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RECEPTORS AND SIGNAL TRANSDUCTION

Coupling of GABA_B receptor GABA_B2 subunit to G proteins: evidence from Xenopus oocyte and baby hamster kidney cell expression system

Departments of ¹Pharmacology and ²Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; and ³Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel

Submitted 8 June 2005 ; accepted in final form 17 August 2005

	ABSTRACT

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Coupling of functional GABA_B receptors (GABA_BR) to G proteins was investigated with an expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescence resonance energy transfer (FRET) analysis of BHK cells coexpressing GABA_B1a receptor (GB_1aR) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABA_B2 receptor (GB₂R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB_1aR-Cerulean and GB₂R-Venus form a heterodimer. The GABA_BR agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying K⁺ currents in a concentration-dependent manner in oocytes expressing GB_1aR and GB₂R, or GB_1aR-Cerulean and GB₂R-Venus, together with G protein-activated inward-rectifying K⁺ channels (GIRKs), but not in oocytes expressing GB_1aR alone or GB₂R alone together with GIRKs. Oocytes coexpressing GB_1aR + G_i2-fused GB₂R (GB₂R-G_i2) caused faster K⁺ currents in response to baclofen. Furthermore, oocytes coexpressing GB_1aR + GB₂R fused to G_qi5 (a chimeric G_q protein that activates PLC pathways) caused PLC-mediated Ca²⁺-activated Cl^– currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB_1aR-G_i2 or GB_1aR-G_qi5 together with GB₂R. BHK cells and Xenopus oocytes coexpressing GB_1aR-Cerulean + a triplet tandem of GB₂R-Venus-G_qi5 caused FRET and Ca²⁺-activated Cl^– currents, respectively, with a similar potency in BHK cells coexpressing GB_1aR-Cerulean + GB₂R-Venus and in oocytes coexpressing GB_1aR + GB₂R-G_qi5. Our results indicate that functional GABA_BR forms a heterodimer composed of GB₁R and GB₂R and that the signal transducing G proteins are directly coupled to GB₂R but not to GB₁R.

fluorescence resonance energy transfer

To determine the subunit of GABA_BR (i.e., GB₁R or GB₂R) that is directly coupled to the G protein, we used Xenopus oocytes coexpressing one of each GABA_BR subunits fused to G_i2, a G protein that preferentially couples to GABA_BR-mediated signaling among G_i/o proteins in the central nervous system (CNS) (18, 19) and its counterpart. Cell signaling via the G protein fused to receptors is preferentially transmitted to the effectors rather than that via G_i/o proteins endogenously expressed in Xenopus oocytes (13, 20, 31). Accordingly, we determined the kinetics of GIRK activation on GABA_BR stimulation in oocytes that expressed both GB₁R and GB₂R fused to G_i2. Furthermore, coupling of the G protein to GABA_BR was also examined in oocytes expressing both GB₁R and GB₂R fused to a chimeric G_q protein, G_qi5, which contains the carboxy-terminal five amino acids of G_i. The chimeric G_qi proteins, including G_qi5, allow G_i protein-coupled receptors to couple to the PLC-mediated signal pathways (6, 9), and such G_qi-mediated signaling has been used for the output of GABA_BR-mediated signaling (7, 9, 10). FRET analysis was used in BHK cells coexpressing Cerulean- or Venus-fused GABA_BR to determine the heterodimerization of the receptors. Thus the objective of the present study was to characterize functionally and morphologically the GABA_BR subunit directly coupled to functional G proteins.

	MATERIALS AND METHODS

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Drugs and chemicals. Baclofen, 3-aminopropylphosphonic acid (3-APPA), ACh, gentamicin, and sodium pyruvate were purchased from Sigma (St. Louis, MO). Other chemicals used in the study were of analytical grade and were obtained from Nacalai Tesuque (Kyoto, Japan).

Construction of cDNAs and preparation for cRNA. cDNAs for rat GIRK1 and mouse GIRK2 were provided by Dr. H. A. Lester (Caltech, Pasadena, CA). GABA_B1a and GABA_B1b receptors (GB_1aR and GB_1bR) and GB₂R were from Dr. N. J. Fraser (Glaxo Wellcome, Stevenage, UK). Human M₂R was from Dr. E. Peralta (Harvard University, Cambridge, MA). Rat G_i2 was from Dr. M. I. Simon (Caltech), and rat µ-opioid receptors were from N. Dascal. The chimeric G_qi5 was a kind gift from Dr. B. R. Conklin (University of California at San Francisco). Cerulean, a brighter variant of cyan fluorescent protein (23) was from Dr. D. W. Piston (Vanderbilt University, Nashville, TN), and Venus, a brighter variant of yellow fluorescent protein (17) was from Dr. T. Nagai (Riken, Wako, Japan). The GB_1aR-G_i2, GB_1bR-G_i2, GB₂R-G_i2, and M₂R-G_i2 tandem cDNAs were created by ligating the receptor cDNA sequences into the EcoRI site of the corresponding G_i2 cDNA. Each receptor-G_qi5 tandem was created by ligation of the receptor cDNA sequences into the NheI site of the corresponding G_qi5. GABA_BR-Cerulean, GABA_BR-Venus, and µ-opioid receptor-Venus were created by ligating the receptor cDNAs into the NotI or BamHI sites of the corresponding Venus or Cerulean sites. The GB₂R-Venus-G_qi5 triplet tandem was created by ligating GB₂R-Venus into the NheI site of corresponding G_qi5. All cDNAs for transfection in BHK cells were subcloned into pcDNA3.1 (Invitrogen). All cDNAs for the synthesis of cRNAs were subcloned into the pGEMHJ vector, which provides 5'- and 3'-untranslated regions of the Xenopus -globin RNA, ensuring a high level of protein expression in the oocytes (31). Each of the cRNAs was synthesized with a mCAP mRNA capping kit and with a T7 RNA polymerase in vitro transcription kit (Ambion, Austin, TX) from the respective linearized cDNAs.

Oocyte preparation and injection. Immature stages V and VI oocytes from Xenopus were enzymatically dissociated as previously described (27, 28). Isolated oocytes were incubated at 18°C in ND-96 medium (in mM: 96 NaCl, 2 KCl, 1 CaCl₂, 1 MgCl₂, and 5 HEPES, pH 7.4) containing 2.5 mM sodium pyruvate and 50 µg/ml gentamicin. For measurement of GIRK currents induced by baclofen or 3-APPA, cRNAs for GIRK1/2 (0.2 ng each) were injected into the oocytes with or without GB_1aR, GB_1bR, GB_1aR-Cerulean, or GB_1bR-Cerulean (5 ng each) and/or GB₂R or GB₂R-Venus (5 ng each). In some oocytes, cRNAs for GB_1aR-G_qi5, GB₂R-G_qi5, GB_1aR-Cerulean-G_qi5, GB₂R-Venus-G_qi5, M₂R, or M₂R-G_qi5 (5 ng each) were injected. The final injection volume was <50 nl in all cases. Oocytes were incubated in ND-96 and used 3–8 days after injection as reported previously (29).

Electrophysiological recordings. Electrophysiological recordings were performed using the two-electrode voltage-clamp method with a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA) at room temperature. Oocytes were clamped at –60 mV and continuously superfused with ND-96 or 49 mM high-K⁺ (HK) solution (in mM: 48 NaCl, 49 KCl, 1 CaCl₂, 1 MgCl₂, and 5 HEPES, pH 7.4) in a 0.25-ml chamber at a flow rate of 5 ml/min, and baclofen or 3-APPA (10^–9 to 10^–3 M) was added to the superfusion solution. Voltage recording microelectrodes were filled with 3 M KCl and had a tip resistance of 1.0–2.5 M. Currents were continuously recorded and stored with MacLab (ADInstruments, Castle Hill, Australia) and a Macintosh computer, as described previously (27, 29). All test compounds applied to oocytes were dissolved in ND-96 or HK media.

Cell culture and transfection. BHK cells were grown in DMEM supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere of 95% air-5% CO₂. For transfection experiments, BHK cells were seeded at a density of 1 x 10⁵ cells/35-mm glass-bottomed culture dish (Fluoro Physiotech) for 24 h. Transient transfection was then performed with Effectene transfection reagent (Qiagen, Tokyo, Japan) in 0.2 µg of each cDNA as described previously (25) and according to the protocol provided by the manufacturer. For titration assay of FRET efficiency, 0.2 µg of GB_1aR-Cerulean and varying concentrations (0.05–0.5 µg) of GB₂R-Venus cDNA were cotransfected. Cells were used in confocal microscopy and FRET analysis 16–20 h after transfection.

Confocal microscopy and FRET analysis. For the analysis of heterodimerization of GABA_BR using the FRET assay, GB₁R and GB₂R were fused through the carboxy terminus to Cerulean and Venus. BHK cells cultured in 35-mm glass-bottomed dishes were cotransfected with a combination of GB_1aR-Cerulean or GB_1bR-Cerulean + GB₂R-Venus or GB_1aR-Venus + GB₂R-Cerulean. In some experiments, Cerulean, Venus, µ-opioid receptor-Venus, the triplet tandem of GB_1aR-Cerulean-G_qi5, or GB₂R-Venus-G_qi5 was also transfected. A x63 magnification, 1.25 numerical aperture oil-immersion objective was used with a pinhole for visualization. Both Cerulean and Venus were excited with a 458-nm laser, and images were obtained by placing the dish onto a stage in a Zeiss LSM510 META confocal microscope (Carl Zeiss, Jena, Germany).

Photobleaching and calculation of FRET efficiency. To confirm FRET between Cerulean and Venus, we monitored acceptor photobleaching analysis in living BHK cells coexpressing Cerulean-fused or Venus-fused GABA_BR. FRET was measured by imaging Cerulean before and after photobleaching Venus with the 100% intensity of a 514-nm argon laser for 2 min, a duration that efficiently bleached Venus with little effect on Cerulean. An increase of donor fluorescence (Cerulean) was interpreted as evidence of FRET from Cerulean to Venus. All experiments were analyzed in at least six cells with three independent regions of interest. As a control, we examined the FRET efficiency of the unbleached area of membrane in the same cell in at least three areas. In some cases we performed the photobleaching assay with fixed BHK cells. Cell fixation was performed as previously described (30).

FRET efficiency was calculated with emission spectra before and after acceptor photobleaching. In our study as well as others, this protocol had almost no effect on Cerulean in the absence of Venus (Ref. 16; see RESULTS). According to this procedure, if FRET is occurring, then photobleaching of the acceptor (Venus) should yield a significant increase in fluorescence of the donor (Cerulean). Increase of donor spectra due to desensitized acceptor was measured by taking the Cerulean emission (at 488 nm) from spectra before and after acceptor photobleaching. FRET efficiency was then calculated using the equation E = 1 – I_DA/I_D, where I_DA is the peak of donor (Cerulean) emission in the presence of the acceptor and I_D is the peak in the presence of the sensitized acceptor, as previously described (22). Before and after this bleaching, Cerulean images were collected to assess changes in donor fluorescence. Any increase in Cerulean fluorescence caused by bleaching of the Venus acceptor could be masked, at least in part, by bleaching of Cerulean related simply to the imaging process itself. To minimize the effect of photobleaching due to imaging, images were collected at 2.0% of the laser intensity, 50 times less than bleach intensity.

Statistical analysis. Data are expressed as means ± SE. Differences between two groups were examined for statistical significance with a paired t-test. GraphPad Prism software was used to analyze data for statistical significance and to analyze data and fit curves for baclofen and 3-APPA dose responses. For comparisons between multiple groups one-way ANOVA was used, followed by Scheffé's test. A P value of <0.05 denotes the presence of a statistically significant difference.

	RESULTS

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FRET and acceptor photobleaching analysis of GABA_B receptors expressed by BHK cells. Expression of GABA_BR composed of GB₁R and GB₂R on the cell surface was examined using FRET analysis in living BHK cells coexpressing GB_1aR fused to Cerulean (GB_1aR-Cerulean) and GB₂R fused to Venus (GB₂R-Venus). Confocal imaging in living cells showed that fluorescence from Cerulean and Venus was colocalized mostly on the cell membrane and in the cytoplasm, if any, of cells coexpressing GB_1aR-Cerulean + GB₂R-Venus (Fig. 1A, top). The same results were obtained with fixed BHK cells (data not shown). Photobleaching analysis demonstrated that Venus fluorescence was reduced by sustained 2-min application of a 514-nm wavelength at 100% intensity of the argon laser power to the indicated area (Fig. 1A, top). This 2-min application did not affect the fluorescence intensity of Cerulean and Venus in unbleached areas (data not shown). Also, this 2-min application had almost no effect on the fluorescence intensity of Cerulean on the cell surface in BHK cells expressing only GB₂R-Cerulean (data not shown). With the use of acceptor photobleaching, Cerulean fluorescence (donor) was increased as the Venus fluorescence decreased (acceptor) (Fig. 1, A, bottom, and B). As shown in Fig. 1C, FRET efficiency was significantly higher in BHK cells coexpressing GB_1aR-Cerulean + GB₂R-Venus or GB_1bR-Cerulean + GB₂R-Venus than in the negative control (pairs of Cerulean + Venus and GB₂R-Cerulean + µ-opioid receptor-Venus). A similar level of FRET efficiency was obtained in BHK cells coexpressing another pair of tandem GABA_BRs, GB_1aR-Venus + GB₂R-Cerulean (Fig. 1C).

View larger version (50K): [in this window] [in a new window]   Fig. 1. Confocal imaging and fluorescence resonance energy transfer (FRET) analysis of the dimerized GABAB1a receptor (GB1aR) and GABAB2 receptor (GB2R) in baby hamster kidney (BHK) cells expressing Cerulean-fused GB1aR (GB1aR-Cerulean) and Venus-fused GB2R (GB2R-Venus). A: visualization of the GB1aR-Cerulean and GB2R-Venus in cotransfected BHK cells (top) and fluorescence changes by acceptor photobleaching (2-min application of 514-nm wavelength; bottom). B: emission spectra from a BHK cell before and after Venus photobleaching as indicated in A. Note the increase of the Cerulean peak emission (488 nm) following the photobleaching of Venus (528 nm). Inset: mathematical equation used to calculate the FRET efficiency (E). IDA, peak of donor emission in presence of acceptor; ID, peak of donor emission in presence of sensitized acceptor. C: FRET efficiency of GB1aR-Cerulean + GB2R-Venus, GB1bR-Cerulean + GB2R-Venus, and GB1aR-Venus + GB2R-Cerulean pairs. The Venus + Cerulean and µ-opioid receptor-Venus + GB2R-Cerulean pairs were used as negative controls for interacting receptors. Each column represents mean ± SE of FRET efficiency in independent experiments using 6 cells with 3 regions of interests per BHK cell (n = 18). D: heterodimerization of GABABR assessment by FRET efficiency titration in living BHK cells. BHK cells were cotransfected with a constant amount (0.2 µg) of GB1aR-Cerulean and an increasing amount (0.05–0.5 µg) of GB2R-Venus. Acceptor photobleaching was performed within the membranes of the cells as indicated in A. The curve was fitted using a nonlinear regression equation assuming a single binding site (GraphPad Prism).

To investigate the effects of the GB₂R-Venus-to-GB_1aR-Cerulean expression ratio on the FRET efficiency, we performed the FRET efficiency titration assay as previously described (30). As shown in Fig. 1D, FRET efficiency was saturable over the ratio (GB₂R-Venus/GB_1aR-Cerulean) of 1, which was in accordance with the results performed with titration of bioluminescence resonance energy transfer assay (30).

In living cells, small movement of cells or movement of organelles within cells might interfere the efficacy of FRET efficiency. Accordingly, we compared FRET efficiency between living and fixed BHK cells coexpressing GB_1aR-Cerulean + GB₂R-Venus. There was almost no difference between FRET efficiency in living and fixed cells (living BHK cells 0.34 ± 0.1, fixed 0.36 ± 0.08; n = 18 each), demonstrating that living cells did not interfere with the efficacy of photobleaching in our experimental system.

Response to GABA_BR agonists in Xenopus oocytes expressing functional GABA_BR. In oocytes coexpressing GB_1aR or GB_1bR together with GB₂R and GIRK1/2, the GABA_BR agonists baclofen and 3-APPA (10^–9 to 10^–3 M) elicited inward-rectifying K⁺ currents in a concentration-dependent manner (Fig. 2 and Table 1). In contrast, both agonists failed to cause any response in oocytes individually coexpressing GB_1aR, GB_1bR, or GB₂R together with GIRK1/2 (Fig. 2A; other data not shown). There were no differences in the time course and peak amplitude of GIRK currents between oocytes expressing GB_1aR + GB₂R and GB_1bR + GB₂R together with GIRK1/2 (Fig. 2B and Table 1). In oocytes coexpressing GB_1aR-Cerulean + GB₂R-Venus together with GIRK1/2, baclofen and 3-APPA also caused a concentration-dependent increase in K⁺ currents with EC₅₀ values essentially similar to those measured in oocytes expressing nonfused GB_1aR + GB₂R together with GIRK1/2, demonstrating that addition of fluorescent proteins to the carboxy terminus of the GABA_BR subunit did not affect the ability to activate GIRK channels (Table 1).

View larger version (14K): [in this window] [in a new window]   Fig. 2. Baclofen- and 3-aminopropylphosphonic acid (3-APPA)-induced G protein-activated inward-rectifying K+ channel (GIRK) currents in Xenopus oocytes coexpressing GB1aR or GB1bR and GB2R together with GIRK1 and GIRK2 (GIRK1/2). A: representative trace of GIRK currents induced by baclofen in oocytes expressing GB1aR alone, GB2R alone, or both together with GIRK1/2 voltage-clamped at –60 mV. Baclofen at 10–4 M was applied as indicated by the horizontal bar for 60 s. HK, high-K+ solution. B: concentration-response curves of baclofen- or 3-APPA-induced GIRK currents in oocytes. Varying concentrations of baclofen or 3-APPA (10–9 to 10–3 M) were applied for 60 s in oocytes as indicated in A, and peak currents were measured. Each point represents mean ± SE of the peak GIRK currents expressed as net GIRK currents in 3–6 oocytes.

Acceleration of GIRK activation processes in Xenopus oocytes expressing GABA_BR fused to G_i2.

View larger version (8K): [in this window] [in a new window]   Fig. 3. ACh-induced GIRK currents in oocytes coexpressing muscarinic M2 receptor (M2R) or M2R fused to Gi2 protein (M2R-Gi2) together with GIRK1/2. A: representative trace of ACh-induced GIRK currents in oocytes coexpressing M2R together with GIRK1/2. ACh at 10–5 M was applied as indicated by the horizontal bar for 30 s. B: comparison of kinetics of activation of ACh-induced GIRK currents (IACh) in oocytes coexpressing M2R or M2R-Gi2 together with GIRK1/2. Representative traces of currents of first 25 s of IACh obtained in the oocytes are shown. Currents were scaled to allow direct comparison of speed of activation as indicated by Ivanina et al. (13). C: summary of measurement of time of 90% of peak (t90%) of IACh. Each bar represents mean ± SE of the t90% from 20 oocytes used in a minimum of 3 independent experiments. *P < 0.05 compared with oocytes coexpressing M2R together with GIRK1/2.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Baclofen-induced GIRK currents in oocytes expressing GABAB receptor subunit (GB1aR and GB2R) fused to Gi2 together with GIRK1/2. Baclofen (10–4 M) was applied for 30 s to the oocytes coexpressing GB1aR + GB2R, GB1aR-Gi2 + GB2R, GB1aR + GB2R-Gi2, or GB1aR-Gi2 + GB2R-Gi2 together with GIRK1/2. A: comparison of kinetics of activation of baclofen-induced GIRK currents (Ibaclofen) in oocytes coexpressing the combination of GB1aR + GB2R with or without fusing Gi2 together with GIRK1/2. Representative traces of currents of first 25 s of Ibaclofen obtained in the oocytes are shown. Currents were scaled to allow direct comparison of speed of activation as shown in Fig. 3. B: summary of baclofen-induced currents. Each bar represents mean ± SE of t90% from 20 oocytes in at least 3 independent experiments. *P < 0.05 compared with oocytes coexpressing GB1aR + GB2R together with GIRK1/2; n.s., not significant compared with oocytes coexpressing GB1aR + GB2R together with GIRK1/2.

Generation of Ca²⁺-activated Cl^– currents in Xenopus oocytes expressing GABA_BR fused to chimeric G_qi5 protein.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Baclofen- or ACh-induced Ca2+-activated Cl– currents in oocytes coexpressing GABABR subunits (GB1aR and/or GB2R) fused to Gqi5 or muscarinic M2R fused to Gqi5, respectively. A, left: representative trace of baclofen-induced currents in oocytes coexpressing GB1aR + GB2R, GB1aR-Gqi5 + GB2R, GB1aR + GB2R-Gqi5, or GB1aR-Gqi5 + GB2R-Gqi5. Baclofen (10–4 M) was applied for 30 s to each group of oocytes as indicated by the bars. Right: summary of baclofen-induced Ca2+-activated Cl– currents. B, left: representative traces of ACh-induced Ca2+-activated Cl– currents in oocytes expressing M2R or M2R-Gqi5. ACh (10–5 M) was applied for 30 s to each oocyte as indicated by the bars. Right: summary of ACh-induced currents. Each bar represents mean ± SE of the peak currents from independent experiments on at least 6 oocytes. *P < 0.05 compared with GB1aR + GB2R-coexpressing oocytes. **P < 0.05 compared with M2R-expressing oocytes.

View this table: [in this window] [in a new window]   Table 2. Effect of fusion of Gqi5 protein or fluorescent proteins to GABAB receptor subunits on FRET efficiency and baclofen-induced Ca2+-activated Cl– currents

	DISCUSSION

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GB₂R plays key roles in the trafficking of the GB₁R subunit to the cell surface (5, 8, 26) and in the mediation of agonist-induced G protein coupling (10, 21). The identity of the GABA_BR subunit, GB₁R and/or GB₂R, that is directly coupled to G proteins in the functional GABA_BR was examined by using each GABA_BR subunit fused to the G protein G_i2. G_i/o protein-mediated responses in Xenopus oocytes are generally obtained by detecting GIRK currents through activation of endogenously expressed G_i/o proteins in GIRK-expressed oocytes (27, 29, 31). Signals via tandem receptors fused to G protein are thought to be preferentially transmitted through the fused G proteins (31). We demonstrated that oocytes coexpressing muscarinic M₂R-G_i2 together with GIRKs caused faster inward-rectifying K⁺ currents in response to ACh compared with oocytes coexpressing nonfused M₂R with GIRKs. The initial GIRK activation process via tandem muscarinic M₂R-G_i3 is also reported to be faster than that via M₂R-mediated activation of G proteins endogenously expressed in oocytes (31). We showed that baclofen elicited faster inward-rectifying K⁺ currents in oocytes coexpressing GB_1aR + GB₂R-G_i2 together with GIRKs compared with oocytes coexpressing GB_1aR + nonfused GB₂R with GIRKs. This type of rapid response to baclofen was not obtained in oocytes coexpressing GB_1aR-G_i2 + GB₂R with GIRKs. These results suggest that G_i2 fused to GB₂R, but not to GB₁R, transduced the signal to GIRKs in our experimental system. A recent detailed three-dimensional structure model demonstrated that homo- or heterodimeric signaling molecules of GPCR-G protein were pentamers composed of two of the dimerized receptors with G, G, and G subunits (2). On the basis of this model, heterodimeric GABA_B receptor-G protein signaling molecules may be composed of two receptors and one trimeric G protein presumably associated with GB₂R, although further investigation is required.

Further experiments were carried out with GB₂R fused to G_qi5 protein, a chimeric G_q protein that enables G_i/o protein-coupled receptors to activate G_q protein-mediated PLC activation (6, 9, 15). We previously demonstrated (15) that activation of G_i/o-coupled -opioid, 5-HT_1A, and somatostatin type 2 receptors resulted in increases in [Ca²⁺]_i and subsequent activation of Ca²⁺-activated Cl^– currents only when coexpressed with G_qi5 in Xenopus oocytes. In the present study, we found that baclofen elicited Ca²⁺-activated Cl^– currents in oocytes coexpressing GB₂R-G_qi5 + GB_1aR but not in those expressing GB_1aR-G_qi5 + GB₂R, indicating that baclofen stimulates GABA_BR and elicits Ca²⁺-activated Cl^– currents through only chimeric G_qi5 fused to GB₂R. FRET analysis demonstrated that such constructions of GB₂R-G_qi5 or GB_1aR-G_qi5 did not influence the heterodimeric properties with GB_1aR or GB₂R on the cell surface, respectively; there was little difference in the FRET efficiency among GB_1aR-Cerulean + GB₂R-Venus, GB_1aR-Cerulean + GB₂R-Venus-G_qi5, and GB_1aR-Cerulean-G_qi5 + GB₂R-Venus in BHK cells (Table 2). Similarly, there were no differences in baclofen-elicited Ca²⁺-activated Cl^– currents between oocytes coexpressing GB_1aR + GB₂R-G_qi5 and GB_1aR + GB₂R-Venus-G_qi5 (Table 2), suggesting that insertion of a fluorescent protein between GB₂R and G_qi5 had no effect on GABA_BR-mediated PLC activation. In a few cases, however, we observed that oocytes coexpressing GABA_B1aR-Cerulean-G_qi5 + GABA_B2R-Venus responded to baclofen, although GB_1aR-G_qi5 + GB₂R did not (Table 2). This may be a promiscuous coupling of G_qi5 with a longer carboxy terminus of GB_1aR. (Inserted Cerulean between GB_1aR and G_qi5 would create the situation in which the G_qi5 may access the G protein binding site for GB₂R.) Nonetheless, the chimeric G_qi5 seems to couple selectively to GB₂R and activate PLC in the functional GABA_BR in our experimental system using Xenopus oocytes. These results indicate that the functional GABA_BR forms a heterodimer with GB₁R and GB₂R and G proteins that transduce signal to the downstream are coupled to GB₂R. Several investigators have shown that intracellular loops 1, 2, and 3 of GB₂R are indispensable for the functional coupling of heterodimeric GABA_BR signaling (7, 10, 11, 14). In addition, they have reported that none of the intracellular loops of GB₁R is linked to the functional GABA_BR-induced signaling (7, 10, 11, 14). Our results are consistent with their conclusions in that only GB₂R-linked G proteins are important for the receptor signaling. These findings suggest that intracellular loops of GB₁R may not physically couple to G proteins. Because some GABA_BR-mediated signaling pathways, such as inhibition of voltage-dependent Ca²⁺ channels, are preferentially mediated via G_o rather than G_i in certain cell types (3, 4), other effector signaling systems, including Ca²⁺ channel inhibition and inhibition and/or activation of adenylate cyclases, should be investigated to further clarify the involvement of dimeric GABA_BR-induced cellular signaling.

In conclusion, we have demonstrated that functional GABA_BR forms a heterodimer composed of GB₁R and GB₂R in living cells and that the G proteins that transduce downstream signals could be coupled to GB₂R but not GB₁R.

	GRANTS

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This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to Y. Uezono, M. Kanaide, K. Sumikawa, and K. Taniyama), Naito Foundation (to Y. Uezono), and Public Health Research Foundation (to M. Kaibara).

	FOOTNOTES

 

Address for reprint requests and other correspondence: Y. Uezono, Dept. of Pharmacology, Nagasaki Univ. Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan (e-mail: uezonoy{at}alpha.med.nagasaki-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
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