TY - JOUR
T1 - The binding of activated Gq to phospholipase C- exhibits anomalous affinity
AU - Navaratnarajah, Punya
AU - Gershenson, Anne
AU - Ross, Elliott M.
N1 - Funding Information:
This work was supported by National Institutes of Health Grants R01GM030355 and R21DC015744 (to E. M. R.) and R01GM060418 (to A. G.). The authors declare that they have no conflicts of interest with the con-tents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Insti-tutes of Health.
Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
PY - 2017/10/6
Y1 - 2017/10/6
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M117.809673
DO - 10.1074/jbc.M117.809673
M3 - Article
C2 - 28842497
AN - SCOPUS:85030769852
SN - 0021-9258
VL - 292
SP - 16787
EP - 16801
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 40
ER -