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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.

Received for publication August 28, 2006.

Accepted for publication January 12, 2007.

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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