Abstract
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.
Highlights
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
We developed a FRET-based binding assay to measure the thermodynamics and kinetics of PLC-3 binding to G␣q
We first describe binding measured using a single G␣q–PLC-3 FRET pair in which cysteine residues introduced in place of Glu249 in G␣q and Gln871 in PLC-3 were labeled with Alexa Fluor 488 and 594 maleimide, respectively
Summary
To study the mechanism of PLC- activation by G␣q and the Gq GAP activity of PLC-, we measured the equilibrium binding of the two proteins and their rates of association and dissociation in comparison with their functional interactions. The data were donor quenching fit to a single-site in the presence of binding equation, yielding a Kd of 160 Ϯ 50 nM In these experiments, G␣q-F was 42% nM G␣q-F–GTP␥S (closed circles) was measured in the presence activated, and of PE/PS/PIP2. In an attempt to approximate the experiment of Runnels and Scarlata [25], we measured FRET from G␣q-F to wild-type PLC-3 that was uniformly labeled (14 cysteine residues) with Alexa Fluor 594 maleimide For this pair, Kd in the presence of PC/PE/PS (1:1:1) vesicles was 560 Ϯ 110 nM (n ϭ 2; supplemental Fig. S1D). Donor quenching was followed upon the addition of PLC-3-Q (150, 300, or 450 nM) to GTP␥S-activated G␣q-F (5 or 25 nM) at 25 °C in the presence of 250 M PE/PS (4:1) vesicles and no added Ca2ϩ (Fig. 5A). This difference alone is not slow enough to account for the nearly 100-fold difference between EC50 and Kd for the interaction of G␣q and PLC-3 but could partially explain the discrepancy between the EC50 and Kd
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