Determination of G-protein-coupled receptor oligomerization by molecular brightness analyses in single cells.

@article{Ibilir2021DeterminationOG,
  title={Determination of G-protein-coupled receptor oligomerization by molecular brightness analyses in single cells.},
  author={Ali Işbilir and Robert Serfling and Jan M{\"o}ller and Romy Thomas and Chiara De Faveri and Ulrike Zabel and Marco Scarselli and Annette G. Beck‐Sickinger and Andreas Bock and Irene Coin and Martin J. Lohse and Paolo Annibale},
  journal={Nature protocols},
  year={2021}
}
Oligomerization of membrane proteins has received intense research interest because of their importance in cellular signaling and the large pharmacological and clinical potential this offers. Fluorescence imaging methods are emerging as a valid tool to quantify membrane protein oligomerization at high spatial and temporal resolution. Here, we provide a detailed protocol for an image-based method to determine the number and oligomerization state of fluorescently labeled prototypical G-protein… 
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References

SHOWING 1-10 OF 114 REFERENCES
Visualization of class A GPCR oligomerization by image-based fluorescence fluctuation spectroscopy
TLDR
The data suggest that the β1-AR appears to be a superior monomeric control than the widely used membrane protein CD86, and the combined image correlation spectroscopy method is a powerful approach to assess the oligomerization behavior of GPCRs in intact cells at high expression levels.
Quantitative Single-Residue Bioorthogonal Labeling of G Protein-Coupled Receptors in Live Cells.
TLDR
It is demonstrated here that the genetic incorporation of trans-cyclooct-2-ene lysine (TCO*) allows achieving quantitative single-residue labeling of the extracellular loops of the β2-adrenergic and the muscarinic M2 class A GPCRs, as well as of the corticotropin releasing factor class B GPCR.
Illuminating the life of GPCRs
TLDR
Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented.
Revealing G‐protein‐coupled receptor oligomerization at the single‐molecule level through a nanoscopic lens: methods, dynamics and biological function
TLDR
Recent evidence from photoactivated localization microscopy (PALM), which allows the visualization of single molecules in dense samples, and single‐molecule tracking (SMT), which determines how GPCRs move and interact in living cells in the presence of different ligands are highlighted.
Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules
TLDR
Total internal reflection fluorescence microscopy in living cells to visualize thousands of individual molecules of a model GPCR, the M1 muscarinic acetylcholine receptor, established the dynamic nature of dimer formation with M1 receptors undergoing interconversion between monomers and dimers on the timescale of seconds.
Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization
TLDR
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 is described.
Using enhanced number and brightness to measure protein oligomerization dynamics in live cells
TLDR
This protocol describes enhanced number and brightness (eN&B), an approach that uses fluorescence fluctuation spectroscopy data to directly measure the oligomerization state and dynamics of fluorescently tagged proteins in living cells.
Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate.
TLDR
It is demonstrated that real-time optical measurements of beta2AR (as well as other GPCR) interactions and dynamics on living cells are feasible and fidelity of the biochemical properties of thebeta2AR/S65T/GFP can be engineered.
Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells
TLDR
An automated, single-molecule protocol based on total internal reflection fluorescence microscopy (TIRFM) imaging that allows protein tracking and subunit counting in living plant cells and other biological specimens visualized by TIRFM or other fluorescence imaging techniques is described.
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