The binding of activated Gα to phospholipase C-β exhibits anomalous affinity.

Upon activation by the G family of Gα subunits, Gβγ subunits, and some Rho family GTPases, phospholipase C-β (PLC-β) isoforms hydrolyze phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC-β isoforms also function as GTPase-activating proteins, potentiating G deactivation. To elucidate the mechanism of this mutual regulation, we measured the thermodynamics and kinetics of PLC-β3 binding to Gα FRET and fluorescence correlation spectroscopy, two physically distinct methods, both yielded values of about 200 nm for PLC-β3-Gα binding. This is 50-100 times greater than the EC for Gα-mediated PLC-β3 activation and for the Gα GTPase-activating protein activity of PLC-β. The measured was not altered either by the presence of phospholipid vesicles, phosphatidylinositol 4,5-bisphosphate and Ca, or by the identity of the fluorescent labels. FRET-based kinetic measurements were also consistent with a of 200 nm We determined that PLC-β3 hysteresis, whereby PLC-β3 remains active for some time following either Gα-PLC-β3 dissociation or PLC-β3-potentiated Gα deactivation, is not sufficient to explain the observed discrepancy between EC and These results indicate that the mechanism by which Gα and PLC-β3 mutually regulate each other is far more complex than a simple, two-state allosteric model and instead is probably kinetically determined.