Hervé Ansanay

Real-time analysis of agonist-induced activation of protease-activated receptor 1/Galphai1 protein complex measured by bioluminescence resonance energy transfer in living cells.

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 Gαi1 protein. A specific constitutive BRET signal can be measured between nonactivated PAR1 and the Gαi1 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 β-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 Gα 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 Gαi1 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.

ACTIVATION OF THE BETA 2-ADRENERGIC RECEPTOR-GALPHA S COMPLEX LEADS TO RAPID DEPALMITOYLATION AND INHIBITION OF REPALMITOYLATION OF BOTH THE RECEPTOR AND GALPHA S

Palmitoylation is unique among lipid modifications in that it is reversible. In recent years, dynamic palmitoylation of G protein α subunits and of their cognate receptors has attracted considerable attention. However, very little is known concerning the acylation/deacylation cycle of the proteins in relation to their activity status. In particular, the relative contribution of the activation and desensitization of the signaling unit to the regulation of the receptors and G proteins palmitoylation state is unknown. To address this issue, we took advantage of the fact that a fusion protein composed of the stimulatory α subunit of trimeric G protein (Gαs) covalently attached to the β2-adrenergic receptor (β2AR) as a carboxyl-terminal extension (β2AR-Gαs) can be stimulated by agonists but does not undergo rapid inactivation, desensitization, or internalization. When expressed in Sf9 cells, both the receptor and the Gαs moieties of the fusion protein were found to be palmitoylated via thioester linkage. Stimulation with the β-adrenergic agonist isoproterenol led to a rapid depalmitoylation of both the β2AR and Gαs and inhibited repalmitoylation. The extent of depalmitoylation induced by a series of agonists was correlated (0.99) with their intrinsic efficacy to stimulate the adenylyl cyclase activity. However, forskolin-stimulated cAMP production did not affect the palmitoylation state of β2AR-Gαs, indicating that the agonist-promoted depalmitoylation is linked to conformational changes and not to second messenger generation. Given that, upon activation, the fusion protein mimics the activated receptor-G protein complex but cannot undergo desensitization, the data demonstrate that early steps in the activation process lead to the depalmitoylation of both receptor and G protein and that repalmitoylation requires later events that cannot be accommodated by the activated fusion protein.

Functional Adhesiveness of the CX3CL1 Chemokine Requires Its Aggregation ROLE OF THE TRANSMEMBRANE DOMAIN

In its native form, the chemokine CX3CL1 is a firmly adhesive molecule promoting leukocyte adhesion and migration and hence involved, along with its unique receptor CX3CR1, in various inflammatory processes. Here we investigated the role of molecular aggregation in the CX3CL1 adhesiveness. Assays of bioluminescence resonance energy transfer (BRET) and homogeneous time-resolved fluorescence (HTRF) in transfected cell lines and in primary cells showed specific signals indicative of CX3CL1 clustering. Truncation experiments showed that the transmembrane domain played a central role in this aggregation. A chimera with mutations of the 12 central transmembrane domain residues had significantly reduced BRET signals and characteristics of a non-clustering molecule. This mutant was weakly adhesive according to flow and dual pipette adhesion assays and was less glycosylated than CX3CL1, although, as we demonstrated, loss of glycosylation did not affect the CX3CL1 adhesive potency. We postulate that cell surfaces express CX3CL1 as a constitutive oligomer and that this oligomerization is essential for its adhesive potency. Inhibition of CX3CL1 self-assembly could limit the recruitment of CX3CR1-positive cells and may be a new pathway for anti-inflammatory therapies.

Differential Coupling of the Vasopressin V1b Receptor through Compartmentalization within the Plasma Membrane

We show here that the rat vasopressin V1b receptor simultaneously activates both the Gq/11-inositol phosphate (IP) and Gs-cAMP pathways when transiently expressed in Chinese hamster ovary, human embryonic kidney (HEK) 293, and COS-7 cells and stimulated with arginine-vasopressin. Higher concentrations of the hormone, however, were needed to trigger the cAMP pathway. The nonmammalian analog arginine-vasotocin and the selective V1b agonist d[Cha4]vasopressin also activated the cAMP and IP pathways, although d[Cha4]-vasopressin elicited the two responses with equivalent potencies. We determined that the V1b receptor is present as a homodimer at the plasma membrane. Treatment of V1b-transfected HEK-293 cells with methyl-β-cyclodextrin, a drug known to dissociate cholesterol-rich domains of the plasma membrane, shifted the EC50 of the vasopressin-induced cAMP accumulation to lower concentrations and, remarkably, increased the hormone efficacy related to the activation of this second messenger system. In parallel, the vasopressin-mediated activation of the IP pathway was slightly reduced without modification of its EC50. These results suggest that, as with many other G protein-coupled receptors, when transfected in heterologous cell systems, the V1b receptor forms dimers that signal differentially through the Gq/11 and Gs proteins depending on the nature of the ligand as well as on its localization within specialized compartments of the plasma membrane. The present study thus illustrates how signal transduction associated with the activation of a G protein-coupled receptor can be versatile and highly dependent on both the cell context and the chemical nature of the extracellular signaling messenger.