Regulation of the rate and extent of phospholipase Cβ2 effector activation by the βγsubunits of heterotrimeric G proteins

The activity of mammalian phosphoinositide-specific phospholipase C beta 2 (PLC-beta(2)) is regulated by the alpha(q) family of G proteins and by beta gamma subunits. We measured the affinity between the laterally associating PLC-beta(2) and G beta gamma on membrane surfaces by fluorescence resonance energy transfer. Using a simple model, we translated this apparent affinity to a bulk or three-dimensional equilibrium constant (K-d) and obtained a value of 3.2 mu M. We confirmed this K-d by separately measuring the on and off (k(f) and k(r)) rate constants. The k(f) was slower than a diffusion-limited value, suggesting that conformational changes occur when the two proteins interact. The off rate shows that the PLC-beta 2.G beta gamma complexes are long-lived (similar to 123 s) and that activation of PLC-beta(2) by G beta gamma would be sustained without a deactivating factor. The addition of alpha(i1)(GDP) subunits failed to physically dissociate the complex as determined by fluorescence. However, enzyme activity studies performed under similar conditions show that the addition of G alpha(i1)(GDP) results in reversal of PLC-beta 2 activation by G beta gamma during the time of the assay (30 s). From these results, we propose that G alpha(GDP) subunits can bind to the PLC-beta 2.G beta gamma complex to allow for rapid deactivation without complex dissociation. In support of this model, we show by fluorescence that G alpha(i1)(GDP).G beta gamma.PLC-beta(2) can form.