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.
Footnotes
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This work was supported by grants from the Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Universités de Montpellier 1 and 2, the Action Concertée Incitative “Biologie Cellulaire, Moléculaire et Structurale” of the French ministry of research and technology (grant BCMS328), the Agence Nationale de la Recherche (ANR-05-PRIB-02502), the European Community (grant LSHB-CT-200-503337), and CisBio International.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.030304.
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ABBREVIATIONS: GPCR, G protein-coupled receptor; PAR, protease-activated receptor; BRET, bioluminescence resonance energy transfer; FRET, fluorescence resonance energy transfer; Rluc, Renilla reniformis luciferase; YFP, yellow fluorescent protein; TRAP, thrombin receptor-activating peptide; PTX, pertussis toxin; GABAb, γ-4-aminobutyric acid receptor type B; ELISA, enzyme-linked immunosorbent assay; SCH79797, N-3-cyclopropyl-7-{[4-(1-methyl-ethyl)phenyl]methyl}-7H-pyrrolo[3,2-f]quinazoline-1,3 diamine; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; IGF, Institut de Génomique Fonctionnelle; TFLLR, Thr-Phe-Leu-Leu-Arg-NH2; SLIGRL, Ser-Leu-Iso-Gly-Arg-Leu-NH2; CGP54626, [S-(R*,R*)]-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxypropyl](cyclohexylmethyl) phosphinic acid.
- Received August 28, 2006.
- Accepted January 12, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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