| The oligomeric status of G proteins, unlike that of G protein coupled receptors, remains unexplored. Dual-colour fluorescence correlation spectroscopy (dcFCS), Forster resonance energy transfer (FRET) and step-wise photobleaching therefore were used to probe for oligomers of fluorescently labelled G proteins (G?i1?1?2) in live CHO cells and purified extracts of Sf9 cells. When G proteins bearing either eGFP or mCherry were co-expressed in CHO cells, the presence of a complex was indicated by a high degree of cross-correlation in dcFCS. Model-based analyses of apparent FRET efficiencies indicated that the complex contained at least four units. Photobleaching of single particles of eGFP-tagged G protein purified from Sf9 cells revealed 4--6 units; this number was essentially unchanged by guanyl nucleotides or agonist-activated M2 receptor, but it was decreased by the combination of both. These results suggest that G proteins form tetramers or larger oligomers that dissociate upon activation. |
