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

Upon activation by the Gq 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 Gq deactivation. To elucidate the mechanism of this mutual regulation, we measured the thermodynamics and kinetics of PLC-β3 binding to Gαq FRET and fluorescence correlation spectroscopy, two physically distinct methods, both yielded Kd values of about 200 nm for PLC-β3-Gαq binding. This Kd is 50-100 times greater than the EC50 for Gαq-mediated PLC-β3 activation and for the Gαq GTPase-activating protein activity of PLC-β. The measured Kd 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 Kd of 200 nm We determined that PLC-β3 hysteresis, whereby PLC-β3 remains active for some time following either Gαq-PLC-β3 dissociation or PLC-β3-potentiated Gαq deactivation, is not sufficient to explain the observed discrepancy between EC50 and Kd These results indicate that the mechanism by which Gαq and PLC-β3 mutually regulate each other is far more complex than a simple, two-state allosteric model and instead is probably kinetically determined.