Single-Molecule Analysis of G Protein-Coupled Receptor Stoichiometry: Approaches and Limitations

Abstract

How G protein-coupled receptors (GPCRs) are organized at the cell surface remains highly contentious. Single-molecule (SM) imaging is starting to inform this debate as receptor behavior can now be visualized directly, without the need for interpreting ensemble data. The limited number of SM studies of GPCRs undertaken to date have strongly suggested that dimerization is at most transient, and that most receptors are monomeric at any given time. However, even SM data has its caveats and needs to be interpreted carefully. Here, we discuss the types of SM imaging strategies used to examine GPCR stoichiometry and consider some of these caveats. We also emphasize that attempts to resolve the debate ought to rely on orthogonal approaches to measuring receptor stoichiometry.

Dimers of serotonin receptors: Impact on ligand affinity and signaling

“…The evidence that class A GPCRs can form homodimers comes mainly from transfected overexpressing cells and a few from intact living cells or animal models. Various techniques that will only be briefly summarized here, have been discussed with more details in other reviews, for example [2,8,30]. Historically, there has been recurrent observations, by Westernblotting against GPCR of immunoreactive bands that could correspond to dimeric or higher order complexes.…”

“…Finally, recent techniques of single-molecule tracking and high-resolution imaging microscopy have initiated the view that GPCRs dimers may only be transient, and can be stabilized by agonists [36,37]. These transient dimers are present at the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes [7,8]. Using single-molecule imaging, dopamine D2 receptor was shown to form transient dimers with a lifetime of 68 ms, which was prolonged upon agonist addition by a factor of ~1.5, suggesting the possibility that dimers are only transiently formed [36].…”

“…Although several studies have asserted the existence of class A GPCR dimers, others have reported that most of them are mainly monomeric [6]. Nevertheless, new techniques of single-molecule analysis suggest that GPCRs are present at the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes [7,8]. The possibility of heterodimers formation has been proposed not only within GPCRs responding to one same ligand, but also across different families responding to different ligands.…”

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

“…There has also been a growing interest in the lateral organization of GPCRs at the cell surface, as advances in single-molecule fluorescence microscopy and single-particle tracking (SPT) have enabled direct observation of the stochastic and dynamic behavior of individual GPCRs in real-time [ 7 , 8 , 9 , 10 , 11 , 12 ]. Current state-of-the-art knowledge derived from SPT studies is that (i) a vast majority of GPCRs maintain a diffusible surface pool [ 13 ], (ii) activation status controls mobility [ 9 , 10 , 14 ], (iii) agonist-dependent global decrease in the diffusion rate is not dependent on the GPCR subfamily or G protein coupling selectivity [ 10 ], and (iv) GPCRs can assemble into transient homo- and heterodimers, although the preponderance and lifetime of such oligomeric assemblies remain controversial [ 8 , 9 , 15 , 16 ]. Nevertheless, these results highlight the need for a continued inquiry into one of the most debated features of GPCR signaling—whether receptors are pre-coupled to signal transducers or engage them via random collisions facilitated by the plasma membrane specialized nanodomains [ 13 , 17 ].…”

Constraints on GPCR Heterodimerization Revealed by the Type-4 Induced-Association BRET Assay

“…This is particularly problematic in the case of heterodimerization because differences in expression rates or subcellular distribution of receptor subunits increases the likelihood of data deviating from pseudolinearity and therefore the risk of reporting false dimers. Arguably, the most powerful tool for examining receptor stoichiometry, that of single-molecule imaging, has yet to be used to demonstrate GPCR heterodimerization, even though it is largely responsible for the present consensus that homodimerization is generally transient and the dominant GPCR stoichiometry is that of monomers ( 8 ). Single-molecule spectroscopic techniques, such as fluorescence cross-correlation spectroscopy, have reported heterodimerization in a number of cases ( 9 ), but these approaches have yet to become routine.…”