Abstract
G protein-coupled receptors are well recognized as being able to activate several signaling pathways through the activation of different G proteins as well as other signaling proteins such as β-arrestins. Therefore, understanding how such multiple GPCR-mediated signaling can be integrated constitute an important aspect. Here, we applied bioluminescence resonance energy transfer (BRET) to shed more light on the G protein coupling profile of trypsin receptor, or protease-activated receptor 2 (PAR2), and its interaction with β-arrestin1. Using YFP and Rluc fusion constructs expressed in COS-7 cells, BRET data revealed a pre-assembly of PAR2 with both Gαi1 and Gαo and a rapid and transient activation of these G proteins upon receptor activation. In contrast, no pre-assembly of PAR2 with Gα12 could be detected and their physical association can be measured with a very slow and sustained kinetics similar to that of β-arrestin1 recruitment. These data demonstrate the coupling of PAR2 with Gαi1, Gαo, and Gα12 in COS-7 cells with differences in the kinetics of GPCR-G protein coupling, a parameter that very likely influences the cellular response. Moreover, this further illustrates that pre-assembly or agonist-induced G protein interaction depends on receptor-G protein pairs indicating another level of complexity and regulation of the signaling of GPCR-G protein complexes and its multiplicity.
Highlights
The study of the interaction of GPCRs with their specific signaling and regulatory proteins has been widely studied using energy transfer-based approaches bioluminescence resonance energy transfer (BRET) and FRET [1,2,3,4,5]
We wanted to investigate the putative coupling of protease-activated receptor 2 (PAR2) with Gαi1, Gαo, and Gα12, as this has been previously demonstrated for PAR1 [6, 7, 27]
We used BRET approach allowing real-time assessment of the receptor-G protein complexes in live cells and BRET measurements were performed in COS-7 cells transiently co-expressing Gα-Rluc and PAR2-YFP fusion proteins and stimulated or not with its specific agonist, trypsin
Summary
The study of the interaction of GPCRs with their specific signaling and regulatory proteins has been widely studied using energy transfer-based approaches BRET and FRET (bioluminescence/fluorescence resonance energy transfer) [1,2,3,4,5] These methods allow the measurements, in real-time and live cells, of either the recruitment of specific proteins One of the important GPCR families is protease-activated receptors (PARs) which is composed by four subtypes, PAR1, protease-activated receptor 2 (PAR2), PAR3, and PAR4, which play crucial roles in a number of physiological processes such as thrombosis, vascular development, cell proliferation, and tumorigenesis [11] They are considered as interesting targets for the treatment of various pathologies like inflammation, cancer, and stroke [12].
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