The binding of activated G?q to phospholipase C-? exhibits anomalous affinity.

ABSTRACT: Upon activation by the Gq family of G? subunits, G?? subunits, and some Rho family GTPases, phospholipase C-? (PLC-?) isoforms hydrolyze phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC-? 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-?3 binding to G?q FRET and fluorescence correlation spectroscopy, two physically distinct methods, both yielded Kd values of about 200 nm for PLC-?3-G?q binding. This Kd 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 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-?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 Kd 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.

SUBMITTER: Navaratnarajah P 

PROVIDER: S-EPMC5633138 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature