Measuring G Protein Activation with Spectrally Resolved Fluorescence Fluctuation Spectroscopy

G protein-coupled receptor signaling has been posited to occur through either collision coupling or pre-assembled complexes with G protein transducers. To investigate the dynamics of G protein signaling, we introduce fluorescence covariance matrix analysis (FCMA), a novel implementation of fluorescence cumulant analysis applied to spectrally resolved fluorescence images. We labeled the GPCR, Gα, and Gβγ units with distinct fluorescent protein labels and we applied FCMA to measure directly the complex formation during stimulation of dopamine and adrenergic receptors. To determine the prevalence of hetero-oligomers, we compared the GPCR data to those from control samples expressing three fluorescent protein labels with known stoichiometries. Interactions between Gα and Gβγ subunits determined by FCMA were sensitive to stimulation with GPCR ligands. However, GPCR/G protein interactions were too weak to be distinguished from background. These findings support a collision coupling mechanism rather than pre-assembled complexes for the two GPCRs studied.