAGCTTCCTCG GGTGATCAGCCTG-3'. A GABA_B1N/2 chimera was generated by ligation of two PCR products (GABA_B1b 1-470 and GABA_B2 479-954). PCR amplification was performed using forward primer 5'-CCCGA ATTCATGGGGCCCGGGGCC-3' and reverse primer 5'-CCCAAGC TTCTGGTCAGCTGGGGGGGAC-3' to generate fragment GABA_B1b 1-470. PCR amplification with forward primer 5'-CCCAAGCTT ATCATCCTGG AGCAGCTGCGGAAG-3' and reverse primer 5'-CCCCTCGAGTTACAGGCCCGAGACCA TGACTC-3' was used to generate fragment GABA_B2479-954. All constructs were cloned into the eukaryotic expression construct pcDNA3.1 (Invitrogen, Paisley, UK). Schematic representations of these constructs are shown in Figure 1c.

View larger version (43K): [in this window] [in a new window]   Figure 1.   Intracellular loop sequence alignments and summary of GABAB1 and GABAB2 constructs. a, Alignment of the second and third intracellular loops of GABAB1 (GABAB1) and GABAB2 (GABAB2) subunits with members of the mGluR family. The alignment was produced in Alscript using HMMALIGN and the G-protein-coupled receptors database (GPCRDB) (Barton, 1993; Horn et al., 1998). The shading indicates different levels of amino acid conservation, in which residues are colored if eight or more residues show conservation of amino acid properties: blue shading, small; yellow shading, hydrophobic; red characters, polar. Transmembrane helices (TMs, dark blue) are displayed for mGluR1 (as assigned in SwissProt, entry MGR1  HUMAN). Boxed letters indicate residues that are negatively charged. Conserved residues discussed in Results are shaded in gray. The alignment has been extracted from a larger alignment of family 3 GPCRs taken from GPCRDB (Horn et al., 1998). b, Enlarged alignment of the second intracellular loop of both GABAB1 and GABAB2 subunits. Bold red letters indicate residues mutated in this study. c, GABAB receptor subunit truncations and chimeras. Epitope tags are marked as red (c-myc) or blue (HA) boxes. Transmembrane domains 1-7 (TMD 1-7) are represented by black striped boxes.. The coiled-coil domains are depicted by green striped boxes.

Transfection. Human embryonic kidney 293 (HEK293) cells were transfected with either wild-type (wt) or mutant GABA_B1- or GABA_B2-containing plasmids using Lipofectamine Plus (Life Technologies, Paisley, UK) according to the instructions of the manufacturer. Cells were maintained in Minimal Essential Medium supplemented with 10% fetal bovine serum and 1% nonessential amino acids (all from Life Technologies). After transfection, cells were left for 24 hr before being subcultured for immunocytochemistry or the Ca²⁺ mobilization assay.

The GABA_B1b/GABA_B2 cell line was generated in Chinese hamster ovary cells as described previously (Hirst et al., 2000). The GABA_B2 cell line was generated in HEK293 cells by transient transfection of the GABA_B2 cDNA as described above and selection of positive cells in 800 µg/ml G418.

Immunocytochemistry. HEK293 cells transfected with either wild-type or mutant constructs were subcultured onto glass coverslips, fixed with 4% paraformaldehyde, and then either permeabilized with 0.1% Triton X-100 for 10 min or washed with PBS. Cells were incubated with primary antibody [anti-c-myc, 1:5000; anti-HA, 1:5000 (Boehringer Mannheim, Brüssel, Belgium); and anti-GABA_B1b, 1:1000] for 60 min and washed, and then secondary antibody [goat anti-mouse IgG FITC for c-myc; goat anti-rat IgG FITC for HA (Sigma, St. Louis, MO); and goat ant-rabbit IgG FITC for GABA_B1b] was used at 1:100 dilution for 60 min. Cells were finally washed, mounted onto glass slides using Citifluor (Citifluor Ltd., London, UK), and viewed using a Leica (Nussloch, Germany) confocal microscope.

Ca²⁺ mobilization assay. Transfected cells were seeded into 96-well plates at a density of 50,000 cells per well and incubated at 37°C in 5% CO₂ for 24 hr before use. The intracellular Ca²⁺ mobilization in response to agonist was then measured as described previously (Calver et al., 2000). The data were iteratively curve fitted using a four parameter logistic model (Bowen and Jerman, 1995) as mean ± SEM (with each data point being determined in triplicate) of one representative experiment. Significance of data were assessed using a one-way ANOVA, followed by post hoc t test (least square difference). Data were considered significant if p < 0.05.

Radioligand binding. Membranes from transfected cells were prepared, and radioligand binding to [³H]CGP54626 was performed as described previously (Calver et al., 2000). The concentration of drug-inhibiting-specific radioligand binding by 50% (IC₅₀) was determined by iterative curve fitting (Wood et al., 2000). pK_i values (the negative log₁₀ of the molar K_i) for receptor binding were then calculated from the IC₅₀ values as described by Cheng and Prusoff (1973) using K_D values determined previously in saturation binding studies (3 nM). B_max values were calculated from the specific bound accounting for receptor occupancy at this radioligand concentration using the Hill-Langmuir adsorption isotherm. Data are expressed as the mean ± SEM of at least three separate experiments from two independent sets of transfections.

[³⁵S]GTPS binding assays. Cells were homogenized in 20 mM HEPES and 10 mM EDTA, pH 7.4 (4°C), and centrifuged at 48,000 × g for 20 min at 4°C. Membrane pellets were rehomogenized in 20 mM HEPES and 0.1 mM EDTA, pH 7.4, (4°C). The pellet was recentrifuged as described above, the supernatant was discarded, and the pellet was resuspended in 20 mM HEPES and 0.1 mM EDTA and stored at 80°C until required.

[³⁵S]GTPS binding assays were performed in a 20 mM HEPES buffer, pH 7.4, containing 3 mM MgCl₂ and 100 mM NaCl. Cell membranes were preincubated with 10 µM GDP, and increasing concentrations of GABA or baclofen for 30 min at 30°C and then 0.1 nM [³⁵S]GTPS was added to each well and incubated for an additional 30 min. Nonspecific binding was determined in the presence of 20 µM GTPS. The incubation was terminated by rapid filtration through J. Whatman glass fiber filters (GF/B) (Semat International, St. Albans, UK) and washed with 3 ml of ice-cold HEPES buffer, pH 7.4, containing 3 mM MgCl₂. Radioactivity was determined by liquid scintillation spectrometry using a Packard (Meridian, CT) TopCount.

cAMP accumulation assays. cAMP levels in cells were determined by radioimmunoasay (SMP004; NEN, Boston, MA) according to the instructions of the manufacturer. In brief, cells were washed once with Ca²⁺-free PBS, scrapped up in the same buffer, and pelleted by a 5 min centrifugation at 400 × g. The pellet resuspended in manufacturers stimulation buffer, and ~50,000 cells were added to the appropriate wells of the NEN flash plates together with 10 µM forskolin, plus increasing concentration of agonist. Plates were incubated for 15 min at 37°C, before the addition of the manufacturers detection mixture containing [¹²⁵I]cAMP tracer (0.16 µCi/ml) to the wells. Plates were covered and left for 24 hr before counting on a Packard TopCount.

Neuron preparation and cDNA injection. Neuron isolation and injection procedures have been described previously (Caulfield et al., 1994; Couve et al., 1998; Filippov et al., 2000). Briefly, single superior cervical ganglion (SCG) neurons were dissociated from 15-to 19-d-old rats and plated on laminin-coated glass coverslips. Five hours after plating, neurons were microinjected into the nucleus with plasmids carrying cDNAs for the GABA_B receptor subunits, in addition to a plasmid encoding green fluorescent protein to subsequently identify successfully injected cells. Electrophysiological recordings were routinely made 16-20 hr after injection at room temperature (20°C).

Ca²⁺ channel current recording. Currents through voltage-gated Ca²⁺ channels were recorded using the conventional whole-cell patch-clamp method as described previously (Caulfield et al., 1994; Couve et al., 1998; Filippov et al., 2000). Ca²⁺ channel currents were routinely evoked every 20 sec with 100 msec depolarizing rectangular test pulse to 0 mV from a holding potential of 90 mV. Ca²⁺ channel current amplitudes were measured isochronally 10 msec from the onset of the rectangular test pulse, i.e., near to the peak of the control current. As reported previously (Filippov et al., 2000), currents were primarily N-type with negligible contribution by L-type channels. A racemic mixture of (+)- and ()-baclofen was used in all experiments. Data are presented as means ± SEM as appropriate. Student's test (unpaired) was applied to determine statistical significance. Difference were considered significant if p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Sequence analysis of the second and third intracellular loops of GABA_B1 and GABA_B2

Using sequence alignments, we compared residues in the second and third intracellular loops of the GABA_B subunits and the related type C mGluRs (Fig. 1a). The basic residue Arg⁷⁷⁵ in the third intracellular loop (il3) of mGluR1 has been shown to be important for coupling to G-proteins (Francesconi and Duvoisin, 1998) and is conserved throughout the mGluR family and also in GABA_B2 (Fig. 1a). In contrast, the corresponding residue in GABA_B1 is a lysine. In il2 of all of the mGluRs, there is a conserved lysine at position 690 (numbered with respect to mGluR1), except for mGluR2 in which the equivalent residue is an arginine, and this basic residue has been shown to be crucial for the interaction of the mGluRs with their respective G-protein (Francesconi and Duvoisin, 1998). Similarly, we noted that GABA_B2 also has a basic lysine at this key position. This is in contrast to the GABA_B1 subunit, in which the acidic amino acid glutamate is substituted for this lysine, resulting in a reversal of charge at this site. These findings may be of functional relevance, because it has been proposed previously that electrostatic interactions between positive charges in the intracellular domains of GPCRs may be important for coupling to a negatively charged face of the G-protein (Fanelli et al., 1998).

Amino acids in the second intracellular loop of GABA_B2 are critical for GABA_B receptor signaling through the chimeric G-protein G_qi5

Based on our sequence analysis of the GABA_B1 and GABA_B2 intracellular loops, we targeted three amino acid residues (K586, M587, and K590) within il2 of GABA_B2 for mutagenesis studies (Fig. 1a,b). These residues were mutated to glutamates either individually or in combination to give a series of single, double, or triple mutants: GABA_B2^K586E, GABA_B2^K590E, GABA_B2^K586E/M587E, and GABA_B2^tripleE (Fig. 2a). When transfected into HEK293 cells in isolation, both wild-type and mutant GABA_B2 subunits could be detected on the cell surface in the absence of membrane permeabilization (Fig. 2b). In addition, coexpression of each GABA_B2 mutant with wtGABA_B1 demonstrated that each mutant retained its ability to traffic the GABA_B1 subunit to the cell surface (Fig. 2b). Having demonstrated that these mutants behaved normally in terms of trafficking to the cell surface, we next tested the ability of the mutated GABA_B2 subunits to functionally couple to G-proteins when coexpressed with the wild-type GABA_B1 subunit. Although GABA_B receptors are known to inhibit adenylyl cyclase activity by preferentially coupling to G_o/i, they can also be made to signal via the phospholipase C pathway when coexpressed with the chimeric G-protein G_qi5 (Franek et al., 1999; Wood et al., 2000). Activation of this pathway results in a mobilization of intracellular Ca²⁺ stores, which can then be measured on a fluorimetric imaging plate reader (FLIPR). Coexpression of wtGABA_B1 and wtGABA_B2 subunits with G_qi5 in HEK293 cells resulted in a robust Ca²⁺ mobilization in response to GABA (pEC₅₀, 7.19 ± 0.06; n = 36). In the same system, expression of wtGABA_B1 with either GABA_B2^K586E or GABA_B2^K590E resulted in significantly reduced pEC₅₀ values when compared with coexpression with wtGABA_B2 (6.35 ± 0.07, n = 11, p < 0.001; and 6.07 ± 0.04, n = 12, p < 0.001, respectively) (Fig. 2c). Expression of wtGABA_B1 with the double-mutant GABA_B2^K586E/M587E resulted in additional reduction of the observed pEC₅₀ in response to GABA (5.40 ± 0.11; n = 10; p < 0.001). In contrast to the other GABA_B2 mutants studied, expression of GABA_B2^tripleE in combination with wtGABA_B1 resulted in no detectable responses in this functional assay (n = 12) (Fig. 2c).

View larger version (29K): [in this window] [in a new window]   Figure 2.   Residues within the second intracellular loop of GABAB2 are critical for agonist-induced GABAB receptor signaling. a, Schematic representation of GABAB2 il2. Arrows indicate residues mutated. b, HEK293 cells were transiently transfected with wtGABAB1 (GB1) and wtGABAB2 (GB2), GB2K586E, GB2K590E, GB2K586E/M587E, or GB2tripleE. Cell surface expression of GABAB subunits was examined by immunofluorescence using an anti-myc antibody for GABAB1 (i-v) and an anti-HA antibody for GABAB2 (vi-x). c, Representative FLIPR analysis of intracellular Ca2+ changes after GABA stimulation of cells transiently transfected with Gqi5, wtGABAB1 (GB1), and wtGABAB2 (GB2) GB2K586E, GB2K590E, GB2K586E/M587E, or GB2tripleE. Expression of single and double GABAB2 mutants resulted in decreased functional responses compared with wtGABAB2, with the GABAB2tripleE mutant showing no response at all. d, Representative FLIPR analysis of intracellular Ca2+ changes after GABA stimulation of cells transiently transfected with Gqi5, wtGABAB1 (GB1), and wtGABAB2 (GB2), GB2K586A, GB2K590A, GB2K586A/M587A, or GB2tripleA. Single and double GABAB2 mutants resulted in decreased functional responses compared with wtGABAB2, with the GABAB2tripleA mutant completely unresponsive to GABA. e, Mutation of residues within the second intracellular loop of GABAB2 do not affect agonist or antagonist ligand binding. Membrane homogenates prepared from cells transiently transfected with wtGABAB1 (GB1) and wtGABAB2 (GB2), GB2K586E, GB2K590E, GB2K586E/M587E, or GB2tripleE specifically bound [3H]CGP54626. This could be completely displaced by GABA (10 mM). Data are expressed as means ± SEM (n = 3).

Based on these results, we next prepared mutations in the second intracellular loop of GABA_B2 that would help us investigate the relative importance of losing or gaining positive charge within this stretch of five amino acids. To do this, we examined the effects of changing residues K586, M587, and K590 to neutral alanines instead of negatively charged glutamates. As with the previous set of glutamate mutants, cell surface expression of each of the alanine mutants in isolation appeared no different to that of the wtGABA_B2 subunit (data not shown). Expression of wtGABA_B1 and G_qi5 in combination with GABA_B2^K586A or GABA_B2^K590A resulted in significantly reduced pEC₅₀ values when compared with coexpression with wtGABA_B2 (6.30 ± 0.13, n = 8, p < 0.001; and 5.75 ± 0.08, n = 10, p < 0.001, respectively) (Fig. 2d). Coexpression with the double-mutant GABA_B2^K586A/M587A resulted in similar significant reduction in pEC₅₀ in response to GABA (6.16 ± 0.07; n = 8; p < 0.001). In contrast to the other GABA_B2 mutants studied, and similar to the GABA_B2^tripleE mutant, expression of GABA_B2^tripleA in combination with wtGABA_B1 resulted in no detectable responses in this functional assay (Fig. 2d).

Mutations in the second intracellular loop of GABA_B2 have no effect on agonist or antagonist binding

Having shown a marked effect on GABA_B receptor signaling after mutation of selected residues within the second intracellular loop of GABA_B2, we wanted to see whether the agonist and antagonist binding properties of the mutant receptor heterodimer were consistent with that of the wild-type receptor. Analysis of membrane homogenates expressing each GABA_B2 mutant in combination with wtGABA_B1 had no effect on the ability of GABA to displace the antagonist CGP54626 in competition binding assays. The potency of GABA observed in homogenates prepared from cells expressing GABA_B2^K586E, GABA_B2^K590E, GABA_B2^K586E/M587E, or GABA_B2^tripleE with wtGABA_B1 subunit was not significantly different from that observed when the wild-type GABA_B2 subunit was coexpressed with the GABA_B1 subunit (pK_i values of 4.57 ± 0.17, 4.22 ± 0.05, 4.48 ± 0.22, 4.84 ± 0.54, and 4.37 ± 0.12, respectively; n = 3) (Fig. 2e). B_max values of [³H]CGP54626 specifically bound did not differ significantly between wild-type and mutants and ranged from 5.6 ± 3.3 to 8.3 ± 3.7 pmol/mg protein.

Negatively charged amino acids in the second intracellular loop of GABA_B1 are not critical for GABA_B receptor signaling through G_qi5

The demonstration that mutation of specific residues within the second intracellular loop of GABA_B2 can have pronounced effects on GABA_B signaling through G_qi5 led us to examine the functional importance of similarly positioned residues within the GABA_B1 subunit. We therefore studied the consequences of mutating the stretch of negative amino acids found in the second intracellular loop of GABA_B1 and in a similar position to those residues mutated previously in GABA_B2 (Fig. 3a). Each of the mutants GABA_B1^E579K, GABA_B1^E583K, GABA_B1^E579K/E580K, and GABA_B1^tripleK were transfected into HEK293 cells with and without wtGABA_B2. In the absence of GABA_B2, neither wt or mutant GABA_B1 variants were expressed at the cell surface (Fig. 3b and data not shown). However, when coexpressed with the GABA_B2 subunit, cell surface expression of all GABA_B1 mutants was observed as expected (Fig. 3b and data not shown). These experiments therefore suggest that mutation of these acidic residues in the second intracellular loop of GABA_B1 do not affect trafficking of the subunit to the surface by GABA_B2. We then tested the ability of these constructs to activate G_qi5 in response to GABA. Coexpression of GABA_B2 with GABA_B1^E579K, GABA_B1^E579K/E580K, GABA_B1^E583K, or GABA_B1^tripleK produced no significant changes in functional response when compared with coexpression with the wtGABA_B1 subunit (pEC₅₀ values for wtGABA_B1, 7.19 ± 0.06, n = 36; GABA_B1^E579K, 7.03 ± 0.13, n = 8; GABA_B1^E579K/E580K, 7.18 ± 0.16, n = 8; GABA_B1^E583K, 7.02 ± 0.13, n = 7; and GABA_B1^tripleK, 7.18 ± 0.16, n = 7) (Fig. 3c). This suggests that these acidic residues are not critical in the signaling of the GABA_B1 receptor dimer through G_qi5.

View larger version (24K): [in this window] [in a new window]   Figure 3.   Acidic residues within the second intracellular loop of GABAB1 are not critical for agonist-induced GABAB receptor signaling. a, Schematic representation of GABAB1 il2. Arrows indicate residues mutated. b, HEK293 cells were transiently transfected with wtGABAB1 (GB1), GB1E579K, GB1E583K, GB1E579K/E580K, GB1tripleK, or GB1C-tripleK alone or with wtGABAB2. Cell surface expression of GABAB1 subunits was examined by immunofluorescence using an anti-myc antibody in the absence (i, iii, iv) or presence (ii, v) of a permeabilizing detergent. GABAB1 is only found at the cell surface when expressed with GABAB2, as shown with the GABAB1tripleK construct (i-iii). The C-terminally truncated GABAB1 is expressed at the cell surface in the absence of GABAB2, as shown with the GABAB1C-tripleK mutant (iv, v). c, Representative FLIPR analysis of cells transiently transfected with Gqi5, wtGABAB2 (GB2), and wtGABAB1 (GB1), GB1E579K, GB1E583K, GB1E579K/E580K, or GB1tripleK. No differences in functional GABA responses were observed between mutant and wild-type GABAB1 subunits. d, Representative FLIPR analysis of cells transiently transfected with Gqi5 and GB1C-tripleK. Expression of GB1C-tripleK alone did not give a functional response.

Addition of positively charged residues to the second intracellular loop of a cell surface-expressed GABA_B1 is not sufficient for receptor function in the absence of GABA_B2

Although mutation of acidic residues in the second intracellular loop of GABA_B1 appeared to have no functional effect with respect to heterodimer signaling, we also wanted to test the function of GABA_B1 mutants when expressed on the cell surface in the absence of GABA_B2. We and others have shown previously that removal of the C-terminal domain of the GABA_B1 subunit results in its cell surface expression in the absence of GABA_B2. However, despite this cell surface expression, the GABA_B1 subunit remains ineffective in coupling to downstream effector systems in the absence of GABA_B2 (Calver et al., 2000; Margeta-Mitrovic et al., 2000). In this set of experiments, we therefore produced four C-terminal truncates: GABA_B1C-^E579K, GABA_B1C-^E579K/E580K, GABA_B1C-^E583K, or GABA_B1C-^tripleK. Expression of these mutants showed that they were all able to reach the cell surface in the absence of GABA_B2 (Fig. 3b and data not shown). We then compared the ability of each GABA_B1 C-terminal truncate mutant to couple to G_qi5 after stimulation by GABA. All of the GABA_B1 mutants tested were nonfunctional when expressed alone (n = 4) (Fig. 3d and data not shown). In contrast, expression of each truncated GABA_B1 mutant gave a robust signal when coexpressed with GABA_B2, no different to that seen with wtGABA_B1 (data not shown). This suggests that removal of negatively charged glutamic acid residues within the second intracellular loop of GABA_B1 is not sufficient to allow signaling and strengthens the argument that the GABA_B2 subunit is absolutely necessary for G-protein signaling.

Positively charged amino acids in the second intracellular loop of GABA_B1 are not critical for GABA_B receptor signaling through G_qi5

We also noted when aligning the GABA_B1 and GABA_B2 il2 sequences that there were five positively charged lysine residues in this region of GABA_B1, although they do not directly align with the positive residues within il2 of GABA_B2 that we implicated in G-protein coupling. We therefore wanted to exclude the possibility that these amino acids might be involved in receptor signaling. We studied the effects of mutating the five lysine residues in il2 of GABA_B1 to neutral alanines and/or negatively charged glutamates, both in pairs and all five together (Fig. 4a). Each of the mutants GABA_B1^K577/578A, GABA_B1^K581/582A, GABA_B1^KQA, and GABA_B1^KQE were transfected into HEK293 cells together with GABA_B2. This resulted in cell surface expression of all of the GABA_B1 mutants as expected (Fig. 4b). We then tested the ability of these constructs to activate G_qi5 in response to GABA. Coexpression of GABA_B2 with GABA_B1^K577/578A, GABA_B1^K581/582A, GABA_B1^KQA, or GABA_B1^KQE resulted in the expression of functional GABA_B receptors. Neither of the double mutations (GABA_B1^K577/578A or GABA_B1^K581/582A) produced any significant changes in the potency of GABA when compared with the wild-type receptor, and although the potency of GABA at the quintuple mutant subunits (GABA_B1^KQA and GABA_B1^KQE) was slightly lower than the wild type, this was a very minor effect when compared with the mutations in GABA_B2 (pEC₅₀ values for wtGABA_B1, 7.19 ± 0.06, n = 36; GABA_B1^K577/578A, 7.28 ± 0.04, n = 9; GABA_B1^K581/582A, 7.17 ± 0.08, n = 9; GABA_B1^KQA, 6.74 ± 0.08, n = 12; and GABA_B1^KQE, 6.76 ± 0.03, n = 9) (Fig. 4c). These data suggest that the basic residues in il2 of GABA_B1 are also not critical for the signaling of the GABA_B receptor dimer through G_qi5.

View larger version (27K): [in this window] [in a new window]   Figure 4.   Basic residues within the second intracellular loop of GABAB1 are not critical for agonist-induced GABAB receptor signaling. a, Schematic representation of GABAB1 il2. Arrows indicate residues mutated. b, HEK293 cells were transiently transfected with wtGABAB2 (GB2) and wtGABAB1 (GB1), GB1K577/578A, GB1K581/582A, GB1KQA, or GB1KQE. GABAB1 is only found at the cell surface when expressed with GABAB2, as shown by immunofluorescence using an anti-myc antibody in the absence (i-v) of a permeabilizing detergent. c, Representative FLIPR analysis of cells transiently transfected with Gqi5, wtGABAB2 (GB2), and wtGABAB1 (GB1), GB1K577/578A, GB1K581/582A, GB1KQA, or GB1KQE. Both mutant and wild-type GABAB1 subunits are functional when coexpressed with wtGABAB2.

Exchanging charged residues between the second intracellular loops of GABA_B1 and GABA_B2 abolishes GABA_B receptor functional coupling to G-proteins

Because GABA_B2^tripleE when coexpressed with the wild-type GABA_B1 produced a nonfunctional receptor, we coexpressed GABA_B2^tripleE with GABA_B1^tripleK in HEK293 cells. Despite both subunits being expressed at the cell surface (Fig. 5a), no functional response was detected in the Ca²⁺ mobilization assay (n = 8) (Fig. 5b). This suggests that GABA_B2 is essential for G-protein coupling in the GABA_B receptor.

View larger version (21K): [in this window] [in a new window]   Figure 5.   Loss of functional coupling after exchange of GABAB1 and GABAB2 second intracellular loop charged residues. a, HEK293 cells were transiently transfected with GABAB1tripleK (GB1tripleK) and GABAB2tripleE (GB2tripleE). Cell surface expression of GABAB subunits was examined by immunofluorescence using an anti-myc antibody for GB1tripleK (i) and an anti-HA antibody for GB2tripleE (ii). b, Representative FLIPR analysis showing no functional response to GABA in cells transiently transfected with Gqi5, GB1tripleK, and GB2tripleE.

Amino acids in the second intracellular loop of GABA_B2 are critical for GABA_B receptor signaling through endogenous G-proteins in superior cervical ganglion cells

It was important to determine that the functional effects observed using our GABA_B2 mutant constructs in conjunction with G_qi5 could be reproduced in an effector system coupled to endogenously expressed G-proteins. We demonstrated previously that SCG neurons express only the GABA_B1 subunit. However microinjection of GABA_B2 constructs into these neurons results in the expression of functional GABA_B receptors that inhibit Ca²⁺ channels (Filippov et al., 2000). Microinjecton of GABA_B2^K590E and GABA_B2^tripleE resulted in cell surface expression of GABA_B2 (Fig. 6a) and GABA_B1 subunits (data not shown). Expression of the wtGABA_B2 subunit and stimulation by baclofen resulted in an ~53 ± 2.02% inhibition of voltage-activated Ca²⁺ currents (Fig. 6b,c), as measured 10 msec after the voltage pulse. Expression of GABA_B2^K590E resulted in significantly reduced inhibition of Ca²⁺ currents when compared with wtGABA_B2 (42 ± 1.92%) inhibition (Fig. 6b-d). In contrast to the other mutants studied, injection of the GABA_B2^tripleE mutant resulted in no baclofen-stimulated inhibition of Ca²⁺ channel opening above that observed in mock-injected neurons. This demonstrates that the GABA_B2^tripleE mutant, in conjunction with endogenous GABA_B1, forms a completely nonfunctional GABA_B receptor (Fig. 6b,c).

View larger version (17K): [in this window] [in a new window]   Figure 6.   Coupling of wtGABAB2 and GABAB2 mutants to N-type Ca2+ channels in sympathetic neurons. a, Cell surface expression of wild-type and mutant GABAB2 subunits examined by immunofluorescence using an anti-HA antibody. b, Currents were recorded by stepping for 100 msec every 20 sec from 90 to 0 mV and leak-corrected by subtracting currents remaining after substituting 5 mM Co2+ for Ba2+. Records show superimposed leak-subtracted currents in the absence (black line) and presence (red line) of 50 µM baclofen for neurons injected 16-24 hr before recordings with 100-150 ng/µl wtGABAB2 (GB2), GB2K590E, or GB2tripleE. c, Bar charts show the mean inhibition of IBa amplitude by 50 µM baclofen, 10 msec after voltage stepping, in neurons injected with GB2, GB2K590E, or GB2tripleE. Error bars show SEM; n indicates number of cells. Note that GABAB2K590E still mediates inhibition of Ca2+ channel current, whereas GABAB2tripleE does not couple to Ca2+ channels. d, Plots show concentration dependence of Ca2+ current inhibition by baclofen in SCG neurons injected with GB2 (n = 5) or GB2K590E (n = 3). Curves were fitted to pooled data points (mean ± SEM) using Origin 5 software to the Hill equation y = ymax [infi] · xnH / (xnH + KnH), where y is observed percentage of inhibition, ymax is the extrapolated maximal percentage of inhibition, x is baclofen concentration (micromolar), K is IC50 (micromolar), and nH is the Hill coefficient. For GABAB2K590E, IC50 of 1.04 ± 0.19 µM; nH of 1.23 ± 0.24; percentage of maximal inhibition, 42.03 ± 1.92%. For GABAB2, IC50 of 0.38 ± 0.06 µM; nH of 1.21 ± 0.19; percentage of maximal inhibition, 52.55 ± 2.02%. Note that GABAB2K590E inhibits Ca2+ current less effectively than GABAB2 and that plots could not be made for GABAB2tripleE because there was no detectable functional response.

The GABA_B2 subunit alone is unable to functionally respond to GABA

It is known that the GABA_B1 subunit contains a binding site for GABA in its N-terminal domain (Malitschek et al., 1999), but it is still unclear from published work as to whether GABA_B2 is capable of binding and responding to GABA on its own. We therefore generated a GABA_B2 stable cell line in HEK293 cells, in which we confirmed GABA_B2 expression on the cell surface by both immunocytochemistry and Western blotting (data not shown). We were unable to observe any [³⁵S]GTPS binding during stimulation with GABA up to a concentration of 1 mM (Fig. 7a). Similarly, we were unable to demonstrate any inhibition of forskolin-stimulated adenylate cyclase activity in these cells in response to GABA at a concentration of up to 100 µM (Fig. 7b). In cell lines expressing both GABA_B1 and GABA_B2 on the other hand, we were readily able to demonstrate functional coupling using both of these techniques with the pharmacology expected from recombinant GABA_B receptors (Fig. 7a,b).

View larger version (16K): [in this window] [in a new window]   Figure 7.   GABAB2 expressed alone is unable to functionally couple to adenylate cyclase or bind GTPS in response to GABA, but substitution of the GABAB2 N terminus with that of GABAB1 abolishes GABAB receptor function. a, Membranes prepared from cells expressing either GABAB2 (GB2) alone (squares) or both GABAB1 (GB1) and GABAB2 together (circles, triangles) were incubated with either GABA (circles, squares) or baclofen (triangles) and subsequently with [35S]GTPS. Data are presented as the percentage of increase in [35S]GTPS binding over basal in response to agonist and are expressed as means ± SEM (n = 3). b, Cells expressing either GABAB2 alone or GABAB1 and GABAB2 together (symbols and legend as above) were incubated with forskolin and then either GABA or baclofen. Data are presented as the percentage of forskolin-stimulated cAMP levels remaining after incubation with agonist and are expressed as means ± SEM (n = 3). c, HEK293 cells were transiently transfected with GABAB1N/2 (GB1N/2) and wtGABAB1 (GB1), and cell surface expression of GABAB subunits was examined by immunofluorescence using either an anti-myc antibody for GABAB1N/2 in the absence (i) or presence (ii) of detergent or an anti-GABAB1 antibody for wtGABAB1 (iii). d, Representative FLIPR analysis showing no functional response to GABA in cells transiently transfected with Gqi5, GABAB1N/2 (GB1N/2), and wtGABAB1 (GB1).

The N-terminal domain of GABA_B2 is also necessary for normal GABA_B receptor signaling

To investigate whether the N-terminal domain of GABA_B2 is important in normal GABA_B receptor function, we made a chimeric GABA_B2 construct by replacing the N-terminal binding domain of GABA_B2 with that of GABA_B1. Expression of this GABA_B1N/2 subunit was clearly observed on the cell surface, and furthermore it retained the ability to traffic wtGABA_B1 to the cell surface (Fig. 7c). However, functional analysis of cells expressing both GABA_B1N/2 and wtGABA_B1 exhibited a complete lack of response to GABA (n = 4) (Fig. 7d). This suggests that the N-terminal domain of GABA_B2 may also be important in the mediation of receptor responses to GABA, despite the fact that this subunit does not actually bind GABA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

GABA_B receptors are the only members of the type C family of GPCRs that have been shown to function as heterodimers. They are also distinct from other reported GPCR heterodimers in which both members of the heterodimer complex are able to form functional receptors when expressed as monomers (Jordan and Devi, 1999; AbdAlla et al., 2000; Gines et al., 2000; Gomes et al., 2000; Rocheville et al., 2000). In this study, we wanted to further analyze the role of each GABA_B subunit with respect to G-protein coupling and signal transduction, focusing on residues within the second and third intracellular loops. Residues within these domains have been demonstrated previously to be critical for G-protein interactions in a number of receptors. More specifically, recent studies on the functional roles of the cytoplasmic domains of rhodopsin have suggested that the third intracellular loop contains sites important in determining G-protein specificity, whereas the second intracellular loop contains regions essential for G-protein activation (Yamashita et al., 2000). Sequence comparison of residues in il2 and il3 for both the GABA_B subunits and the related mGluRs highlighted clear differences between GABA_B1 and GABA_B2 of potential relevance for G-protein coupling (Fig. 1a), suggesting that it may be GABA_B2 rather than GABA_B1 that is involved in the coupling of the GABA_B receptor to G-proteins and downstream signaling. Furthermore, comparison of 46 il2 sequences from various species demonstrated that all of the mGluRs and GABA_B2 contained fewer than two negatively charged residues within this loop. This is in sharp contrast to GABA_B1, which contains four negatively charged residues. This may be of particular relevance in the context of modeling studies by Fannelli et al. (1998), who investigated the electrostatic complimentarity of receptor-G-protein complexes. These studies favor a model whereby "opening" of the cytosolic domains of the receptor allows for an interaction between the electrostatically positive surface of the domains of the receptor and the negatively charged surface of the G subunit (Higgs and Reynolds, 2001). In this respect, it is of interest that the electrostatic potential of G_o, reported to be the predominant G-protein interacting with GABA_B receptors (Leaney and Tinker, 2000), is proposed to be one of the most negative of the G subunits (C. Reynolds, personal communication). Based on these observations, we can speculate that the negatively charged residues of the GABA_B1 subunit would make it a less attractive candidate for G-protein coupling than GABA_B2.

To determine the functional role that il2 residues play in GABA_B receptor function, we made a series of amino acid substitution mutants at K⁵⁸⁶, M⁵⁸⁷, and K⁵⁹⁰ in GABA_B2. Single or double substitutions of these residues to glutamate or alanine did not affect the ability of GABA_B2 to reach the cell surface itself or to traffic GABA_B1 to the cell surface but led to a significant reduction in agonist potency when compared with the wild-type receptor heterodimer. In addition, when all three residues were substituted by glutamates or alanines, it resulted in a receptor that was completely unresponsive to agonists. These changes in agonist efficacy were not attributable to aberrant folding of the receptor subunits or changes in binding affinity because both agonist and antagonist binding was unaltered when compared with wild-type receptor. Moreover, our binding data were consistent with immunocytochemical data and suggested that there was no significant effect on receptor expression levels. Importantly, these mutants also interfered with the physiological coupling of GABA_B receptors to Ca²⁺ channels in SCG neurons, confirming that mutation of these residues in il2 of GABA_B2 was sufficient to abolish GABA_B receptor signaling. These data therefore identify key residues important in G-protein coupling and activation of the receptor heterodimer and is in agreement with previous proposals suggesting that the heptahelical domains of GABA_B2, and not GABA_B1, contain these molecular determinants (Calver et al., 2001; Galvez et al., 2001).

To further investigate the role of il2 residues in the heterodimer, we substituted the corresponding negatively charged residues in GABA_B1, E⁵⁷⁹, E⁵⁸⁰, and E⁵⁸³, to lysines. These GABA_B1 mutants were all expressed on the cell surface after coexpression with GABA_B2, and all responded to agonist as effectively as the wild-type receptor heterodimer. Similarly, GABA_B1 mutants in which all five positively charged lysine residues in this region were changed to either neutral or negatively charged amino acids are completely functional within heterodimers with wild-type GABA_B2. Furthermore, a number of other GABA_B1 mutants we expressed all showed a complete lack of responsiveness to GABA in the absence of GABA_B2. Although this data does not completely discount the possibility that G-proteins may be interacting with il2 of the GABA_B1 subunit, it does suggest that it is less likely that there are electrostatic interactions between GABA_B1 and G-proteins. A recent study however has reported that replacement of the entire seven transmembrane and intracellular domains of GABA_B1 with that of GABA_B2 (termed GB1/2) reduces the efficacy of G-protein coupling to a "GB1/2 GB2" receptor (Galvez et al., 2001). Although this may be indicative of a modulatory role for GABA_B1 in G-protein coupling, we would suggest that it is more likely as a result of less efficient signaling between GABA_B1 and its "downstream" partner, GABA_B2.

Molecular modeling studies have identified residues in the GABA_B1 subunit that are critical for binding of GABA_B agonists and antagonists, and these residues are absent from the GABA_B2 subunit (Galvez et al., 2000a,b). In this study, we demonstrated that GABA_B2 expressed in the absence of GABA_B1 in HEK293 cells is unable to respond functionally to GABA, by either inhibition of adenylate cyclase activity or binding of [³⁵S]GTPS. However, the absence of a ligand binding site does not necessarily mean that GABA_B2 is not important in signal transduction. Indeed, the recent solving of the crystal structures for the N-terminal binding domain of mGluR1 demonstrate that movements between all four globular lobes of the N-terminal domains of the dimer are likely to cause shifts in transmembrane and intracellular domains, resulting in receptor activation (Kunishima et al., 2000). These findings may help to explain results from this and other studies in which deletion or substitution of substantial parts of the GABA_B2 N-terminal domain results in a complete loss of GABA_B receptor function (Jones et al., 2000; Galvez et al., 2001). This therefore suggests that the N-terminal domain of the GABA_B2 subunit, although unable to bind GABA itself, may instead be involved in the conformational changes induced after ligand binding to the GABA_B1 subunit.

In summary, our study has determined three residues within il2 of the GABA_B2 subunit critical for G-protein signaling of the GABA_B receptor heterodimer, demonstrating that this signaling absolutely requires the presence of the GABA_B2 protein. Although our data do not rule out a possible involvement of GABA_B1 in the signaling process, these studies, together with current knowledge of this receptor heterodimer, strongly suggest a novel mechanism of "sideways" signal transduction, whereby agonist binds to the GABA_B1 subunit, resulting in a conformational change that is in some way passed on to the GABA_B2 subunit, perhaps through its N-terminal and/or transmembrane domains. This in turn results in the receptor forming an activated state suitable for G-protein recruitment, via a direct interaction with GABA_B2, and thus downstream signal transduction (Fig. 8). Such novel sideways signaling may allow increased complexity of receptor signal transduction, which may also be applicable to other GPCR heterodimers.

View larger version (32K): [in this window] [in a new window]   Figure 8.   Proposed sideways signaling mechanism for the GABAB receptor heterodimer. GABAB1 (orange) and GABAB2 (green) subunits form heterodimers via C-terminal coiled-coil interactions, as well as N-terminal and possibly transmembrane domain interactions. The agonist GABA binds to the ligand binding domain within the GABAB1 N terminus, resulting in a conformational change in the GABAB2 subunit. This activated state of the receptor heterodimer allows recruitment of G-proteins, at least in part via an interaction with the positively charged residues in il2 of GABAB2 and subsequent activation of downstream signal transduction cascades.

	FOOTNOTES

Received May 18, 2001; revised July 26, 2001; accepted July 27, 2001.

A.C. and S.J.M. are supported by the Wellcome Trust and the Medical Research Council. A.K.F. and D.A.B. are supported by the Wellcome Trust. We thank Prof. Derek Middlemiss and Prof. Gary Price for helpful discussion and comments on this manuscript.

M.J.R and A.R.C. contributed equally to this work.

Correspondence should be addressed to Menelas N. Pangalos, Department of Neurology CEDD, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK. E-mail: menelas_n_pangalos{at}gsk.com.

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