Real-Time Analysis of Agonist-Induced Activation of Protease-Activated Receptor 1/G alpha sub(i1) Protein Complex Measured by Bioluminescence Resonance Energy Transfer in Living Cells

• Published in: Molecular pharmacology Vol. 71; no. 5; pp. 1329 - 1340
• Main Authors: Ayoub, Mohammed A, Maurel, Damien, Binet, Virginie, Fink, Michel, Prezeau, Laurent, Ansanay, Herve, Pin, Jean-Philippe
• Format: Journal Article
• Language: English
• Published: 01.05.2007
• ISSN: 0026-895X

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 alpha sub(i1) protein. A specific constitutive BRET signal can be measured between nonactivated PAR1 and the G alpha sub(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 G alpha 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 alpha sub(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.