Real-time analysis of agonist-induced activation of protease-activated receptor 1/Gαi1 protein complex measured by bioluminescence resonance energy transfer in living cells

Mohammed A. Ayoub, Damien Maurel, Virginie Binet, Michel Fink, Laurent Prézeau, Hervé Ansanay, Jean Philippe Pin

Research output: Contribution to journalArticlepeer-review

76 Citations (Scopus)

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 [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.

Original languageEnglish
Pages (from-to)1329-1340
Number of pages12
JournalMolecular Pharmacology
Volume71
Issue number5
DOIs
Publication statusPublished - May 2007
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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