Revealing G‐protein‐coupled receptor oligomerization at the single‐molecule level through a nanoscopic lens: methods, dynamics and biological function

Abstract

The introduction of super-resolution fluorescence microscopy has allowed the visualization of 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 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. PALM has demonstrated that GPCR oligomerization depends on the receptor subtype, the cell type, the actin cytoskeleton, and other proteins. Conversely, SMT has revealed the transient dynamics of dimer formation, whereby receptors show 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 dimers/oligomers is also supported by well-known techniques, such as resonance energy transfer 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 the function and druggability of homo-oligomers and hetero-oligomers.

Structural assemblies of the di- and oligomeric G-protein coupled receptor TGR5 in live cells: an MFIS-FRET and integrative modelling study

“…Moreover, as described by Scarselli et al 46,. PALM experiments using a class A GPCR suggested that oligomerization remains unchanged by the addition of the agonist.…”

G protein-coupled receptor-receptor interactions give integrative dynamics to intercellular communication

“…For instance, combined BRET/FRET and complementation studies have revealed that the assemblage of dopamine D 2 receptors by symmetrical interfaces at TM4 and TM1 can lead to a complex composed of at least four protomers in the plasma membrane of living mammalian cells (Guo et al, 2008). Moreover, based on an analysis of PALM data, it has been proposed that direct RRI could lead to the formation of high-order oligomers (tetramers, octamers, and larger-sized complexes) depending on the specific membrane microenvironment (Scarselli et al, 2016). -The existence of multiple interaction interfaces opens the possibility that the assemblage of a given set of receptor molecules to form a complex could occur in a number of different geometrical arrangements (Agnati et al, 2009a) depending on a number of conditions including not only the physical properties of the interacting proteins (surface charge, hydrophobicity, etc.)…”

G Protein-Coupled Receptor Signaling

“…We and others have previously described distinct single-molecule imaging modalities used to study GPCRs, and the advantages and limitations of each of these methods. Thus, we refer the reader to the following previous reviews (6,22). Here we will discuss our methodology of the super-resolution imaging technique of photoactivatable localisation microscopy (PALM) using photoactivatable dyes (PD-PALM), within the context of applying this technology to study GPCR heteromers and the reorganization of these complexes following ligand activation.…”