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. Author manuscript; available in PMC: 2011 Nov 12.
Published in final edited form as: Science. 2010 Oct 21;330(6006):974–980. doi: 10.1126/science.1193438

Fig. 5.

Fig. 5

Contribution of the EF hand region to GAP activity of PLC-β3. (A) The GAP activity of purified wild-type PLC-β3 is compared with that of mutant PLC-β3 isozymes. Steady-state GTP hydrolysis was quantified with phospholipid vesicles reconstituted with purified P2Y1 receptor, Gαq, and Gβ1γ2. Assays were in the presence of the P2Y1 receptor agonist 2MeSADP (3 μM) and the indicated concentrations of purified PLC-β3; PLC-β3(δEF); PLC-β3(V262A); or PLC-β3(N260A), PLC-β3(N260G), or PLC-β3(N260S) [all designated as PLC-β3(N260*)] as described in (41). Data are plotted as percent of maximal response obtained with PLC-β3. Data are mean ± SEM of three experiments. (B) Deficiency in termination of Gαq-stimulated PLC activity of a GAP-deficient mutant of PLC-β3. PLC activity was quantified with [3H]PtdIns(4,5)P2-containing phospholipid vesicles reconstituted with purified P2Y1 receptor, Gαq, and Gβ1γ2. Vesicles were incubated with 300 nM PLC-β3 or PLC-β3(δEF) in the absence (open circles) or presence of the P2Y1 receptor agonist 2MeSADP (300 nM; black squares) and either 30 μM GTP or 100 nM GTPγS for 90 s before addition of P2Y1 receptor antagonist MRS2500 (50 μM; red squares) or vehicle. Incubations were continued for an additional 165 s. Data are plotted as percent of the maximal response observed with either PLC-β3 or PLC-β3(δEF) in the presence of agonist plus GTPγS. (C) Delayed termination of the photoresponse in Drosophila expressing a GAP-deficient mutant of PLC-β. Electro-retinograms from flies harboring wild-type PLC-β (NORPA, blue) or a mutant form (NORPAN262A, red) deficient in capacity to accelerate the GTPase activity of Gαq. Flies ~1 day posteclosion were dark-adapted for 2 min before exposure to 5-s pulses of orange light indicated by the event marker below each electroretinogram. At right are plotted deactivation rates and maximal amplitudes for the average of ten individual electroretinograms. Error bars indicate SEM. Expression of the norpA transgenes was confirmed by immunoblot (gel) of head extracts prepared from flies ~1 day posteclosion. (D) GAP activity of a mutant of PLC-β found in pancreatic cancer. PLC-β3 was mutated at a position (R258) (21) equivalent to a homozygous substitution identified in PLC-β4 during genome-wide profiling of pancreatic cancers (30). GAP activity of PLC-β3(R258Q) was compared with that of PLC-β3 as described in (A) above. Data are mean ± SEM of three experiments.