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. 2016 Dec 2;291(49):25608-25616.
doi: 10.1074/jbc.M116.740407. Epub 2016 Oct 14.

Potent and Selective Peptide-based Inhibition of the G Protein Gαq

Thomas H Charpentier  1   2 , Gary L Waldo  1 , Emily G Lowery-Gionta  1 , Krzysztof Krajewski  3   4 , Brian D Strahl  3   4 , Thomas L Kash  1 , T Kendall Harden  1 , John Sondek  5   2   3
Affiliations

Potent and Selective Peptide-based Inhibition of the G Protein Gαq

Thomas H Charpentier et al. J Biol Chem. .

Abstract

In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gαq binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gαq within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gαq in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gαq A representative peptide was specific for active Gαq because it did not bind inactive Gαq or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ1γ2 In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gαq; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gαq in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gαq-dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gαq in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gαq in cells.

Keywords: G protein; inhibitor; p63; peptide interaction; phospholipase C.

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Figures

FIGURE 1.
FIGURE 1.
Gαq uses a conserved mechanism to engage effectors. A, a helix-turn-helix (Hα1/Hα2, cylinders) in either PLC-β3 (blue) or p63RhoGEF (yellow) engages a shallow groove on activated Gαq (green with switch regions in red) between switch II and α3. The structures of Gαq bound to either PLC-β3 (PDB code 3OHM) (30) or p63RhoGEF (PDB code 2RGN) (31) were superimposed using Gαq. For clarity, only a single Gαq is shown. The expanded region highlights residues in PLC-β3 that directly contact Gαq; contacts boxed in the corresponding sequence alignment. C-term, C terminus; N-term, N terminus. B and C, peptides derived from the helix-turn-helix of PLC-β3 selectively bind active Gαq. B, fluorescence polarization (ΔmP, change in millipolarization) was used to measure the binding of the indicated Gα subunits to either a TAMRA-labeled peptide (27-mer(I860A)) derived from the helix-turn-helix of PLC-β3 or the equivalent peptide also containing L859E (27-mer(L859E+I860A)). All Gα subunits were activated with aluminum fluoride except where indicated (no AlF4). C, the above assay was used to measure affinities of active Gαq for the TAMRA-labeled peptides listed. Substitutions include I860A (red) and M869Nle (underlined in red).
FIGURE 2.
FIGURE 2.
Competition assay to measure affinities of peptides and effectors for Gαq. A and B, individual unlabeled peptides were titrated into a solution of Gαq bound to TAMRA-27-mer(I860A), and fluorescence polarization was measured. Ki values were calculated using an equation to convert IC50 to Ki values in fluorescence-based competition assays (38). The sequence alignment is annotated as in Fig. 1. Inset, circular dichroism spectra for select peptides. Substitutions include I860A (red) and M869Nle (underlined in red). C, competition assay used to measure affinities of the indicated effectors for Gαq activated with aluminum fluoride.
FIGURE 3.
FIGURE 3.
27-mer(I860A) inhibits active Gαq in lipid vesicles. A and B, lipid vesicles containing [3H]PIP2 were reconstituted with PLC-β3 and Gαq prior to measuring hydrolysis of PIP2 in the presence of increasing concentrations of the indicated peptides. 120 nm Gαq was used in B. C, TAMRA-27-mer(I860A) did not inhibit the capacity of Gβ1γ2 to activate PLC-β3 in lipid vesicles but prevented synergy with Gαq.
FIGURE 4.
FIGURE 4.
Peptides derived from the helix-turn-helix of PLC-β3 inhibit the capacity of Gαq to activate p63RhoGEF. RhoA (100 nm) preloaded with BODIPY-GDP was incubated with unlabeled GDP (2 μm), Gαq (100 nm), aluminum fluoride, and the indicated amounts of 27-mer(I860A) prior to measuring nucleotide exchange using fluorescence (cps, counts per second). p63RhoGEF (100 nm) was added as indicated. The trace marked RhoA only contains no Gαq or p63RhoGEF and indicates spontaneous nucleotide exchange.
FIGURE 5.
FIGURE 5.
Gαq-dependent PLC-β3 activity in HEK293 cells is specifically inhibited by genetically encoded versions of the helix-turn-helix of PLC-β3. A–C, HEK293 cells were co-transfected with expression vectors encoding the 5HT2A receptor (100 ng) and increasing amounts of PLC-β3 (A), YFP-27mer-CFP (B), or YFP-27mer-CFP-CAAX (C) variants prior to metabolic labeling of inositide pools, receptor activation with DOI (2 μm), and quantification of inositol phosphates. Protein expression and cellular totals (actin) were verified with Western blotting as shown. Cells without addition of agonist (−DOI) were also tested.
FIGURE 6.
FIGURE 6.
27-mer(I860A) inhibits Gαq-mediated neuronal depolarization in the prefrontal cortex of mice. A, pretreatment of a representative prefrontal cortex neuron with 27-mer(L859E+I860A) had no effect on action potential firing in response to CCh (10 μm) (top panel). In contrast, depolarization was lost when 27-mer(I860A) was used (center panel) or the M1 receptor antagonist pirenzepine (2 μm) was applied with 27-mer(L859E+I860A) (bottom panel). B, the inward current induced by carbachol after indicated pretreatments. Sample sizes included six neurons for the active and inactive peptides and four neurons for the active peptide with agonist. *, p < 0.05 from baseline. **, p < 0.05 between the different experiments n.s., not significant.
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