Signal transduction through G␣ q involves stimulation of phospholipase C␤ (PLC␤) that results in increased intracellular Ca 2؉and activation of protein kinase C. We have measured complex formation between G␣ q and PLC␤ 1 in vitro and in living PC12 and HEK293 cells by fluorescence resonance energy transfer. In vitro measurements show that PLC␤ 1 will bind to G␣ q (guanosine 5-3-O-(thio)triphosphate) and also to G␣ q (GDP), and the latter association has a different protein-protein orientation. In cells, image analysis of fluorescent-tagged proteins shows that G␣ q is localized almost entirely to the plasma membrane, whereas PLC␤ 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 nontransfected cells, we found that under our conditions overexpression should not significantly promote complex formation. G␣ q -PLC␤ 1 complexes are observed in both single cell measurements and measurements of a large (i.e. 10 6 ) cell suspension. The high level (ϳ40% maximum) of FRET is surprising considering that G␣ q is more highly expressed than PLC␤ 1 and that not all PLC␤ 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.

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