Diversity of GABA B receptor signaling through G protein variability

combinations and their multiple splice variants control diverse signal transduction pathways. The expression of the G protein subtypes in the developing brain is

age-dependent and characteristic for a specific brain region. Qualitative and quantitative analysis of the Gα protein subtypes in mouse brainstem revealed diverse expression patterns for the Gα protein subtypes of our interest. The expression levels of the Gαi/o

are equal in all three investigated brain areas (cortex, cerebellum and brainstem) at both developmental stages (newborn and adult). The same result was revealed from the analysis of the Gαq/11. The examination of the Gαs proteins was more complicate because of the numerous isoforms produced by the rather complex genomic locus of GNAS. Western blot analysis with Gαs antibody showed up six bands with a developmental regulated expressions pattern. Two of them, clearly identified as the short and long isoforms of the Gαs, showed strong expression levels in the immature brainstem that were decreased during maturation. On the other side, the two isoforms Gs1 and Gs2, which are almost missing in the newborn give a strong signal in adult brainstem, and represent two XLαs isoforms of the Gαs. The other two variants Gs5 and Gs6, was not possible to be distinguished. Thus, although the complexity of the GNAS locus and the continuously new data did not permit us to identify all six Gαs

isoforms, we could distinguish between the two Gαs (short and long) which show a decreas in expression, and the two extra long Gαs which show an increase in expression during mouse brainstem maturation. Although it is known, like in the case of β2-adrenoreceptor, that the Gαs short and long variants, which differ in only 15 amino acids, have different consequences for the functional properties of a G-protein-coupled receptor (Seifert, Wenzel-Seifert et al. 1998), it is not known how the interaction between the XLαs isoforms and the GABAB receptor influences the function of the receptor. The paternal expressed XLαs contains all structural domains of Gαs, except the long amino-terminal extension encoded within its specific first exon. Even though in vitro studies have shown that XLαs forms a heterotrimer with βγ subunits, binds GTP and mediates receptor-stimulated cAMP production, the biological role(s) of XLαs within the cell remains obscure. There are also some conflict reports, based on the ability of XLas to activate a receptor. Moreover, recent findings indicate that the GNAS products are capable of modulating the activity/expression of each other, something that makes more difficult to predict which influence will have these interactions in the GABAB receptors signaling. For example, it is known that XLαs and Gαs exert antagonistic functions and that the

structurally unrelated protein to the G proteins, and is also encoded in the GNAS locus, has the ability to interact with XLαs, and inhibits its adenylyl cyclase-stimulating function.

The brainstem cAMP measurement at newborn and adult mice after activation with baclofen, revealed no significant values that could possibly be explained through the antagonistic effect of the GNAS locus products (e.g. Nesp, Alex, XLN1), or the antagonistic function of the Gα and Gβγ subunits, or even through cross-talk with other pathways.

We can only speculate on the GABAB receptor-mediated signaling through the GNAS locus products. One explanation could be that the GABABR-Gαs (short and long) Figure 4.1. Schematic representation of the activation of different signaling pathways through the GABAB receptors in immature and adult mouse brainstem.

interaction in the immature brainstem is hindered in the adult brainstems through a sterical interference or general an antagonistic function of the XLas variants (or other by other proteins expressed in the GNAS locus).

The distinctive characteristic of GABABR, the formation of a heterodimers, could influence additional receptor signaling. GABAB1 with the two isoforms GABAB1a and GABAB1b which contain the ligand binding site and GABABR2 with the G protein coupling site(s), regulate the function of the receptor based on the organism, the age, the brain region, the microdomain organisation and the receptor composition.

Although it is generally accepted that coexpression of the two GABAB receptors subunits (GABABR1 and GABABR2) is required for a fully functional receptor, thus GABABR2 is necessary for the GABABR1 transportation, there is evidence that also GABABR1 (1a and 1b) and GABABR2 alone are able to couple to K⁺ channels or adenylyl cyclase, although at low efficiency (Kaupmann, Huggel et al. 1997;

Kaupmann, Schuler et al. 1998; Kuner, Kohr et al. 1999; Urwyler, Mosbacher et al.

2001). Thus, in our cloning experiments (GABABR1a and GABABR2 expression in HEK 293 cells) because of the weak signal observed in the cells and the GABABR2 expresion in the intracellular areas of the cell, we can conclude it concerns monomeric GABABR1a receptors.

Additionally, although the GABAB1 intracellular loops are not necessary for G protein coupling they may be involved in signaling pathways not involving G proteins or in the regulation of subcellular GABAB receptor localization. For example GABAB1

subtypes interact directly with CREB2/ATF4, USF, 14-3-3 and through subtype-selective interacting proteins for the GABAB1a and GABAB2 (see chapter 1.6.3.1).

These associated proteins are able to regulate subtype-specific expression of the GABAB1a&b subunits, receptor heterodimerization and cross-talks between different systems, such as Tamalin and NSF.

The distinct subcellular distribution and behaviour of the GABAB1 subunits with GABAB1a dominant in neonatal brain and GABAB1b predominant in adults could play an additional role to the GABAB receptor mediated signaling diversity.

Another layer of complexity arises from the ability of the GPCRs to activate simultaneously multiple G protein pools. Dual coupling to Gαs and Gαi/o has been reported, as in the case of the β2-adrenoceptor (Daaka, Luttrell et al. 1997), where the

pathway. In some cases, a single receptor has been found to simultaneously activate members of three or even four unrelated classes of G protein (Gαs, Gαi/o, Gαq/11, and Gα12) (Laugwitz, Allgeier et al. 1996). The interaction of these receptors with multiple G protein classes produces additional interference between the products of each pathway, through the generation of different Gα and Gβγ subunits.

Thus, although we do not know if the interaction of the GABAB receptors in the immature brainstem to the Gαs and Gαi/o proteins is a simultaneous coupling of the two G proteins or if there is a factor that controls a Gαs-Gαi/o switching, we can conclude that in general the GABAB-receptor binding to Gαs proteins will initiates a new set of signaling events.

These observations together lead us to the conclusion that the interaction of GABAB

receptor to the Gαs and Gαi/o proteins in the immature brainstem and only to the Gαi/o

proteins in the mature brainstem, are probably regulated by additional factors e.g. the GABABR1 subunit, the way of interaction with the G proteins (simultaneously or switching) and via the organization of all these proteins (e.g. receptors, G proteins, associated proteins) within specialized microdomains.

4.2 Changes in the GABAA receptor signaling in the developing mouse