The introduction of super resolution fluorescence microscopy has allowed to visualize single proteins in their biological environment. Recently, these techniques have been applied to determine the organization of class A G protein-coupled receptors (GPCRs), and to determine whether they exist as monomers, dimers and/or higher-order oligomers. On this subject, this review highlights recent evidence coming from Photoactivated Localization Microscopy (PALM) that allows the visualization of single molecules in dense samples, and Single-Molecule Tracking (SMT) that determines how GPCRs move and interact in living cells in the presence of different ligands. PALM has demonstrated that GPCR oligomerization depends on the receptor subtype, cell-type, actin cytoskeleton and other proteins. Conversely, SMT has revealed the transient dynamics of dimer formation, where receptors display a monomer-dimer equilibrium characterized by rapid association and dissociation. At steady state, depending on the subtype, approximately 30-50% of receptors are part of dimeric complexes. Notably, the existence of many GPCR di-/oligomers is also supported by using well-known techniques, such as Resonance Energy Transfer (RET) methodologies, and by approaches that exploit fluorescence fluctuations, such as Fluorescence Correlation Spectroscopy (FCS). Future research using single-molecule methods will deepen our knowledge related to function and druggability of homo- and hetero-oligomers. This article is protected by copyright. All rights reserved.
|Titolo:||Revealing GPCR oligomerization at the single-molecule level through a nanoscopic lens: methods, dynamics and biological function|
|Data di pubblicazione:||2016|
|Appare nelle tipologie:||1.1 Articolo in rivista|