Received for publication August 29, 2006.
Revised January 11, 2007.
Accepted for publication January 12, 2007.

Real-time analysis of agonist-induced activation of protease-activated receptor 1/Gi1 protein complex measured by BRET in living cells

Abstract

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 (FRET and BRET) were recently shown to be powerful methods to monitor GPCRs-G proteins association in living cells. Here, we used a BRET technique to monitor the coupling between the protease-activated receptor 1 (PAR1) and Gi1 protein. A specific constitutive BRET signal can be measured between non-activated PAR1 and the Gi1 protein expressed at physiological level. This signal is insensitive to Pertussis toxin (PTX) and likely reflects the pre-assembly 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 pre-assembled complex with Gi1 protein, resulting either from a direct interaction between these partners or from their co-localization in specific microdomains, and that receptor activation likely results in rearrangements within such complexes.

Key words: Thrombin/PAR, Gi family, Desensitization/uncoupling, GRKs, barrestins, Molecular dynamics, Fluorescence techniques

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