No Ligand Binding in the GB2 Subunit of the GABA_BReceptor Is Required for Activation and Allosteric Interaction between the Subunits

Abstract

The GABA(B) receptor plays important roles in the tuning of many synapses. Although pharmacological differences have been observed between various GABA(B)-mediated effects, a single GABA(B) receptor composed of two subunits (GB1 and GB2) has been identified. Although GB1 binds GABA, GB2 plays a critical role in G-protein activation. Moreover, GB2 is required for the high agonist affinity of GB1. Like any other family 3 G-protein-coupled receptors, GB1 and GB2 are composed of a Venus Flytrap module (VFTM) that usually contains the agonist-binding site and a heptahelical domain. So far, there has been no direct demonstration that GB2 binds GABA or another endogenous ligand. Here, we have further refined the GABA-binding site of GB1 and characterized the putative-binding site in the VFTM of GB2. None of the residues important for GABA binding in GB1 appeared to be conserved in GB2. Moreover, mutation of 10 different residues, alone or in combination, within the possible binding pocket of GB2 affects neither GABA activation of the receptor nor the ability of GB2 to increase agonist affinity on GB1. These data indicate that ligand binding in the GB2 VFTM is not required for activation. Finally, although in either GB1 or the related metabotropic glutamate receptors most residues of the binding pocket are conserved from Caenorhabditis elegans to human, no such conservation is observed in GB2. This suggests that the GB2 VFTM does not constitute a binding site for a natural ligand.

The activation mechanism of class-C G-protein coupled receptors

“…Accordingly, it has been proposed that the closure of only one VFTM within such dimeric receptor would be sufficient to activate the receptor. In agreement with this possibility, we found that the agonist GABA binds exclusivelly within the GABA B1 VFTM , and indeed, we even get data that strongly support the absence of natural ligand interacting with the GABA B2 VFTM (Kniazeff et al, 2002). This is based on the absence of selective conservation of the putative binding site of the GABA B2 VFTM during evolution, in contrast to the high degree of conservation of the equivalent site in GABA B1 and mGluRs.…”

“…In contrast to this variety of mGluRs even further increased by the existence of various isoforms generated by alternative splicing (Pin and Duvoisin, 1995;Conn and Pin, 1997;Pin et al, 2003), only two GABA B encoding genes have been identified (Marshall et al, 1999) (Fig.1). Among these only GABA B1 appears to bind GABA B ligands (Kniazeff et al, 2002), the GABA B2 protein being required for normal functioning of the GABA B1 subunit, as we will see later (Marshall et al, 1999). Only one gene encoding a Ca 2+ -sensing receptor is known (Brown and MacLeod, 2001) (Fig.1).…”

“…Although this subunit is retained in the ER due to a retention signal in its C-terminal tail (Couve et al, 1998;Pagano et al, 2001), no function can also be observed with a subunit mutated in the ER retention signal , whereas robust responses can be obtained in the presence of GABA B2 . Indeed, this second subunit, although unlikely to bind any ligand (Kniazeff et al, 2002), i) unmasks the ER retention signal of GABA B1 allowing the heterodimer to reach the cell surface ; ii) increases agonist affinity on GABA B1 (Kaupmann et al, 1998); C) plays a major role in G-protein coupling of the heteromer (Duthey et al, 2002). By analyzing the function of a number of chimeric subunits in which the VFTMs were swapped between GABA B1 and GABA B2 we found that none of the chimeras express alone were functional, whereas many combinations of subunits were able to activate G-proteins (Galvez et al, 2001).…”

Des dimères et des oligomères de récepteurs couplés aux protéines G, oui mais pourquoi ?

“…En effet, seule la sous-unité GABA B1 lie le GABA, alors que GABA B2 est nécessaire à l'activation des protéines G [5,6], indiquant un fonctionnement asymétrique d'un dimère de RCPG, décrit depuis pour d'autres RCPG hétérodimériques, mais aussi homodimériques [7]. Chacune de ces sous-unités est constituée d'un Les récepteurs couplés aux protéines G (RCPG) sont des acteurs importants de la communication cellulaire.…”

L’hétérodimérisation des récepteurs couplés aux protéines G

“…L'hétérodimérisation des deux sous-unités dans le RE masque le signal de rétention sur GABA B1 et favorise ainsi l'expression de l'hétérodimère à la surface cellulaire [26,27]. Une fois le dimère exprimé à la surface, GABA B1 peut lier le ligand et GABA B2 peut transduire l'effet du ligand en couplage avec les protéines G. La sous-unité GABA B2 possédant un domaine de liaison semblable à GABA B1, mais qui est incapable de lier le ligand, peut être considérée comme un récepteur orphelin à 7TM [28] (Figure 2). Ce mécanisme d'hétérodimérisation semble être conservé au cours de l'évo-lution puisqu'une étude réalisée chez la drosophile a confirmé le caractère obligatoire de la dimérisation pour la fonctionnalité des récepteurs D-GABA B1 et D-GABA B2 [29].…”