Pharmacological characterisation of a cell line expressing GABAB1b and GABAB2 receptor subunits

doi:10.1016/S0006-2952(02)01658-1

Biochemical Pharmacology

Volume 65, Issue 7, 1 April 2003, Pages 1103-1113

https://doi.org/10.1016/S0006-2952(02)01658-1 Get rights and content

Abstract

The γ-aminobutyric acid (GABA_B) receptor has been shown to be a heterodimer consisting of two receptor subunits, GABA_B1 and GABA_B2. We have stably co-expressed these two subunits in a CHO cell line, characterised its pharmacology and compared it to the native receptor in rat brain membranes. Radioligand binding using [ $^{3} H$ ]CGP54626A demonstrated a similar rank order of potency between recombinant and native receptors: CGP62349>CGP54626A>SCH $50911>3- aminopropylphosphinicacid (3- APPA)> GABA > baclofen > saclofen > phaclofen$ . However, differences were observed in the affinity of agonists, which were higher at the native receptor, suggesting that in the recombinant system a large number of the receptors were in the low agonist affinity state. In contrast, [ $^{35} S$ ]GTPγS binding studies did not show any differences between recombinant and native receptors with the full agonists GABA and 3-APPA. Measurement of cAMP accumulation in the cells revealed a degree of endogenous coupling of the receptors to G-proteins. This is most likely to be due to the high expression levels of receptors (B_max=22.5±2.5 pmol/mg protein) in this experimental system. There was no evidence of GABA_B2 receptors, when expressed alone, binding [ $^{3} H$ ]CGP54626A, [ $^{3} H$ ]GABA, [ $^{3} H$ ]3-APPA nor of GABA having any effect on basal [ $^{35} S$ ]GTPγS binding or cAMP levels.

Introduction

GABA is the major inhibitory neurotransmitter in the mammalian CNS. GABA mediates its effects through ligand-gated chloride channels (GABA_A/C receptors) to produce fast synaptic inhibition (reviewed by [1]) and metabotropic GABA_B receptors to produce slow, prolonged inhibitory signals [2], [3], [4]. GABA_B receptors are thought to be involved in a number of physiological and disease processes, including nociception, cognition, epilepsy, depression and drug addiction [3], [5].

The GABA_B receptor was first identified pharmacologically as a bicuculline-insensitive binding site with affinity for both baclofen and GABA [6], [7], [8]. GABA_B receptor activation inhibits adenylyl cyclase activity and causes prolonged synaptic inhibition through restriction of pre-synaptic calcium channel activity and activation of post-synaptic potassium channels [3], [5], [9], [10]. These effects are inhibited by pertussis toxin suggesting the involvement of G_i and G_o proteins, although selective antisense oligonucleotide studies suggest a greater role for the G_o protein [9]. Native receptors have been shown to be activated by baclofen [6] and selectively antagonised by phaclofen and saclofen [11]. The discovery of more potent and selective antagonists [12], [13], [14] and the subsequent radiolabelling of two of these compounds, [ $^{125} I$ ]CGP64213 and [ $^{125} I$ ]CGP71872, enabled the expression cloning of two GABA_B1 receptor splice variants, GABA_B1a and GABA_B1b[15]. Although this receptor displayed many of the antagonist binding characteristics of the native GABA_B receptor, it did not couple efficiently to adenylyl cyclase or potassium channels [15]. Further studies suggested that the lack of functional coupling to a wide variety of effector pathways was due to a lack of cell surface expression of the GABA_B1a and GABA_B1b proteins [16], [17]. A GABA_B2 subunit, with 35% sequence homology and 54% sequence similarity to the GABA_B1 subunits, was subsequently identified and studies demonstrated the functional requirement of heterodimerisation between the GABA_B1 and GABA_B2 receptor subunits (reviewed in [18]). The heterodimerisation of GABA_B1 and GABA_B2 receptor subunits occurs, at least in part, by interactions between homologous α-helical coiled-coil domains present in the intracellular C-termini of both GABA_B1 and GABA_B2[19], [20], [21]. In situ hybridisation, immunohistochemical and autoradiographic studies have shown that the GABA_B receptors are distributed throughout the brain. Intriguingly, there are many reports of differential expression patterns, not only between the GABA_B1 splice [22], [23], [24] but also between the GABA_B1 and GABA_B2 receptor subunits [25], [26] where, for example, in the striatum GABA_B2 mRNA and protein are barely detectable but there are abundant levels of the GABA_B1 receptor subunits. These observations, together with previous studies [27], [28] have led to speculation of the existence of pharmacologically distinct receptor subtypes. However, recent reports on a GABA_B1 receptor knockout demonstrate that this subunit is essential for all GABA_B receptor-mediated mechanisms and cast doubt on the existence of non-GABA_B1-based subtypes [29], [30].

We have generated a Chinese Hamster Ovary (CHO) cell line stably expressing the GABA_B1b and GABA_B2 receptor subunits which will be used both in experiments to further our understanding of GABA_B receptor biology and particularly in the drug discovery process to identify novel compounds acting at these receptors. In this study, we extend previously reported data describing the ligand binding characteristics of recombinant heterodimeric GABA_B receptors [31] to focus on the functional characterisation of a recombinant cell line GABA_B receptor subunits and compare its pharmacology to that of native GABA_B receptors in rat cerebellar membranes. A preliminary account of the data presented here has been published in abstract form [32].

Section snippets

Materials

GABA, (R,S)-baclofen, GTPγS and GDP were purchased from Sigma. [S-(R^∗,R^∗)]-[3-[[1-(3,4-Dichlorophenyl)ethyl]amino]-2-hydroxypropyl]-(cyclohexylmethyl)phosphinic acid (CGP54626A), saclofen and phaclofen were purchased from Tocris-Cookson. 3-APPA, SKF-97530 and CGP27492 and 3-[1-(R)-[[2(S)-hydroxy-3-[hydroxy[(4-methoxyphenylmethyl)]phosphinyl]propyl]amino]-ethyl]-benzoic acid (CGP62349) were manufactured by GlaxoSmithKline. All cell culture reagents were from Life Technologies. HEPES, EDTA,

Radioligand binding studies

Saturation analysis of [ $^{3} H$ ]CGP54626A binding to CHO cells stably transfected with the GABA_B1b and GABA_B2 subunits (1E4 cell line) revealed a single binding site (Fig. 1A) with a K_D of 4.2±0.8 nM and a B_max of 22.5±2.5 pmol/mg protein. No specific binding of any of the radioligands used was found in cells transfected with only the GABA_B2 subunit (data not shown). At a radioligand concentration of 3 nM, the concentration used in the competition binding experiments, specific binding represented

Discussion

We have previously described further two other stable cell lines in which the calcium sensing properties of the GABA_B receptor were investigated [39]. We also reported that the ligand binding characteristics of each of the major isoforms of recombinantly expressed GABA_B receptors were identical [31]. However, these studies were not extended to complete a thorough comparison with native receptors. The present study describes the pharmacological characterisation of a CHO DG44 cell line stably

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Changes in the properties of allosteric and orthosteric GABA<inf>B</inf> receptor ligands after a continuous, desensitizing agonist pretreatment
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There are other reports in which silent GABAB receptor antagonists have been claimed to be inverse agonists. Grünewald et al. (2003) have found that CGP54626 and SCH50911 increased cAMP formation and Hirst et al. (2003) have seen this with CGP54626, SCH50911 and CGP62349. In a study on constitutively active GABAB receptor mutants inverse agonistic properties were observed for CGP54626, CGP52432 and CGP55845 (Mukherjee et al., 2006).
It has been estimated that only 15% of the compounds classified as silent G protein-coupled receptor antagonists are indeed devoid of either positive or negative intrinsic efficacy. Considering that 40% of all drugs on the market target G protein-coupled receptors mainly as orthosteric ligands, elucidating their intrinsic properties is becoming increasingly important. While agonism can be demonstrated using appropriately sensitive experimental setups, the detection of inverse agonism can be limited by a low degree of constitutive activity in many assay systems. In this study, changes in ligand behavior upon a lasting pretreatment with γ-aminobutyric acid (GABA), that induced receptor desensitization, were observed, measuring the second messenger cyclic AMP (cAMP) in a GABA_B receptor-expressing recombinant cell line. The GABA_B receptor partial agonist 2-OH-saclofen lost its ability to inhibit 7β-forskolin-induced cAMP production upon GABA-pretreatment. The “silent” receptor antagonists CGP62349, CGP52432, CGP56999 and SCH50911, on the other hand, stimulated 7β-forskolin-induced cAMP production under these conditions. The inverse agonism of CGP56999 was inhibited by the efficacy-deficient 2-OH-saclofen, proving it was truly mediated through the orthosteric site of the GABA_B receptor. Finally, the positive allosteric modulator GS39783, which previously only marginally inhibited cAMP production, suppressed it by 60% both alone and in the presence of the competitive receptor antagonist 2-OH-saclofen, thus GS39783 became an allosteric receptor agonist at desensitized GABA_B receptors. These changes likely reflect adaptations in the mechanisms of GABA_B receptor function following desensitization and may be important in the elucidation of intrinsic ligand efficacies as well as for the consequences of continuous drug treatment.
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3H]CGP54626 binding in bullfrog brain membranes was of high affinity (KD = 2.97 nM) and comparable to that found in rat brain membranes (KD = 1.7 nM, Bischoff et al., 1999; KD = 2.3 nM, Hirst et al., 2003) and two human GABAB receptor splice variants expressed in mammalian cells (KD = 1.51 nM, 0.86 nM or 4.2 nM; Green et al., 2000; Hirst et al., 2003). The density of the binding sites was also similar in bullfrog brain membranes (Bmax = 2619 fmol/mg protein) and rat brain membranes (Bmax = 3003 or 1950 fmol/mg protein; Bischoff et al., 1999; Hirst et al., 2003). This supports the hypothesis that there is a significant structural similarity in vertebrate GABAB receptors and suggests that the distribution of receptors across the CNS might be similar.
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¹: These authors contributed equally to this work.

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Pharmacological characterisation of a cell line expressing GABAB1b and GABAB2 receptor subunits

Abstract

Introduction

Section snippets

Materials

Radioligand binding studies

Discussion

Brain Res. Rev.

Mol. Cell. Neurosci.

Int. Rev. Neurobiol.

Curr. Opin. Neurobiol.

Pharmacol. Ther.

Brain Res.

Eur. J. Pharmacol.

Neuropharmacology

Trends Pharmacol. Sci.

J. Biol. Chem.

Trends Pharmacol. Sci.

Neuron

Neuroscience

Trends Pharmacol. Sci.

Neuropharmacology

Mol. Cell. Neurosci.

Neuron

Anal. Biochem.

Trends Pharmacol. Sci.

Neuropharmacology

Trends Pharmacol. Sci.

Neuropharmacology

J. Biol. Chem.

Trends Pharmacol. Sci.

Mol. Cell. Neurosci.

Pharmacological characterisation of a cell line expressing GABA_B1b and GABA_B2 receptor subunits