Bioluminescence resonance energy transfer (BRET) is increasingly being used to monitor protein-protein interactions and cellular events in cells. However, the ability to monitor multiple events simultaneously is limited by the spectral properties of the existing BRET partners. Taking advantage of newly developed Renilla luciferases and blue-shifted fluorescent proteins (FPs), we explored the possibility of creating novel BRET configurations using a single luciferase substrate and distinct FPs. Three new (to our knowledge) BRET assays leading to distinct color bioluminescence emission were generated and validated. The spectral properties of two of the FPs used (enhanced blue (EB) FP2 and mAmetrine) and the selection of appropriate detection filters permitted the concomitant detection of two independent BRET signals, without cross-interference, in the same cells after addition of a unique substrate for Renilla luciferase-II, coelentrazine-400a. Using individual BRET-based biosensors to monitor the interaction between G-protein-coupled receptors and G-protein subunits or activation of different G-proteins along with the production of a second messenger, we established the proof of principle that two new BRET configurations can be multiplexed to simultaneously monitor two dependent or independent cellular events. The development of this new multiplexed BRET configuration opens the way for concomitant monitoring of various independent biological processes in living cells.

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2 receptors (V2R) associate with both Gs and G12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gs complexes, V2R-G12 complexes are not destabilized by guanine nucleotides and do not promote G12 activation. Activating V2R does not lead to signaling responses downstream of G12 activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12 heterotrimers. Overexpressing G12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G12 complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

G protein-coupled receptors (GPCRs) have been shown to activate the mitogen-activated protein kinases, ERK1/2, through both G protein-dependent and -independent mechanisms. Here, we describe a G protein-independent mechanism that unravels an unanticipated role for β-arrestins. Stimulation of the V2 vasopressin receptor (V2R) in cultured cells or in vivo in rat kidney medullar collecting ducts led to the activation of ERK1/2 through the metalloproteinase-mediated shedding of a factor activating the insulin-like growth factor receptor (IGFR). This process was found to be both Src- and β-arrestin–dependent. Whereas Src was found to act upstream of the metalloproteinase activation and be required for the release of the IGFR-activating factor, β-arrestins were found to act downstream of the IGFR transactivation. Unexpectedly, the engagement of β-arrestins by the IGFR but not by the V2R was needed to promote the vasopressin-stimulated ERK1/2 activation, indicating that a pool of β-arrestins distinct from those β-arrestins recruited to the V2R acts downstream of the receptor tyrosine kinase to activate ERK1/2. Such a dual site of action for β-arrestins helps explain the pleiotropic actions of this scaffolding protein. Given the role that V2R-stimulated ERK1/2 plays in kidney cell proliferation, this transactivation mechanism may have important implications for renal pathophysiology. Still, the role of β-arrestins downstream of a transactivation event is not limited to the V2R, because we observed a similar involvement for an unrelated GPCR (the platelet-activating factor receptor), indicating that it may be a general mechanism shared among GPCRs.