AbstractG protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors conveying extracellular information into intracellular signals. Many GPCRs have been shown to be able to oligomerize and it is firmly established that Class C GPCRs (e.g. metabotropic glutamate receptors) function as obligate dimers. However, the oligomerization capability of the larger Class A GPCRs (e.g. comprising the β-adrenergic receptors (β-ARs)) is still, despite decades of research, highly debated.Here we assess the oligomerization behavior of three prototypical Class A GPCRs, the β1-ARs, β2-ARs, and muscarinic M2Rs in single, intact cells. We combine two image correlation spectroscopy methods based on molecular brightness, i.e. the analysis of fluorescence fluctuations over space and over time, and thereby provide an assay able to robustly and precisely quantify the degree of oligomerization of GPCRs. In addition, we provide a comparison between two labelling strategies, namely C-terminally-attached fluorescent proteins and N-terminally-attached SNAP-tags, in order to rule out effects arising from potential fluorescent protein-driven oligomerization. The degree of GPCR oligomerization is expressed with respect to a set of previously reported as well as newly established monomeric or dimeric control constructs. Our data reveal that all three prototypical GPRCs studied display, under unstimulated conditions, a prevalently monomeric fingerprint. Only the β2-AR shows a slight degree of oligomerization.From a methodological point of view, our study suggests three key aspects. First, the combination of two image correlation spectroscopy methods allows addressing cells transiently expressing high concentrations of membrane receptors, far from the single molecule regime, at a density where the kinetic equilibrium should favor dimers and higher-order oligomers. Second, our methodological approach, allows to selectively target cell membrane regions devoid of artificial oligomerization hot-spots (such as vesicles). Third, our data suggest that the β1-AR appears to be a superior monomeric control than the widely used membrane protein CD86.Taken together, we suggest that our combined image correlation spectroscopy method is a powerful approach to assess the oligomerization behavior of GPCRs in intact cells at high expression levels.
Influence of FRET and fluorescent protein maturation on the quantification of binding affinity with dual-channel fluorescence cross-correlation spectroscopy
