G-protein-coupled receptor oligomers: two or more for what? Lessons from mGlu and GABAB receptors.

G-protein-coupled receptors (GPCRs) are key players in the precise tuning of intercellullar communication. In the brain, both major neurotransmitters, glutamate and GABA, act on specific GPCRs [the metabotropic glutamate (mGlu) and GABA(B) receptors] to modulate synaptic transmission. These receptors are encoded by the largest gene family, and have been found to associate into both homo- and hetero-oligomers, which increases the complexity of this cell communication system. Here we show that dimerization is required for mGlu and GABA(B) receptors to function, since the activation process requires a relative movement between the subunits to occur. We will also show that, in contrast to the mGlu receptors, which form strict dimers, the GABA(B) receptors assemble into larger complexes, both in transfected cells and in the brain, resulting in a decreased G-protein coupling efficacy. We propose that GABA(B) receptor oligomerization offers a way to increase the possibility of modulating receptor signalling and activity, allowing the same receptor protein to have specific properties in neurons at different locations.

GABAB receptor: a site of therapeutic benefit.

Although the presence of functional GABAB receptors in mammalian brain has been known for more than 20 years, there is still only one therapeutic agent in use, baclofen, which mediates its effects directly via this receptor. However, activation of this receptor can produce numerous effects that might be amenable to drug development. Evidence from preclinical studies also suggests that antagonism of the GABAB receptor produces beneficial clinical effects.

Don't worry 'B' happy!: a role for GABA(B) receptors in anxiety and depression.

GABA, the main inhibitory neurotransmitter in the brain, regulates many physiological and psychological processes. Thus, dysfunction of the GABA system is implicated in the pathophysiology of several neuropsychiatric disorders, including anxiety and depression. However, the role of GABA(B) receptors in behavioural processes related to these disorders has not been resolved. GABA(B) receptors are G-protein-coupled receptors that function as heterodimers of GABA(B(1)) and GABA(B(2)) subunits. In addition to highly selective agonists and antagonists, novel GABA(B) receptor tools have been developed recently to further assist elucidation of the role of GABA(B) receptors in CNS function. These include mice that lack functional GABA(B) receptors, and novel positive modulators of the GABA(B) receptor. In this review, we discuss evidence that points to a role of GABA(B) receptors in anxiety and depression.

Pathway-specific action of gamma-hydroxybutyric acid in sensory thalamus and its relevance to absence seizures.

The systemic injection of gamma-hydroxybutyric acid (GHB) elicits spike and wave discharges (SWDs), the EEG hallmark of absence seizures, and represents a well established, widely used pharmacological model of this nonconvulsive epilepsy. Despite this experimental use of GHB, as well as its therapeutic use in narcolepsy and its increasing abuse, however, the precise cellular mechanisms underlying the different pharmacological actions of this drug are still unclear. Because sensory thalamic nuclei play a key role in the generation of SWDs and sleep rhythms, and because direct injection of GHB in the ventrobasal (VB) thalamus elicits SWDs, we investigated GHB effects on corticothalamic EPSCs and GABAergic IPSCs in VB thalamocortical (TC) neurons. GHB (250 microm-10 mm) reversibly decreased the amplitude of electrically evoked EPSCs and GABAA IPSCs via activation of GABAB receptors; however, approximately 60% of the IPSCs were insensitive to low (250 microm-1.0 mm) GHB concentrations. The putative GHB receptor antagonist NSC 382 applied alone had a number of unspecific effects, whereas it either had no action on, or further increased, the GHB-elicited effects on synaptic currents. Low GHB concentrations (250 microm) were also effective in increasing absence-like intrathalamic oscillations evoked by cortical afferent stimulation. These results indicate that low concentrations of GHB, similar to the brain concentrations that evoke SWDs in vivo, differentially affect excitatory and inhibitory synaptic currents in TC neurons and promote absence-like intrathalamic oscillations. Furthermore, the present data strengthen previous suggestions on the GHB mechanism of sleep promotion and will help focus future studies on the cellular mechanisms underlying its abuse.

Ethanol modulates GABA(B) receptor expression in cortex and hippocampus of the adult rat brain.

Using in situ hybridization, RNase protection assay and Western blot, we studied the effects of ethanol on the expression levels of GABA(B) receptor mRNA and protein in the cortex and hippocampus from adult rat brain. The results showed that ethanol significantly increased GABA(B1) and GABA(B2) receptor protein expression in the cortex, whereas only GABA(B2) was increased in the hippocampus. GABA(B) receptor agonist baclofen could partially reverse the effect of ethanol. Further studies of the mRNA levels defined that GABA(B1) mRNA levels were significantly increased in the hippocampus, with no significant changes of GABA(B2) mRNA levels. Moreover, GABA(B1) and GABA(B2) receptor mRNA levels were increased on 3-week ethanol treatment. Finally, GABA(B) agonist baclofen and antagonist phaclofen showed significant decreasing effects on GABA(B1) receptor mRNA levels in the cortex, but not in the hippocampus. These results were further confirmed by in situ hybridization. Thus, the present results showed the effects of ethanol on GABA(B) receptors in the cortex and hippocampus, implying the possible role of GABA(B) receptor in ethanol effects. The effects of GABA(B) receptor agonist and antagonist suggested that the possible mechanisms underlying that GABA(B) receptor modulated the behavioral effect induced by ethanol.

Changes in GABA(B) receptor mRNA expression in the rodent basal ganglia and thalamus following lesion of the nigrostriatal pathway.

Loss of striatal dopaminergic innervation in Parkinson's disease (PD) is accompanied by widespread alterations in GABAergic activity within the basal ganglia and thalamus. Accompanying changes in GABA(B) receptor binding have been noted in some basal ganglia regions in parkinsonian primates, suggesting that plasticity of this receptor may also occur in PD. However, the molecular mechanisms underlying the changes in receptor binding and the manner and extent to which different GABA(B) receptor mRNA subunits and splice-variants are affected remain unknown. This study used in situ hybridisation to examine the full profile of changes in expression of the known rat GABA(B) receptor genes and gene variants in the basal ganglia and thalamus of rats, brought about by degeneration of the nigrostriatal tract. All of the GABA(B) mRNA species examined showed unique expression patterns throughout the basal ganglia and thalamus. In addition, all exhibited a marked loss of expression (between 46 and 80%) in the substantia nigra pars compacta of animals bearing a complete 6-hydroxydopamine-induced lesion of the nigrostriatal tract, confirming the presence of these variants in dopaminergic neurones in this region. Further analysis of autoradioagrams revealed additional changes only in GABA(B(1a)) mRNA in discrete anatomical regions. Expression of the GABA(B(1a)) variant was significantly increased in the substantia nigra pars reticulata (33+/-2%), entopeduncular nucleus (26+/-1%) and the subthalamic nucleus (16+/-1%). Since these regions all receive reduced GABAergic innervation following nigrostriatal tract lesioning, it is possible that the increased expression occurs as a compensatory measure. In conclusion, these data demonstrate that GABA(B) receptor genes exhibit regional- and subunit/variant-specific plasticity at the molecular level under parkinsonian conditions.

Functional pharmacology of cloned heterodimeric GABAB receptors expressed in mammalian cells.

1. In this study we report a new assay of heterodimeric gamma-amino-butanoic acid subtype B (GABAB) receptors where either GABABR1a or GABABR1b are co-expressed with GABABR2 and the chimeric G-protein Galphaq-z5 in tsA cells. In this manner we obtained a robust response to GABAB agonists measured as increase in phosphoinositide hydrolysis. 2. We used this assay to characterize a number of commonly used GABAB receptor ligands. Both splice variants displayed the same rank order of agonist potency; 3-aminopropyl(methyl)phosphinic acid (SKF-97541)>GABA>(R)-4-amino-3-(4-chlorophenyl)butanoic acid ((R)-baclofen)>(RS)-4-amino-3-(5-chloro-2-thienyl)butanoic acid (BCTG)>3-aminopropylphosphonic acid (3-APPA) and furthermore, the absolute agonist potency values were very close to each other. 3. 3-APPA was a partial agonist displaying maximal responses of 41 and 61% compared to GABA at GABABR1a and GABABR1b, respectively. The antagonist (RS)-3-amino-2-(4-chlorophenyl)-2-hydroxypropylsulphonic acid (2-OH-saclofen) displayed KB values of 15 and 7.8 microM at GABABR1a and GABABR1b, respectively. 4. The rank order of agonist potency as well as the absolute ligand potencies correspond very well with those previously reported in different tissues, and this study thus provides a functional assay of cloned GABAB receptors which should be a valuable tool for further characterization of GABAB ligands. Finally, we can conclude that the functional pharmacological profiles of the two GABABR1 splice variants are very similar.

[Long-term alteration of gamma-aminobutyric acid B receptor subunits in immature rats after recurrent febrile seizures].

OBJECTIVE: Febrile seizure (FS) is closely related to an altered transmission of gamma-aminobutyric acid (GABA). GABA exerts its effects through ionotropic receptors (GABA(AR) and GABA(CR)) and metabotropic receptors (GABA(BR)). GABA(BRs) are located at pre- and postsynaptic sites. Stimulation of postsynaptic receptors generates long-lasting inhibitory postsynaptic potentials (IPSPs) that are important for the fine-tuning of inhibitory neurotransmission and caused by an increase in K(+) conductance. At presynaptic sites, GABA(BRs) mediate a suppression on the release of neurotransmitters such as of GABA or glutamate by inhibiting voltage-sensitive Ca(2+) channels. The present study aimed to explore the long-term changes of GABA(B) receptor subunits in immature rats after recurrent febrile seizures. METHODS: Rats were randomly divided into control group and hyperthermia treatment group. The control rats (n = 64) were put into 37 degrees C water for 5 minutes. Rats with hyperthermia treatment were put into 44.8 degrees C water for 5 minutes. If a rat in hyperthermia treatment group showed seizure within 5 min, the rat was taken out of the water as soon as the seizure occurred. Water-immersion was carried out 10 times, once every 2 days. Rats showing 10 seizures (FS(10), n = 64) were studied. Rats exposed to hyperthermia for 10 times without seizure were also studied as hyperthermia-only (H, n = 64) group. Rats showing one seizure at the last time of 10 times of hyperthermia treatment were studied as one-seizure group (FS(1), n = 64). The other rats were studied for other research. The changes of GABA(B)R(1) and GABA(B)R(2) co-localization were detected by double fluorescence;the quantitative alteration of GABA(B)R(1) and GABA(B)R(2) were detected by quantitative RT-PCR; the binding of GABA(B)R(2) to GABA(B)R(1) was detected by immunoprecipitation/Western blot. RESULTS: GABA(B)R(1), GABA(B)R(2), and the binding of GABA(B)R(2) to GABA(B)R(1) decreased after the last febrile seizure in FS(10) group, the expression of GABA(B)R(1) returned to normal in later phase while GABA(B)R(2) and the binding of them did not. CONCLUSION: Recurrent FS down-regulated the expression of GABA(B)R subunits in a long term.

International Union of Pharmacology. XXXI. Recommendations for the nomenclature of multimeric G protein-coupled receptors.

A receptor is defined by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) as a protein, or a complex of proteins, which recognizes physiologically relevant ligands that can regulate the protein to mediate cellular events (Ruffolo et al., 2000). This definition does not include associated proteins, which are not required for agonist recognition and/or receptor assembly. Thus, G proteins are not included in the nomenclature of G protein-coupled receptors (GPCRs). Similarly, proteins which modify receptor disposition, such as proteins with a PDZ domain (Sheng and Sala, 2001), and which associate with the cytosolic portion of the receptor are not included. The question arises, however, as to the way to name multimeric receptors where subunits influence receptor assembly and agonist recognition. The essential issue is whether to name the individual proteins or the association of proteins? NC-IUPHAR recommends that, where possible, the functional receptor complex be given a different name from that of the subunits.

The metabotropic GABA receptor: molecular insights and their functional consequences.

Recent years have seen rapid and significant advances in our understanding of the G-protein-coupled gamma-amino butyric acid, B-type (GABA(B)) receptor, which could be a therapeutic target in conditions as diverse as epilepsy and hypertension. This progress originated with the ground-breaking work of Bernhard Bettler's team at Novartis who cloned the DNA encoding a GABA(B) receptor in 1997. Currently, the receptor is thought to be an unusual, possibly unique, example of a heterodimer composed of homologous, seven-transmembrane-domain (7TMD) subunits (named GABA(B) R1 and GABA(B) R2), neither of which is fully functional when expressed alone. The large N-terminal domain of the GABA(B) R1 subunit projects extracellularly and contains a ligand binding site. The similarity of the amino acid sequence of this region to some bacterial periplasmic amino acid-binding proteins of known structure has enabled structural and functional modelling of the N-terminal domain, and the identification of residues whose substitution modulates agonist/antagonist binding affinities. The intracellular C-terminal domains of the R1 and R2 subunits appear to constitute an important means of contact between the two subunits. Alternative splice variants, a common and functionally important feature of 7TMD proteins, have been demonstrated for the R1 subunit. Notably GABA(B) R1a differs from GABA(B) R1b by the possession of an N-terminal extension containing two complement protein modules (also called SCRs, or sushi domains) of unknown function. The levels at which each of the respective variants is expressed are not equal to one another, with variations occurring over the course of development and throughout the central nervous system. It is not yet clear, however, whether one variant is predominantly presynaptically located and the other postsynaptically located. The existence of as yet unidentified splice variants, additional receptor subtypes and alternative quaternary composition has not been ruled out as a source of receptor heterogeneity.

gamma-aminobutyric acid(B) receptors: first of the functional metabotropic heterodimers.

Activation of the metabotropic gamma-aminobutyric acid(B) (GABA(B)) receptor increases K(+) conductance and decreases Ca(2+) channel activity in neuronal membranes. Studies with a number of new GABA(B) receptor agonists and antagonists reveal that in addition to their muscle relaxant effects, agonists display analgesic activity and reduce the craving for cocaine. With regard to GABA(B) receptor antagonists, preclinical data suggest they improve cognitive performance and possess antidepressant and antiepileptic potential. With a high-affinity GABA(B) antagonist, the structural properties of the receptor were characterized through expression cloning. Moreover, it has been found that expression of a fully functional GABA(B) receptor requires coupling between two separate and distinct gene products: GABA(B) R1 and GABA(B) R2. Besides being the first example of a functional heterodiameric metabotropic receptor, the components and molecular configuration of the GABA(B) receptor suggest novel mechanisms for producing pharmacologically distinct subtypes of G protein-coupled receptors.