Intracellular Retention of Recombinant GABABReceptors

Abstract

␥-Aminobutyric acid type B (GABA B ) receptors mediate the transmission of slow and prolonged inhibitory signals in the central nervous system. Two splice variants of GABA B receptors, GABA B R1a and GABA B R1b, were recently cloned from a mouse cortical and cerebellar cDNA library. As predicted, these receptors belong to the G protein-coupled receptor superfamily. We have used epitope-tagged versions of GABA B R1a receptors to study the cellular distribution of these proteins in a variety of non-neuronal and neuronal cell types. Here we report that recombinant GABA B receptors fail to reach the cell surface when expressed in heterologous systems and are retained in the endoplasmic reticulum when introduced into COS cells. In addition, we prove that recombinant GABA B receptors are excluded from the cell surface when overexpressed in ganglion neurons and we further demonstrate that they fail to activate in superior cervical ganglion neurons. Together our observations suggest that recombinant GABA B receptors require additional information for functional targeting to the plasma membrane.

The Heptahelical Domain of GABAB2 Is Activated Directly by CGP7930, a Positive Allosteric Modulator of the GABAB Receptor

“…Indeed, the GABA B receptor can activate efficiently phospholipase C when co-expressed with the chimeric G-protein Gqi9 (47). As noticed previously, the IP assay is much more sensitive than the [␥- 35 S]GTP assay as indicated by the 100-fold lower EC 50 value measured for GABA compare with the [␥- 35 S]GTP assay. This probably results from the large amplification of the signaling cascade between G-protein activation and IP formation.…”

“…The dose-response curves were fitted using the Kaleidagraph program and the equation y ϭ [(y max Ϫ y min )/(1 ϩ (x/EC 50 ) nH )) ϩ y min , where the EC 50 is the concentration of the compound necessary to obtain 50% of the maximal effect and n H is the Hill coefficient. EC 50 for GABA was increased 3-and 10-fold in the presence of 0.1 and 1 mM CGP7930, respectively (42.0 Ϯ 11.0, 30.0 Ϯ 7.5, and 4.7 Ϯ 1.04 M in the absence or the presence of 0.1 and 1 mM CGP7930, respectively). A small increase in the binding of [␥- 35 S]GTP binding also was observed in the absence of GABA, suggesting that CGP7930 also may be able to slightly activate the receptor.…”

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

“…Whereas GABA B agonists and competitive antagonists interact in the GABA B1 VFTM (Galvez et al, 1999;Galvez et al, 2000), this subunit expressed alone does not give rise to consistent responses. 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).…”

L’hétérodimérisation des récepteurs couplés aux protéines G

“…En revanche, la sous-unité GABA B2 est correctement exprimée à la surface, mais elle est en revanche incapable de lier le ligand et donc de déclencher l'activation des voies de signalisation intracellulaires. 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).…”