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

Punya Navaratnarajah, Anne Gershenson, Elliott M. Ross

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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-bisphos-phate 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 Gq. FRET and fluorescence correlation spectroscopy, two physically distinct methods, both yielded Kd values of about 200 nM for PLC-3–Gq binding. This Kd is 50 –100 times greater than the EC50 for Gq-mediated PLC-3 activation and for the Gq 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 Gq–PLC-3 dissociation or PLC-3–potentiated Gq deactivation, is not sufficient to explain the observed discrepancy between EC50 and Kd. These results indicate that the mechanism by which Gq and PLC-3 mutually regulate each other is far more complex than a simple, two-state allosteric model and instead is probably kinetically determined.

Original languageEnglish (US)
Pages (from-to)16787-16801
Number of pages15
JournalJournal of Biological Chemistry
Volume292
Issue number40
DOIs
StatePublished - Oct 6 2017

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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