title: Interaction of Protease-Activated Receptor 2 with G Proteins and β-Arrestin 1 Studied by Bioluminescence Resonance Energy Transfer.
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.
authors.affiliation: Département de Pharmacologie Moléculaire, Institut de Génomique Fonctionnelle , Montpellier , France ; UMR5203, Centre national de la recherche scientifique, Universités Montpellier 1 & 2 , Montpellier , France ; U661, Institut national de la santé et de la recherche médicale, Universités Montpellier 1 & 2 , Montpellier , France.
authors.forename: Mohammed Akli
authors.initials: MA
authors.lastname: Ayoub
authors.affiliation: Département de Pharmacologie Moléculaire, Institut de Génomique Fonctionnelle , Montpellier , France ; UMR5203, Centre national de la recherche scientifique, Universités Montpellier 1 & 2 , Montpellier , France ; U661, Institut national de la santé et de la recherche médicale, Universités Montpellier 1 & 2 , Montpellier , France.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Pin
keywords: BRET; G proteins; PAR2; pre-assembly; trypsin; β-arrestin