Constitutive interaction of the P2Y2 receptor with the hematopoietic cell-specific G protein Gα16 and evidence for receptor oligomers

Hematopoietic cells uniquely express Gα16, a G protein α-subunit of the Gq-type. Gα16 is obligatory for P2Y2 receptor-dependent Ca2+-mobilization in human erythroleukemia cells and induces hematopoietic cell differentiation. We tested whether P2Y2 receptors physically interact with Gα16. Receptor and G protein were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein (GFP), respectively. When expressed in K562 leukemia cells, the fusion proteins were capable of triggering a Ca2+-signal upon receptor stimulation, demonstrating their functional integrity. In fluorescence resonance energy transfer (FRET) measurements using confocal microscopy, a strong FRET signal from the plasma membrane region of fixed, resting cells was detected when the receptor was co-expressed with the G protein as the FRET acceptor, as well as when the CFP-tagged receptor was co-expressed with receptor fused to YFP. We conclude that, under resting conditions, Gα16 and P2Y2 receptors form constitutive complexes, and that the P2Y2 receptor is present as an oligomer.