The GABAB (γ-aminobutyric acid-B) receptor is composed of two subunits, GABAB1 and GABAB2. Both subunits share structural homology with other class-III G-protein-coupled receptors. They contain two main domains, a heptahelical domain typical of all G-protein-coupled receptors and a large ECD (extracellular domain). It has not been demonstrated whether the association of these two subunits is always required for function. However, GABAB2 plays a major role in coupling with G-proteins, and GABAB1 has been shown to bind GABA. To date, only ligands interacting with GABAB1-ECD have been identified. In the present study, we explored the mechanism of action of CGP7930, a compound described as a positive allosteric regulator of the GABAB receptor. We have shown that it can weakly activate the wild-type GABAB receptor, but also the GABAB2 expressed alone, thus being the first described agonist of GABAB2. CGP7930 retains its weak agonist activity on a GABAB2 subunit deleted of its ECD. Thus the heptahelical domain of GABAB2 behaves similar to a rhodopsin-like receptor. These results open new strategies for studying the mechanism of activation of GABAB receptor and examine any possible role of GABAB2.
The human body is composed of billions of cells. Since these cells are linked and communicate in very well-organized systems, they constitute organisms. The nervous system is by far the most complex organ of the body. A primary aim in the field of neuroscience is to understand better the communication between neuronal cells by the action of chemical messengers (neurotransmitters) on their membrane proteins (receptors). The amino acid GABA (γ-aminobutyric acid) is considered to be the main inhibitory neurotransmitter in the adult mammalian brain. Its action is mediated by either ionotropic or metabotropic receptors . The latter, called GABAB receptors, couple with intracellular G-proteins and are abundant throughout both central and peripheral nervous systems. This suggests that they play important roles in the physiology of the nervous system. Moreover, they are involved in various cerebral and neurodegenerative diseases including spasticity, pain , epilepsy [3,4], anxiety and depression , schizophrenia  and drug addiction . So, the more we learn about GABAB receptors, the more we can understand several physiological and pathological processes, thereby allowing us to propose new therapeutical applications.
The GABAB receptor belongs to Class-III GPCRs (G-protein-coupled receptors)
The GPCRs form a large family of membrane proteins responsible for transduction of various external signals into intracellular responses through heterotrimeric G-proteins. Five main classes have been described based on their structural features. The GABAB receptor is a class-III GPCR, as are the receptors for glutamate, Ca2+, pheromones and umami and sweet taste compounds. In addition to the typical GPCR HD (heptahelical domain), class-III GPCRs possess a large ECD (extracellular domain) responsible for ligand recognition. Furthermore, most of these receptors are known to form dimers. However, to form a GABAB receptor capable of activating G-proteins, the association of two class-III GPCRs, called GABAB1 and GABAB2, is required, meaning that GABAB is an obligatory heterodimeric receptor. While the GABAB1-ECD has been shown to bind GABA, the GABAB2-HD is responsible for coupling with G-proteins (Figure 1). Today, it is well established that interaction between the GABAB1-ECD and the GABAB2-ECD is essential for optimal ligand binding , and that closure of the GABAB1-ECD lobes after ligand binding is sufficient to keep the receptor in its active state [9,10] by inducing or stabilizing the active conformation of the HD. Nonetheless, the molecular mechanisms which underlie the active conformation in the HD are not understood.
Schematic representation of the GABAB receptor
Recently, ligands acting directly on class-III GPCR HDs have been identified. For example, the action of positive and negative allosteric modulators of mGluR5 (metabotropic glutamate receptor 5) has been characterized. Moreover, in the absence of the ECD, mGluR5-HD functions as a ‘rhodopsin-like’ GPCR as it is activated or inhibited by the positive and negative allosteric modulators respectively . Accordingly, it was important to analyse the mechanism of action of a newly identified GABAB receptor allosteric modulator, CGP7930 .
CGP7930 is a partial agonist of the GABAB receptor and an activator of the GABAB2-HD
The mechanism of action of CGP7930 was explored on GABAB receptors transiently co-expressed in HEK-293 (human embryonic kidney) cells with a chimaeric G-protein (Gqi9) allowing its positive coupling with phospholipase C. As shown by Urwyler et al. , CGP7930 is a positive allosteric modulator of the GABAB receptor, since it increases the maximal effect and the potency of GABA in assays measuring both the GTP[35S] binding on G-proteins and inositol phosphate production .
Of particular interest, we showed that CGP7930 alone dose-dependently stimulates the GABAB receptor, as measured in the inositol phosphate production assay. However, only a partial activity on the wild-type receptor is detected (the EC50 for CGP7930 is 30 times higher than that of GABA, and the maximal activity observed with CGP7930 was 50–60% of that of GABA).
To determine the location at which CGP7930 acts in the receptor, its effect was examined on different combinations of chimaeric GABAB receptor subunits, in which the ECD was swapped. Of interest, only the combinations possessing one or two GABAB2-HD were sensitive to CGP7930. This indicated that the GABAB1 and GABAB2-ECD are not necessary for CGP7930 to exerts its effects. As confirmation of this hypothesis, CGP7930 was found to be capable of activating receptors lacking both the ECDs (GABAB1-HD and GABAB2-HD only).
Thus CGP7930 binds to the HD of the GABAB receptor. Moreover, because of the ability of the GABAB2 subunit to couple with G-proteins, we observed that CGP7930 acts directly on the GABAB2-HD. However, this does not necessary rule out the possibility that CGP7930 may also bind the GABAB1-HD, but there is currently no available assay to measure activation of the GABAB1-HD.
In any case, the determination of the precise binding site of CGP7930 in GABAB2-HD would be very informative for the activation mechanism of GABAB receptor.
Apart from the fact that the positive allosteric modulator CGP7930 is an excellent pharmacological tool to explore the molecular mechanisms of HD activation, its original binding at a site distinct from that of GABA and the large implication of the GABAB receptor in physiology and pathology suggest that such compounds are promising drugs for therapeutical application.
Signalling Outwards and Inwards: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by J. Challiss (Leicester, U.K.), A. Harwood (University College London, U.K.), M. Humphries (Manchester, U.K.), C. Isacke (Institute of Cancer Research, London, U.K.), R. Liddington (Burnham Institute, La Jolla, CA, U.S.A.), T. Palmer (Glasgow, U.K.), K. Siddle (Cambridge, U.K.), C. Sutherland (Dundee, U.K.), H. Wallace (Aberdeen, U.K.) and M. Welham (Bath, U.K.).