Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization

@article{Maurel2008CellsurfacePI,
  title={Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization},
  author={Damien Maurel and Laetitia Comps-Agrar and Carsten Brock and Marie-Laure Rives and Emmanuel Bourrier and Mohammed Akli Ayoub and Herv{\'e} Bazin and Norbert Tinel and Thierry Durroux and Laurent Prézeau and Eric Trinquet and Jean-Philippe Pin},
  journal={Nature Methods},
  year={2008},
  volume={5},
  pages={561-567}
}
Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput–compatible format. Using this… 
Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to G protein-coupled receptor oligomerization.
TLDR
A technology is developed, which combines TR-FRET detection with a new labeling method called SnapTag, which has allowed us to address the oligomeric state of many GPCRs.
Methods used to study the oligomeric structure of G-protein-coupled receptors
TLDR
The latest developments that have been made in the methods used to study GPCR oligomerization are summarized and an overview of their application is given.
Single-molecule FRET imaging of GPCR dimers in living cells.
TLDR
Generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells are reported and show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers.
In-Cell Detection of Conformational Substates of a G Protein-Coupled Receptor Quaternary Structure: Modulation of Substate Probability by Cognate Ligand Binding.
TLDR
The significantly improved FRET spectrometry technique allowed us to detect multiple distinct quaternary conformational substates of Ste2 oligomers, and to assess how the α-factor ligand altered the proportion of such substates.
Single-molecule analysis of fluorescently labeled G-protein–coupled receptors reveals complexes with distinct dynamics and organization
TLDR
The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.
Probing Self-Assembly of G Protein-Coupled Receptor Oligomers in Membranes Using Molecular Dynamics Modeling and Experimental Approaches
TLDR
Recent insights gained from a multidisciplinary research approach using computational coarse grain molecular dynamics (CGMD) simulations and experiments facilitated by molecular and chemical biology approaches are reviewed.
In-cell detection of conformational sub-states of a GPCR quaternary structure: Modulation of sub-state probability by cognate ligand binding
TLDR
The significantly improved FRET spectrometry technique allowed us to detect multiple distinct quaternary conformational substates of Ste2 oligomers, and to assess how the α-factor ligand altered the proportion of such substates.
Contributions of fluorescence techniques to understanding G protein-coupled receptor dimerisation
TLDR
The challenges of studying multicomponent membrane protein systems have led to the development and refinement of many fluorescence assays, allowing the functional consequences of receptor dimerisation to be investigated and individual protein molecules to be imaged in the membranes of living cells.
Time resolved FRET strategy with fluorescent ligands to analyze receptor interactions in native tissues: application to GPCR oligomerization.
TLDR
This work developed a new experimental approach based on the labeling of receptors with high affinity fluorescent ligands compatible with time-resolved energy transfer measurements that allows the direct identification of wild-type GPCR oligomers in native tissues.
BRET and Time-resolved FRET strategy to study GPCR oligomerization: from cell lines toward native tissues
TLDR
A fluorescent ligand-based GPCR labeling strategy combined to a TR-FRET approach has been successfully used to prove the existence of G PCR oligomerization in native tissues.
...
...

References

SHOWING 1-10 OF 37 REFERENCES
A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein
TLDR
It is demonstrated that a prototypical GPCR, the β2-adrenergic receptor (β2AR), can be incorporated into a reconstituted high-density lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs.
FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells
  • B. Meyer, J. Segura, H. Vogel
  • Biology, Chemistry
    Proceedings of the National Academy of Sciences of the United States of America
  • 2006
The lateral organization of a prototypical G protein-coupled receptor, the neurokinin-1 receptor (NK1R), was investigated in living cells by fluorescence resonance energy transfer (FRET) microscopy,
Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction
TLDR
It is demonstrated by pharmacological and hydrodynamic experiments that purified neurotensin receptor NTS1, a class A GPCR, dimerizes in detergent solution in a concentration-dependent manner, with an apparent affinity in the low nanomolar range.
Oligomerization of G-protein-coupled transmitter receptors
  • M. Bouvier
  • Biology, Chemistry
    Nature Reviews Neuroscience
  • 2001
TLDR
The evidence supporting the existence of G-protein-coupled-receptor dimerization is reviewed and its functional importance is discussed to have important implications for the development and screening of new drugs.
A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer
TLDR
The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein–coupled receptors proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments.
Real-Time Analysis of Agonist-Induced Activation of Protease-Activated Receptor 1/Gαi1 Protein Complex Measured by Bioluminescence Resonance Energy Transfer in Living Cells
TLDR
The results show that BRET between GPCRs and Gα proteins can be used to monitor the receptor activation in real time and in living cells and revealed that PAR1 can be part of a preassembled complex with Gαi1 protein, and that receptor activation probably results in rearrangements within such complexes.
Asymmetric conformational changes in a GPCR dimer controlled by G‐proteins
TLDR
It is shown that a receptor dimer with only a single agonist‐occupied subunit can trigger G‐protein activation, and the two subunits of the receptors dimer in the G‐ protein‐coupled state differ in their conformation, even when both are liganded by the agonist.
The α1b-Adrenoceptor Exists as a Higher-Order Oligomer: Effective Oligomerization Is Required for Receptor Maturation, Surface Delivery, and Function
TLDR
Data indicate that the α1b-adrenoceptor is able to form oligomeric rather than only simple dimeric complexes and that disruption of effective oligomerization by introducing mutations into transmembrane domain IV has profound consequences for cell surface delivery and function.
Molecular tinkering of G protein‐coupled receptors: an evolutionary success
TLDR
Indirect studies have led to a useful model of a common ‘central core’, composed of seven transmembrane helical domains, and its structural modifications during activation of G protein‐coupled receptors.
...
...