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gamma-aminobutyric acid type B (GABA(B)) receptor is an allosteric complex made of two subunits, GABA(B1) and GABA(B2). GABA(B2) plays a major role in the coupling to G protein whereas GABA(B1) binds GABA. It has been shown that GABA(B) receptor activates ERK(1/2) in neurons of the central nervous system, but the molecular mechanisms underlying this event are poorly characterized. Here, we demonstrate that activation of GABA(B) receptor by either GABA or the selective agonist baclofen induces ERK(1/2) phosphorylation in cultured cerebellar granule neurons. We also show that CGP7930, a positive allosteric regulator specific of GABA(B2), alone can induce the phosphorylation of ERK(1/2). PTX, a G(i/o) inhibitor, abolishes both baclofen and CGP7930-mediated-ERK(1/2) phosphorylation. Moreover, both baclofen and CGP7930 induce ERK-dependent CREB phosphorylation. Furthermore, by using LY294002, a PI-3 kinase inhibitor, and a C-term of GRK-2 that has been reported to sequester Gbetagamma subunits, we demonstrate the role of Gbetagamma in GABA(B) receptor-mediated-ERK(1/2) phosphorylation. In conclusion, the activation of GABA(B) receptor leads to ERK(1/2) phosphorylation via the coupling of GABA(B2) to G(i/o) and by releasing Gbetagamma subunits which in turn induce the activation of CREB. These findings suggest a role of GABA(B) receptor in long-term change in the central nervous system.

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.

International audience; Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B(4) receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G(i2) protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human alpha(5) integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB(4) binding in the presence of the purified G protein G alpha(i2). The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5'-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified G alpha(i2) beta(1) gamma(2) protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.

The stromal cell-derived factor-1/CXCL12 chemokine engages the CXCR4 and CXCR7 receptors and regulates homeostatic and pathologic processes, including organogenesis, leukocyte homeostasis, and tumorigenesis. Both receptors are widely expressed in mammalian cells, but how they cooperate to respond to CXCL12 is not well understood. Here, we show that CXCR7 per se does not trigger Gαi protein–dependent signaling, although energy transfer assays indicate that it constitutively interacts with Gαi proteins and undergoes CXCL12-mediated conformational changes. Moreover, when CXCR4 and CXCR7 are coexpressed, we show that receptor heterodimers form as efficiently as receptor homodimers, thus opening the possibility that CXCR4/CXCR7 heterodimer formation has consequences on CXCL12-mediated signals. Indeed, expression of CXCR7 induces conformational rearrangements within preassembled CXCR4/Gαi protein complexes and impairs CXCR4-promoted Gαi-protein activation and calcium responses. Varying CXCR7 expression levels and blocking CXCL12/CXCR7 interactions in primary T cells suggest that CXCR4/CXCR7 heterodimers form in primary lymphocytes and regulate CXCL12-promoted chemotaxis. Taken together, these results identify CXCR4/CXCR7 heterodimers as distinct functional units with novel properties, which can contribute to the functional plasticity of CXCL12.

International audience; Familial hemiplegic migraine type 1 (FHM-1) is caused by mutations in CACNA1A, the gene encoding for the Ca(v)2.1 subunit of voltage-gated calcium channels. Although various studies attempted to determine biophysical consequences of these mutations on channel activity, it remains unclear exactly how mutations can produce a FHM-1 phenotype. A lower activation threshold of mutated channels resulting in increased channel activity has been proposed. However, hyperactivity may also be caused by a reduction of the inhibitory pathway carried by G-protein-coupled-receptor activation. The aim of this study is to determine functional consequences of the FHM-1 S218L mutation on direct G-protein regulation of Ca(v)2.1 channels. In HEK 293 cells, DAMGO activation of human mu-opioid receptors induced a 55% Ba(2+) current inhibition through both wild-type and S218L mutant Ca(v)2.1 channels. In contrast, this mutation considerably accelerates the kinetic of current deinhibition following channel activation by 1.7- to 2.3-fold depending on membrane potential values. Taken together, these data suggest that the S218L mutation does not affect G-protein association onto the channel in the closed state but promotes its dissociation from the activated channel, thereby decreasing the inhibitory G-protein pathway. Similar results were obtained with the R192Q FHM-1 mutation, although of lesser amplitude, which seems in line with the less severe associated clinical phenotype in patients. Functional consequences of FHM-1 mutations appear thus as the consequence of the alteration of both intrinsic biophysical properties and of the main inhibitory G-protein pathway of Ca(v)2.1 channels. The present study furthers molecular insight in the physiopathology of FHM-1.

Mu and delta opioid receptors (MORs and DORs) were co-expressed as fusion proteins between a receptor and a pertussis insensitive mutant Galpha(i/o) protein in human embryonic kidney 293 cells. Signalling efficiency was then monitored following inactivation of endogenous Galpha(i/o) proteins by pertussis toxin. Co-expression resulted in increased delta opioid signalling which was insensitive to the mu specific antagonist d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2. Under these conditions, mu opioid signalling was also increased and insensitive to the delta specific antagonist Tic-deltorphin. In this latter case, however, no G protein activation was observed in the presence of the delta specific inverse agonist N,N(CH3)2-Dmt-Tic-NH2. When a MOR fused to a non-functional Galpha subunit was co-expressed with the DOR-Galpha protein fusion, delta opioid signalling was not affected whereas mu opioid signalling was restored. Altogether our results suggest that increased delta opioid signalling is due to enhanced DOR coupling to its tethered Galpha subunit. On the other hand, our data indicate that increased mu opioid signalling requires an active conformation of the DOR and also results in activation of the Galpha subunit fused the DOR.

International audience; Opioid efficacy on mu-receptor may be influenced by various Gi/o-G-protein subunits interacting with intracellular face of receptor. Pertussis toxin-insensitive Galphai1 and Galphai2 subunits tethered with mu-receptor were stably transfected into AtT20 cells to (i) determine coupling of different alpha-subunits on opioid efficacy, and (ii) determine coupling to downstream effectors, for example, calcium and potassium channels. After pertussis toxin, stimulation of [35S]GTP-gamma-S incorporation persisted. Both constructs were able to couple to native calcium and potassium channels, with endomorphins 1 and 2 equally effective. However, pertussis toxin abolished opioid actions on calcium and potassium channels suggesting strong coupling to endogenous G-proteins, and that differences in coupling efficacy to Galphai1 and Galphai2 previously observed are restricted to initial step of signaling cascade.

The effects of a non-toxic fraction, called M1, from Buthus occitanus tunetanus (Bot) scorpion were studied on rat cardiac contraction and calcium transient and current. A decrease in both rate and tension on isolated intact hearts as well as in calcium transient induced by depolarizing 100 K(+) solution on isolated ventricular cardiomyocytes was firstly observed. Studies with the whole cell patch clamp method showed that M1 decreased the L-type calcium current (ICa(L)) in a dose-dependent manner with an IC50 of 0.36 microg/mL and a Hill coefficient of 0.95. This effect was blocked and reversed by the specific muscarinic receptors antagonist atropine, 1 microM, and was completely prevented when cardiomyocytes were pretreated with Pertussis toxin, 1 microg/mL, to block the alpha subunit of the PTX-sensitive G proteins. These results show that M1 fraction of Bot inhibits basal calcium current by interacting with muscarinic receptors and suggest that this inhibition could be attributed to inhibition of adenylate cyclase activity by a mechanism involving PTX-sensitive G proteins.