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Real-Time Analysis of Agonist-Induced Activation of Protease-Activated Receptor 1/G_i1 Protein Complex Measured by Bioluminescence Resonance Energy Transfer in Living Cells

Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Montpellier, France (M.A.A., D.M., V.B., L.P., J.P.P.); Institut National de la Santé et de la Recherche Médicale U661, Montpellier, France (M.A.A., D.M., V.B., L.P., J.P.P.); Université de Montpellier 1, Montpellier, France (M.A.A., D.M., V.B., L.P., J.P.P.); Université de Montpellier 2, Montpellier, France (M.A.A., D.M., V.B., L.P., J.P.P.); Institut de Génomique Fonctionnelle, Département de Pharmacologie Moléculaire, Montpellier, France (M.A.A., D.M., V.B., L.P., J.P.P.); and CisBio International, Bagnols-sur-Cèze, France (M.F., H.A.)

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 G_i1 protein. A specific constitutive BRET signal can be measured between nonactivated PAR1 and the G_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 -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 G 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 G_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.

Address correspondence to: Dr. Jean-Philippe Pin, Institut de Génomique Fonctionnelle; Département de Pharmacologie Moléculaire; 141, rue de la Cardonille, Montpellier F-34094 Cedex 5, France. E-mail: jppin{at}igf.cnrs.fr

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