The ␥-aminobutyric acid, type B (GABA B ) receptor is well recognized as being composed of two subunits, GABA B1 and GABA B2 . Both subunits share structural homology with other class-III G-protein-coupled receptors. They are composed of two main domains: a heptahelical domain (HD) typical of all G-protein-coupled receptors and a large extracellular domain (ECD). Although GABA B1 binds GABA, GABA B2 is required for GABA B1 to reach the cell surface. However, it is still not demonstrated whether the association of these two subunits is always required for function in the brain. Indeed, GABA B2 plays a major role in the coupling of the heteromer to G-proteins, such that it is possible that GABA B2 can transmit a signal in the absence of GABA B1 . Today only ligands interacting with GABA B1 ECD have been identified. Thus, the compounds acting exclusively on the GABA B2 subunit will be helpful in analyzing the specific role of this subunit in the brain. Here, we explored the mechanism of action of CGP7930, a compound described as a positive allosteric regulator of the GABA B receptor. We showed that it activates the wild type GABA B receptor but with a low efficacy. The GABA B2 HD is necessary for this effect, although one cannot exclude that CGP7930 could also bind to GABA B1 . Of interest, CGP7930 could activate GABA B2 expressed alone and is the first described agonist of GABA B2 . Finally, we show that CGP7930 retains its agonist activity on a GABA B2 subunit deleted of its ECD. This demonstrates that the HD of GABA B2 behaves similar to a rhodopsin-like receptor, because it can reach the cell surface alone, can couple to G-protein, and be activated by agonists. These data open new strategies for studying the mechanism of activation of GABA B receptor and examine any possible role of homomeric GABA B2 receptors.

G protein-coupled receptors transmit extracellular signals into the cells by activating heterotrimeric G proteins, a process that is often followed by receptor desensitization. Monitoring such a process in real time and in living cells will help better understand how G protein activation occurs. Energy transfer-based approaches [fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET)] were recently shown to be powerful methods to monitor the G protein-coupled receptors (GPCRs)-G protein association in living cells. Here, we used a BRET technique to monitor the coupling between the protease-activated receptor 1 (PAR1) and Galpha(i1) protein. A specific constitutive BRET signal can be measured between nonactivated PAR1 and the Galpha(i1) protein expressed at a physiological level. This signal is insensitive to pertussis toxin (PTX) and probably reflects the preassembly of these two proteins. The BRET signal rapidly increases upon receptor activation in a PTX-sensitive manner. The BRET signal then returns to the basal level after few minutes. The desensitization of the BRET signal is concomitant with beta-arrestin-1 recruitment to the receptor, consistent with the known rapid desensitization of PARs. The agonist-induced BRET increase was dependent on the insertion site of fluorophores in proteins. Taken together, our results show that BRET between GPCRs and Galpha proteins can be used to monitor the receptor activation in real time and in living cells. Our data also revealed that PAR1 can be part of a preassembled complex with Galpha(i1) protein, resulting either from a direct interaction between these partners or from their colocalization in specific microdomains, and that receptor activation probably results in rearrangements within such complexes.

Whereas most membrane receptors are oligomeric entities, G‐protein‐coupled receptors have long been thought to function as monomers. Within the last 15 years, accumulating data have indicated that G‐protein‐coupled receptors can form dimers or even higher ordered oligomers, but the general functional significance of this phenomena is not yet clear. Among the large G‐protein‐coupled receptor family, class C receptors represent a well‐recognized example of constitutive dimers, both subunits being linked, in most cases, by a disulfide bridge. In this review article, we show that class C G‐protein‐coupled receptors are multidomain proteins and highlight the importance of their dimerization for activation. We illustrate several consequences of this in terms of specific functional properties and drug development.

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