Subcellular Imaging of Dynamic Protein Interactions by Bioluminescence Resonance Energy Transfer

Abstract

Despite the fact that numerous studies suggest the existence of receptor multiprotein complexes, visualization and monitoring of the dynamics of such protein assemblies remain a challenge. In this study, we established appropriate conditions to consider spatiotemporally resolved images of such protein assemblies using bioluminescence resonance energy transfer (BRET) in mammalian living cells. Using covalently linked Renilla luciferase and yellow fluorescent proteins, we depicted the time course of dynamic changes in the interaction between the V2-vasopressin receptor and beta-arrestin induced by a receptor agonist. The protein-protein interactions were resolved at the level of subcellular compartments (nucleus, plasma membrane, or endocytic vesicules) and in real time within tens-of-seconds to tens-of-minutes time frame. These studies provide a proof of principle as well as experimental parameters and controls required for high-resolution dynamic studies using BRET imaging in single cells.

GKAP-DLC2 interaction organizes postsynaptic scaffold complex to enhance synaptic NMDA receptor activity

“…We further characterized the association between GKAP and DLC2 at the subcellular level by imaging protein-protein interactions on single hippocampal neuron using BRET imaging (Coulon et al, 2008;Perroy, 2010). The BRET signal between RLuc8-GKAP and Venus-DLC2 was homogeneous in the cell body and punctiform in dendrites ( Fig.…”

“…Identification of the new GKAP interactor, DLC2, raised the question of its functional interaction in the organization and activity of the glutamate receptors. Using recent developments in single-cell BRET imaging (Coulon et al, 2008;Perroy, 2010), we have examined this issue by studying the spatiotemporal dynamics of GKAP-DLC2 interaction in living hippocampal neurons. We found that GKAP-DLC2 interaction was prominent in dendritic spines and could be increased by sustained synaptic activity.…”

“…BRET measurements in cell populations were performed as previously described (Perroy et al, 2004). Single cell BRET imaging in cultured hippocampal neurons to study the subcellular localization of protein-protein interactions was performed according to previously published protocols (Coulon et al, 2008;Perroy, 2010). Briefly, images were obtained using a Plan-Apochromat 636/1.40 oil M27 objective, at room temperature.…”

Nanoscale organization of β2-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

“…Fusion proteins of bARs have provided useful tools for studies of adrenergic receptor biology and the development of assays for ligand discovery based on bioluminescence and Forster resonance energy transfer (BRET and FRET, respectively) [2,[15][16][17][18][19][20][21][22][23][24]. Functional fusion proteins of b 2 ARs have been accomplished with FLAG tags for immunofluorescence and immunoprecipitation experiments as well as green fluorescent proteins (GFPs) and their analogues [15][16][17][18]25].…”

“…bAR signaling represents an important pharmacological target [14] and many assays have been developed to aid in the drug discovery process [15][16][17][18]. Herein we investigate the conditions required for mimicking the nanoscale distributions and local environment observed in primary cells from the mammalian heart using a conditionally expressed fusion protein of b 2 AR in HEK293 cells.Fusion proteins of bARs have provided useful tools for studies of adrenergic receptor biology and the development of assays for ligand discovery based on bioluminescence and Forster resonance energy transfer (BRET and FRET, respectively) [2,[15][16][17][18][19][20][21][22][23][24]. Functional fusion proteins of b 2 ARs have been accomplished with FLAG tags for immunofluorescence and immunoprecipitation experiments as well as green fluorescent proteins (GFPs) and their analogues [15][16][17][18]25].…”