The binding of activated G_q to phospholipase C- exhibits anomalous affinity

Upon activation by the G_q family of G subunits, G subunits, and some Rho family GTPases, phospholipase C- (PLC-) isoforms hydrolyze phosphatidylinositol 4,5-bisphos-phate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC- isoforms also function as GTPase-activating proteins, potentiating G_q deactivation. To elucidate the mechanism of this mutual regulation, we measured the thermodynamics and kinetics of PLC-3 binding to G_q. FRET and fluorescence correlation spectroscopy, two physically distinct methods, both yielded K_d values of about 200 nM for PLC-3–G_q binding. This K_d is 50 –100 times greater than the EC₅₀ for G_q-mediated PLC-3 activation and for the G_q GTPase-activating protein activity of PLC-. The measured K_d was not altered either by the presence of phospholipid vesicles, phosphatidylinositol 4,5-bisphosphate and Ca², or by the identity of the fluorescent labels. FRET-based kinetic measurements were also consistent with a K_d of 200 nM. We determined that PLC-3 hysteresis, whereby PLC-3 remains active for some time following either G_q–PLC-3 dissociation or PLC-3–potentiated G_q deactivation, is not sufficient to explain the observed discrepancy between EC₅₀ and K_d. These results indicate that the mechanism by which G_q and PLC-3 mutually regulate each other is far more complex than a simple, two-state allosteric model and instead is probably kinetically determined.