REGULATION OF THE RATE AND EXTENT OF PHOSPHOLIPASE-C-BETA(2) EFFECTORACTIVATION BY THE BETA-GAMMA-SUBUNITS OF HETEROTRIMERIC G-PROTEINS

The activity of mammalian phosphoinositide-specific phospholipase C be ta 2 (PLC-beta(2)) is regulated by the alpha(q) family of G proteins a nd by beta gamma subunits. We measured the affinity between the latera lly associating PLC-beta(2) and G beta gamma on membrane surfaces by f luorescence resonance energy transfer. Using a simple model, we transl ated 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 constant s. 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 w ould be sustained without a deactivating factor. The addition of alpha (i1)(GDP) subunits failed to physically dissociate the complex as dete rmined by fluorescence. However, enzyme activity studies performed und er similar conditions show that the addition of G alpha(i1)(GDP) resul ts in reversal of PLC-beta 2 activation by G beta gamma during the tim e 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 fo r 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.