Stable association between Gαq and phospholipase Cβ1 in living cells

Signal transduction through G alpha(q) involves stimulation of phospholipase C beta (PLC beta) that results in increased intracellular Ca2+ and activation of protein kinase C. We have measured complex formation between G alpha(q) and PLC beta(1) in vitro and in living PC12 and HEK293 cells by fluorescence resonance energy transfer. In vitro measurements show that PLC beta(1) will bind to G alpha(q)(guanosine 5 '-3-O-(thio) triphosphate) and also to G alpha(q) (GDP), and the latter association has a different protein- protein orientation. In cells, image analysis of fluorescent-tagged proteins shows that G alpha(q) is localized almost entirely to the plasma membrane, whereas PLC beta(1) has a significant cytosolic population. By using fluorescence resonance energy transfer, we found that these proteins are pre-associated in the unstimulated state in PC12 and HEK293 cells. By determining the cellular levels of the two proteins in transfected versus non-transfected cells, we found that under our conditions overexpression should not significantly promote complex formation. G alpha(q)-PLC beta(1) complexes are observed in both single cell measurements and measurements of a large (i. e. 10(6)) cell suspension. The high level (similar to 40% maximum) of FRET is surprising considering that G alpha(q) is more highly expressed than PLC beta(1) and that not all PLC beta(1) is plasma membrane-localized. Our measurements suggest a model in which G proteins and effectors can exist in stable complexes prior to activation and that activation is achieved through changes in intermolecular interactions rather than diffusion and association. These pre-formed complexes in turn give rise to rapid, localized signals.