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. 2018 Apr 27;293(17):6387-6397.
doi: 10.1074/jbc.RA118.002354. Epub 2018 Mar 13.

G Protein βγ Subunits Directly Interact With and Activate Phospholipase Cϵ

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Free PMC article

G Protein βγ Subunits Directly Interact With and Activate Phospholipase Cϵ

Jerry C Madukwe et al. J Biol Chem. .
Free PMC article

Abstract

Phospholipase C (PLC) enzymes hydrolyze membrane phosphatidylinositol 4,5 bisphosphate (PIP2) and regulate Ca2+ and protein kinase signaling in virtually all mammalian cell types. Chronic activation of the PLCϵ isoform downstream of G protein-coupled receptors (GPCRs) contributes to the development of cardiac hypertrophy. We have previously shown that PLCϵ-catalyzed hydrolysis of Golgi-associated phosphatidylinositol 4-phosphate (PI4P) in cardiac myocytes depends on G protein βγ subunits released upon stimulation with endothelin-1. PLCϵ binds and is directly activated by Ras family small GTPases, but whether they directly interact with Gβγ has not been demonstrated. To identify PLCϵ domains that interact with Gβγ, here we designed various single substitutions and truncations of WT PLCϵ and tested them for activation by Gβγ in transfected COS-7 cells. Deletion of only a single domain in PLCϵ was not sufficient to completely block its activation by Gβγ, but blocked activation by Ras. Simultaneous deletion of the C-terminal RA2 domain and the N-terminal CDC25 and cysteine-rich domains completely abrogated PLCϵ activation by Gβγ, but activation by the GTPase Rho was retained. In vitro reconstitution experiments further revealed that purified Gβγ directly interacts with a purified fragment of PLCϵ (PLCϵ-PH-RA2) and increases PIP2 hydrolysis. Deletion of the RA2 domain decreased Gβγ binding and eliminated Gβγ stimulation of PIP2 hydrolysis. These results provide first evidence that Gβγ directly interacts with PLCϵ and yield insights into the mechanism by which βγ subunits activate PLCϵ.

Keywords: G protein; Ras homolog gene family; Ras protein; member A (RhoA); phosphatidylinositol signaling; phospholipase C.

Conflict of interest statement

The authors declare that they have no conflicts of interest regarding the contents of this article

Figures

Figure 1.
Figure 1.
Regulation of PLCϵ by G proteins. A, PLCϵ WT and corresponding domain boundaries are illustrated. B, COS-7 cells were transfected with PLCϵ (300 ng) in the presence or absence of varying concentrations of Gβ1 and Gγ2 plasmids. Total [3H]inositol phosphate accumulation was quantified as described under “Experimental Procedures.” The data shown are mean ± S.E. for triplicate samples. The experiment was done thrice with similar results. C, effect of Gαi1 on PLCϵ activation by Gβγ. PLCϵ (300 ng) was co-transfected with Gβ1 (200 ng) and Gγ2 (200 ng) in the presence or absence of Gαi1 (200 ng). The data shown are mean ± S.E. for three independent experiments and analyzed by one-way ANOVA with Dunnett's post test. *, p < 0.05 versus Gβγ.
Figure 2.
Figure 2.
RA2 domain deletion and mutations does not completely inhibit PLCϵ activation by Gβγ. A, schematic of PLCϵ WT, PLCϵ Lys-Glu (K2150/2152E), and PLCϵ ΔRA2 (1–2113) constructs. B, a representative Western blot showing expression of the PLCϵ constructs. C, COS-7 cells were transfected with 300 ng PLCϵ WT or PLCϵ Lys-Glu in the presence or absence of 200 ng Gβ1 and 200 ng Gγ2, or constitutively active small GTPases (RhoG14V (200 ng) and RasG12V (100 ng)) and total [3H]inositol phosphate accumulation was measured. D, COS-7 cells were transfected with PLCϵ WT or PLCϵ ΔRA2 in the presence or absence of 200 ng Gβ1 and 200 ng Gγ2 or constitutively active small GTPases (RhoG14V (200 ng) and RasG12V (100 ng)) and total [3H]inositol phosphate accumulation was measured. The data shown are mean ± S.E. for at least three independent experiments and analyzed by one-way ANOVA with Dunnett's post test. *, p < 0.05 versus Gβγ; ns, not significant.
Figure 3.
Figure 3.
N-terminal domain deletions do not completely inhibit PLCϵ activation by Gβγ. A, schematic of PLCϵ WT, CDC25-RA2 (394–2281), PH-RA2 (837–2281), and EF-RA2 (1198–2281) constructs. B, a representative Western blot showing expression of the PLCϵ constructs. C, COS-7 cells were transfected with PLCϵ WT or CDC-RA2 (300 ng) in the presence or absence of 200 ng Gβ1 and 200 ng Gγ2 or constitutively active Rho (RhoG14V) (200 ng) and total [3H]inositol phosphate accumulation was measured. D, COS-7 cells were transfected with 300 ng WT PLCϵ or PH-RA2 in the presence or absence of 200 ng Gβ1 and 200 ng Gγ2 or 200 ng RhoG14V and total [3H]inositol phosphate accumulation was measured. E, COS-7 cells were transfected with PLCϵ WT or EF-RA2 (300 ng) in the presence or absence of 200 ng Gβ1 and 200 ng Gγ2 or 200 ng RhoG14V and total [3H]inositol phosphate accumulation was measured. The data shown are mean ± S.E. for at least three independent experiments and analyzed by one-way ANOVA with Dunnett's post test. **, p < 0.01; *, p < 0.05 versus Gβγ.
Figure 4.
Figure 4.
Simultaneous deletion of the N-terminal and the RA2 domains of PLCϵ blocks activation by Gβγ. A, schematic of PLCϵ WT, CDC25-RA1 (394–2113), PH-RA1 (837–2113), and EF-RA1 (1198–2113) constructs. B, a representative Western blot showing relative expression of the PLCϵ constructs. C, COS-7 cells were transfected with 300 ng PLCϵ WT or CDC25-RA1 in the presence or absence of 200 ng Gβ1 and 200 ng Gγ2 or 200 ng RhoG14V and total [3H]inositol phosphate accumulation was measured. D, COS-7 cells were transfected with 300 ng PLCϵ WT or PH-RA1 in the presence or absence of 200 ng Gβ1 and 200 ng Gγ2 or 200 ng RhoG14V and total [3H]inositol phosphate accumulation was measured. E, COS-7 cells were transfected with 300 ng PLCϵ WT or EF-RA1 in the presence or absence of 200 ng Gβ1 and 200 ng Gγ2 or 200 ng RhoG14V and total [3H]inositol phosphate accumulation was measured. The data shown are mean ± S.E. for at least three independent experiments and analyzed by one-way ANOVA with Dunnett's post test. **, p < 0.01; *, p < 0.05 versus Gβγ; ns, not significant.
Figure 5.
Figure 5.
Constitutive membrane localization of PLCϵ EF-RA1 rescues activation by Rho but not Gβγ. A, schematic of EF-RA1 (1198–2113) and EF-RA1 CAAX (contains CAAX tag from Kras4B) constructs. B, a representative Western blot showing expression of the PLCϵ constructs. C, COS-7 cells were transfected with 300 ng EF-RA1 or EF-RA1 CAAX in the presence or absence of 200 ng Gβ1 and 200 ng Gγ2 or 200 ng RhoG14V and total [3H]inositol phosphate accumulation was measured. The data shown are mean ± S.E. for at least three independent experiments and analyzed by one-way ANOVA with Dunnett's post test. **, p < 0.01; *, p < 0.05 versus Gβγ; #, p < 0.05 verses EF-RA1; ns; not significant.
Figure 6.
Figure 6.
PLCϵ-PH-RA2 directly binds to Gβγ. A, Coomassie Blue–stained polyacrylamide gel showing purified proteins. B, PLCϵ-PH-RA2 binding to Gβγ. Biotinylated Gβ1γ2 was used to pull down PLCϵ mutants in the presence of neutravidin beads. Representative blot showing direct pulldown of PLCϵ-PH-RA2 with Gβ1γ2 and inhibition of this interaction by Gαi1-GDP. 50 ng of purified PLCϵ-PH-RA2 was mixed with purified 15 nm biotinylated Gβ1γ2 in the presence or absence of Gαi1-GDP for 2 h. 15 μl 50% magnetic neutravidin bead slurry was added and the mix was incubated for another 2 h. Pellet was washed thrice and subjected to gel electrophoresis and Western blotting. Input is 1/20 of supernatant. C, quantitation of the PLCϵ band densities from three experiments as in B (PH-RA2 binding was normalized to 100%). The data shown are mean ± S.E. and analyzed by Student's t test. ***, p < 0.001 versus PH-RA2 binding in the absence of Gαi1-GDP. D, 300 nm1γ2 alone or with 600 nmi1-GDP was incubated with 10 ng PLCϵ PH-RA2. The effect on PLCϵ mutant activity is shown. Values are the mean ± S.E. of triplicate determinations and representative of three independent experiments and analyzed by one-way ANOVA with Dunnett's post test.
Figure 7.
Figure 7.
Deletion of the PLCϵ RA2 domain inhibits binding and activation by Gβγ. A, schematic of PLCϵ RA domain mutants. B, representative Western blot showing direct pulldown of PLCϵ-PH-RA2 but not PH-RA1 by biotinylated Gβ1γ2. Input is 1/10 of supernatant. C, PLCϵ band densities are quantitated from three independent experiments as in B. The data shown are mean ± S.E. *, p < 0.05 versus PH-RA2 and analyzed by Student's t test. D, representative assay of PLC enzymatic activity for the indicated purified PLCϵ protein fragments reconstituted with the indicated concentrations of purified Gβγ subunits performed in triplicate, repeated three times with similar results. The data shown are mean ± S.E. and analyzed by one-way ANOVA with Dunnett's post test. *, p < 0.05 versus basal PLCϵ activity.
Figure 8.
Figure 8.
Model of PLCϵ activation by Gβγ. A, in the first scenario, free Gβγ interacts with and activates PLCϵ by binding to the N-terminal CDC25 domain and the C-terminal RA2 domain at two independent sites. Interaction with both sites is required for full activation. B, alternatively, the CDC25 and RA2 domains could be in close proximity to one another in the three-dimensional structure of PLCϵ, forming a single binding site for Gβγ.

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