Real-Time Analysis of Agonist-Induced Activation of Protease-Activated Receptor 1/Gα_i1Protein Complex Measured by Bioluminescence Resonance Energy Transfer 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 [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 Galpha(i1) protein. A specific constitutive BRET signal can be measured between nonactivated PAR1 and the Galpha(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 Galpha 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 Galpha(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.

PAR1 and PAR2 Couple to Overlapping and Distinct Sets of G Proteins and Linked Signaling Pathways to Differentially Regulate Cell Physiology

“…In our studies, we assessed receptor/G protein complex formation, inhibition of adenylyl cyclase-directed cAMP production, and PTX-sensitive ERK1/2 activation. These findings are consistent with previous reports indicating that PAR1-directed phosphatidylinositol 3-kinase signaling and platelet activation is mediated by PTX-sensitive G i/o signaling (Voss et al, 2007), that PAR1 preassembles with G i1 in bioluminescence resonance energy transfer studies (Ayoub et al, 2007), and that G o mediates PAR1-directed intracellular calcium signaling and cytoskeletal rearrangements in endothelial cells (Vanhauwe et al, 2002). It is significant that our findings suggest that at least in the cells examined in these studies, PAR2 does not couple to G o or to G i family members.…”

Functional Selective Oxytocin-derived Agonists Discriminate between Individual G Protein Family Subtypes

“…Upon OT activation, OTRs are phosphorylated by GRK2, bind ␤-arrestin, and are endocytosed via clathrin-coated vesicles (33,42,43); after internalization, they recycle back to the plasma membrane via the Rab4/Rab5 short recycling pathway (22). Because neither atosiban nor DNalOVT promoted ␤-arrestin1 or ␤-arrestin2 recruitment and receptor internalization, we suggest that active OTR conformations coupling to G i do not efficiently recruit ␤-arrestins, which would be in line with published data showing that the recruitment of ␤-arrestins is also G␣ i -independent in prostaglandin E2 receptors (44) and protease-activated receptor 1 (45,46). Whether these active conformations correspond to phosphorylated or unphosphorylated forms remains to be established.…”

P2X2 and P2X5 Subunits Define a New Heteromeric Receptor with P2X7-Like Properties

“…3). The G-proteincoupled receptor PAR1-YFP and ATP-gated channel P2X4-YFP were used to assess the specificity of BRET signals (Ayoub et al, 2007). As shown in Figure 3A, when P2X5-Rluc was coexpressed with P2X5-YFP, or P2X2-YFP, a specific BRET signal was observed, characterized by a hyperbolic increase of the signal.…”